DECIPHERING THE GENETIC BASIS OF SOLANUM CHACOENSE MEDIATED COLORADO POTATO BEETLE (LEPTINOTARSA DECEMLINEATA) RESISTANCE AND SELF-FERTILITY IN A DIPLOID SOLANUM CHACOENSE RECOMBINANT INBRED LINE POPULATION By Natalie Kaiser A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Breeding, Genetics and Biotechnology—Crop and Soil Sciences—Doctor of Philosophy 2021 ABSTRACT DECIPHERING THE GENETIC BASIS OF SOLANUM CHACOENSE MEDIATED COLORADO POTATO BEETLE (LEPTINOTARSA DECEMLINEATA) RESISTANCE AND SELF-FERTILITY IN A DIPLOID SOLANUM CHACOENSE RECOMBINANT INBRED LINE POPULATION By Natalie Kaiser The Colorado potato beetle (Leptinotarsa decemlineata) is the most widespread and destructive insect defoliator pest of potato and its control has historically been achieved through the use of insecticide. The diploid potato species Solanum chacoense has been utilized for over four decades in an attempt to introgress glycoalkaloid-based insect resistance into cultivated tetraploid potato. Despite these efforts, insect resistant cultivars have not been achieved, due in part to the complex genetics underlying the trait. The creation of inbred diploid lines would allow more efficient examination and deployment of this economically important trait. We introduced self-compatibility into diploid insect resistant S. chacoense germplasm and developed the first potato recombinant inbred line (RIL) population to study, understand and deploy this mechanism of host-plant insect resistance in cultivated, diploid breeding lines. We first examined the genetic features underlying leptine glycoalkaloid mediated Colorado potato beetle host plant resistance in the F2 generation derived from a cross between S. chacoense lines USDA8380-1 (80-1) and M6. Using biparental linkage mapping, a major overlapping QTL region with dominant effects was identified on chromosome 2 explaining 49.3% and 34.1% of the variance in Colorado potato beetle field resistance and leptine accumulation, respectively. Bulk segregant whole genome sequencing of the same F2 population detected QTL associated with Colorado potato beetle resistance on chromosomes 2, 4, 6, 7, and 12. Candidate genes within these QTL regions were identified by weighted gene co-expression network analysis of parental lines and resistant and susceptible F2 individuals. Second, we exploited M6-mediated self-compatibility and established vigorous, F5 inbred diploid lines to further examine loci associated with Colorado potato beetle resistance and explore the practicality of inbreeding in diploid potato. F5 inbred lines carrying Colorado potato beetle resistance equivalent to the resistant donor parent were created without field selection during the inbreeding process. We report that the ratio of acetylated to non-acetylated glycoalkaloids measured under greenhouse conditions is a powerful metabolite marker to predict field performance without incurring the costs of conducting a Colorado potato beetle field trial. Leptine production was successfully introduced into diploid breeding germplasm. Single nucleotide polymorphism (SNP) genotyping coupled with stylar analysis of pollen tube growth and self- fertility phenotyping of the F4 and F5 generations revealed that multiple factors mediate the self- compatible response in this RIL population. Third, we assessed the initial transcriptional and metabolite response to Colorado potato beetle herbivory in beetle resistant and beetle susceptible S. chacoense lines over a 48-hour time course. To facilitate genome editing modification of the leptine biosynthesis pathway, we characterized the allelic variation between S. chacoense 80-1 and M6 in a candidate leptine biosynthesis gene identified by transcriptional profiling. This work highlights the challenges of establishing inbred germplasm, reinforces the complexity of selecting for self-fertility in diploid potato, and lays the foundation for optimization of potato RIL development. The availability of highly homozygous Colorado potato beetle resistant lines will enable further genomic inquiry of the loci contributing to this trait and will facilitate rapid deployment of beetle resistant diploid potato varieties. ACKNOWLEDGEMENTS I would like to thank my primary advisor Dr. David Douches who embodies the ideal mentor and is a supreme role model in his strength of character. He is truly an extraordinary individual and it has been my privilege, pleasure, and honor to receive his guidance. I am immensely grateful to the members of my guidance committee Dr. Robin Buell, Dr. Christina DiFonzo, Dr. Robert Last for dedicating their time and counsel to develop my scientific and professional competence. I would also like to extend my sincere gratitude to Dr. Courtney Hollender for kindly evaluating my thesis and participating in my thesis defense as a proxy committee member. My heartfelt gratitude goes out to the members of the Douches Potato Breeding and Genetics Program who all made Michigan feel like home during my tenure at Michigan State University. I would like to thank Dr. Felix Enciso for genome editing, comparative genomics and molecular biology instruction and for his enduring friendship. I am grateful to Dr. Norma Manrique-Carpintero for her mentorship in glycoalkaloid metabolism and genetic analysis. I am most grateful for Joe Coombs who bequeathed to me his wealth of Colorado potato beetle knowledge and instilled in me the skills to critically analyze data. I would like to thank Greg Steere, Matt Zuehlke, Nick Garrity and Azamat Sardarbekov for patiently instructing me in the technical aspects of the breeding program and providing the most cheerful and expert assistance in countless experiments. I am grateful to Sylvia Morse for her comprehensive tissue culture instruction. I wish to thank Dr. Daniel Zarka for always offering invaluable counsel on matters of molecular biology and experimental design. I am especially grateful for the friendship and advice of Kelly Zarka who iv was the most excellent officemate during my graduate career. I would like to extend my gratitude to Kate Shaw and Chen Zhang for their support in laboratory, greenhouse and field experiments but also for their camaraderie that made me look forward to each day at work. I am grateful for the peer mentorship and friendship of past and present graduate students at Michigan State University Dr. Maher Alsahlany, Dr. Ben Mansfeld, Thilani Jayakody and William Behling. I would like to also thank Anthony Schilmiller, Assistant Core Manager of the Michigan State University Mass Spectrometry and Metabolomics Core facility, for his guidance on methods. I would like to thank Dr. Shelley Jansky, who exemplifies the scientist and geneticist I aspire to be, for her counsel in the projects comprising this dissertation. I would like to thank the Michigan State University Plant, Soil and Microbial Department, Michigan AgBioResearch, and the United States Department of Agriculture for supporting my research projects. I am enormously grateful for my husband, Luca Kaiser, for his endless support and encouragement. Finally, I would like to thank my parents Mary and Jonathan Kirkwyland for igniting in me a passion for plant cultivation and equipping me with the skills to pursue a career in this field. v TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES ...................................................................................................................... xv KEY TO ABBREVIATIONS ....................................................................................................... xx CHAPTER 1 ................................................................................................................................... 1 INTRODUCTION .......................................................................................................................... 1 Potato, a world food crop ............................................................................................................ 1 Potato production is hampered by the Colorado potato beetle ................................................... 1 Remarkable adaptability of the Colorado potato beetle results in widespread insecticide resistance ..................................................................................................................................... 2 Exploiting host plant resistance to the Colorado potato beetle in wild diploid germplasm ....... 3 Diploid potato breeding offers unprecedented opportunities ...................................................... 4 Dissertation Organization and Objectives .................................................................................. 5 REFERENCES ........................................................................................................................... 8 CHAPTER 2 ................................................................................................................................. 14 THE ROLE OF CONVENTIONAL PLANT BREEDING IN ENSURING SAFE LEVELS OF NATURALLY OCCURRING TOXINS IN FOOD CROPS ....................................................... 14 Abstract ..................................................................................................................................... 15 Introduction ............................................................................................................................... 16 Conventional Breeding Practices Used by Plant Breeders ................................................... 17 Naturally occurring plant toxins in food crops ..................................................................... 22 Crop category 1: Crop plants with no significant plant-produced toxins ................................. 23 Case Study: Maize ................................................................................................................ 24 Crop Category 2: Crop plants with known plant-produced natural toxins ............................... 27 Crop plants with allergenicity potential ................................................................................ 27 Crops with known toxins in the non-consumed portion ....................................................... 28 Case Study: Apple ................................................................................................................. 29 Crop plants with plant-produced toxins in the consumed portion can broadly affect human health ......................................................................................................................................... 36 Case study: Potato ................................................................................................................. 38 Conclusion ................................................................................................................................ 50 APPENDICES .......................................................................................................................... 51 APPENDIX A: Chapter 2 Tables ............................................................................................. 52 APPENDIX B: Chapter 2 Figures ............................................................................................ 53 APPENDIX C: Chapter 2 Copyright Permission ..................................................................... 58 REFERENCES ......................................................................................................................... 59 CHAPTER 3 ................................................................................................................................. 79 vi MAPPING SOLANUM CHACOENSE MEDIATED COLORADO POTATO BEETLE (LEPTINOTARSA DECEMLINEATA) RESISTANCE IN A SELF‑COMPATIBLE F2 DIPLOID POPULATION ............................................................................................................................. 79 Abstract ..................................................................................................................................... 80 Introduction ............................................................................................................................... 81 Materials and Methods .............................................................................................................. 84 Plant Material ........................................................................................................................ 84 Developmental Resistance Profiling of Parental Lines ........................................................ 85 Glycoalkaloid Analysis ......................................................................................................... 86 Field Trial Colorado Potato Beetle Phenotyping .................................................................. 87 SNP Genotyping and Linkage Analysis ............................................................................... 90 Phenotypic Validation of Colorado Potato Beetle Resistance Extremes in vitro ................. 91 Whole Genome Sequencing Bulk Segregant Analysis ......................................................... 93 Gene Expression of Colorado Potato Beetle Resistance Extremes ...................................... 95 Molecular Marker Development and fine mapping of QTL region ..................................... 96 Results ....................................................................................................................................... 98 S. chacoense Colorado potato beetle resistance is tissue and age-dependent ....................... 98 Phenotypic Evaluation of the S. chacoense F2 Population ................................................... 98 Linkage Map Construction ................................................................................................... 99 Identification of QTL Associated with Colorado Potato Beetle Field Resistance and Leptine Accumulation ...................................................................................................................... 100 Validation of the F2 Colorado Potato Beetle Resistance Phenotypic Extremes ................. 101 Detection of QTL Associated with Colorado Potato Beetle Resistance by BSA-Seq ........ 102 Fine Mapping of Candidate QTL on Chromosome 2 ......................................................... 102 Identification of Candidate Genes Within the QTL Region on Chromosome 2 ................. 103 Discussion ............................................................................................................................... 106 S. chacoense Colorado Potato Beetle Resistance is Tissue and Age-Dependent ............... 106 Phenotypic Evaluation of the S. chacoense F2 Population ................................................. 107 Genotyping the F2 Population and Distorted Segregation Analysis ................................... 108 QTL and candidate gene identification ............................................................................... 110 APPENDICES ........................................................................................................................ 113 APPENDIX A: Chapter 3 Tables ........................................................................................... 114 APPENDIX B: Chapter 3 Figures .......................................................................................... 118 APPENDIX C: Chapter 3 Supplementary Data ...................................................................... 124 APPENDIX D: Chapter 3 Copyright Permission ................................................................... 222 APPENDIX E: Tuber Yield .................................................................................................... 223 REFERENCES ....................................................................................................................... 231 CHAPTER 4 ............................................................................................................................... 244 ASSESSING THE CONTRIBUTION OF SLI TO SELF-COMPATIBILITY IN NORTH AMERICAN DIPLOID POTATO GERMPLASM USING KASPTM MARKERS ................... 244 Abstract ................................................................................................................................... 245 Introduction ............................................................................................................................. 246 Materials and Methods ............................................................................................................ 248 Plant Material ...................................................................................................................... 248 DNA Isolation and KASPTM Genotyping ........................................................................... 250 SNP genotyping .................................................................................................................. 250 vii Self-compatibility Phenotyping .......................................................................................... 251 Data analysis ....................................................................................................................... 251 Results ..................................................................................................................................... 252 KASPTM Haplotypes of Eight SC diploids ......................................................................... 252 KASPTM Analysis of a Diploid Recurrent Selection Population ........................................ 253 Connecting SNP genotyping to self-compatibility phenotyping ........................................ 254 Sli Alleles in S. tuberosum Dihaploids ............................................................................... 255 KASPTM Analysis of a Diploid Backcross Population ....................................................... 255 Discussion ............................................................................................................................... 256 Self-compatibility Donors without Sli SC haplotypes ........................................................ 256 Sli Linkage to a Deleterious Allele ..................................................................................... 257 Sli Contributes to Self-compatibility in a Diploid Recurrent Selection Population ........... 257 Sli Contribution to Self-compatibility in Dihaploids Extracted from Cultivated Potato .... 259 Challenges of Increasing Sli-based Self-compatibility in a Backcross Population ............ 260 Prospects of Improving Self-fertility in Diploid Potato ...................................................... 260 Conclusion .............................................................................................................................. 260 APPENDICES ........................................................................................................................ 262 APPENDIX A: Chapter 4 Tables ........................................................................................... 263 APPENDIX B: Chapter 4 Figures .......................................................................................... 265 APPENDIX C: Chapter 4 Supplementary Data ...................................................................... 268 APPENDIX D: Chapter 4 Copyright Permissions.................................................................. 295 REFERENCES ....................................................................................................................... 296 CHAPTER 5 ............................................................................................................................... 301 SELF-FERTILITY AND RESISTANCE TO THE COLORADO POTATO BEETLE (LEPTINOTARSA DECEMLINEATA) IN A DIPLOID SOLANUM CHACOENSE RECOMBINANT INBRED LINE POPULATION ................................................................... 301 Abstract ................................................................................................................................... 302 Introduction ............................................................................................................................. 302 Materials and Methods ............................................................................................................ 305 Plant material ...................................................................................................................... 305 Self-fertility phenotyping .................................................................................................... 306 Glycoalkaloid analysis ........................................................................................................ 308 Colorado potato beetle resistance phenotyping .................................................................. 310 Single Nucleotide Polymorphism (SNP) genotyping ......................................................... 311 Linkage Analysis of F4 individuals ..................................................................................... 312 Statistical analysis and SNP marker trait association ......................................................... 313 Distorted segregation analysis ............................................................................................ 315 Heterozygosity retention analysis ....................................................................................... 315 Sli KASP marker genotyping .............................................................................................. 316 Results ..................................................................................................................................... 316 Producing the F4 and F5 generation ..................................................................................... 316 Characterizing self-fertility traits ........................................................................................ 317 Foliar glycoalkaloid content of greenhouse grown plants .................................................. 319 Colorado potato beetle field defoliation resistance ............................................................. 319 Glycoalkaloid content of foliar and tuber tissue of field-grown plants .............................. 320 V3 22K SNP Array genotyping of the RIL population ...................................................... 320 viii SNP marker loci associated with self-fertility .................................................................... 321 Sli KASPTM marker genotyping .......................................................................................... 322 SNP marker loci associated with glycoalkaloid content ..................................................... 322 SNP marker loci associated with Colorado potato beetle resistance .................................. 323 Genetic map construction ................................................................................................... 324 V4 32K SNP Array genotyping of parental lines ............................................................... 324 Heterozygosity retention ..................................................................................................... 325 Distorted segregation .......................................................................................................... 325 Discussion ............................................................................................................................... 327 Creating diploid inbred lines for use in potato breeding ..................................................... 327 Self-fertility ......................................................................................................................... 328 Utility of RILs in potato genetics ........................................................................................ 329 Colorado potato beetle host plant resistance in F5 inbred lines .......................................... 331 Introducing Colorado potato beetle resistance to diploid breeding lines ............................ 332 Limitations of utilizing the SNP Array platform to genotype small populations ............... 332 Conclusion .............................................................................................................................. 333 APPENDICES ........................................................................................................................ 335 APPENDIX A: Chapter 5 Tables ........................................................................................... 336 APPENDIX B: Chapter 5 Figures .......................................................................................... 341 APPENDIX C: Chapter 5 Supplementary Data ...................................................................... 349 APPENDIX D: HT-B Sequence Analysis .............................................................................. 398 APPENDIX E: S-RNase Sequence Analysis .......................................................................... 401 REFERENCES ....................................................................................................................... 404 CHAPTER 6 ............................................................................................................................... 411 CHARACTERIZING THE TRANSCRIPTIONAL AND GLYCOALKALOID RESPONSE TO COLORADO POTATO BEETLE INFESTATION IN SOLANUM CHACOENSE .................. 411 Abstract ................................................................................................................................... 412 Introduction ............................................................................................................................. 412 Materials and Methods ............................................................................................................ 415 Plant Material ...................................................................................................................... 415 Whole Plant Colorado potato beetle time course bioassay ................................................. 415 RNA extraction, quantification and sequencing ................................................................. 416 Identification of allelic variation in candidate leptine biosynthesis gene ........................... 418 Results ..................................................................................................................................... 418 Whole plant Colorado potato beetle time course bioassay ................................................. 418 Identification of allelic variation in a candidate leptine biosynthesis gene ........................ 420 Discussion ............................................................................................................................... 421 Beetle infestation does not alter S. chacoense foliar glycoalkaloid profile ........................ 421 Transcriptional response to beetle infestation differs between S. chacoense genotypes .... 421 Allelic variation in a candidate leptine biosynthesis gene .................................................. 422 APPENDICES ........................................................................................................................ 423 APPENDIX A: Chapter 6 Tables ........................................................................................... 424 APPENDIX B: Chapter 6 Figures .......................................................................................... 426 APPENDIX C: Chapter 6 Supplementary Data ...................................................................... 430 REFERENCES ....................................................................................................................... 433 ix CHAPTER 7 ............................................................................................................................... 438 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS .................................................. 438 Refining the genetic landscape of glycoalkaloid-mediated host plant resistance ................... 439 Achieving sustainable Colorado potato beetle management .................................................. 440 Understanding and deploying self-fertility in diploid potato breeding ................................... 442 Opportunities to accelerate genetic gain in diploid potato ...................................................... 444 Funding ................................................................................................................................... 445 REFERENCES ....................................................................................................................... 446 x LIST OF TABLES Table 2.1. Example crops, traits and natural compounds measured for each of two proposed plant breeding crop safety categories. .................................................................................................... 52 Table 3.1. Mean, range and standard deviation (SD) of measured glycoalkaloids in the diploid Solanum chacoense USDA8380-1 x M6 F2 population (n = 233) ............................................. 114 Table 3.2. Spearman’s rank correlation coefficients among measured traits in the Solanum chacoense USDA8380-1 x M6 F2 population ............................................................................ 115 Table 3.3. Summary of single nucleotide polymorphism (SNP) marker information for individual chromosomes of the Solanum chacoense USDA8380-1 x M6 F2 population linkage map ....... 116 Table 3.4. Summary of QTLs detected by MQM in MapQTL® 6 Software in a diploid Solanum chacoense USDA8380-1 x M6 F2 population ............................................................................ 117 Table S3.1. Phenotypes of the 20 F2 individuals from the Solanum chacoense USDA8380-1 x M6 population used for bulk segregant analysis of Colorado potato beetle resistance. .................... 140 Table S3.2. Insertion/deletion (Indel) markers on chromosome 2 designed from Solanum chacoense USDA8380-1 and M6 whole genome sequence data ................................................ 141 Table S3.3. Genetic map of the Solanum chacoense USDA8380-1 x M6 F2 population. ......... 142 Table S3.4. Number of loci with distorted and expected segregation based on a Chi-square test using three thresholds of significance (5%, 1%, and 0.1%) in the 754 mapped SNPs of the Solanum chacoense USDA8380-1 x M6 F2 population ............................................................................ 172 Table S3.5. Spearman’s rank correlation coefficients among measured traits in 20 F2 individuals from the Solanum chacoense USDA8380-1 × M6 F2 population used for bulk segregant analysis of Colorado potato beetle resistance ........................................................................................... 173 Table S3.6. Significant QTL identified by G' analysis of SNPs generated from alignment to Solanum tuberosum clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03), corrected with Solanum chacoense M6 whole genome sequence data, using the QTLseqr package in R (Mansfeld BN, Grumet R (2018) QTLseqr: An R package for bulk segregant analysis with next-generation sequencing. bioRxiv:208140. https://doi.org/10.1101/208140) ................................................. 174 Table S3.7. Genes Differentially Expressed between Resistant and Susceptible Individuals using DESeq2 package. ........................................................................................................................ 175 Table S3.8. Number of genes in each of the 29 modules detected by weighted gene co-expression network analysis. ......................................................................................................................... 216 xi Table S3.9. Hub genes in the darkgrey, grey60, midnightblue and red modules detected by weighted gene co-expression analysis. ....................................................................................... 217 Table S3.10 Tuber yield of greenhouse grown plants in the Solanum chacoense F2 mapping population. .................................................................................................................................. 223 Table 4.1. KASPTM marker genotypes of eight self-compatible (SC) diploid clones at six tested marker loci (homozygous SC genotype (AA), heterozygous genotype (AB) and homozygous self- incompatible genotype (BB)) (NC = No Call). ........................................................................... 263 Table 4.2. Chi-squared likelihood ratio testing of association between the genotype at six marker loci and a self-compatible phenotype in a diploid recurrent selection population. .................... 264 Table S4.1. Pedigree and self-compatibility data of 241 lines from a diploid recurrent selection population used in this study....................................................................................................... 270 Table S4.2. Pedigree and self-compatibility data of 86 lines from a diploid backcross population used in this study. ........................................................................................................................ 282 Table S4.3. Kompetitive Allele Specific PCR Primers used in this study. ................................ 286 Table S4.4. Self-compatibility phenotype and SNP marker genotype at four SNP loci from the Illumina Infinium V1 8.3K Array in 164 recurrent selection clones .......................................... 287 Table S4.5. Self-compatibility phenotype and SNP marker genotype at eleven SNP loci from the Illumina Infinium V3 22K Array in 31 recurrent selection clones ............................................. 293 Table 5.1. Spearman’s rank correlation coefficients among measured traits in the F4 (left) and F5 (right) generations of a diploid recombinant inbred line population derived from Solanum chacoense lines USDA8380-1 and M6. ...................................................................................... 336 Table 5.2. Significant single nucleotide polymorphisms (SNPs) associated with fertility traits by Kruskal-Wallis testing in the F4 and F5 generations of a diploid recombinant inbred line population derived from Solanum chacoense lines USDA8380-1 and M6. ................................................. 337 Table 5.3. Summary of the Solanum chacoense USDA8380-1 x M6 F4 population linkage map based on 97 individuals and 288 single nucleotide polymorphism (SNP) markers ................... 338 Table 5.4. Individual mean, minimum (Min) and maximum (Max) genotype frequencies at 1,020 SNP markers segregating in the F2 (N = 236), F4 (N = 113) and F5 (N=80) generations of a Solanum chacoense recombinant inbred line population. .......................................................................... 339 Table 5.5. The number of loci exhibiting distorted segregation at the p<1e-8 level of significance in the F2, F4 and F5 generations of a Solanum chacoense recombinant inbred line population. . 340 Table S5.1. Selfed Fruit per flower and seed per fruit data for the 48 F4 recombinant inbred lines evaluated under greenhouse conditions in 2019 and 2020. ........................................................ 356 xii Table S5.2. Self-fertility trait data for the F4 and F5 recombinant inbred line individuals measured during the 2020 greenhouse season. ........................................................................................... 358 Table S5.3. Summary of the number of individuals (N) used for single nucleotide polymorphism (SNP) marker-trait association in the F4 and F5 generations of a Solanum chacoense recombinant inbred line population in 2020. ................................................................................................... 363 Table S5.4. Primer information for the two KASPTM markers used to genotype the Sli candidate region in this study. ..................................................................................................................... 364 Table S5.5. Foliar glycoalkaloid data for greenhouse grown individuals from the F4 and F5 generations of the recombinant inbred line population. .............................................................. 365 Table S5.6. Field Colorado potato beetle defoliation data for genotypes in the F4 and F5 generations of the recombinant inbred line population evaluated at the Montcalm Research Center in 2019 and 2020. ........................................................................................................................ 375 Table S5.7. Leaf and tuber glycoalkaloid data of field-grown F5 inbreds and MSHH786B hybrids evaluated for Colorado potato beetle field resistance in 2020. ................................................... 379 Table S5.8. Graphical genotype data for F2, F4, and F5 recombinant inbred line individuals at the 1020 V3 SNP loci used for analysis in this study. ...................................................................... 380 Table S5.9. KASPTM marker genotyping data of parental lines, their F1 hybrid, F4 recombinant inbred lines and F5 recombinant inbred lines at two loci within the Sli candidate region associated with self-compatibility. ............................................................................................................... 381 Table S5.10. SNPs significantly associated with leptine I/II, alpha-solanine, alpha-chaconine, the ratio of acetylated to non-acetylated compounds, and the presence of leptines in the F4 and F5 generation of the recombinant inbred line population. ............................................................... 383 Table S5.11. Genetic map based on 97 F4 individuals from the recombinant inbred line population and 288 SNPs. ............................................................................................................................. 386 Table S5.12. Excessively heterozygous loci in F4 and F5 individuals of the recombinant inbred line population. ........................................................................................................................... 392 Table 6.1. Differentially expressed genes between Solanum chacoense USDA8380-1 (80-1) and the F2 line EE501F2_093 in response to Colorado potato beetle feeding at 24 hrs and 48 hrs post beetle infestation. ........................................................................................................................ 424 Table S6.1 The presence of leptines (1 = leptines present; 0 = no foliar leptine content) and the Soltu.DM.02G006530 ORF marker(‘+’ = band present; ‘-‘ = no band present) designed in this study. ........................................................................................................................................... 431 Table S6.2 Gene names and representative transcript ID for the doubled monoploid (DM) v6.1 assembly of glycoalkaloid genes inspected in the RNAseq experiment. .................................... 432 xiii Table S6.3 Percent defoliation caused by adult Colorado potato beetle feeding on Solanum chacoense USDA8380-1 (80-1) and EE501F2_093 whole plants at T2 (48 hrs after beetle placement). .................................................................................................................................. 432 xiv LIST OF FIGURES Figure 2.1. A general framework of the conventional breeding process that is comprised of three stages: 1. Trait Mapping; 2. Trait Introgression and; 3. Field Testing. ........................................ 53 Figure 2.2. Maize breeding testing practices for the processed maize kernels industry. .............. 54 Figure 2.3. Overview of the Washington State University Apple Breeding Program traditional breeding operations. ...................................................................................................................... 55 Figure 2.4. Overview of a conventional tetraploid potato breeding cycle. ................................... 56 Figure 2.5. Colorado potato beetle detached leaf assays after 5 days of feeding ......................... 57 Figure S2.1 Elsevier Copyright Permission for inclusion of Chapter 2 in this dissertation. ........ 58 Figure 3.1. Colorado potato beetle larval defoliation (%) of Solanum chacoense parental lines M6 and USDA8380-1 (80-1) at three time points post-transplant from tissue culture. .................... 118 Figure 3.2. Distribution of Colorado potato beetle resistance in 151 F2 Solanum chacoense progeny and parental lines under field conditions expressed in relative area under the defoliation curve multiplied by 100 (RAUDC x 100) ................................................................................... 119 Figure 3.3. The QTL regions on chromosome 2 associated with Colorado potato beetle resistance under field conditions and foliar concentration of glycoalkaloids identified by bi-parental linkage mapping ....................................................................................................................................... 120 Figure 3.4. Colorado potato beetle resistance phenotypic validation of the F2 phenotypic extremes ..................................................................................................................................................... 121 Figure 3.5. Weighted gene co-expression network module associations with three traits: Defoliation (RAUDC), Total Leptines (mg/g DW) and Ratio (the ratio of acetylated compounds to non-acetylated compounds accumulated) ............................................................................... 122 Figure 3.6. Interaction of hub genes in the midnight blue module visualized using Cytoscape 3.7 software ....................................................................................................................................... 123 Figure S3.1. Analysis of network topology for increasing soft-thresholding powers ................ 124 Figure S3.2. Distribution of 754 mapped single nucleotide polymorphisms along the 12 chromosomes .............................................................................................................................. 125 Figure S3.3. Distribution of segregation rates of maternal (Solanum chacoense USDA8380-1;red), paternal (Solanum chacoense M6; black) and heterozyogous (purple) genotype of 754 mapped single nucleotide polymorphisms along the 12 chromosomes of the genetic map. .................... 126 xv Figure S3.4. Means trait values of maternal Solanum chacoense USDA8380-1 alleles (mu_A), paternal Solanum chacoense M6 alleles (mu_B), and heterozygous genotypes (mu_H) plotted along the genetic map (x-axis, cM) of chromosome 2. ............................................................... 127 Figure S3.5. The minor QTL region on chromosome 7 associated with Colorado potato beetle resistance under field conditions identified by MQM mapping ................................................. 128 Figure S3.6. Distribution of significant QTL along physical positions (Mb) of the 12 chromosomes identified by alignment of whole genome sequence from bulked beetle resistant and bulked beetle susceptible F2 progeny to the Solanum tuberosum clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03), corrected with Solanum chacoense M6 whole genome sequence data, and G’ analysis ........................................................................................................................................ 129 Figure S3.7. Significant QTL associated with Colorado potato beetle resistance identified by G’ analysis plotted on the physical position (Mb) of the Solanum tuberosum clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03), corrected with Solanum chacoense M6 whole genome sequence reads ............................................................................................................................ 130 Figure S3.8. Genetic map of chromosome 2 and a QTL associated with Colorado potato beetle resistance under field conditions in 96 additional Solanum chacoense F2 individuals .............. 131 Figure S3.9. Principal component analysis of differentially expressed genes between resistant and susceptible lines produced within the R package DESeq2 ......................................................... 132 Figure S3.10. Distribution of significantly (padj < 0.001) down- (left) and up- (right) regulated transcripts in leaf tissue of Solanum chacoense USDA8380-1 and three Colorado potato beetle resistant F2 progeny across the 12 chromosomes ....................................................................... 133 Figure S3.11. Distribution of significantly (padj < 0.001) down- (left) and up- (right) regulated transcripts in leaf tissue of Solanum chacoense 80-1 and three Colorado potato beetle resistant F2 progeny across chromosome (Chr) 2 (Solanum tuberosum DM Pseudomolecule PGSC Version 4.03) physical positions (Mb) ..................................................................................................... 134 Figure S3.12. Weighted gene co-expression network module significance values of each of the 29 modules for three traits measured in Colorado potato beetle resistant and susceptible lines ..... 135 Figure S3.13. Weighted gene co-expression network analysis clustering .................................. 136 Figure S3.14. Interaction of 9 hub genes in the darkgrey module visualized using Cytoscape 3.7 software ....................................................................................................................................... 137 Figure S3.15. Interaction of 55 hub genes in the red module visualized using Cytoscape 3.7 software ....................................................................................................................................... 138 Figure S3.16. Interaction of 23 hub genes in the red module visualized using Cytoscape 3.7 software ....................................................................................................................................... 139 Figure S3.10 Springer Copyright Permission for inclusion of Chapter 3 in this dissertation. ... 222 xvi Figure 4.1. Physical position of nine KASPTM markers and two SNP markers reported in this study on chromosome 12 of the potato doubled monoploid S. phureja clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03) ...................................................................................... 265 Figure 4.2. The frequency of the homozygous self-compatible (SC) genotype (AA) at six KASPTM marker loci spanning the Sli region on chromosome 12 (58,960,090-59,040,898 bp) increased over the course of five generations in a recurrent selection population ..................... 266 Figure 4.3. Genotype frequencies of individuals from the diploid backcross population at KASPTM marker Sli_561........................................................................................................... 267 Figure S4.1. Proportion of flowers that set fruit upon selfing under greenhouse conditions plotted against the marker genotype [homozygous self-compatible genotype (AA), heterozygous genotype (AB) and homozygous SI genotype (BB)] of six KASPTM markers in 178 individuals of a diploid recurrent selection population (18 cycle 0 clones, 15 cycle 1 clones, 42 cycle 2 clones, 74 cycle 3 clones, and 29 cycle 4 clones). .................................................................................................... 268 Figure S4.2. Stacked bar graphs show the frequency of the homozygous self-compatible (SC) (AA; black), heterozygous (AB; blue), homozygous self-incompatible (BB; grey), and No Call (magenta) genotype at six KASPTM marker loci in the backcross population .......................... 269 Figure 5.2. Self-fertility in the F4 and F5 generation of a Solanum chacoense recombinant inbred line (RIL) population. ................................................................................................................. 342 Figure 5.3. Pollen tube growth in the style 48 hrs post pollination of self-incompatible parent Solanum chacoense 80-1 (a), an F5 recombinant inbred line (095_04_01_01) that failed to set fruit upon selfing (b), an F5 recombinant inbred line (199_02_01_01) that produced only parthenocarpic fruit (c), and an F5 recombinant inbred line (495_01_05_04) that set fruit and seed (d) ........... 343 Figure 5.4. Foliar glycoalkaloid content of greenhouse grown plants and Colorado potato beetle resistance under field conditions. ................................................................................................ 344 Figure 5.5. Loci associated with leptine I/II accumulation in the F5 recombinant inbred line population (N =26) ...................................................................................................................... 345 Figure 5.6. Excessive heterozygosity (red) at 229 loci in the F4 generation (a) and 307 loci in the F5 generation (b) of a Solanum chacoense recombinant inbred line population are plotted according to their absolute position on the 12 S. tuberosum clone DM1-3 516 R44 PGSC v4.03 pseudomolecules ......................................................................................................................... 347 Figure 5.7. Physical location of 1020 SNPs segregating in the F2, F4 and F5 generations of a Solanum chacoense recombinant inbred line population ............................................................ 348 Figure S5.1. Distribution of average fruit weight in grams (grey) and the fraction of flowers setting fruit (black) in the F4 and F5 generation under greenhouse conditions in 2020. ....................... 349 Figure S5.2. Foliar glycoalkaloid content in mg/g dry weight (DW) of greenhouse grown F4 (N = 62) (a) and F5 (N = 74) (b) individuals ....................................................................................... 350 xvii Figure S5.3. a) Distribution of Colorado potato beetle resistance under field conditions, represented by the mean relative area under the defoliation progression curve (RAUDC), in the 15 MSHH786B F1 individuals ......................................................................................................... 351 Figure S5.4. Mean tuber glycoalkaloid content expressed as total mg% fresh weight of Colorado potato beetle resistant F5 recombinant inbred lines, resistant parent Solanum chacoense 80-1, and an F1 hybrid (MSHH786_01) ...................................................................................................... 352 Figure S5.6. Genetic map of the F4 recombinant inbred line population based on 97 individuals and 288 SNP markers. ................................................................................................................. 354 Figure S5.7. Locus genotype frequency (a) and individual genotype frequency (b) of the two homozygous genotypes (AA:grey, BB:black) and the heterozygous genotype (AB:red) at 1,020 SNPs segregating in 236 F2 individuals, 113 F4 individuals, and 80 F5 individuals. ............... 355 Figure S5.8. Genotype frequency of the M6 parental (grey), recombinant (black) and 80-1 parental (blue) genotype of 519 SNPs plotted against their physical position on the PGSC v4.03 pseudomolecules ......................................................................................................................... 355 Figure. S5.9. Sequence analysis of the high-top B (HT-B) open reading frame (ORF) in parental lines Solanum chacoense USDA8380-1 (80-1) and Solanum chacoense M6. ........................... 399 Figure S5.10. Predicted protein sequences generated from genomic high-top B (HT-B) sequence in parental lines Solanum chacoense USDA8380-1 (80-1) and S. chacoense M6. .................... 400 Figure S5.11. Sequence analysis of the S-RNase open reading frame (ORF) in parental lines Solanum chacoense USDA8380-1 (80-1) and Solanum chacoense M6. .................................... 402 Figure S5.12. Predicted protein sequences generated from genomic S-RNase sequence in parental lines Solanum chacoense USDA8380-1 (80-1) and S. chacoense M6. ...................................... 403 Figure 6.1. Foliar glycoalkaloid profiling of resistant and susceptible Solanum chacoense genotypes in response to Colorado potato beetle infestation under growth chamber conditions. ..................................................................................................................................................... 426 Figure 6.2. Principal component analysis of log2 transformed counts of transcriptome data of Colorado potato beetle resistant diploid Solanum chacoense USDA8380-1 (80-1; green) and a susceptible F2 line (EE501F2_093; orange) from the S. chacoense recombinant inbred line population (Kaiser et al., 2021) in response to adult Colorado potato beetle feeding. ............... 427 Figure 6.3. Differential expression observed in leaf tissue of Colorado potato beetle resistant diploid Solanum chacoense USDA8380-1 (80-1) and a susceptible F2 line (EE501F2_093) from the S. chacoense recombinant inbred line population (Kaiser et al., 2021) in response to adult Colorado potato beetle feeding 24hrs and 48 hrs after infestation. ............................................ 428 Figure 6.4 Gene structure and sequence of Soltu.DM.02G006530. ........................................... 429 xviii Figure S6.1 Pearson correlation of log2 transformed gene counts between samples in the RNAseq experiment. .................................................................................................................................. 430 xix KEY TO ABBREVIATIONS 80-1 Solanum chacoense USDA8380-1 ANOVA One-way analysis of variance KO Knock-out HT-B High top protein, isoform B MS Male sterile NF Non-flowering plant RIL Recombinant inbred line SC Self-compatible Sli SI S-locus inhibitor gene Self-incompatible SNP Single Nucleotide Polymorphism TPS True potato seeds xx CHAPTER 1 INTRODUCTION Potato, a world food crop Cultivated potato, Solanum tuberosum L. Group Tuberosum (2n=4x=48), is currently the fourth most important food crop worldwide, with an annual production of 370 million tons following wheat (766 million tons), rice (755 million tons), and maize (1.1 billion tons) (FAOSTAT, 2019) and is grown in most countries across a diverse array of environments. Potato is highly productive on a per unit area basis with a potato crop producing 54% more protein per unit of land area than wheat and 78% more than rice. Potato ranks second only to soybean in protein produced per acre among the major crops (Kaldy, 1972). Additionally, a single potato provides 50% of the recommended daily allowance of vitamin C, 21% of potassium, 12% of fiber (Kolasa, 1993). Today, the United States ranks fifth in world potato production (FAOSTAT, 2019), where potatoes are on 0.9 million acres and deliver $3.9 billion in farm value (NASS, 2020). Potato production is hampered by the Colorado potato beetle The Colorado potato beetle (Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) is the most widespread and destructive insect defoliator of potato crops, inflicting yield losses of 30-50% (Alyokhin et al., 2012a; Vreugdenhil et al., 2007) and covering a range spanning 16 million km2 in North America and Eurasia (Weber, 2003). While potato is the preferred host, the Colorado potato beetle also causes considerable defoliation to other Solanaceous crops, such as eggplant and pepper (Maharijaya & Vosman, 2015). In addition to a robust appetite, the Colorado potato beetle is characterized by high fecundity (Ferro et al., 1985). Offspring are distributed across space, as adults are capable of walking several hundred meters and flying several kilometers (Weber et al., 1994), and over time via diapause. Short-day photoperiod induces diapause in adult 1 beetles (de Kort, 1995) which become unresponsive to external stimuli for approximately 3 months. However, a variable proportion of adults may remain in extended diapause for up to 3 years (Alyokhin, 2008). As a result, the risk posed by traditional control mechanisms such as pesticide application or crop rotation to this pest is diminished. Financial losses attributed to Colorado potato beetle feeding are rarely published, most likely a function of the fact that commercial fields are rarely attacked by a singular pest and a lack of controlled, replicated experiments on the subject. However, research conducted in Michigan in 1994 determined that control of and yield losses due to the Colorado potato beetle resulted in financial expenditures of 14.4 million dollars in the state of Michigan during that growing season (Grafius, 1997). Remarkable adaptability of the Colorado potato beetle results in widespread insecticide resistance Non-chemical cultural Colorado potato beetle control practices, such as trapping, border sprays, trap crops and propane flamers, are too time- and labor-intensive to be feasible for commercial production (Alyokhin, 2008). Thus, control of the Colorado potato beetle has historically relied heavily upon the use of pesticides (Alyokhin et al., 2015; Maharijaya & Vosman, 2015). Unfortunately, failure of chemical control against Colorado potato beetle has been reported for most major classes of synthetic insecticides and for over 50 different active ingredients (Szendrei et al., 2012). Resistance to multiple insecticides is common in an individual Colorado potato beetle (Alyokhin et al., 2008; Alyokhin et al., 2007; Mota-Sanchez et al., 2006). Resistant populations of Colorado potato beetle are found across the entirety of its range but are most prevalent in North America (Whalon et al., 2008), due in part to intensive pesticide application (Alyokhin, 2009) and the high adaptability of the insect. Although the neonicotinoid insecticides 2 introduced in 1995 provided excellent Colorado potato beetle control, their efficacy has been declining over the past decade, necessitating higher application rates and the development of new chemistries (Mota-Sanchez et al., 2006). The release of new chemistries presents increasing costs to growers and may introduce increased environmental hazards compared to the existing chemicals (Alyokhin et al., 2012b). Increasing dosage of currently available insecticides alleviates insect pressure in the short-term but accelerates the rate of resistance development in the population and such concentrations of pesticide pose consumer health risks (Maharijaya & Vosman, 2015). The limitations of currently employed control strategies demand incorporation of additional control mechanisms to combat this menacing pest. Exploiting host plant resistance to the Colorado potato beetle in wild diploid germplasm Host plant resistance to Colorado potato beetle has the potential to maintain the efficacy of insecticides and mitigate environmental impacts by reducing the number of insecticide applications. Wild species relatives of potato produce potent glycoalkaloids, leptines and leptinines, that effectively reduce Colorado potato beetle feeding and reproduction through a cholinesterase inhibiting and cell membrane disruption mechanism (Sanford et al., 1994; Sanford et al., 1996; Sinden et al., 1980). Specifically, the high leptine-producing diploid Solanum chacoense accession USDA8380-1 (80-1) has demonstrated strong antibiosis properties against the Colorado potato beetle (Sinden et al., 1986). Attempts to introgress 80-1 mediated Colorado potato beetle resistance into cultivated potato in the past several decades has not been successful for several reasons. First, numerous studies point to multiple loci contributing to leptine production and recessive inheritance of key functional and/or regulatory genes in the leptine biosynthesis pathway (Boluarte-Medina et al., 2002; Hutvágner et al., 2001; Manrique-Carpintero et al., 2014; Ronning et al., 1998; Ronning et al., 1999; Sagredo et al., 2009; Sagredo et al., 2006). Second, 3 efforts to understand the inheritance and expressivity of beneficial leptine alleles in cultivated potato backgrounds are stymied by the tetraploid nature of commercial potato varieties (Lorenzen et al., 2001; Sanford et al., 1997; Yencho et al., 2000). Diploid potato breeding offers unprecedented opportunities Unlike self-compatible grain crops, cultivated potato is a heterozygous tetraploid outcrossing species that is vegetatively propagated as tubers. Conducting potato improvement at the diploid level allows implementation of tools, technologies and breeding approaches that are not possible or are inefficient at the tetraploid level. The creation of diploid inbred lines in potato offers a strategy to address many limitations faced by current potato breeding methods. Although the road to homozygosity is faster, many diploids are self-incompatible due to a gametophytic self- incompatible system. The S-locus on chromosome 1 contains tightly linked genes encoding the female (S-locus RNase (S-RNase)) and male (S-locus F-box (SLF)) determinants (McClure et al., 1989; Takayama & Isogai, 2005). The pistil-expressed S-RNase inhibits self-pollen tube growth by degrading pollen RNA (Kubo et al., 2015). The pollen-expressed SLF mediates ubiquitination, and subsequence degradation, of non-self S-RNase which facilitates the growth of non-self pollen tubes (Kubo et al., 2015; Sijacic et al., 2004). In self-incompatible plants, the SLF fails to recognize its own S-RNase and self pollen tube growth is inhibited (Hua et al., 2008). In the S. chacoense diploid inbred line M6, however, the self-incompatibility system is inactivated by the dominant allele of the S-locus inhibitor gene Sli on the most distal end of chromosome 12 (Jansky et al., 2014). M6-mediated introduction of self-compatibility affords the opportunity to conduct fine mapping in recombinant inbred lines, identify markers for desirable traits harbored in wild germplasm, and more efficiently introgress these traits into cultivated material. In addition to self- compatibility, diploid potato inbred line development depends on the concurrent improvement of 4 self-fecundity traits such as fruit set, seed set, pollen viability, and synchronized flowering time. The genetic basis of these traits in diploid potato remains to be determined (Peterson et al., 2016). The Michigan State University Potato Breeding and Genetics Program has substantially invested in creating diploid potato breeding germplasm with desirable agronomic traits through recurrent selection and backcross breeding (Alsahlany, 2019; Alsahlany et al., 2020). The power of the gene editing tool CRISPR/Cas9 can also be leveraged in the simpler genetic system of diploid potatoes for rapid validation of candidate genes and targeted introduction of genes from wild relatives without bringing along unadapted traits (Enciso-Rodriguez et al., 2019; Nadakuduti et al., 2019). Dissertation Organization and Objectives The purpose of this study was to characterize the causative genetic, genomic and molecular features of host plant resistance and self-fertility in S. chacoense with the aim to develop germplasm and genetic resources that will be used to breed improved diploid varieties. The dissertation is organized into an introductory chapter, four research chapters and a concluding chapter. The introductory Chapter 2 is a published journal article entitled “The role of conventional plant breeding in ensuring safe levels of naturally occurring toxins in food crops” that contextualizes plant breeding efforts to both reduce and leverage plant-produced toxins (Kaiser, Douches, et al., 2020). Chapter 3 entitled, “Mapping Solanum chacoense mediated Colorado potato beetle (Leptinotarsa decemlineata) resistance in a self‑compatible F2 diploid population” is a published research article (Kaiser, Manrique-Carpintero, et al., 2020). The objectives of Chapter 3 are as follows: 5 3.1 Create a diploid F2 population segregating for Colorado potato beetle resistance and glycoalkaloid production 3.2 Employ bi-parental linkage mapping and whole genome bulk segregant analysis to identify genetic regions associated with host plant resistance and glycoalkaloid production 3.3 Conduct gene expression profiling of beetle resistant and susceptible F2 individuals to define high confidence candidate genes involved in host plant resistance Chapter 4, entitled “Assessing the contribution of Sli to self-compatibility in North American diploid potato germplasm using KASPTM markers” is a published research article (Kaiser et al., 2021). The objectives of Chapter 4 are as follows: 4.1 Determine the genotype at six marker loci in the candidate Sli region in a diverse set of self-compatible diploid breeding lines 4.2 Appraise the transmission of Sli in a diploid recurrent selection population and a diploid backcross population 4.3 Determine the feasibility of using Sli markers to predict a self-compatible phenotype The objectives of Chapter 5 entitled, “Self-fertility and resistance to the Colorado potato beetle (Leptinotarsa decemlineata) in a diploid Solanum chacoense recombinant inbred line population” are as follows: 5.1 Create vigorous, self-fertile inbred diploid potato lines 5.2 Examine heterozygosity and segregation distortion patterns in recombinant inbred lines 5.3 Identify loci associated with Colorado potato beetle resistance and self-fertility 6 5.4 Evaluate transmission of leptine production and Colorado potato beetle resistance to diploid breeding lines The objectives of Chapter 6 entitled, “Characterizing the transcriptional and glycoalkaloid response to Colorado potato beetle infestation in Solanum chacoense” are as follows: 6.1 Assess the transcriptional and glycoalkaloid response to Colorado potato beetle herbivory in beetle resistant and beetle susceptible S. chacoense lines over a 48-hour observation period 6.2 Clarify the allelic sequences of Soltu.DM.02G006530 in the high-leptine producing line S. chacoense USDA8380-1 and S. chacoense M6, which does not produce leptines The conclusion Chapter 7 summaries the findings of the dissertation research and provides prospects for future investigation of Colorado potato beetle host plant resistance and self-fertility in diploid potato. 7 REFERENCES 8 REFERENCES Alsahlany, M. (2019). Redesigning diploid potato breeding with self-compatibility. (PhD). Michigan State University, Alsahlany, M., Enciso-Rodriguez, F., Lopez-Cruz, M., Coombs, J., & Douches, D. (2021). Developing self-compatible diploid potato germplasm through recurrent selection. Euphytica. In Review Alyokhin, A. (2009). Colorado potato beetle management on potatoes: current challenges and future prospects. Fruit, Vegetable and Cereal Science and Biotechnology, 3(1), 10-19. Alyokhin, A., Baker, M., Mota-Sanchez, D., Dively, G., & Grafius, E. (2008). Colorado Potato Beetle Resistance to Insecticides. American Journal of Potato Research, 85(6), 395-413. doi:10.1007/s12230-008-9052-0 Alyokhin, A., Dively, G., Patterson, M., Castaldo, C., Rogers, D., Mahoney, M., & Wollam, J. (2007). Resistance and cross-resistance to imidacloprid and thiamethoxam in the Colorado potato beeetle Leptinotarsa decemlineata. 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Resistance and cross-resistance to neonicotinoid insecticides and spinosad in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Pest Management Science, 62, 30-37. doi:10.1002/ps.1120 Nadakuduti, S. S., Starker, C. G., Voytas, D. F., Buell, C. R., & Douches, D. S. (2019). Plant Genome Editing with CRISPR Systems. 1917, 183-201. doi:10.1007/978-1-4939-8991-1 NASS. Potatoes 2019 (2020). Retrieved https://www.nass.usda.gov/Publications/Todays_Reports/reports/pots0920.pdf Summary. from Peterson, B. A., Holt, S. H., Laimbeer, F. P. E., Doulis, A. G., Coombs, J., Douches, D. S., Hardigan, M. A., Buell, C. R., & Veilleux, R. E. (2016). Self-fertility in a cultivated diploid potato population examined with infinium 8303 potato single-nucleotide polymorphism array. The Plant Genome, 9, 0. doi:10.3835/plantgenome2016.01.0003 the Ronning, C. M., Sanford, L. L., Kobayashi, R. S., & Kowalsld, S. P. (1998). Foliar leptine production in segregating F1, inter-F1, and backcross families of Solanum chacoense Bitter. American Journal of Potato Research, 75, 137-143. Ronning, C. M., Stommel, J. R., Kowalski, S. P., Sanford, L. L., Kobayashi, R. S., & Pineada, O. (1999). Identification of molecular markers associated with leptine production in a population of Solanum chacoense Bitter. Theoretical and Applied Genetics, 98, 39-46. doi:10.1007/s001220051037 Sagredo, B., Balbyshev, N., Lafta, A., Casper, H., & Lorenzen, J. (2009). A QTL that confers resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]) in tetraploid potato populations segregating for leptine. Theoretical and Applied Genetics, 119, 1171-1181. doi:10.1007/s00122-009-1118-y 11 Sagredo, B., Lafta, A., Casper, H., & Lorenzen, J. (2006). Mapping of genes associated with leptine content of tetraploid potato. Theoretical and Applied Genetics, 114, 131-142. doi:10.1007/s00122-006-0416-x Sanford, L. L., Deahl, K. L., & Sinden, S. L. (1994). Glycoalkaloid content in foliage of hybrid and backcross populations from a Solanum tuberosum X S. chacoense cross. American Potato Journal, 71, 225-235. Sanford, L. L., Kobayashi, R. S., Deahl, K. L., & Sinden, S. L. (1996). Segregation of leptines and other glycoalkaloids in Solanum tuberosum (4x)× S. chacoense (4x) crosses. American Potato Journal, 73, 21. Sanford, L. L., Kobayashi, R. S., Deahl, K. L., & Sinden, S. L. (1997). Diploid and Tetraploid Solanum chacoense genotypes that synthesize leptine glycoalkaloids and deter feeding by Colorado potato beetle. American Potato Journal, 74, 15-21. Sijacic, P., Wang, X., Skirpan, A., Wang, Y., Dowd, P., McCubbin, A., Huang, S., & Kao, T. (2004). Identification of the pollen determinant of S-RNase-mediated self-incompatibility. Nature, 429, 302-305. Sinden, S. L., Sanford, L. L., Cantelo, W. W., & Deahl, K. L. (1986). Leptine glycoalkaloids and resistance to the Colorado potato beetle (Coleoptera: Chrysomelidae) in Solanum chacoense. Environmental Entomology, 15, 1057-1062. Sinden, S. L., Sanford, L. L., & Osman, S. F. (1980). Glycoalkaloids and resistance to the Colorado potato beetle in Solanum chacoense Bitter. American Potato Journal, 57, 331-343. doi:10.1007/BF02854028 Szendrei, Z., Grafius, E., Byrne, A., & Ziegler, A. (2012). Resistance to neonicotinoid insecticides in field populations of the Colorado potato beetle (Coleoptera: Chrysomelidae). Pest Management Science, 68, 941-946. doi:10.1002/ps.3258 Takayama, S., & Isogai, A. (2005). Self-incompatibility in plants. Annual Review of Plant Biology, 56(1), 467–489. doi:https://doi.org/10.1146/annurev.arplant.56.032604.144249 Vreugdenhil, D., Bradshaw, J., Gehardt, C., Govers, F., Mackerron, D. K. L., A.Taylor, M., & A.Ross, H. (2007). Potato biology and biotechnology: Advances and perspectives. 91-111. Weber, D. (2003). Colorado Beetle: Pest on the move. The Royal Society of Chemistry 256-259. doi:10.1039/b314847p Weber, D. C., Ferro, D. N., Buonaccorsi, J., & Hazzard, R. V. (1994). Disrupting spring colonization of Colorado potato beetle to nonrotated potato fields. Entomologia experimentalis et applicata, 73(1), 39-50. Whalon, M. E., Mota-Sanchez, D., & Hollingworth, R. M. (2008). Analysis of global pesticide resistance in arthropods. In Global pesticide resistance in arthropods (pp. 5-31). Wallingford: CABI. 12 Yencho, G. C., Kowalski, S. P., Kennedy, G. G., & Sanford, L. L. (2000). Segregation of leptine glycoalkaloids and resistance to Colorado potato beetle (Leptinotarsa decemlineata (Say)) in F2 Solanum tuberosum (4x) x S. chacoense (4x) Potato progenies. American Journal of Potato Research, 77, 167-178. 13 CHAPTER 2 THE ROLE OF CONVENTIONAL PLANT BREEDING IN ENSURING SAFE LEVELS OF NATURALLY OCCURRING TOXINS IN FOOD CROPS This chapter is a published research article (Kaiser, et al., 2020). As the author of this document, the publisher Elsevier affords Natalie Kaiser the right to reproduce the document in this thesis (Appendix C). 14 Abstract Background The process of selecting superior performing plants for food, feed and fiber products dates back more than 10,000 years and has been substantially refined in the last century. While the perceived risks posed by genetically engineered crop plants has been extensively addressed, the extant levels of naturally occurring plant toxins in food crops has received far less attention. Scope and Approach This review discusses how conventional breeding practices are used by plant breeders to develop safe new food crop varieties. Crops are grouped into two categories: 1) crop plants with no significant plant-produced toxins; and 2) crop plants with known plant-produced natural toxins. Examples and crop case studies from each category are used to illustrate the safety considerations of breeding these economically important crops and how plant breeding practices are adjusted prior to commercialization, depending on whether the crop produces known natural toxin(s). Key Findings and Conclusions Conventional breeding practices, such as cross- or self-pollinating, shuffle genetic allelic combinations to produce new progeny varieties without giving rise to novel uncharacterized biosynthetic pathways. Therefore, plant breeders can fine tune their practices depending on the crop and specific known natural toxins inherent to that crop species, thereby ensuring a safe food supply for consumers. Breeders often select different varieties of a single food crop for use in disparate markets, each with unique breeding selection practices depending on the desirable characteristics and safety considerations for the portion of the plant that is consumed and the nature of the particular processing industry. 15 Introduction The vast majority of food crops the consumer encounters in grocery store aisles are the product of conventional plant breeding. Even varieties such as seedless watermelon, pluots, apriums, and tangelos, which are often mistakenly thought to be a product of modern genetic engineering technologies, are products of conventional breeding practices (Judkis, 2018; Sousa, 2013). In fact, varieties resulting from genetic engineering, defined by the USDA as a process that utilizes modern biotechnology tools to introduce, eliminate or rearrange specific genes (USDA, 2013), are available only for a small portion of food crops such as maize, soybean, canola, rice, potato, papaya, squash and apple (ISAAA, 2018). By comparison, hundreds of new crop varieties are released every year by commercial conventional breeding to improve crop productivity, bolster food security, enhance nutrition, and expand consumer choice (Evenson & Gollin, 2002). Conventional plant breeding involves identifying parent plants with desirable characteristics to create favorable combinations in the next generation. The process of selecting superior performing plants for food, feed and fiber products dates back more than 10,000 years and has been substantially refined in the last century (Doebley, Gaut, & Smith, 2006; Smith, 2001). Early farmers relied on extant genetic variation in wild plant populations and selected individual plants with desired traits. Plant breeders today expand upon existing genetic variation by selecting genetically diverse plants as parents, which may or may not sexually reproduce in nature due to obstacles such as geographic isolation or differences in maturity. In order to identify the best individuals in the resulting offspring, plant breeders select plants for traits of interest and use well-established scientific methods to characterize parameters important for each crop. 16 Consumers expect foods from conventionally bred crops to be safe and nutritious, although few foods have been systematically assessed for whether or not any harm might occur when foods are consumed (Constable, et al., 2007). This consumer expectation of crop plants providing safe foods is based on either their own personal history of safely eating such foods and/or their knowledge that throughout history people have been preparing and eating foods from a given crop without evidence of harm or adverse consequences. Many factors contribute to foods having a “history of safe consumption” including: the period of time the food has been consumed, strategies to prevent post-harvest accumulation of toxins, knowledge of whether the crop has endogenous plant toxins, and if present, accepted preparation methods to ensure safe consumption. This review focuses specifically on how plant breeding practices deliver improved crops while maintaining safe levels of naturally occurring plant toxins. Conventional Breeding Practices Used by Plant Breeders The process of conventional breeding has evolved over time, creating an effective framework that not only improves crop performance, but also supports development of foods that are safe and nutritious to consume. Plant breeding is a process of making decisions- which parents to choose, which parents to cross pollinate and which progeny to advance. Plant breeding, unlike animal breeding, benefits from the ability to create very large populations (depending upon the crop, into the tens of thousands), in which the vast majority of plants (often >99%) are discarded while selecting the few individual plants with the desired characteristics to advance to future breeding rounds. This ability to select a few individuals from large populations is a critical contributor to the plant breeding process and is applied during many stages of the process, including trait mapping, trait introgression and field testing (Figure 1). 17 The purpose of trait mapping is to identify and confirm the genetic basis of the trait of interest by finding the DNA region linked to the trait (Falconer & Mackay, 1996). Since the genetic basis of plant phenotypic differences is not always readily apparent, breeders identify a set of DNA markers that differentiate both parent plants. One common breeding strategy for trait mapping is to cross pollinate parent plants with extremes of the trait of interest (e.g., high vs. low disease resistance or presence vs. absence of the trait of interest) to produce progeny. This allows the trait of interest to segregate in the progeny plants in subsequent rounds of self-pollination and/or cross pollination. Trait mapping is a statistically iterative process to correlate measurement of the trait of interest (phenotype) with DNA markers (genotype). DNA from all progeny plants at each generation is assayed with each plant’s parental marker set to produce genotype information. Simultaneously, plant breeders test for the trait of interest in all progeny. A correlation between phenotype and genotype informs the breeder which markers co-segregate with the trait of interest at each generation. The first generation (F2) of progeny assessed for phenotype-genotype correlation maps the trait of interest at the chromosome level (Figure 1.1). Identification of the precise location of genes underlying the trait of interest within the identified chromosome is achieved over the subsequent 5-6 generations of progeny plants. The number of progeny plants, number of markers, and the number of advanced generations of self-pollination and/or cross pollination must increase in order to obtain more exact localization of the DNA region (gene(s) or causal locus) responsible for the trait of interest (phenotype). Using maize as an example, a breeder might need to grow 20,000 maize plants over 5-6 generations to select 200-300 plants co- segregating for the trait and marker, to map the genetic locations for the trait to a region of ~200,000 base pairs within one of the ten chromosomes (Figure 1.1). 18 After mapping the genetic basis for the trait of interest within a chromosomal region, a trait- linked DNA marker that segregates, or is consistently co-inherited, with the trait has now been identified to be genetically linked to the trait. This trait-linked marker is then used to develop a DNA marker-based assay. DNA marker-based assays allow breeders to conduct rapid molecular screening assays for the genetic basis of the trait of interest in thousands of progeny plants, replacing more laborious and resource intensive phenotyping methods. The DNA marker-based assay is now ready to be used by breeders for the next stage of breeding of trait introgression to identify and select individual plants with the trait of interest. The purpose of trait introgression is to introduce the trait of interest from the source parent plant into the germplasm of parental varieties that are well characterized for additional traits suitable for commercialization (e.g., uniform yield performance, adaptability to different environments). Breeders use two types of DNA markers for trait introgression, the trait-linked marker developed from trait mapping and genome-wide markers from the commercial-track varieties (Figure 1.2). Trait introgression is a two-step process that begins with cross pollinating a plant from the trait mapping stage that carries the trait of interest with plants from one or a set of varieties with commercially suitable, well characterized traits (Figure 1.2). First, breeders use the marker-based assay developed during the trait mapping stage to screen progeny plants, selecting plants that carry the trait of interest. This step is called marker-assisted selection. Second, over successive breeding cycles, a plant breeder continuously cross pollinates progeny carrying the trait of interest with the same parental commercial-track varieties used in step one (Figure 1.2). This step is called marker- assisted backcrossing. Breeder uses a genome wide marker set of the parental commercial-track varieties to screen and select plants with that genetic background. By continuously cross- 19 pollinating progeny with the same commercial-track parent over 5-6 generations, breeders are able to shift the genetic background towards a greater proportion of genes from the commercial-track variety (varieties) (Figure 1.2). Selection and screening performed during both steps ensures: 1) elimination of plants with genetic backgrounds conferring undesirable traits and; 2) retention of the gene(s) associated with the trait of interest as the genetic background of commercial-track varieties becomes progressively more dominant in the progeny genome through the successive breeding cycles (Glenn et al, 2017). A commercial maize breeder in the US, for example, would typically screen approximately 1000 plants during the two-step process of trait introgression to generate 10-15 plants to introgress the trait of interest into one commercially competitive genetic background. Since a breeder usually introgresses the trait of interest into multiple commercially competitive backgrounds that are adapted to grow in different environments and/or geographies (e.g., within a state or in different states in the US), this can quickly multiply to screening several thousands of plants in total. The few plants selected from the trait mapping and trait introgression steps are then used as the parental plants for the final step in conventional breeding practices needed to make a commercial variety. Using commercial maize in the US as an example, the selected 10-15 parental plants are cross pollinated to generated more than 150,000 progeny plants (Figure 1.3). This large number of progeny plants are evaluated for many agronomic and quality parameters over the course of approximately 6-7 years at an increasing number of geographic or environmentally diverse locations in this “Field Testing” stage of the process (Glenn et al 2017). Plants that do not meet the pre-defined performance criteria are discarded, thereby removing unintended or off-type effects that might become apparent under environmentally diverse cultivation conditions. For maize breeders, as an example, by the end of field testing, they have eliminated more than 99.9% of the progeny plants to identify commercially competitive varieties 20 suitable to grow in different locations (Glenn, et al., 2017) (Figure 1.3). At the final stage of the field testing process, a breeder must use field data to show that the characteristics of a new variety are distinct and stably and uniformly inherited. In the United States, this data is submitted to the U.S. Department of Agriculture (USDA) to receive plant variety protection (PVP) certification. The PVP system is administered by the USDA PVP Office to provide intellectual property protection to breeders of new varieties to help manage the use by other breeders and to ensure legal protection of their work (USDA, 2019). In the United States, further oversight is administered by the U.S. Food and Drug Administration (FDA), which is responsible for ensuring that all food and feed products (with the exception of specific red meat, poultry and egg products regulated, instead, by the US Department of Agriculture) (FDA, 2017) are safe for human and animal consumption (FDA, 2011). The breeding process framework described above is universally applied by both public and industrial breeding programs across crops that address intrinsic and extrinsic factors related to crop improvement such as: 1) agronomic parameters (e.g., yield, biotic and abiotic stress resistance); 2) consumer preferences (e.g., flavor, appearance); 3) allergens (e.g., Mal d 1); 4) plant-produced toxins (e.g., glycoalkaloids) and; 5) nutrition. It is standard practice of breeding programs to fortify their germplasm collection with disease resistance traits to protect yield against prevalent bacterial, viral and fungal diseases. Protecting crops plants from disease can also help ensure a safe food supply since some diseases, such as fungal ear rot, are associated with mycotoxin contamination of foods. Breeders have applied plant selection practices in diverse crops to enhance the content of desirable compounds (e.g., antioxidant in tomato) (Abbadi & Leckband, 2011; Duvick, 2005; Hanson, et al., 2004) while maintaining a safe food supply. The rare reported cases of a new variety posing a food safety risk have been observed with crop species already known to have the 21 metabolic pathways present to make plant toxins (Berkley, et al., 1986; Seligman, et al., 1987; Zitnak & Johnston, 1970). In contrast, there are no documented examples where conventional breeding has resulted in production of a random, novel toxicant or a novel toxin metabolic pathway that was not previously known to be present in a given crop (Steiner, et al., 2013; Weber, et al., 2012). This review discusses how conventional breeding practices are used by plant breeders to bring forward desirable new traits while ensuring that naturally occurring plant-produced toxins remain at safe levels during the plant breeding processes that bring new varieties to market. Naturally occurring plant toxins in food crops Plants naturally synthesize and accumulate a wide array of chemical compounds, some with toxic or antinutritional properties. In order to help understand how plant breeders can fine tune their practices to ensure a safe food supply for consumers, two categories of crops are proposed, according to the type of compound present throughout crop production, harvest and processing. Crop case studies for each category are used to further describe how plant breeders adjust breeding practices to ensure food derived from conventionally bred crops are safe for consumption. Crop Category 1: Crop plants with no significant plant-produced toxins or allergens: Crops in this category, such as maize, have long histories of safe consumption across millennia of domestication and breeding practices (Table 1). The framework of conventional breeding practices (Figure 1) is used to incorporate traits that improve yield, enhance nutrition and improve abiotic (e.g., drought) and biotic (e.g., microbial infection) stress tolerance of crops in this category. Breeders of crops in this category focus on agronomic parameters as advancement criteria to evaluate variety performance under different environmental conditions, such as varied geographic location and soil type, and management practices (e.g. irrigation, nutrition, plant density). 22 Crop Category 2: Crop plants with known plant-produced natural toxins: Crop examples include celery, cassava, potato and rapeseed (Table 1). The breeding practices of such crops include advancement criteria for the same agronomic plant testing and selection practices used for Category 1 crops. Additionally, the presence and quantity of specific known toxins are monitored throughout the many stages of the breeding process (Figure 1), with toxin production and accumulation serving as pivotal selection criteria to ensure toxin levels do not exceed an acceptable range as recommended by food safety authorities (e.g., Food Standards Australia New Zealand (FSANZ), Food Safety Authority of Ireland). Crop category 1: Crop plants with no significant plant-produced toxins Many plant crops are contained within this category. Breeding of crops in this category includes a series of tests and selection for a range of quality parameters (e.g., taste, size, shape, appearance and nutrient levels) in addition to agronomic traits important for crop growers. When applicable, breeders of these crops also monitor and select for compounds correlated with characteristics integral to improved food processing, consumer preference and/or human nutrition (Table 1). For example, to improve quality traits of interest to consumers, carrot breeders select for pigment (e.g., carotenoids and anthocyanins) and flavor (e.g., volatile terpenoids) compounds (Simon, 2019). Since Category 1 crops, by definition, lack significant known toxins or allergens, the only other type of food safety concern associated with these crop plants primarily stem from whether the plants have properties that mitigate mycotoxin contamination. To that end, plant breeders indirectly reduce mycotoxin contamination in the food supply by developing disease resistant varieties. For example, the presence of aflatoxin contamination in grains and nuts infected with various Aspergillus species can make a crop legally unmarketable in developed countries (Sarma, 23 Bhetaria, Devi, & Varma, 2017) and pose a significant public health risk in developing countries (Brown, et al., 2013; Groopman, Kensler, & Wild, 2008; Wild, 2007). Aspergillus resistance is, therefore, a target trait for plant breeders who work on these crops (Abbas, 2005; Brown, et al., 2013), although physical and chemical aflatoxin decontamination measures often complement the use of host plant resistant varieties (Ismail, et al., 2018; Jalili, 2016; Pankaj, Shi, & Keener, 2018). Since mycotoxin contamination in the food supply, resulting from infection of certain fungal plant pathogens during plant development, harvest or storage, has been thoroughly and recently reviewed by others, it will not be extensively discussed in this review (Anfossi, Giovannoli, & Baggiani, 2016; DeVries, Trucksess, & Jackson, 2012; Moretti, Logrieco, & Susca, 2017; Wu, 2019). However, highlights of disease resistance plant breeding criteria to helps to reduce mycotoxin contamination in foods is included in the following case study of maize (a Category 1 crop) since maize breeding includes significant efforts aimed at incorporating host plant antifungal resistance against mycotoxigenic fungi. Case Study: Maize Maize (Zea mays) is a widely consumed and an economically significant crop domesticated more than 8,700 years ago in Central America from teosinte, a wild grass ancestor (Doebley, et al., 2006; Smith, 2001; Wesley, Helliwell, & Smith, 2001; Yang, et al., 2019). After Europeans were introduced to maize by the indigenous peoples of the Americas (Staller, Tykot, & Benz, 2006; Wills, 1988), maize has been widely cultivated worldwide for both food and feed uses. Maize breeders primarily focus on improving traits such as yield and abiotic and biotic stress tolerance using the breeding framework illustrated in Figure 1. The breeding process employs large numbers of parental plants that factorially result in an order of magnitude higher set of hybrid pairings that 24 are then subjected to selective breeding practices. Breeders use an array of agronomic parameters as advancement criteria to test all maize plants prior to variety release. Depending on the end user for maize, plant breeders adjust their breeding practices. For instance, breeders perform additional testing when maize is to be processed into food items by the maize processing industry (Figure 2A). All maize varieties are subjected to agronomic characterization testing, such as yield, disease resistance and standability (Glenn, et al., 2017). A small proportion of maize varieties that meet the agronomic performance criteria are further tested in analytical labs using near infrared spectroscopy (NIR) for a variety of kernel characteristics including density, and composition (e.g., carbohydrate, protein, and fat) (Egesel & Kahrıman, 2012). Kernel hardness is tested by image analysis for a subset of these maize varieties that meet an acceptable density threshold (Figure 2B). Ultimately, what differentiates food grade maize from feed grade maize is typically kernel density or hardness which results from horneous endosperm. The higher percentage of horneous endosperm directly contributes to higher mill yield for food processors and are, thus, more profitable and less wasteful for this industry. Hence, breeders assess maize kernels for desired grain quality prior to variety release. Maize was domesticated from teosinte (Ramos-Madrigal, 2016). Regulatory assessment of teosinte did not find any scientific report on teosinte that would point to a safety concern (European Food Safety Authority, 2016). The Task Force for the Safety of Novel Foods and Feeds of the Organization of Economic Co-operation and Development (OECD) developed consensus documents that define the nutrients, anti-nutrients and/or toxicants relevant to the food and feed safety of novel varieties of crops. In the OECD consensus document for maize, the only compounds identified as needing to be assessed as an anti-nutrient (or toxicant) were: phytic acid (because phytate binds phosphorus preventing it from being nutritionally available in animal feed), 25 raffinose (which, if not removed by food/feed processing, can cause uncomfortable flatulence, but is not a toxicant) and DIMBOA (2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one) (Organization for Economic Co-operation and Development, 2002). The glycoside of DIMBOA (plus other defense-related phytochemicals such as terpenoid phytoalexins) are present in a variety of plant tissues (Ahmad, et al., 2011; Engelberth, Alborn, Schmelz, & Tumlinson, 2004; Schmelz, et al., 2011). However, these plant defense phytochemicals are predominantly present in green aerial and root tissues and, therefore, are only of a safety concern for animal feed silage (in which tissues from the whole plant are fed to ruminants), but they are not present in the kernel tissues used to make human food (Organization for Economic Co-operation and Development, 2002). Field and post-harvest conditions that promote fungal growth on maize grain resulting in mycotoxin contamination represent the primary food safety concern for this crop (Nuss & Tanumihardjo, 2010; WHO, 2018). The mycotoxins that occur most frequently in maize and are associated with the most detriment to human health are aflatoxins (produced by Aspergillus flavus and A. parasiticus), deoxynivalenol (DON, produced by Fusarium graminearum), and fumonisins (produced primarily by Fusarium verticillioides and F. proliferatum) (Munkvold, 2003). Many studies have used genetic mapping, genomics, transcriptomics and/or proteomics to identify candidate genes associated with resistance to aflatoxin accumulation or Aspergillus infection (Brown, et al., 2013; Gaikpa & Miedaner, 2019; Hawkins, et al., 2018). As a result, potential biochemical and genetic resistance markers have been developed and are utilized in maize breeding programs as selectable markers (Cleveland, Dowd, Desjardins, Bhatnagar, & Cotty, 2003). Genomic selection is widely implemented in maize and represents a valuable tool to select simultaneously for the many minor-effect alleles that contribute to resistance of certain mycotoxin 26 producing pathogens (Chen, et al., 2016) and has been implemented in maize to predict resistance (Han, et al., 2018; Riedelsheimer, et al., 2013). Crop Category 2: Crop plants with known plant-produced natural toxins Crop plants with allergenicity potential The extensive topic of food allergies has been previously well reviewed (Békés, et al., 2017; Breiteneder & Mills, 2005; Cianferoni & Spergel, 2009; Helm & Burks, 2000; Jouanin, et al., 2018; Mills, Madsen, Shewry, & Wichers, 2003; Sicherer & Sampson, 2018; Tsuji, Kimoto, & Natori, 2001; Zuidmeer, et al., 2008) and, therefore, is not a focus for this review. The presence of crop plant allergens is often not a stringent selection criterion, comparable to other plant toxins, especially given that food allergens are almost always specific proteins of large protein families, that have complex inheritance in plant breeding. Therefore, although screening germplasm to identify individuals with significantly reduced or null allergen content is laborious, conventional breeding efforts toward hypoallergenic varieties have been undertaken in wheat, soybean, peanut and apple. The gluten in hexaploid bread wheat is comprised of many different proteins, predominated by the glutenin and gliadin classes of protein. Glutenins are integral to baking quality while gliadins contain the majority of fragments (epitopes) associated with coeliac disease. Old hexaploid bread and tetraploid durum wheat varieties with few epitopes linked to gluten intolerance have been identified, but creating favorable combinations of gluten genes to satisfy baking quality requirements in a polyploid is challenging (Gilissen, van der Meer, & Smulders, 2014). Similarly, screening soybean and peanut germplasm collections has resulted in the identification of lines with zero to low allergen content (Riascos, Weissinger, Weissinger, & Burks, 2010). Additionally, genetic engineering of the specific target gene encoding the allergenic protein has been adopted as an efficient alternative in peanut (Chandran, Chu, Maleki, & Ozias- 27 Akins, 2015; Dodo, Konan, Chen, Egnin, & Viquez, 2008), soy (Herman, Helm, Jung, & Kinney, 2003) and cereals (Becker, et al., 2012; Gil-Humanes, Pistón, Tollefsen, Sollid, & Barro, 2010; Gilissen, et al., 2014). Crops with known toxins in the non-consumed portion An understanding of plant biochemistry of the consumed portion of a crop plant is crucial to develop crop varieties, and their resulting food products, that are safe and nutritious for human consumption. For example, fruits belonging to the Rosaceae family, such as apples, almonds, apricots, peaches and cherries, are known to produce a natural undesirable bitter compound in the seed called amygdalin, high levels of which can cause cyanide poisoning when ingested (Arrázola, Sánchez, Dicenta, & Grané, 2012; Chaouali, et al., 2013; Conn, 1980; Dicenta, et al., 2002; Franks, et al., 2008; Kolesár, Halenár, Kolesárová, & Massányi, 2015; McCarty, Lesley, & Frost, 1952; Poulton & Li, 1994; Sánchez-Pérez, Jørgensen, Olsen, Dicenta, & Møller, 2008). As a seed crop, potential new almond varieties must be screened for amygdalin and those that have unacceptable seed bitterness are discarded (Gradziel, 2009). In contrast, humans generally only consume the flesh and peel of other members of the Rosaceae family. Therefore, apple, apricot, peach and cherry breeders do not screen new fresh market varieties for the toxin since amygdalin is not present in the consumed fleshy parts of the fruit. The target market sector for the food crop also informs the breeder’s selection criteria. For instance, apple juice processing routinely involves the entire fruit, including the seeds which may disintegrate and contaminate the juice. However, analysis of apple juice found that processing reduced the amygdalin content drastically, ranging from 0.01 mg/m to 0.08 mg/ml, which is unlikely to present any health problems (Bolarinwa et al., 2015). 28 Expansion of a food crop into new markets may also be predicated on breeding efforts for reduced production of a plant toxin. Although apricot seeds are a source of dietary protein, (Nout, Tuncel, & Brimer, 1995) fiber and oil (Femenia, Rossello, Mulet, & Canellas, 1995), the use of apricot seeds for human consumption is constrained by the availability of cultivars with low amygdalin seed levels (Gómez, Burgos, Soriano, & Marín, 1998). Case Study: Apple Apple, (Malus domestica Borkh.) is the most economically important crop species of the Rosaceae family, with over 83 million tons of fruit produced worldwide in 2017 (FAOSTAT, 2017). Although the center of origin of apple can be traced back to the Neolithic age (11,200 BCE), archeological evidence for the gathering of wild Malus species indicates that cultivation of apple began circa 2000 BCE (Zohary & Hopf, 2000). The modern cultivated varieties of apple are proposed to have originated from natural hybridization between four species - the Tien Shan wild apple (M. sieversii (Ledeb.) M.Roem.) followed by M. baccata (L.) Borkh., M. orientalis Uglitzk., and M. sylvestris (L.) Mill. (Cornille, Giraud, Smulders, Roldán-Ruiz, & Gladieux, 2014). These species were collectively hybridized into the modern domesticated apple (M. pumila/domestica) which has been the progenitor of various cultivated landraces through cloning, grafting and further hybridization. Successive selection has led to the development of modern cultivars such as ‘Honey Crisp’, ‘Gala’, ‘Fuji’, ‘Pink Lady’ and most recently, ‘Cosmic Crisp’ that represent a range of juiciness, sweetness, crispiness, crunchiness, colors, firmness, size, time of harvest, and overall eating experience (Velasco, et al., 2010). Currently, there are over 10,000 apple cultivars documented across 25-30 species of Malus, with at least six typically non-commercial subspecies colloquially termed ‘crabapple’ (Gardiner & Folta, 2009; Janick & Moore, 1996). 29 Many of the world’s prominent varieties were sourced from chance seedlings until the mid- 20th century (Janick & Moore, 1996) and from cider apple seeds around the end of 19th century (Janick & Moore, 1996) until Thomas Andrew Knight performed the first controlled cross breeding of multiple varieties with the English dessert apple ‘Golden Pippen’ (Morgan & Richards, 2002). Most apple cultivars are diploid (n=17; allotetraploid), although triploid (3x=51; e.g., ‘Jonagold’, ‘Gravenstein’, and ‘Roxbury Russet’) and tetraploid (4x=68; e.g., ‘Gala’) cultivars also exist (Spengler, 2019). Breeders will sometimes seek triploid progeny in their programs, knowing that triploids often have larger fruits. (Ferree & Warrington, 2003). Apple fruit is consumed as fresh, or processed for use in pies, jams, and sauces, or the juice from the fruit is often distilled into brandy or fermented into cider, from which vinegar is also made (Hummer & Janick, 2009). Apple flesh is mostly water, carbohydrates, and simple sugars (at roughly 75-80%, 13% and 10% total weight, respectively), but also contains a considerable amount of dietary fiber (~3% total weight) along with phytonutrients such as quercetin, catechin and chlorogenic acid that have been associated with human health (Boyer & Liu, 2004). Seed-produced amygdalin is the only known toxin in apple (Organization for Economic Co-operation and Development, 2019). Genes conferring any flesh-specific toxic secondary metabolites were most likely eliminated during domestication. However, because flesh flavor is a quantitative trait, controlled by many genes, individual plants producing apple fruit with offensive flavors or undesirable organoleptic profiles may arise through the process of cross breeding. These individuals are eliminated in the early stages of sensory testing. The only deliberate example of modifying a pre-existing biochemical pathway in apple is the development of the transgenic non- browning Arctic® apple to reduce the levels of an already present enzyme-polyphenol oxidase 30 enzyme (Carter, 2012). Prior to commercialization of the Arctic® apple, regulatory agencies reviewed data showing that the metabolic change did not affect the food safety and nutritional quality of the fruit, and that the transgenic apple was substantially equivalent to the parental variety (Carter, 2012; Stowe & Dhingra, 2019). Ingestion of apple flesh can trigger oral allergy syndrome (OAS) in some individuals, manifested as a contact allergic reaction of the oral mucosa, lips, throat and tongue. The prevalence of a perceived OAS reaction was estimated to be 0.5% in adults (Europe, the United States, Australia and New Zealand) and 0.9% to 8.5% in European children (Organization for Economic Co-operation and Development, 2019). The most prevalent OAS reaction to apples is noted for individuals sensitive to the birch tree (Betula spp.) pollen protein, Bet v1. Such individuals will experience an immunoglobin-E-mediated (IgE) cross-reaction with the Bet v1 Malus homologue, Mal d 1(Wagner, Szwed, Buczylko, & Wagner, 2016). Mal d 1 protein content varies among different apple cultivars, but can vary inconsistently among apples of the same variety. The Mal d 1 protein is readily denatured by processing, such as in Pasteurized juices, stewed fruit and cakes, such that individuals allergic to raw apples can tolerate these apple-containing processed foods. A less common (predominantly seen in the Mediterranean area), although symptomatically more severe allergic reaction to apples is observed in some individuals sensitive to the Mal d 3 protein. Parallel to the allergic reaction to Mal d 1, the allergic reaction to Mal d 3 is observed in individuals that have previously been sensitized to the peach allergen, Pru p 3, and suffer an IgE-mediated cross-reaction to Mal d 3 in apples. Unlike Mal d 1, however, Mal d 3 is very stable and resistant to heating. The topic of apple allergens and allergic reactions are well reviewed (Geroldinger- Simic, et al., 2011; Gilissen, et al., 2005; Wagner, et al., 2016). Unlike toxins, screening for allergens is not routinely conducted during the apple breeding process although certain cultivars 31 with low allergenicity potential have been identified (Vlieg-Boerstra, et al., 2013). However, the possibility to reduce or eliminate clinical allergenicity to apples was recently demonstrated in a study reducing the gene expression of Mal d 1 in apples (Dubois, et al., 2015). The most significant post-harvest apple food safety concern is the development of blue mold in apple caused by Penicillium expansum. Contamination of infected applies with the carcinogenic mycotoxin, patulin, is a concern in fresh and processed apple products but can be mitigated through management of storage conditions, fungicide application, physical removal of infected tissue, and processing (Ioi, Zhou, Tsao, & Marcone, 2017; Vidal, et al., 2019). Although there are currently no commercial cultivars with blue mold resistance, DNA regions contributing to variation in resistance have been mapped in a wild Malus sieversii accession PI613981 (Norelli, et al., 2017) and differentially expressed genes identified in these resistant genotypes respond to pathogen infection (Ballester, et al., 2017). This work lays the foundation for incorporating resistance into apple breeding programs. An additional food safety concern for apples, albeit unrelated to apple breeding practices, is the association of bacterial contamination (Listeria monocytogenes) from packing houses and production lines, with processed foods from apples, such as apple juice, leading to food recalls on occasion (Pietrysiak, Smith, & Ganjyal, 2019). Apple breeders screen and advance promising apple selections primarily based on fruit quality parameters, such as juiciness, crispiness, firmness, storability along with some diseases such as scab, fireblight and powdery mildew (Baumgartner, Patocchi, Frey, Peil, & Kellerhals, 2015; Laurens, et al., 2018). The apple breeding process, with respect to selection and field testing, is similar to that shown in Figure 1. However, apple breeders have fine-tuned the process for the apple crop, by incorporating: 1) screening for powdery mildew resistance, and 2) using two breeding methods in tandem (cross pollination and clonal propagation). The first step in apple 32 tandem breeding involves cross pollination of plants, followed by clonally propagating with root stock and scion. With advanced molecular biology genomics tools and whole genome sequencing approaches available today, apple breeders can use genomic methods to distinguish with precision between individuals or cultivars, or cultivars from somatic sports (Hewitt, et al., 2017; Lee, et al., 2016; Nybom, 1990). Genomic approaches have resulted in significant advances in speed, accuracy and effectiveness of the breeding process (Ru, Main, Evans, & Peace, 2015). However, the basic principles of breeding, and the process itself, remain the same. Apple breeders, during the typical breeding process, first generate hybrid (F1) seeds from cross pollinating two parental plants. Hybrid seeds are then germinated in greenhouses and subjected to multiple rounds of selection for powdery mildew resistance. Breeders perform a mandatory plant health screening practice that is required throughout all apple breeding programs in the US (Brown, 2012), where apple plantings are screened against susceptibility to infection by powdery mildew (Podosphora leucotricha), and various other diseases. Next, the promising seedling selections are grafted onto rootstocks (the root and lower stem section of a plant) for clonal propagation (Koepke & Dhingra, 2013). Physical traits, such as dwarfing and floriferousness, are transmitted through the rootstock while additional morphological and foliar disease resistance traits are conferred by the scion (the aerial bud or shoot of a plant), resulting in a composite tree with characteristics imparted by both (Janick & Moore, 1996). Apple breeders can use both natural (e.g., cross pollination, spontaneous somatic mutations) and induced genetic variation (e.g., mutagenesis) in apple breeding. For instance, mutational breeding has led to darker red apple skin and compact tree stature (van Harten, 1998). Of the 13 listed commercial apple varieties in the International Atomic Energy Agency Mutant Varieties Database (IAEA, 2019), none are tested for any toxins because of mutagenesis. Spontaneous somatic mutations with 33 distinct phenotypic differences from the mother tree, called budsports (or “sports”), are another source of genetic diversity in apple. For example, the conventionally bred variety ‘Delicious’ has produced sport clones with more desirable characteristics and have acquired new names that have entirely replaced the original cultivar. Similarly, a sport of the favored variety ‘Jonagold’ (a crossbreed of ‘Golden Delicious’ and ‘Jonathan’), referred to as ‘Jonagored’, was discovered in Belgium in 1986 and is now quite popular because of its more intense red coloring (van Harten, 1998). A crossbreeding strategy developed by the Washington State University (WSU) Apple Breeding Program (WABP) is shown in (Figure 3) to illustrate a representative fruiting scion selection process. Primary breeding targets for selection include fruit texture, appearance, storability, yield, and lack of blemish (such as russet). In Year One of the WABP, approximately 20,000 seeds, from ~200 to ~3000 open pollinated progenies, are produced. Year Two begins with seedling germination in a greenhouse in January/February. Seedlings are visually screened for mildew endemic sources in the Pacific Northwest and susceptible individuals are eliminated. In scion breeding programs in general, one of the major goals, along with fruit quality traits, is resistance to various disease such as fireblight, scab and powdery mildew. As apple breeding became more organized, powdery mildew (PM) resistance became a concern for plant health in the mid-90s. Breeders started the practice of breeding for PM resistance which is now a mandatory part of scion breeding (Brown, 2012). Seedlings are then transferred to the nursery in late May to early June and are screened once again for PM susceptibility and subsequently budded onto dwarfing rootstocks in Year Three. Dwarfing rootstocks, such as the widely used M.9, generally reach maximum heights of 2-2.5m and are easier to prune than rootstocks that are not dwarfed. By Year Five, trees are transitioned into Phase 1 of three selection steps. Since no food safety concerns 34 exist for the flesh and skin of apple fruit, selection at this point in the program is focused on assessing food quality items, such as starch levels and eating quality, and the appearance of the fruit. Phase 1 trees are planted at WSU’s research orchard where they are subjected to industry standard spraying and irrigation regimen. While spraying and irrigation are not direct selection criteria, trees that do not perform well under these standard cultivation practices may still be discarded. Individual plants with desirable fruit phenotypic characteristics (e.g., appearance, taste) starts at Year Six and carries into Years Seven and Eight. Fruit characteristics are assessed immediately after harvest, as well as after two- and four-months storage at 4oC. Both instrumental and sensory assessments are conducted on fruit selected from Phase One. Fruit weight, size, firmness and crispness metrics are measured with a penetrometer, while starch levels, titratable acidity, and Brix from each fruit is also recorded. Room temperature fruit samples are rated on appearance and sensory traits. The top performing individuals are grafted onto M.9 rootstocks and advance to Phase 2 of selection. Phase 2 begins at Year Nine, with five trees from each top performing selection planted in randomized blocks at multiple diverse orchards in Washington State. These trees are managed as local grower norms dictate. Fruit selection and assessment continues as in Phase 1, but with larger sample sizes from fruit harvested at weekly intervals until year 13. Individual tree selections made at this stage are deemed ‘elite’, more are grafted onto M.9 rootstocks, and advanced to Phase 3. In Phase 3, four unique and geographically diverse grower sites receive approximately 75 trees of each ‘elite’ selection made in Phase 2, where harvest, storage and packing line tests are conducted with the aid of the Washington Tree Fruit Research Commission (WTFRC) until year 18. Fruit from Phases 2 and 3 are subject to the same assessment as Phase 1 fruit as well as sensory analysis by a trained professional and untrained consumer panel (Evans, 2013). 35 Apple breeding programs in the public and private sector are abundant throughout the developed world. Breeding objectives may be tailored toward local grower and consumer demands or focus on broader traits, such as tree architecture and precocity. Recent advances in gene editing methods have allowed apple breeders to consider their use as supplemental technologies in breeding programs and provide an example of contemporary apple breeding techniques employed across the world to overcome breeding obstacles. For instance, the long juvenile phase in Malus species hampers breeding progress by extending time requirements and resource needs to obtain fruit from prospective seedlings. Researchers at the Julius Kühn Institute of Breeding Research on Fruit Crops (Dresden) are implementing a transgenic approach to bypass the protracted generation cycle in apple by overexpressing a member of the APETALA1/FRUITFULL group of MADS genes in a popular German apple cultivar ‘Pinova’(Flachowsky et al., 2011). The BpMADS4 gene from silver birch (Betula pendula) is responsible for inflorescence initiation in Betula species and was reported by this German research team to induce early flowering upon over expression in apple (Elo et al., 2007; Flachowsky et al., 2007). The ‘Pinova’ apple transformed to overexpress BpMADS4 reduced the juvenile phase to under 18 months to flower, a trait not previously observed in apple breeding programs. It is hoped that the genetic background of this apple may help accelerate conventional breeding practices, such as the integration of new traits from wild Malus species, a process that can take five or more generations to accomplish with each generation cycle taking between four and ten years (Elo et al., 2007). Crop plants with plant-produced toxins in the consumed portion can broadly affect human health Breeders of crops in this category monitor the content of known toxins throughout the selection process and in some cases have labored for decades to reduce toxin levels of otherwise 36 valuable plants to improve food security. One such example is the reduction of the neurotoxin β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP) in grass pea (Lathyrus sativus L.), a staple legume food and feed crop of economic significance to South Asia and Sub-Saharan Africa. Although grass pea agriculture excels in harsh climatic conditions, fixing soil nitrogen and providing an important source of balanced protein, prolonged consumption results in neurological disorders in humans (Kumar, 2011). Genetic variation for ODAP content was identified, allowing concentrated breeding efforts to result in high-yielding, low ODAP (<0.2% w/w) varieties through both hybridization of existing varieties and adaptation of wild low toxin landraces (Dixit, Parihar, Bohra, & Singh, 2016). However, the stability of low ODAP content across environments still presents a challenge (Fikre, 2008; Girma, 2012). Furthermore, the genetic purity of low ODAP producing varieties can be difficult to maintain due to insect-mediated outcrossing. For this reason, it is beneficial for grass pea breeders to co-select for traits that promote self-pollination such as small flowers (Kumar, 2011). Another proteinaceous grain crop with potential to improve food security and environmental sustainability is lupin (Lupinus spp.). Four species of lupine play an important role in agronomic production world-wide: L. albus L. in the Mediterranean, L. angustifolius L. in Australia, L luteus L. in Europe and L. mutabilis L. in South America. The presence of toxic quinolizidine alkaloids (QA) in all tissues of this crop presents an impediment to consumption of this crop and QA reduction is therefore a key breeding target (Gulisano, Alves, Neves Martins, & Trindade, 2019). Selection for ‘sweet lupin’ began in the 1930s in Germany and has resulted in significantly lower QA content of all modern L. albus, L. angustifolius, L luteus, and L. mutabilis L. cultivars compared to their wild counterparts (Frick, Kamphuis, Siddique, Singh & Foley, 2017). Development of low alkaloid L. angustifolius varieties by Dr. John Gladstones in the 1970s 37 enabled the establishment of the modern Australian lupin industry that currently supplies the majority of the world’s lupin grain for human and livestock consumption (Cowling & Gladstones, 2000). To date, the natural variants with low QA levels in lupine are inherited in a recessive manner, which presents a fundamental challenge transmitting the trait in breeding populations and maintaining the purity of released lines in the field (Baer, 2011; Gross, et al., 1988; Santana & Empis, 2001; Williams, Harrison, & Jayasekera, 1984). One of these recessive mutations, the pauper locus, is particularly effective in reducing QA levels and has been incorporated in many lupin breeding programs (Gladstones, 1970; Harrison & Williams, 1982). Expression of QA by lupine provides important defense and competitive fitness functions for the plants by inhibiting bacterial and fungal multiplication, deterring herbivores, and inhibiting competitor plant growth (Dreyer, Jones, & Molyneux, 1985; Waller & Nowacki, 1978; Wink, 1985, 1987). Thus, a major drawback to reducing lupine QA content is increased pest susceptibility. An understanding of how QA are translocated within the plant will facilitate the development of genotypes with low QA levels in the consumed seed while maintaining sufficient foliar levels to prevent pest damage. Other examples of crops in this category include plants in the Brassicaceae and Cucurbit families, celery, rapeseed, lettuce, cassava, and grapefruit (Table 1). Potato is presented as a case study. Case study: Potato Domestication of potato: An economically important food crop Plants in the Solanaceae family produce an array of the naturally occurring compounds called alkaloids and glycoalkaloids that have likely evolved to protect the plant from pest herbivory, many of these compounds are toxic to humans and animals. Consequently, the economically important Solanaceous food crops, potato, tomato and capsicum pepper, have a complex history of human cultivation. These crop members of the Solanaceae family originated 38 in South America and their cultivation was initially met with skepticism in Europe due to their morphological similarity to Eurasia natives, such as deadly nightshade, known to be toxic when consumed and, consequently, long associated with spells and witchcraft (Daunay, Laterrot, & Janick, 2008). This fear was not unfounded. Potato indeed produces toxic glycoalkaloids in all plant tissues including the consumed underground storage organ called the tuber. In high doses, these glycoalkaloids confer a bitter taste and can induce nausea, vomiting, diarrhea and even loss of consciousness. Toxicity is dependent on the ratio and combination of specific glycoalkaloids (Rayburn, Friedman, & Bantle, 1995; Roddick & Rijnenberg, 1987; Roddick, Rijnenberg, & Osman, 1988). Cultivated potato, Solanum tuberosum L. Group Tuberosum (2n=4x=48), was originally domesticated 8,000 - 10,000 years ago from wild diploid species native to the Andes of southern Peru (Spooner, McLean, Ramsay, Waugh, & Bryan, 2005). There is both chemical and genomic evidence for selection against total glycoalkaloid content during the domestication process (Hardigan, et al., 2017; Johns & Alonso, 1990). Indeed, tuber glycoalkaloid levels in the over 100 extant wild, tuber-bearing relatives of potato can be as high as 3500 mg/kg (Gregory, Sinden, Osman, Tingey, & Chessin, 1981). The predominant glycoalkaloids present in cultivated potato are chaconine and solanine, but wild relatives contain unique profiles of a diverse array of glycoalkaloids with largely unknown toxicity (Schreiber, 1968). Because glycoalkaloids are largely heat-stable and water-insoluble, they are not destroyed in common food preparation methods, such as boiling, baking and frying (Bushway & Ponnampalam, 1981). To combat the toxic effects of early landrace tubers, Andean and native North American consumers dipped potato tubers in edible clay to bind the glycoalkaloids and allow for more efficient excretion (Johns, 1986). Bitter tubers were also somewhat detoxified in a process that consisted of repeatedly drying 39 in the sun, squeezing out residual liquid, and subsequent boiling (Johns & Kubo, 1988). These techniques may have permitted growth and consumption of successive generations necessary for selection of more palatable tubers. Consequently, selection against bitter tubers has resulted in decreased tuber flesh glycoalkaloid levels and tuber glycoalkaloids are predominantly localized in the tuber skin of modern potato varieties (Friedman, Roitman, & Kozukue, 2003; Kozukue, Kozukue, & Mizuno, 1987), Cultivation of the potato was crucial to the establishment of early civilizations in the Altiplano where high altitudes, variable temperatures and droughts restrict the growth of maize and other staple grain crops. Scarce arable land also favored the cultivation of potato, which produces 54% and 78% more protein per unit of land area than wheat and rice, respectively, and potato has an impressive nutritional profile. A single potato provides 50% of the recommended daily human allowance of vitamin C, 21% of potassium, and 12% of fiber (Kolasa, 1993). Low in fat, the potato also offers several of the daily required micro-elements and a suite of antioxidants (Brown, 2005; Zehra, 2011). The Highland people leveraged the harsh Andean climate to conserve potatoes as a freeze-dried product, known as chuño, that could be stored up to ten years in a sealed container (Lee, 2006). Chuño later provided the primary fuel for the growth of the Incan empire, as it was easily collected as a tax and utilized to feed labor gangs toiling on the many infrastructural feats of this imperial society (Zuckerman, 1999). Upon their arrival to Potosí, Bolivia, in 1545, Spaniards bought up vast quantities of chuño to resell at inflated rates to miners conscripted to mine silver (Peñarrieta, Juan Antonio Alvarado, Bravo, & Bergenståhl, 2012). Yet it was not until approximately two decades later that the potato was first brought to Spain by ship, perhaps accidentally. Regarded as an inferior crop fit only for indigenous peoples, early European adoption of potato was in peasant gardens for animal feed. The potato further 40 suffered from a rumor surfacing in 1620 that it spread leprosy, and its cultivation was briefly banned by the French Parliament (Zuckerman, 1999). However, in the eighteenth century the potato began to receive more widespread acceptance after Frederick the Great of Prussia recognized the potential of human potato consumption and commanded his subjects to cultivate and eat them (De Jong, 2016). Later, French pharmacist Antoine-Augustin Parmentier, who credited the potato for his survival as a prisoner of war in Prussia during the Seven Years War, encouraged King Louis XVI and Queen Marie Antoinette to endorse the potato as a “fashionable” food, thereby building public acceptance of potatoes as a low-cost safeguard against grain crop failure and food scarcity in wartimes (Salaman & Burton, 1949). Subsequent selection for short- day photoperiod adaption has permitted widespread global potato cultivation in the last 300 years. This complex history of the potato has been recently reviewed by others (Campos & Ortiz, 2020; Sood, Bhardwaj, Pandey, & Chakrabarti, 2017). Today, potato is the fourth most important food crop worldwide, with an annual production of 388 million tons following rice (770 million tons), wheat (771 million tons), and maize (1.1 billion tons) (FAOSTAT, 2017) and is grown in most countries across a diverse array of environments. The potato is utilized not only for fresh market consumption but also is the raw ingredient for the French fry, multiple snack chips and for starch processing (used both in foods and non-food industrial applications). In response to evolving consumer preferences, approximately 65% of US potato production is currently used in the processing market (NASS, 2019). Modern potato breeding and genetics Unlike its wild progenitors, cultivated potato is a tetraploid. Although tetraploid S. tuberosum is not an obligate outbreeder, selfing results in severe inbreeding depression and, as such, modern cultivars are considered outbreeders (Shimelis, 2015). Consequently, cultivated 41 potatoes are highly heterozygous, making it difficult to fix desirable alleles through inbred lines (Bradshaw, 2017; Lindhout, et al., 2011). To circumnavigate inbreeding depression, potato breeders made phenotypic selections on the approximately 40 important traits segregating in the F1 generation and appraised these selections clonally over 10-15 years (Hirsch, et al., 2013; Lindhout, et al., 2011). Moreover, backcrossing to add or stack traits cannot be employed because it will destroy the unique allelic combination within a preferred clone. Potato breeding for all market classes (e.g., chip processing, French fry, table) in the US is primarily conducted in the public sector. In contrast, new European potato varieties are developed by private breeding companies and/or public-private partnerships (Almekinders, Mertens, Van Loon, & van Bueren, 2014). While disease and pest resistance traits are common breeding objectives for all programs, institutions tend to focus varietal development efforts on the on the market class and unique production challenges that predominate in specific geographic regions. For instance, breeding programs in the Midwest select for round, white tubers with high starch content suitable for the potato chip processing market. A representative breeding cycle using chip processing is presented below as an example. Like many crops, development of new potato varieties must address grower, processor and consumer demands as well as anticipate emerging production challenges and consumer preferences. To ensure profitable yield, growers require varieties resistant to pests and diseases, that mature in less than 120 days, and efficiently utilize soil nutrients. Processors have several requirements for potato varieties. One key processor requirement for potato varieties is to produce tubers suitable for cold storage. The majority of potatoes grown for the chip processing market are placed in post-harvest cold storage to ensure year-round availability. While cold storage reduces undesirable sprouting and disease incidence, it also prompts the conversion of starch to 42 reducing sugars, glucose and fructose. When processed at high temperatures, reducing sugars form dark pigments and an undesirable bitter taste through the Maillard reaction, resulting in a potato chip that is unacceptable to the consumer. More problematically, the Maillard reaction of reducing sugars and amino acids generates acrylamide, a neurotoxin and a potential human carcinogen (Mottram, Wedzicha, & Dodson, 2002). Other quality traits essential to processors include resistance to tuber internal defects, tuber bruising throughout harvest, transportation and storage, and oxidative browning upon tuber slicing. Processors also dictate strict requirements for uniform tuber size and shape. Consumer preferences that potato breeders much consider include flavor, texture and white flesh color. Few of these numerous traits are controlled by a single gene, necessitating the generation of large breeding populations to select varieties with most favorable combinations of traits required by growers, producers and consumers (Bradshaw, Hackett, Pande, Waugh, & Bryan, 2008). A typical tetraploid breeding cycle begins by selecting high performing potato varieties (with acceptable tuber glycoalkaloid levels) and generating 100 -1000 crosses in the greenhouse during the winter (Figure 4). Between 100 and 1,000 true seeds are then extracted from each mature fruit of these segregating F1 populations, and the resulting 20,000 -100,000 seedlings are grown to produce tubers over the summer months. These tubers are harvested, bulked as a family and planted in the field the following year. Selection of individuals occurs at harvest in the fall and is based largely on tuber type and tuber internal characteristics. Depending on the market class and stringency of standards for tuber shape and skin type, only 1-3% of first year material is selected to advance in the breeding program as resource constraints dictate that each successive year fewer lines are evaluated more exhaustively for more traits. In the spring of the third year, 12 clones of the single individual selections from the previous year are planted in the field. Selection in the fall 43 is chiefly based on examination of disease and pest resistance potential donated by the parents/grandparents in addition to the observed tuber characteristics. The approximately 300 selected lines can then be subjected to a variety of tests that are too intensive in terms of cost, time, and labor to implement more widely in earlier generations. High throughput DNA extraction allows screening for markers linked to known disease resistance genes. Resistance to commercially relevant diseases and pests are appraised in inoculated field trials. Important processing traits, such as starch content, chip frying color, and bruising susceptibility, are also measured. This data is integrated in the following field season to select approximately 50 lines. These advanced lines are entered into a national 9-location trial that functions to rapidly identify lines performing well in multiple environments. It is at this point in the potato breeding program that tuber glycoalkaloid content is quantified to ensure further resources are not invested in high-glycoalkaloid producing lines. Seed of approximately 10 promising lines is then increased to assess large-scale production performance on farmers’ fields and in storage. Wild potato species introgression: Glycoalkaloid implications Plant toxins, like glycoalkaloids commonly found in Solanaceae plants, are synthesized through complex, multistep pathways. The staggering diversity of these compounds is the result of coordinated regulation of many enzymatic reactions at each step of the biosynthetic pathway. The natural genetic variation of genes encoding these enzymes or regulatory elements in germplasm used by crop breeders can result in quantitative and structural changes of the compounds produced from known pathways (Keurentjes, et al., 2006; Wink, 2010). This is evidenced in potato breeding, as described below, where functional genes necessary for the production of the specialized leptine glycoalkaloids are present only in a single species. The production of leptines, however, is predicated on the extant Solanaceae glycoalkaloid biosynthetic 44 pathway, which has been present in the genome for millennia. Importantly, although conventional breeding practices, such as cross- or self-pollinating, reshuffle genetic allelic combinations to produce new progeny varieties, these breeding practices do not give rise to unfamiliar biosynthetic pathways that produce novel toxins. Plant breeders are thus attuned to the biochemical profile of their crop and track the potential for novel decoration of a known toxin structure when introducing new germplasm. Implementation of affordable genomic sequencing technologies in many crops has also led to the characterization of biochemical pathways (Gupta, Karkute, Banerjee, Meena, & Dahuja, 2017; Patra, Schluttenhofer, Wu, Pattanaik, & Yuan, 2013; Pichersky & Gang, 2000; Xiao, et al., 2013), identifying sequence variation of genes involved in the production of plant toxins, and facilitating the development of genetic markers linked to these genes. Advances in high-throughput metabolite analysis also enables profiling of hundreds of previously uncharacterized compounds in parallel. The breeding heritage of modern North American cultivars is grounded on a narrow genetic base due to a limited number of initial European introductions from South America and subsequent population reduction by devastating late blight outbreaks in the mid-19th century (Hirsch, et al., 2013). Breeders have traditionally attempted to generate sufficient genetic variation and introgress agronomic and biotic/abiotic stress resistance traits through interspecific crosses with wild relatives. Extraction of haploids (2x) from adapted tetraploid S. tuberosum (4x) permits hybridization with diploid wild species (2x) and the capture of these desirable alleles (Carputo, Barone, & Frusciante, 2000). However, because the potential for total glycoalkaloid content in potatoes is highly heritable (Sanford & Sinden, 1972) careful consideration must be given to the glycoalkaloid levels of parental material when developing varieties. At least one accession of each of the wild species routinely used in breeding programs has been assessed for glycoalkaloid levels, 45 and to a lesser extent, composition (Gregory, 1984; Gregory, et al., 1981; Osman, Herb, Fitzpatrick, & Schmiediche, 1978; Schmiediche, Hawkes, & Ochoa, 1980; Schreiber, 1963; Schreiber, 1968; Tingey, Mackenzie, & Gregory, 1978; Tingey & Sinden, 1982). However, glycoalkaloid profiles differ drastically between individuals within an accession, necessitating profiling of the specific individuals used in each breeding program (McCollum & Sinden, 1979; Osman, Herb, Fitzpatrick, & Sinden, 1976). This principle is illustrated by the release and subsequent withdrawal from the market of the potato variety, Lenape, due to elevated glycoalkaloid levels stemming from wild species Solanum chacoense ancestry (Akeley, Mills, Cunningham, & Watts, 1968). Use of S. chacoense accessions has increased recently in contemporary breeding programs in parallel with efforts to restructure potato breeding to a diploid inbred/F1 hybrid variety system using self-compatible diploid germplasm to overcome the current limitations of potato breeding at the tetraploid level (Jansky, et al., 2016). Breeding issues, such as limited recombination, long breeding cycles, and vegetative propagation, are removed. Although the road to homozygosity is faster, many diploids are self-incompatible. In the S. chacoense diploid inbred line M6, however, the self-incompatibility system is inactivated (Jansky, Chung, & Kittipadukal, 2014). Ample use of the M6 line to donate self-compatibility in recurrent selection and recombinant inbred line populations has inadvertently led to elevated levels of glycoalkaloids in breeding germplasm. Fortunately, backcrossing to S. tuberosum material to reduce glycoalkaloid levels in these lines is a viable option at the diploid level. At the diploid level, backcrossing M6-derived diploid potatoes to S. tuberosum material reduces tuber glycoalkaloid content to levels well within standards suitable for human consumption in a single breeding cycle. Although not commonly practiced in tetraploid breeding programs, two cycles of backcrossing were sufficient to reduce progeny 46 glycoalkaloid content to levels comparable with the S. tuberosum parent in a S. chacoense Í S. tuberosum tetraploid population (Sanford, Deahl, & Sinden, 1994). Monitoring, and leveraging, potato glycoalkaloid levels in the breeding process The industry standard for glycoalkaloid levels in tubers intended for human consumption is <200 mg/kg fresh weight and concentrations of glycoalkaloids range from 100-150 mg/kg fresh weight in commercially released potato variety tubers (Sinden, Sanford, & Webb, 1984). The majority of tuber glycoalkaloids are located in the skin (Friedman, et al., 2003; Kozukue, et al., 1987), which presents particular concern for processed potato products with high skin/flesh ratios, such as fries and wedges. Potato breeders utilize pedigrees to monitor potential high glycoalkaloid levels in breeding germplasm and directly quantify glycoalkaloids in advanced selections. The most popular method for glycoalkaloid quantification is high-performance liquid chromatography (HPLC) using extractions from freeze-dried tuber tissue. Since the cultivated potato glycoalkaloid profile is primarily composed of solanine and chaconine, quantification of these compounds is used as a proxy for total glycoalkaloids. Solanine and chaconine concentrations are calculated using a standard curve generated from pure standards. Samples are submitted to laboratories in the public and private sector or processed in-house, depending on the technical capacity of each breeding program. Advanced selections with glycoalkaloid levels determined by chromatography analysis to be <200 mg/kg, are sometimes subject to additional bitterness taste testing, since bitterness can result in rejection from commercial markets. As is the case for many plant produced toxins, glycoalkaloid content of potato tubers is also strongly influenced by environmental factors. Climatic variation in growing environments can lead to drastic differences in glycoalkaloid content of tubers from the same variety (Gosselin, Mondy, & Evans, 1988; Mondy & Munshi, 1990; Morris & Petermann, 1985; Sinden, et al., 1984; 47 Slanina, 1990; Van Gelder & Dellaert, 1988). Although significant interactions between genotype and environment have been reported (Sinden & Webb, 1972), high glycoalkaloid accumulation in one environment is typically predictive of even higher levels under stress conditions (Lepper, 1949; Sinden, et al., 1984). In addition, glycoalkaloid levels can increase significantly post-harvest in response to storage, temperature, mechanical wounding (Friedman & McDonald, 1999; Mondy & Gosselin, 1988; Mondy, Leja, & Gosselin, 1987) and light exposure (Friedman, 2006). Light stress also prompts chlorophyll production, commonly referred to as ‘tuber greening.’ Thus, green tubers are often associated with increased glycoalkaloid levels. Tubers not properly covered by soil in the field are exposed to sunlight and can receive additional artificial light stress during the storage, grading, and packaging process. For this reason, as an additional checkpoint to ensure a safe food supply, U.S. potato grading standards regard potatoes as “damaged” or “seriously damaged” if 5% or 10% of the total weight must be removed due to greening, respectively (USDA- ARS, 2011). Additional light exposure can occur in the retail market, where tubers are often displayed in mesh or clear plastic packaging to afford the consumer product visibility, risking additional stress response increases in glycoalkaloid levels. To mitigate environmentally induced high glycoalkaloid levels, breeders select for genetic backgrounds with low glycoalkaloid production potential. There are breeding objectives for which it is desirable to actually select for specific glycoalkaloids in the breeding germplasm. For instance, several accessions of Solanum chacoense produce and accumulate the specialized leptine glycoalkaloids (Hutvágner, et al., 2001; Mweetwa, et al., 2012; Ronning, Sanford, Kobayashi, & Kowalsld, 1998; Ronning, et al., 1999; Sagredo, Lafta, Casper, & Lorenzen, 2006; Sanford, Kobayashi, Deahl, & Sinden, 1996), which deter Colorado potato beetle feeding through a cholinesterase inhibiting mechanism (Rangarajan, 48 Miller, & Veilleux, 2000; Sanford, Kobayashi, Deahl, & Sinden, 1997; Sinden, Sanford, Cantelo, & Deahl, 1986; Sinden, Sanford, & Osman, 1980), similar to that of organophosphate insecticides. The Colorado potato beetle is the most widespread and destructive insect defoliator pest of potato crops and, uncontrolled, can reduce yield up to 80% (Alyokhin, Vincent, & Giordanengo, 2012). Unlike chaconine and solanine, commonly found in all plant organs of cultivated potato, leptines are only produced in aerial tissues and therefore do not pose a food hazard to human health (Mweetwa, et al., 2012). These novel glycoalkaloids can be extracted from foliar tissue implementing simple protocols akin to those for total glycoalkaloid extraction and quantified with HPLC using the known molecular weights of these compounds. The S. chacoense host plant resistance is introduced into beetle susceptible, adapted material by crossing, and the progeny inexpensively screened for resistance in the lab by observing feeding of Colorado potato beetle larvae on detached leaves in petri dishes (Figure 5). Lines that demonstrate superior resistance and low tuber glycoalkaloids are then selected for field appraisal using natural populations of Colorado potato beetle. The genetic control of glycoalkaloid content and composition has been increasingly elucidated in recent years (Cárdenas, et al., 2016; Itkin, et al., 2013; Mariot, et al., 2016; Sawai, et al., 2014). Development of markers linked to genes responsible for glycoalkaloid biosynthesis would facilitate marker-assisted selection in potato breeding programs for varieties with low levels of glycoalkaloids, as opposed to the current reliance on phenotypic characterization. Transgenic tools also stand to help breeders develop potato lines with reduced glycoalkaloid levels. Recent silencing of key genes in the glycoalkaloid biosynthetic pathway has resulted in tetraploid lines with reduced foliar solanine and chaconine accumulation (Paudel, et al., 2017) and altered glycoalkaloid partitioning in tubers to mitigate accumulation of the more potent chaconine 49 (McCue, Breksa, Vilches, & Belknap, 2018). Tuber-specific silencing of known regulatory transcription factors in the glycoalkaloid biosynthesis pathway (Cárdenas, et al., 2016; Mariot, et al., 2016) could reduce total tuber toxin levels while leaving foliar insect protectant functions intact. Conclusion Conventional plant breeding has a long history of improving crop productivity, food security and safety. Although similar practices are employed for the breeding of most crops, selection criteria are modulated to account for the unique challenges of each crop. The advent of molecular and genomic tools has allowed breeders to track specific genes known to influence traits of interest and concern in addition to characterizing more broadly the genetic landscape of new varieties. Importantly, although conventional breeding practices, such as cross- or self-pollinating, reshuffle genetic allelic combinations to produce new progeny varieties, these breeding practices do not give rise to unfamiliar biosynthetic pathways that produce novel toxins. Therefore, plant breeders can fine tune their practices depending on the crop and specific known natural toxins inherent to that crop species, thereby ensuring a safe food supply. Furthermore, as consumers, plant breeders themselves are the recipients of the food supply system and as such have a vested interest in producing safe crops for themselves and their families. Taken together, generations of historical knowledge that includes breeding selection practices coupled to a robust set of industry standards and governmental review procedures ensure the safety of new crop varieties brought to market. 50 APPENDICES 51 APPENDIX A: Chapter 2 Tables Table 2.1. Example crops, traits and natural compounds measured for each of two proposed plant breeding crop safety categories. Purpose Reference Nutrition, Food processing Consumer preference Consumer preference Consumer preference Nutrition Consumer preference Consumer preference, Nutrition Safety Safety Safety, Consumer preference Safety Safety Safety, Consumer preference Safety (Keilwagen, et al., 2017; Simon, 2019) (Egesel & Kahrıman, 2012; Glenn, et al., 2017) (Clark, Shaw, Wright, & McCallum, 2018) (Naves, et al., 2019) (Wang, et al., 2019) (Acharya, Dutta, Dutta, & Chattopadhyay, 2018; Bai & Lindhout, 2007) (Folta & Klee, 2016; Manoharan, et al., 2017; Zhu, et al., 2018) (Ceballos, Iglesias, Pérez, & Dixon, 2004; Zidenga, Siritunga, & Sayre, 2017) (Yang & Quiros, 1993) (Shang, 2014; Zhang, et al., 2012) (Fidel, et al., 2016) (Dixit, et al., 2016; Lambein, Travella, Kuo, Van Montagu, & Heijde, 2019) (Drewnowski & Gomez- Carneros, 2000) (Gulisano, et al., 2019) (Ginzberg, et al., 2009) (Abbadi & Leckband, 2011) Maize Onion Pepper Tomato Crop Traits Category 1. Crops containing no significant natural toxins Carrot Compound Terpene, carotene, carotenoids, nitrate Carbohydrate, protein and fat Color, vitamin content, flavor Kernel quality Pungency, sweetness Pyruvate, fructans, glucose, sucrose Capsaicinoids Carotene, Carotenoids Vitamin content Citric acid and fructose; Flavor Color Carotenoids Color Category 2. Crop plants with known plant-produced natural toxins Cassava Toxin content Cyanogen Celery Cucurbits Psoralens Cucurbitacins Grapefruit Furanocoumarins Grass pea Lettuce Lupine Potato Rapeseed β-N-oxalyl-l-α,β- diaminopropionic acid (L-ODAP) Terpenes Quinolizidine Alkaloids Glycoalkaloids Erucic Acid Toxin content Bitterness Intestinal enzyme inhibition Neurotoxin Bitterness Toxin Toxin content Toxin content Safety Safety 52 APPENDIX B: Chapter 2 Figures Figure 2.1. A general framework of the conventional breeding process that is comprised of three stages: 1. Trait Mapping; 2. Trait Introgression and; 3. Field Testing. The approximate time needed for each stage is shown, using maize which has a 3-4 months of generation time as an example. The approximate number of plants and field locations in the Field Testing stage is also representative of a maize breeding program. The symbols « and u indicate the genetic markers for the Trait of Interest or genomic mapping marker, respectively, on representative chromosomes (Trait Mapping) or the whole plant genome (Trait Introgression and Field Testing). The open bars (¨) and filled bars (n) represent the chromosomes from the respective parental varieties in the Trait Mapping stage, and the respective parental whole plant genomes in the other two stages of breeding. 53 Figure 2.2. Maize breeding testing practices for the processed maize kernels industry. Panel A shows that all maize varieties are subjected to agronomic characterization testing, such as yield, disease resistance and standability, while a small proportion of varieties continue for near infrared (NIR) testing of kernel attributes (e.g., density) and proximate composition (e.g., starch, protein, and oil content, density. Panel B shows image analysis for kernel hardness for a subset of maize varieties that meet a density threshold. Horneous of kernel endosperm is digitized by analysis of transmitted light kernel images. 54 Figure 2.3. Overview of the Washington State University Apple Breeding Program traditional breeding operations. In Year 1, ~20,000 seeds are harvested and between 200-3000 are germinated in Year 2. Trees tolerant to Podosphaera leucotricha are progressed through the program in Years 3 and 4 and scions taken from these selections are propagation onto M.9 rootstock. Grafted compound trees are planted in Phase 1 orchard evaluation. Selections from Phase 1 are then propagated for replicated trial in three Phase 2 sites before being advanced to Phase 3 multi-site grower trials. Adapted with permission from (Evans, 2013). 55 Figure 2.4. Overview of a conventional tetraploid potato breeding cycle. After making crosses between select parental lines, between 20,000 and 100,000 genetically unique F1 individuals are evaluated in the field in Year 1. Selection for agronomic traits and disease and insect resistance testing reduces the number of individuals to approximately 50 lines by year 5. These lines are subject to quality evaluation and regional testing as well as glycoalkaloid testing. A subset of approximately 10 lines then advance to large-scale agronomic testing on growers’ fields in years 6-8. Ultimately this process produces between 1-3 varieties. 56 Figure 2.5. Colorado potato beetle detached leaf assays after 5 days of feeding demonstrates resistance of Solanum chacoense leptine-producing line 80-1 (below) compared to tetraploid commercial cultivar Atlantic (above). 57 APPENDIX C: Chapter 2 Copyright Permission Figure S2.1 Elsevier Copyright Permission for inclusion of Chapter 2 in this dissertation. 58 REFERENCES 59 REFERENCES Abbadi, A., & Leckband, G. (2011). Rapeseed breeding for oil content, quality, and sustainability. European Journal of Lipid Science and Technology, 113, 1198-1206. Abbas, H. K. (2005). Aflatoxin and food safety. Boca Raton: Taylor & Francis CRC Press.1420028170. Acharya, B., Dutta, S., Dutta, S., & Chattopadhyay, A. (2018). Breeding tomato for simultaneous improvement of processing quality, fruit yield, and dual disease tolerance. International Journal of Vegetable Science, 24, 1-17. https://doi.org/10.1080/19315260.2018.1427648. 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The publisher Springer Nature permits the use of the full article (Appendix D). 79 Abstract We examined the genetic features underlying leptine glycoalkaloid mediated Colorado potato beetle (Leptinotarsa decemlineata) host plant resistance in a diploid F2 mapping population of 233 individuals derived from Solanum chacoense lines USDA8380-1 and M6. The presence of foliar leptine glycoalkaloids in this population segregated as a single dominant gene and displayed continuous distribution of accumulated quantity in those individuals producing the compound. Using biparental linkage mapping, a major overlapping QTL region with partial dominance effects was identified on chromosome 2 explaining 49.3% and 34.1% of the variance in Colorado potato beetle field resistance and leptine accumulation, respectively. Association of this putative resistance region on chromosome 2 was further studied in an expanded F2 population in a subsequent field season. Loci significantly associated with leptine synthesis colocalized to chromosome 2. Significant correlation between increased leptine content and decreased Colorado potato beetle defoliation suggests a single QTL on chromosome 2. Additionally, a minor QTL with dominance effects explaining 6.2% associated with Colorado potato beetle resistance donated by susceptible parent M6 was identified on chromosome 7. Bulk segregant whole genome sequencing of the same F2 population detected QTL associated with Colorado potato beetle resistance on chromosomes 2, 4, 6, 7, and 12. Weighted gene co-expression network analysis of parental lines and resistant and susceptible F2 individuals identified a tetratricopeptide repeat containing protein with a putative regulatory function and a previously uncharacterized acetyltransferase within the QTL region on chromosome 2, under the control of a regulatory Tap46 subunit within the minor QTL on chromosome 12. 80 Introduction The Colorado potato beetle (Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) is the most widespread and destructive insect defoliator of potato crops worldwide. Both larvae and adult beetles consume potato leaves and stems, inflicting yield losses of 30-50% (Alyokhin et al., 2012; Vreugdenhil et al., 2007), depending on the timing of the defoliation within the plant growth cycle (Hare, 1980). Control of the Colorado potato beetle historically relied heavily on the use of insecticides (Grafius & Douches, 2008), beginning with the use of arsenical compounds in 1871 (Riley, 1871). However, Colorado potato beetle resistance is reported for most major classes of synthetic insecticides and over 50 different active ingredients (APRD, 2019) necessitating more frequent sprays and higher application rates (Mota-Sanchez et al., 2006). Resistant populations of Colorado potato beetle are found across the entirety of its range but are most prevalent in its North American region of origin (Izzo et al., 2018; Whalon et al., 2008), where greatest insect genetic diversity has been exposed to the longest period of intensive pesticide application (Alyokhin et al., 2008). Availability and effective implementation of Colorado potato beetle resistant varieties could reduce both the environmental impact and the economic burden to growers of repeated insecticide applications. Cultivated potato Solanum tuberosum Group Tuberosum L (2n=4x=48) naturally produces secondary metabolites in the form of glycoalkaloids with antimicrobial and insecticidal properties (Lachman et al., 2001; Tingey, 1984). Host plant resistance efficacy is dependent on both total leaf glycoalkaloid accumulation and the production of specific glycoalkaloids. For example, the common glycoalkaloids a-chaconine and a-solanine are present in leaves of Colorado potato beetle susceptible varieties in insufficient amounts to inhibit insect feeding (Friedman & McDonald, 1997; Sinden et al., 1980). Accessions of the wild, diploid species S. chacoense 81 produce the potent glycoalkaloids leptines I/II that deter Colorado potato beetle feeding through a cholinesterase inhibiting and cell membrane disruption mechanism (Sanford et al., 1996; Sinden et al., 1986; Sinden et al., 1980; Tingey, 1984). Unlike a-chaconine and a-solanine, leptines are present only in aerial tissue and thus do not pose a hazard to human health (Mweetwa et al., 2012). While a dose-dependent, neuroreceptor specific to leptine I has been identified in Colorado potato beetle (Hollister et al., 2001), the minimum foliar leptine concentration required to reduce Colorado potato beetle feeding has yet to be established (Deahl et al., 1991; Rangarajan et al., 2000). Leptines were first implicated in Colorado potato beetle resistance by Sinden et al. (1986) in the S. chacoense accession USDA8380-1 (80-1). Leptines I and II are acetylated forms of the ubiquitous glycoalkaloids a-chaconine and a-solanine, respectively, and are hypothesized to share a common precursor (Ronning et al., 1998). The leptinines I/II also present in 80-1 are proposed to be intermediates between a-chaconine/a-solanine and leptines I/II but do not exhibit strong Colorado potato beetle antifeedant properties (Lorenzen et al., 2001; Stürekow & Löw, 1961; Yencho et al., 2000). Glycoalkaloids are composed of a cholesterol-derived skeleton, or aglycone, and a glycosidic group. Cholesterol, produced via the mevalonate pathway, is converted to solanidine which is then glycosylated in two separate reactions to produce a-chaconine and a- solanine (Kumar et al., 2017). It has been proposed that the aglycone solanidine is first modified to generate the leptinine aglycone leptinidine by a hydroxylation at C-23 (Lawson et al., 1993; Osman et al., 1987; Silhavy et al., 1996). Acetylation of the resulting hydroxyl group is proposed to yield the leptine aglycone acetyl-leptinidine (Lawson et al., 1993; Osman et al., 1987). Subsequent glycosylation of each aglycone would give rise to leptinines and leptines. 82 Several recessive genes are hypothesized to control the presence of leptines (Boluarte- Medina et al., 2002; Hutvágner et al., 2001; Manrique-Carpintero et al., 2014; Ronning et al., 1999; Sagredo et al., 2006). Loci associated with leptine synthesis were identified on potato chromosomes 1, 2, 7, and 8 (Manrique-Carpintero et al., 2014) and two complimentary epistatic loci associated with the synthesis of aglycones leptinidine and acetyl-leptinidine were mapped to chromosomes 2 and 8, respectively (Sagredo et al., 2006). Yet despite decades of research, the genetic underpinnings of leptine biosynthesis and accumulation remain elusive and introgression of this trait into cultivated material has not been achieved (Ginzberg et al., 2009; Grafius & Douches, 2008; Manrique-Carpintero et al., 2014). Consequently, there are currently no Colorado potato beetle resistant cultivars with commercial acreage. Investigation of S. chacoense derived Colorado potato beetle resistance at the diploid level using interspecific populations is difficult (Boluarte-Medina et al., 2002; Veilleux & Miller, 1998). Interspecific hybridization may disrupt unique beneficial allelic combinations underlying this trait in S. chacoense and could affect recombination rates as reported by (Manrique-Carpintero et al., 2016). Moreover, diploid potatoes are largely self-incompatible due to a gametophytic system which precludes the generation of large F2 or recombinant inbred line mapping populations. In the S. chacoense diploid inbred line M6, however, the self-incompatibility system is inactivated by the dominant allele of the S-locus inhibitor gene Sli on chromosome 12 (Jansky et al., 2014). We propose that M6-mediated introduction of self-compatibility into Colorado potato beetle resistant germplasm provides an ideal system to study, understand and exploit this mechanism of host-plant insect resistance in potato. To that end, we employed combined bi-parental linkage mapping and whole-genome sequencing bulk segregant analysis in a diploid F2 S. chacoense population. Here we describe the identification of a major QTL region on the long arm of chromosome 2 explaining 83 49.3% and 34.1% of the variance in Colorado potato beetle field resistance and leptine accumulation, respectively. Minor QTL on chromosomes 4, 6, 7, and 12 associated with Colorado potato beetle field resistance are also discussed. Materials and Methods Plant Material Twenty F1 hybrids were generated from a cross between the S. chacoense clone USDA8380-1 (PI 458310, 80-1) and the S. chacoense self-compatible inbred line M6 (Jansky et al., 2014). Clone 80-1 is largely homozygous, based on SNP genotyping described below, produces high levels of leptines, comprising 90% of total leaf glycoalkaloids (Sanford et al., 1996) and is resistant to Colorado potato beetle defoliation (Sanford et al., 1997) while M6 does not produce leptines and is susceptible to Colorado potato beetle defoliation (Crossley et al., 2018). A single F1 individual demonstrating robust self-compatibility, Colorado potato beetle resistance and high heterozygosity based on SNP marker genotype was selected for self-pollination to produce 700 diploid F2 seedlings. Of these, 325 individuals grew and developed and 305 were determined to be self-compatible. Self-compatibility was evaluated by a maximum of 50 self-pollinations of each F2 individual under greenhouse conditions for the purpose of creating a recombinant inbred line population for future genetic studies. A total of 233 F2 self-compatible individuals were randomly selected to constitute the mapping population. All plant material was maintained in tissue culture by nodal propagation in modified Murashige and Skoog media (MS salts at 8.8g/L, 3% sucrose, pH 5.8 and 0.6% plant agar) at 25°C under a 16-hr photoperiod, permitting replicated experiments of these individuals in Michigan because the parental lines, F1 and F2 progeny do not readily tuberize under long day conditions of northern latitudes. 84 Developmental Resistance Profiling of Parental Lines Host plant resistance to Colorado potato beetle larval feeding was assessed using a detached-leaf no-choice assay. No-choice assays are a tool to measure insect behavior such as feeding, oviposition, and larval survival to maturity when exposed to plant material from a single genotype. Newly hatched Colorado potato beetle larvae have limited mobility and thus their food source is largely dependent on adult maternal oviposition preference. No-choice neonate assays mimic this situation where the insect must feed or starve. Minimal, or a complete lack of, adult feeding in a no-choice setting also provides strong evidence that the genotype is not a suitable host. However, feeding on a sub-optimal host may be artificially inflated by virtue of the containment. No-choice detached leaf assays of parental lines were conducted using Colorado potato beetle first instars. Eggs from a colony initially collected from Long Island, NY and maintained in culture for use in laboratory bioassays were purchased from French Agricultural Research Incorporated (Lamberton, MN). Eggs and newly hatched neonates were maintained on foliage of S. tuberosum commercial cultivar ‘Atlantic’ plants grown in greenhouse facilities at Michigan State University. Nine plants each of 80-1 and M6 were grown in the greenhouse from in vitro plantlets (N = 18) that had been repropagated on the same day. At seven weeks post-transplant, the first six fully-expanded leaves were harvested from three randomly chosen plants of each line. Each leaf was placed separately in a 6.35 cm floral tube (Tezula Plants, FL) on filter paper in a 150 mm diameter Petri dish. Five neonates were placed on separate leaflets of each leaf using a paintbrush. Petri dishes were arranged on shelves under fluorescent lights (30 umol m-2 s-1) and 16-hr photoperiod. Percent defoliation, larval mortality, and larval development was visually assessed at 5 timepoints: 1, 3, 5, 7, and 9 days after neonates were placed on the leaves. Percent defoliation was estimated visually as the percent of the total foliage consumed by the larvae from 85 0-100% in increments of 5%. For lines with minimal feeding between 0-1% estimates were as follows: 0.0% = no feeding, 0.5% = pinhole feeding that did not penetrate the leaf, 1% to 4% = one or more pinholes of 1mm diameter that completely penetrated the leaf. Larval mortality was calculated as the number of dead larvae in each Petri dish at the time of observation divided by five and multiplied by 100%. The developmental instar of each living larva was determined according to estimated head width, body length and pronotum coloration. The no-choice assay was repeated at 12- and 15-wks post-transplant using three previously unsampled plants for each genotype, using the same method. This resulted in 54 individual assays per line (6 leaf positions x 3 time points x 3 replicates) using a total of 540 larvae. Statistical analyses of differences in larval mortality and defoliation between lines and leaf age were accomplished using Tukey’s HSD (a = 0.05) in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). Glycoalkaloid Analysis Sample Preparation and Extraction. Glycoalkaloid content is strongly influenced by environmental factors (Mondy & Gosselin, 1988; Mondy et al., 1987; Mondy & Munshi, 1990; Morris & Petermann, 1985; Sinden et al., 1984; Sinden & Webb, 1972; Slanina, 1990; Van Gelder & Dellaert, 1988). To mitigate environmentally induced variation, foliar glycoalkaloids were extracted from greenhouse grown plants. One plant each of the two parental lines (M6 and 80-1), their F1 hybrid, and the 233 F2 individuals (N = 236) were grown in the greenhouse under a 16-hr photoperiod at 20°C in 3.8 L pots. Since accumulation of the novel glycoalkaloids leptine I/II in leaves of the resistant parental line, S. chacoense 80-1, increases with maturity (Mweetwa et al., 2012) foliar tissue samples were taken 15-wks post-germination for analysis to ensure glycoalkaloid detection. Five leaflets from the fourth fully-expanded leaf were pooled for each genotype in a 15 mL Corning tube, flash frozen and stored at -80°C prior to lyophilization for 72 86 hrs. The freeze-dried tissue was ground, and 50 mg of powder was extracted in 1 mL of solution (49% HPLC grade methanol, 49% sterile water, 1% glacial acetic acid, 0.1% formic acid). The samples were briefly vortexed and incubated at 60°C for 30 minutes before centrifugation for one minute at 14,000 rpm. The supernatant was filtered through a 0.22 um Corning® Costar® Spin- X® centrifuge tube and diluted 1:100 in extraction solution containing internal standard Telmisartan at a final concentration of 0.5 uM. Glycoalkaloid Quantification. Glycoalkaloids were analyzed using Waters Acquity (Waters Corporation, MA, USA) high performance liquid chromatography (Quattro Micro) coupled with tandem mass spectrometry (HPLC–MS/MS). Compounds were separated on a C-18 reverse-phase column. Glycoalkaloids were eluted in a binary gradient system composed of Solvent A (LC-MS grade water, 0.1% formic acid) and Solvent B (LC-MS grade acetonitrile) at a flow rate of 0.3 mL/min at 25 °C. The following stepwise gradient was implemented: 90% A, 10% B; 2:00 min, 40% A, 60% B; 2:01, 0% A, 100%B; 3:01, 90% A, 10% B. Each sample was injected at a volume of 10 uL in triplicate (N = 678). The mass spectrometer was operated in positive ion mode. Mass spectroscopy data were acquired by the Waters MassLynx software and processed using Waters Quanlynx MS Software. Molar concentrations were determined using standard curves of purified α-solanine and α-chaconine (Sigma-Aldrich) in a range from 0.01-40.0 uM. The response factors for α-chaconine and α-solanine were used for leptine I and leptine II, respectively. Field Trial Colorado Potato Beetle Phenotyping Field trials were conducted in 2017 and 2018 at the Michigan State University Montcalm Research Center (Lakeview, MI) in a field planted annually with untreated, susceptible potato plants and naturally infested with overwintering Colorado potato beetles for at least four decades (Coombs et al., 2003). The beetle population on this research farm has a history of resistance to 87 commonly-used potato insecticides (Ioannidis et al., 1991; Ioannidis et al., 1992; Szendrei, 2014). To provide food for emerging beetles prior to transplanting research lines, tuber seed pieces of the commercial cultivar ‘Atlantic’ was planted around, and in alternate rows of, the study area on 19 April 2017 and 25 April 2018. These untreated, susceptible plants also serve to maintain high beetle densities uniformly throughout the field. Adult beetles emerged from the soil within the field the weeks of 21 May 2017 and 28 May 2018. The trial area and borders were fertilized and irrigated according to best management practices, but no insecticides were applied to the field. Damage by other potato insect pests, such as potato leafhopper, were not observed at this field location in 2017 or 2018. In 2017, nine in vitro plantlets of each research line (2 parental lines, their F1 hybrid, and 151 F2 individuals) and susceptible check ‘Atlantic’ were transplanted to trays in the greenhouse, grown for six weeks (16-hr photoperiod, 20°C) and transplanted in the field on 2 June 2017 (N=1395). At this time, all transplants were intentionally the approximate same age and maturity of the ‘Atlantic’ spreader rows. A randomized complete block design consisting of three replications of three plants was used. Percent defoliation of each three-plant plot was assessed visually each week, beginning on 7 June 2017 and continuing for a total of seven weeks, at which point the ‘Atlantic’ check was completely defoliated and beetles left the plots. Over the seven weeks, defoliation was caused by overwintered adults, first-generation larvae, and second- generation adults and larvae. Defoliation data were used to calculate the area under the defoliation curve (AUDC), comparable to the area under the disease progression curve (Shaner & Finney, 1977). To determine the relative AUDC (RAUDC) for each plot over the seven-week observational period, the AUDC for each plot was divided by the maximum defoliation observation for that plot (e.g. 4900 if 100% 88 of the plot was defoliated by the 49th day of the trial). Data were analyzed in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). Levene’s test revealed unequal variances (P <0.0001) and accordingly non-parametric Kruskal Wallis tests were used for pairwise comparison (α = 0.05). Phenotypic extremes (the ten-most susceptible and ten-most resistant F2 individuals) were selected for field evaluation the following year. In 2018, five replications of five plants each of the parental lines, the F1 hybrid, and the 20 phenotypic extremes from the F2 generation were transplanted to the field on 11 June. ‘Atlantic’ was again included as a susceptible check and ‘Atlantic NewLeaf,’ a deregulated genetically modified clone expressing the Bacillus thuringiensis Cry3A protein, was used as a resistant check (N = 625). Two complete beetle generations were observed during the field season. Beetle pressure in 2018 was observed to be less than in 2017. Beginning on 11 June 2018, defoliation data were collected weekly for five weeks, after which beetles were no longer feeding. This shorter experimental duration period was determined by the beetle development pattern and environmental conditions in this year. The RAUDC was calculated for each plot, using a similar method as in 2017. To account for the non-normality of the data, we used Spearman’s rank correlation coefficients in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC) to test the relationship between measured traits for both years. The mean of technical replicates was used to calculate correlations coefficients for compound concentrations (mg/g DW) of individual measured glycoalkaloids, total measured glycoalkaloids and the ratio of acetylated glycoalkaloids to nonacetylated glycoalkaloids [mean total leptines (mg/g DW)]/ [mean α-chaconine (mg/g DW) + mean α-solanine (mg/g DW)]. Means of biological replicates (plots) were used to calculate correlation coefficients for field defoliation (RAUDCx100) for each individual in both years. 89 SNP Genotyping and Linkage Analysis Genomic DNA was extracted from freeze-dried leaf tissue of the two parental lines, their F1 hybrid, and the 233 F2 individuals following the Mag-BindÒ Plant DNA Plus 96 Kit protocol (Omega Bio-tek, Norcross, GA). SNP genotyping was performed using the Illumina Infinium Potato 22K V3 Array, including a genome wide marker selection from different sources (Felcher et al., 2012; Hamilton et al., 2011; Vos et al., 2015) at Michigan State University. Filtering removed SNPs that were monomorphic for all individuals, SNPs with >10% progeny missing genotype, and SNPs with missing parental genotypes. This resulted in 754 segregating markers distributed across the 12 chromosomes. Joinmap Ò 5.1 (Van Ooijen, 2006) was used to create the 12 linkage groups using the F2 population type. The physical position of mapped SNPs from the Illumina V3 Array on the potato doubled monoploid S. tuberosum clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03) was used to compare genetic and physical maps. The physical length of each chromosome was calculated by subtracting the first megabase (Mb) position of mapped loci on each chromosome from the last position. Total physical map length was the sum of the physical map lengths for each of the 12 chromosomes. Map coverage for each chromosome was reported as the total distance in Mb covered by SNP positions divided by the total length of each DM Version 4.03 assembled chromosome. Total map coverage was reported as the total distance (Mb) covered by all 12 chromosomes divided by the total distance of all 12 DM Version 4.03 assembled chromosomes. Average distances between loci mapped in each chromosome was calculated by summing all the individual interlocus intervals in cM and divided by the total number of intervals, and the average from chromosome average intervals for the overall genome. Concordance between the reference DM genome (PGSC Version 4.03) and the linkage map was tested by creating Marey maps comparing the genetic position (cM) to the physical 90 position (Mb) of each SNP in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). Deviation from the expected 1:2:1 (homozygous:heterozygous:homozygous) genotypic class frequencies was calculated by chi-squared tests of each SNP at three significance levels (P<0.05, P<0.01, P<0.001). The significance of distorted segregation was reported as the minus logarithm of the chi-square test p-value. QTL analysis was performed in MapQTL® 6 software (Van Ooijen & Kyazma, 2009) using interval mapping to first identify major QTL for Colorado potato beetle defoliation (mean RAUDC x 100), accumulation of leptine I, leptine II, total leptines, a-solanine, a-chaconine, and total glycoalkaloids as well as the ratio of acetylated to non-acetylated glycoalkaloids. A 1000x permutation test was run for each trait to establish the LOD threshold corresponding to a genome wide false positive rate of 5%. Markers with LOD scores exceeding this threshold were used as cofactors in multiple-QTL-mapping (MQM). Final reported LOD scores were determined by MQM and MapChart (v 2.32) (Voorrips, 2002) was used to visualize the results. To identify QTL associated with leptine synthesis, leptine accumulation data were converted to presence/absence and coded 1/0. QTL were detected using the nonparametric Kruskal-Wallis test and significance threshold of P = 0.0001 in MapQTL® 6 software (Van Ooijen & Kyazma, 2009). Phenotypic Validation of Colorado Potato Beetle Resistance Extremes in vitro Detached-Leaf Choice Bioassays with adults. Host plant resistance to Colorado potato beetle adult feeding was assessed using a detached-leaf choice assay. Choice assays, where insects are confined with multiple host genotypes, offered insight into Colorado potato beetle host preferences under field conditions. Assays on the 20 phenotypic extremes (the ten-most susceptible and ten-most resistant) of the F2 generation (Table S1) were conducted using adult beetles collected from the Montcalm Research Center and undamaged leaves collected from plants in the 91 2018 field trial. An undamaged, fourth fully-expanded leaf was selected from three plants of each line four weeks after transplanting to the field. Leaves were placed in a 2.35 cm floral tube (Tezula Plants, FL) filled with distilled water. Each choice arena consisted of a 25.4 x 30.5 cm disposable foil cake pan with a fitted plastic lid (Gordon Food Service, Grand Rapids, MI), with a piece of moist cheese cloth placed in the bottom. Each arena was large enough to hold ten leaves. The single leaves from five resistant and five susceptible lines were randomly selected to be tested in each arena; this resulted in a total of six arenas (pans) to test each of the 20 lines in triplicate. Ten adult beetles were placed on the cheesecloth in the center of each arena. Pans were held at room temperature under fluorescent lights (30 umol m-2 s-1) on a 16-hr photoperiod. After 48 hrs, defoliation was visually scored as the percent of each leaf consumed. Detached Leaf No-Choice Bioassays with larvae. Three plants of the parental lines and the 20 resistance extremes were also grown in the greenhouse in 2018 from in vitro plantlets (N = 66). No-choice first instar detached leaf assays were conducted using the fourth fully-expanded leaf of each plant according to methodology described for developmental profiling of parental lines at 12-wks post-transplant. Non-parametric Kruskal Wallis tests were used for pairwise comparison of defoliation among lines (α = 0.05) in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC) in both choice and no-choice bioassays. Spearman’s rank correlation coefficients (ρ) were used to compare both bioassay phenotypes to the beetle resistance phenotypes observed in the two field seasons and measured glycoalkaloids for these 20 F2 individuals. Glycoalkaloid Profiling. At the same time the no-choice bioassays were conducted, glycoalkaloids were extracted from a pooled sample of the terminal leaflet from three biological replicates of the third fully-expanded leaf for each genotype (N = 22) and quantified as described above. 92 Whole Genome Sequencing Bulk Segregant Analysis Sample Preparation and Sequencing. Tissue was harvested by hole punching a young, terminal leaf of the two parental lines and the 20 phenotypic extremes of the F2 generation (Table S1). A single hole punch from the ten susceptible and ten resistant individuals were bulked to form a susceptible sample and a resistant sample prior to DNA extraction using the Qiagen DNAeasy Plant Mini Kit. Illumina libraries were prepared using the TruSeq DNA sample prep kit and sequenced in paired-end mode (150nt) on the Illumina Hiseq platform to 30x depth for parental lines and 10x depth for resistant and susceptible bulks. Read Alignment and Variant Calling. Raw reads were processed with Trimmomatic (v0.35) (MINLEN = 100, LEADING = 3, TRAILING = 3, SLIDINGWINDOW = 4,20) to remove adapters, primers and low-quality bases (Bolger et al., 2014). As currently only 508 Mb of the 826 Mb S. chacoense M6 assembly (Leisner et al., 2018) is anchored to the 12 chromosomes, we aligned trimmed reads from bulk samples to an alternate M6 assembly built from the more complete Solanum tuberosum DM pseudomolecules (PGSC Version 4.03) (henceforth referred to as the M6-corrected DM assembly). First, cleaned M6 reads were aligned to the DM genome using BWA-mem (v0.7.12.r1044) (Li, 2013) in paired-end mode. The resulting alignments were sorted, depurated from duplicates and indexed using Picard tools (v1.113) ("Picard tools,") ("Picard tools,") and indel realignment performed with the Genome Analysis Toolkit (GATK, v3.5.0) (DePristo et al., 2011). SNPs and indels were subsequently called using GATK HaplotypeCaller, employing a standard minimum confidence threshold for calling of 20.0. Called variants were subject to hard filtering using the GATK VariantFiltration tool with the following parameters: quality by depth <10.0, mapping quality < 40, strand bias estimated by Fisher’s exact test >60.0, Haplotype Score > 13.0, mapping quality rank sum test less than -12.5, read position rank sum test 93 less than -8.0. Using the FastaAlternateReferenceMaker utility in GATK, bases in the DM assembly were replaced at variation sites with bases supplied by this SNP set. Alignments of resistant and susceptible bulk reads to this M6-corrected DM assembly were then processed, and variants called and filtered as described for M6, with the exception that SNPs and indels were called jointly in all samples using GATK HaplotypeCaller and GATK GenotypeGVCF. Bulk-segregant Analysis. A total of 11,070,484 SNPs resulting from alignment of bulk samples to the M6-corrected DM assembly were imported into R (v1.1.423) and further filtered by reference allele frequency to remove 5,000,179 SNPs over or under-represented in both bulks, SNPs with read depth discrepancies >100 between bulks, and SNPs with total depth <10 and >200. We independently assessed the differences in allele frequency between the resistant and susceptible bulks using the G’ statistic method proposed by Magwene et al. (2011), which accounts for linkage disequilibrium and minimizes SNP calling error noise, within the R package QTL-seqr (Mansfeld & Grumet, 2018). Briefly, SNPs were counted in a one Mb sliding window and a tri- cubed DSNP-index calculated within each window. A SNP-index is the proportion of reads with a SNP that differs from the sequence of the susceptible reference genome. A SNP-index value of 0 indicates that all read sequences in the bulk contain the reference allele. Conversely, if all reads contain the non-reference allele the SNP-index = 1. The DSNP-index is the difference in SNP- index between resistant and susceptible pools and is expected to be 0 in regions not associated with the trait of interest. Both p-values and genome-wide Benjamini-Hochberg FDR (Benjamini & Hochberg, 1995) adjusted p-values are calculated for each SNP. We used an absolute DSNP- index value of 0.1 to filter outlier regions and an FDR of 0.01 to identify significant QTL associated with Colorado potato beetle resistance. 94 Gene Expression of Colorado Potato Beetle Resistance Extremes Sample Preparation and RNA Sequencing. Three strongly resistant and three susceptible F2 individuals were selected from the 20 characterized phenotypic extremes for expression profiling. The resistant individuals selected contained leptines and the susceptible individuals did not contain leptines (Table S1). Three in vitro plants of each F2 individual and the two parental lines were grown in a growth chamber (20°C, 16-hr photoperiod, 70% relative humidity) (N = 24). RNA was isolated from leaf tissue at 16-wks post-transplant using the Qiagen RNeasy Plant Mini Kit. Samples were Turbo DNase (Thermo Fisher Scientific, Waltham, MA, USA) treated, and RNA concentration and quality were measured using Qubit 2.0 Fluorometer (Life Technologies, Inc., Carlsbad, CA, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All samples had a minimum RNA integrity number (RIN) score of 8. For each genotype, three biological replicates were prepared from equal quantities of leaflets from the fourth fully-expanded leaf. Libraries were prepared using the Illumina TruSeq Stranded mRNA Library Preparation Kit and approximately 30,000,000 50nt single-end Illumina reads were generated for each sample on the Illumina HiSeq 4000 platform. Raw reads were processed with Trimmomatic (v0.35) (MINLEN = 36, LEADING = 20, TRAILING = 20) to remove low-quality bases, adapters, and primers (Bolger et al., 2014). RNAseq Read-Mapping and Differential Expression Analysis. Cleaned reads were aligned to the DM genome (PGSC Version 4.03) using STAR (v2.6.0) (Dobin et al., 2013). Reads aligning to annotated DM reference genes were counted using subread (v1.6.2) package featureCounts in reverse stranded mode (Liao et al., 2013). Counts were then analyzed using the R package DESeq2 to determine normalized expression values (Love et al., 2014). Analyzed genes 95 were required to have counts greater than 10 in at least 3 individuals. Spearman’s rank correlation coefficients (ρ) were calculated between individual candidate transcripts and measured traits. Weighted Gene Co-expression Network Analysis. The weighted gene co-expression network analysis (WGCNA) package was used to conduct co-expression analysis in R (Langfelder & Horvath, 2008), with a soft-thresholding power of 22 (Fig. S1A). The varianceStabilizingTransformation function was used to transform counts for the 21,996 genes passing the count threshold (>10 in at least 3 individuals). Modules within the signed network were identified in a single block using the BlockwiseConsensusModule function with a minimum module size of 30 and medium sensitivity (deepSplit value of 2). Module eigengenes, the first principal component of the expression matrix in the module, were correlated with defoliation (RAUDC), total leptine accumulation (mg/g DW) and the ratio of acetylated to nonacetylated glycoalkaloids measured. Average gene significance was plotted for each module and trait. Hub genes within interesting modules were required to have absolute module membership values (kME) greater than 0.9, indicative of strong intra-module connectivity, and an absolute gene significance for the trait of interest greater than 0.2. Networks of selected modules were visualized separately in Cytoscape 3.7 (Kohl et al., 2011), in the Attribute Circle Layout with log2FoldChange as the selected attribute. Data Availability. Raw whole genome sequence data is available at NCBI SRA accession # SRR10197400; SRR10197399 and raw expression and count data is available at NCBI GEO (accession # GSE138184). Molecular Marker Development and fine mapping of QTL region Design of PCR indel markers. The .bam file containing SNPs and indels identified between 80-1 and M6 from whole genome sequence data were visualized using the Integrative 96 Genome Viewer (IGV, v.2.4.9). Candidate indel markers flanking and within the QTL region were required to be homozygous in both parents and greater than 15 bp in length to permit adequate size separation on an electrophoresis gel. Primers were designed in house using Primer3 (Rozen & Skaletsky, 2000) and validated using genomic DNA extracted from young leaf tissue of 80-1, M6 and the F1 hybrid plants. All reactions were performed in a 15 ul volume using 7.5 ul of GoTaq Green Master Mix (Promega, Madison, WI), 0.5ul of each primer, 2ul of DNA and 4ul of distilled water following manufacturer protocols. Cycling conditions included an initial denaturing step of 3 minutes at 94°C followed by 34 cycles of 30 seconds denaturing 94°C, 30 seconds annealing 50°C, 1 minute elongation 72°C and a final elongation step of 5 minutes at 72°C. PCR products were run on a 2.5% agarose gel stained with SYBR Safe (Thermo Fisher Scientific, Waltham, MA, USA) at 120 V for 45 minutes. Screening additional F2 progeny to identify recombinants. Two validated PCR primers within the QTL region (Table S2) were then tested on an additional 406 F2 individuals generated from the F1 hybrid and grown in greenhouse conditions under a 16-hr photoperiod at 20°C. The F2 individuals with recombination in the QTL region (n=96), as well as the two parental lines and their F1 hybrid, were replicated by stem cuttings in the greenhouse to produce nine biological replicate plants (N=891). Rooted stem cuttings were planted as transplants in the Colorado potato beetle nursery at the Montcalm Research Center on 21 June 2019 in a randomized complete block design of three replicates of three plants. Percent defoliation of each three-plant plot was assessed visually each week, for a total of five weeks and the RAUDC calculated as described previously. Fine mapping of candidate QTL region on chromosome 2. Eleven validated PCR markers (Table S2) within the candidate QTL region were then used to genotype the 96 F2 individuals phenotyped for Colorado potato beetle resistance in the field. Linkage map 97 construction was achieved with JoinmapÒ 5.1 (Van Ooijen, 2006) using the F2 population type. Permutation testing to establish a LOD threshold and MQM mapping were conducted in MapQTL® 6 (Van Ooijen & Kyazma, 2009) as described above to confirm the association of PCR markers with the Colorado potato beetle field resistance phenotype. Results S. chacoense Colorado potato beetle resistance is tissue and age-dependent Younger leaves (1-2) of both S. chacoense parental lines 80-1 and M6 were more defoliated by Colorado potato beetle larvae than older leaves (5-6) throughout plant development (a= 0.05) (Fig. 1). In addition to leaf age, plant age was observed to impact defoliation resistance. Defoliation of 80-1 leaves (1-5) was least at seven weeks post-transplant and increased in the subsequent sample time points (Fig. 1). This timing has practical significance because Colorado potato beetles typically emerge before or with potato plants. However, the larval mortality was higher on old leaves (3-6) than on young leaves (1-2) of young 80-1 plants (seven weeks post-transplant) (p = 0.0036). Phenotypic Evaluation of the S. chacoense F2 Population Leptine I/II was detected in 162 F2 progeny, ranging from 0.1-25.9 and 0.1-41.6 mg/g DW, respectively (Table 1). Presence of leptines in this population was determined to be not significantly different from a 3:1 ratio by chi-squared test (c2 = 0.053). Leptine levels exceeded 8.6 mg/g DW, the concentration previously reported to reduce larval feeding (Sanford et al., 1997), in 130 F2 individuals; 40 individuals had greater leptine content than parent 80-1. All F2 progeny contained a-chaconine and a-solanine. The average foliar concentration of a-chaconine (21.8 mg/g DW) and a-solanine (28.5 mg/g DW) was higher than leptines (14.9 mg/g DW) (Table 1). 98 In individuals containing leptines, leptine concentration was weakly and inversely correlated with a-chaconine levels, but not correlated with a-solanine (Table 2). In field trials under natural beetle pressure, the susceptible parent M6 and cv. Atlantic were completely defoliated. In contrast, 80-1 and the F1 hybrid exhibited minimal feeding (RAUDC < 2.9). Field defoliation among F2 lines was continuously distributed, with RAUDC ranging from 0.0 (no damage) to 54.3 (Fig. 2). Increased leptine I, II and total leptine concentration were moderately correlated to decreased field defoliation while a-chaconine and a-solanine content were moderately correlated with increased field defoliation (Table 2). The ratio of acetylated compounds to nonacetylated compounds was the best predictor of field defoliation (Table 2). Linkage Map Construction To identify QTL underlying this observed Colorado potato beetle resistance, we SNP genotyped the mapping population of 233 F2 individuals to generate a linkage map with 12 chromosomes covering 97% of the 12 assembled S. tuberosum clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03) (Table 3, Table S3). The resultant map spanned a genetic distance of 1193.8 cM with an average of 63 SNP markers per chromosome distributed at an average distance of 1.6 cM between SNPs (Table 3). Comparison of the genetic location and physical position on the DM pseudomolecules of SNPs used for mapping showed good concordance (Fig. S2). The percentage of distorted segregation in the F2 progeny was assessed at three levels of significance. Distorted segregation at the 5% and 1% conservative significance levels was detected on 56% and 40% of all mapped loci. At the 0.1% significance level, corresponding to highly distorted loci, 26% of all mapped loci exhibited distorted segregation and were located on all 99 chromosomes except 4, 5 and 10 (Table S4). The chromosome with the greatest number of loci with distorted segregation was chromosome 12 with 90% of distorted loci at the 0.1% significance level. Plotting the maternal, paternal and heterozygous genotypic frequency at each SNP position along the 12 chromosomes showed preferential inheritance of M6 alleles on chromosomes 1, 3 and 12 (Fig. S3). Compared to other chromosomes, chromosome 8 exhibited a high preferential inheritance of heterozygous genotypes (Fig. S3). Identification of QTL Associated with Colorado Potato Beetle Field Resistance and Leptine Accumulation Two QTLs were detected for Colorado potato beetle field defoliation resistance by MQM analysis of the F2 mapping population. A major QTL on chromosome 2 with partial dominance effect (the 80-1 allele contributing to lower RAUDC) explained 49.3% of the variance (Fig. 3, S4, Table 4). The 1.5 LOD interval of this QTL region is delimited by SNP markers solcap_snp_c2_4521 and PotVar0039036 which corresponds to positions 7,676,939 and 22,151,711 bp on the DM genome (PGSC, 2011; Sharma et al., 2013) (Fig. 3). A second, minor QTL located on chromosome 7 explained 6.2% of phenotypic variation with overdominance effect from M6 that contributed to decreased Colorado potato beetle defoliation (Fig. S5, Table 4). Chromosome 2 also contained a major QTL with partial dominance effects that explained between 29.1%-34.3% of variation, with 80-1 contributing to higher accumulation values for leptine I, leptine II, and total leptine while decreasing α-chaconine (Fig. 3, S4, Table 4). Loci significantly associated with leptine synthesis also colocalized to chromosome 2 and peaked at 4.1-4.5 cM which corresponds to 22.1-22.4 Mb (Kruskal-Wallis test P-value <0.0001). An overlapping minor QTL with partial dominance effects was also present on chromosome 2 and explained 38.2% 100 variation in acetylated to non-acetylated glycoalkaloids (Fig. 3, S5, Table 4). Significant QTL were not detected for solanine or total glycoalkaloids. Validation of the F2 Colorado Potato Beetle Resistance Phenotypic Extremes We validated the Colorado potato beetle resistance phenotype in 20 F2 individuals exhibiting high resistance or susceptibility in a second field trial as well as choice and non-choice detached leaf assays. There was high positive correlation between the phenotypes evaluated in each of these experiments (Table S5). The ten putatively resistant F2 lines demonstrated significantly less defoliation (mean RAUDC = 1.3) over the five-week field observational period than the ten putatively susceptible F2 individuals (mean RAUDC = 51.3), the susceptible parent M6 (mean RAUDC = 60.3) and susceptible check cultivar ‘Atlantic’ (mean RAUDC = 84.8) (P = 0.0002, a=0.05) (Fig. 4a). The ten field susceptible F2 lines were also significantly more defoliated than resistant parent 80-1 and resistant F2 lines in both no-choice larval detached leaf assays (P = 0.0003, a=0.05) and choice adult detached assays (P = 0.0002, a=0.05). Percent larval mortality recorded after nine days in detached leaf conditions was much more variable than percent defoliation between resistant and susceptible lines. Larval development was impeded on resistant lines evidenced by significantly more larvae progressing to third instar on susceptible lines (mean number of third instar larvae = 3.7) than resistant lines (mean number of third instar larvae = 2.7) (P = 0.0092). Furthermore, resistant line foliar mean concentration of leptine I (mean mg/g DW = 5.8, P = 0.001), leptine II (mean mg/g DW = 8.2, P = 0.0045), a-chaconine (mean mg/g DW = 17.4, P = 0.0073) but not a-solanine (mean mg/g DW = 32.6) differed significantly from susceptible lines (Fig. 4b). Resistant lines also had a significantly higher ratio of acetylated/non- acetylated glycoalkaloids (mean ratio = 0.031) than susceptible lines (mean ratio = 0.05) (P = 0.0017). 101 Detection of QTL Associated with Colorado Potato Beetle Resistance by BSA-Seq Whole genome sequencing parental lines and bulked resistant and susceptible F2 samples yielded 353,005,026 total reads to provide ~29x depth of coverage. G’ analysis identified QTL associated with Colorado potato beetle resistance on five chromosomes. The most significant QTL region contains two peaks between 7.2-31.7 Mb on chromosome 2 and is donated by resistant parent 80-1 (Fig. S6, Table S6). In addition, a QTL contributed by susceptible parent M6 on chromosomes 7 spanning 0.1-2.2 Mb was detected (Fig. S6, S7). Both of these intervals correspond to the physical positions of the QTL identified by biparental linkage mapping. However, QTL contributed by resistant parent 80-1 not identified by biparental mapping were also mapped to chromosomes 4, 6 and 12 using the BSA-seq approach (Fig. S7, Table S6). Two peaks between 60.7-62.3 Mb and 63.5-68.7Mb reside on chromosome 4 and a single peak was located on chromosome 6 between 54.6-59.5Mb (Fig. S6, Table S6). Chromosome 12 also contains two peaks on opposite chromosomal arms (Fig. S7, Table S6). The glycoalkaloid metabolism genes (GAME4, GAME12) located on chromosome 12, but not the reported glycoalkaloid genes on chromosome 7 (GAME11, GAME6, SGT1, and SGT3), fall within these minor QTL (Table S6) (Itkin et al., 2013). Fine Mapping of Candidate QTL on Chromosome 2 In an expanded set of 406 additional F2 progeny, we identified 96 recombinant individuals between custom PCR indel markers ch02_5451852 and ch02_29095760 spanning the candidate QTL region on chromosome 2. Linkage map construction with nine additional PCR indel markers within the QTL region and MQM mapping identified a QTL peak at marker ch02_21096852 with a dominant effect decreasing defoliation (6.76) explaining 27.5% of phenotypic variation (Fig. S8). This fine mapping narrowed the QTL region interval to a region of 7.3 Mb. 102 Identification of Candidate Genes Within the QTL Region on Chromosome 2 A total of 351 genes, were predicted within the QTL region (15.1-22.4 Mb) on chromosome 2 identified by BSA-seq and biparental linkage mapping (PGSC, 2011; Sharma et al., 2013). We investigated differential gene expression of the parental lines, three resistant and three susceptible F2 individuals to narrow this slate of candidate genes. After filtering, a total of 21,996 genes were used for expression analysis. Analysis of the mapped reads showed that 923 transcripts are differentially expressed between resistant and susceptible plants at 16-wks post-transplant and distributed across the 12 chromosomes (Table S7). A principal component analysis reveals a clear pattern of expression differentiating resistant and susceptible plants (Fig. S9). Of these, 360 transcripts are down-regulated while 563 are upregulated in the resistant F2 lines and parent 80-1 (Fig. S10). Chromosome 2 is enriched for downregulated transcripts which are distributed toward the distal end of the long arm (Fig. S10, 11). Four of the five upregulated transcripts and two of the five downregulated transcripts with the highest magnitude of log2foldchange are located on chromosome 2 (Table S7). Differentially expressed genes were then grouped into 29 color-coded modules with similar patterns of expression by hierarchical average linkage clustering for network analysis (Fig. S13a). The size of modules ranged from 45 genes (skyblue module) to 8499 genes (grey module) (Table S8). We identified modules with eigengenes correlated to Colorado potato beetle defoliation, total leptine accumulation and the ratio of acetylated to non-acetylated compounds (Fig. 5). Defoliation was strongly positively (0.94, P = 8.22 e-12) and leptine accumulation negatively correlated (- 0.77, P = 1.19e-05) to the midnightblue module, respectively. The darkgrey module was strongly negatively correlation with beetle resistance (-0.74 P = 3.0 e-05) and moderately correlated with gene significance for the total leptines (0.53, P = 4e-06). The lightcyan module was also strongly 103 positively correlated with leptine accumulation (0.66 P = 5.0 e-04). For the ratio of acetylated to non-acetylated compounds, two related modules grey60 and red were the most highly correlated with this trait (0.91, P = 5.2 e-10; 0.83, P = 6.4e-7) (Fig. 5, S13b). We also examined individual gene significance within these interesting modules for the traits leptine accumulation, Colorado potato beetle defoliation resistance and the glycoalkaloid ratio. Gene significance for leptine accumulation and Colorado potato beetle defoliation were correlated with module membership in the midnightblue (0.39, P = 3.8e-09; 0.79, P = 1.0e-46) and darkgrey (0.53, P = 4.0e-06; 0.65, P = 2.6e-09) modules. Gene significance for glycoalkaloids ratio was strongly correlated with module membership in the red and grey60 modules (0.68, P = 1.5 e- 61; 0.68, P = 5.8e-22). We then focused on the core hub genes of these modules, identifying 55 genes in the red module, 9 in the darkgrey module, 23 in the grey60 module and 27 in the midnightblue module (Table S8). To better understand the connectivity between hub genes within modules, the chromosome location, proximity to a QTL region, co-expression relationships, and metabolism associated were analyzed. Genes in the midnightblue module were located on chromosomes 00, 1, 2, 3, 4, 8 and 12, and all the most strongly upregulated genes in resistant lines resided on chromosome 2 (Table S9, Fig. 6). Interestingly an upregulated gene on chromosome 12 (PGSC0003DMG400004286) within our minor QTL annotated as a regulatory subunit Tap46 was found to be highly connected to six upregulated genes within our major QTL on chromosome 2 (PGSC0003DMG400015505, PGSC0003DMG400013094, PGSC0003DMG400042914, PGSC0003DMG400012650, PGSC0003DMG400017873, and PGSC0003DMG400004521) (Fig. 6). One of these genes (PGSC0003DMG400017873) is annotated as a tetratricopeptide repeat 5 oligo-binding fold domain containing protein spanning chromosome 2 positions 18,356,198 to 104 18,362,279. Tetratricopeptide repeats function as protein-protein interaction motifs (Blatch & Lässle, 1999) and have recently been implicated in hormone signaling in plants (Schapire et al., 2006). Notably, counts of transcripts aligning to this gene were only detected in counts <2.5 in susceptible lines and based on publicly available expression data, this transcript is expressed not at all in Solanum tuberosum DM tubers and at levels >0.5 FPKM in other tissues (Hirsch et al., 2014). Another of these coordinately up-regulated genes (PGSC0003DMG400015505) on chromosome 2 is annotated as an anthranilate N-benzoyltransferase protein in SpudDB (Hirsch et al., 2014) and sharing 100% identity with a S. tuberosum predicted uncharacterized acetyltransferase protein, At3g50280-like (LOC102606194), which is significantly and substantially up-regulated in resistant lines (Fig 6). The 1.56 kb genic sequence spans positions 21,003,553 to 21,005,147 on chromosome 2. Expression of this gene is positively correlated with foliar leptine I (ρ = 0.7306, P < 0.0396), leptine II (ρ = 0.7319, P < 0.0396), and the ratio of acetylated/non-acetylated glycoalkaloids (ρ = 0.8571, P < 0.0065). Moreover, this gene is also not expressed in susceptible lines (Table S7) or Solanum tuberosum DM tissues (Hirsch et al., 2014). This subnetwork also contained genes involved in biotic stress response (Table S9). The hub genes for the darkgrey module, correlated with defoliation and leptine accumulation, were located on chromosomes 3, 6, 7, 8 and 9 and all up-regulated in resistant lines (Table S9, Fig. S14). This module contained genes associated with plant defense (Table S9). The red and grey60 modules associated with the ratio of acetylated to non-acetylated glycoalkaloids contained highly interconnected nodes (Fig. S15, S16). The red module was enriched for genes without annotated function and there were no intuitive candidate genes within the hub genes of these modules (Table S9). 105 We then searched for differential expression of known genes involved in the synthesis of glycoalkaloid precursors and genes previously associated with leptine accumulation. We identified a glycosyltransferase (PGSC0003DMG402004500) within the chromosome 2 QTL region downregulated in resistant lines and negatively correlated with leptine I/II content (ρ = -0.7306, P < 0.0396; ρ = -0.8051, p < 0.0159) and the ratio of acetylated/non-acetylated glycoalkaloids (ρ = 0.8333, P < 0.0102). We did not detect changes in expression between resistant and susceptible lines of genes 3-hydroxy-3-methylglutaryl coenzyme A reductase 2, sterol-C5(6)-desaturase, or squalene epoxidase on chromosome 2 involved in the early stages of glycoalkaloid biosynthesis. Genes GAME4 and GAME12 on chromosome 12 as well as GAME7 and GAME 11 on chromosome 7 were upregulated in resistant lines (Table S7). Discussion S. chacoense Colorado Potato Beetle Resistance is Tissue and Age-Dependent The resistance of old leaves on young M6 plants, which do not produce leptines, in a no- choice context observed in this study provides evidence for another resistance mechanism. Colorado potato beetles can detect leaf age (De Wilde et al., 1969) and prefer young to mature foliage (Noronha & Cloutier, 2006), but will feed on mature S. tuberosum leaves in a choice setting (Mitchell & Low, 1994). Structural characteristics, such as leaf toughness, of mature leaves can serve as a mechanical deterrent to beetle herbivory (Larson & Csiro, 1988; RAUPP, 1985; Tanton, 1962). Yet, that the differences in Colorado potato beetle feeding by leaf age in M6 disappear over time suggests instead a mechanism with transient properties. The production of leaf surface compounds, volatiles and cuticular waxes, is a key determinate of Colorado potato beetle feeding preference (Szafranek et al., 2006; Szafranek et al., 2008; Visser et al., 1979) and is dependent on plant developmental stage and tissue type (Agelopoulos et al., 2000; Szafranek & Synak, 2006). 106 Although the role of leaf volatile compounds in Colorado potato beetle host plant acceptance and rejection has been characterized in S. tuberosum (Bolter et al., 1997; Dickens, 2002; Landolt et al., 1999; Martel et al., 2007), their effect on beetle feeding preferences in S. chacoense is incomplete (Hufnagel et al., 2017). Phenotypic Evaluation of the S. chacoense F2 Population While all F2 individuals accumulated the non-acetylated glycoalkaloids α-solanine and α- chaconine, accumulation of the acetylated leptine glycoalkaloids was detected only in a subset of the population (70%). There was strong positive correlation between accumulation of α-solanine and α-chaconine and between leptine I and leptine II. There was a modest negative correlation between accumulation of α-chaconine and individual leptines I and II as well as total leptines. Interestingly, there was no significant correlation between accumulation of α-solanine and leptine I and a slight negative correlation between accumulation of α-solanine and leptine II. This observation supports differential regulation of the accumulation of non-acetylated and acetylated glycoalkaloids, which has been previously proposed (Manrique-Carpintero et al., 2014; Sanford et al., 1996). Although previous studies using mapping populations with a S. tuberosum parent have implicated recessive genes in leptine synthesis (Hutvágner et al., 2001; Manrique-Carpintero et al., 2014; Ronning et al., 1999; Sagredo et al., 2006; Sanford et al., 1996), the presence of leptines in this S. chacoense population followed a single dominant gene model which has been previously observed by (Rangarajan et al., 2000) in a S. phureja x S. chacoense population. Production of the aglycone leptinidine and leptinines by both parental lines in the population used in this study offers the unique opportunity to examine the segregation of downstream glycoalkaloid derivatives alone (Cárdenas et al., 2019). It is possible that other recessive genes involved in leptine biosynthesis are fixed in the two largely homozygous parental lines of this population. Variability in the 107 concentration of acetylated and non-acetylated glycoalkaloids in the lines accumulating all four compounds is indicative of previously described polygenic control of the glycoalkaloid biosynthesis pathway. The F2 population exhibited a range of resistance to Colorado potato beetle defoliation under field conditions. We observed transgressive segregation for both foliar leptine content and Colorado potato beetle field defoliation resistance in the F2 population, which suggests the contribution of alleles controlling leptine accumulation and beetle resistance from both parental lines. However, the F2 lines with greater leptine content than resistant parent 80-1 were not significantly more beetle resistant than 80-1 and lines transgressively segregating for beetle resistance do not contain more leptines than 80-1. We also identified several F2 lines intermediately susceptible to defoliation with leptine levels not significantly different than 80-1 and resistant lines with low leptine accumulation, which casts doubt on the necessity of leptines alone for resistance (Lorenzen et al., 2001; Sagredo et al., 2009). Interestingly, the best predictor of field defoliation resistance was not leptine concentration but rather the ratio of acetylated/non-acetylated glycoalkaloids (i.e. a higher ratio of acetylated/non-acetylated glycoalkaloids is significantly correlated with lower field defoliation). Genotyping the F2 Population and Distorted Segregation Analysis The modest number of informative SNPs (754) generated from genotyping the F2 population with the Illumina Infinium V3 22K SNP array may be attributable to a high degree of similarity between the two S. chacoense parental lines and ascertainment bias inherent in interrogating individuals with divergent genetic landscapes relative to those used to develop the SNP array. We observed distorted segregation of mapped SNPs within the range previously reported (6-40%) in diploid potato (Bonierbale et al., 1988; Felcher et al., 2012; Gebhardt et al., 108 1991; Jacobs et al., 1995; Kreike & Stiekema, 1997; Manrique-Carpintero et al., 2016; Rivard et al., 1996). The regions with the greatest distorted segregation were located on chromosome 1, 3 and 12 with preferential inheritance of M6 alleles, the self-compatible male parent of this population. Because of the self-compatibility selection of the F1 and F2, strong distorted segregation patterns were expected toward regions associated with self-compatibility and associated mechanisms from M6. The greatest distorted segregation observed in this population was located on the long arm of chromosome 12, where the frequency of the 80-1 genotype decreases to zero in some regions. The preferential inheritance of the paternal M6 genotype in the distal region of chromosome 12 is most likely explained by the presence of the Sli locus, associated with self-compatibility in S. chacoense (Hosaka & Hanneman, 1998b). Linkage of Sli to recessive lethal genes on chromosome 12 has also been proposed (Hosaka & Hanneman, 1998a). Transmission of a recessive lethal allele on chromosome 12 from maternal parent 80-1 to the F1 hybrid could be responsible for the preferential inheritance of the heterozygous genotype in the pericentromeric region, where reduced recombination could prevent the purging of deleterious alleles as mentioned by Zhang et al. (2019). However, this distinct pattern of heterozygous genotype retention is indicative of an independent region of segregation distortion which could be driven by genomic interactions between the two S. chacoense parental lines (Moyle & Graham, 2006). The self-incompatibility multiallelic locus (S) on chromosome 1 is the most common source of distorted segregation in potato and contains tightly linked S-RNase and F-box (SLF/SFB) genes expressed in the style and pollen, respectively (Enciso-Rodriguez et al., 2019; Ye et al., 2018). The distorted segregation on chromosome 1 observed in this population is most likely a product of gametic selection in the self-pollinated F1 hybrid against pollen with the 80-1 S-allele, 109 resulting in the absence of individuals with the homozygous 80-1 SLF/SFB genotype in the F2 progeny. (Zhang et al., 2019) similarly reported segregation distortion resulting from gametic selection of S-locus alleles on chromosome 1 in S. stenotomum. Our observation of selection against the homozygous 80-1 genotype on chromosome 1 suggests that Sli in M6 does not completely inactivate the gametophytic incompatibility reaction. The possibility that other factors contribute to transmission of self-compatibility has important ramifications for the introduction of M6-mediated self-compatibility into diploid potato breeding programs. The distorted segregation identified in the population did not interfere with QTL detection since major QTL were located on different chromosomes. Interestingly, the distorted segregation on chromosome 3 favoring M6 alleles points to another potential region associated with self- compatibility. Preferential inheritance of heterozygous loci on chromosome 8 could be associated with zygotic selection driven by sublethal and meiotic mutant alleles hypothesized to reside on chromosome 8 (Jacobs et al., 1995). QTL and candidate gene identification A major QTL was detected for Colorado potato beetle resistance and leptine accumulation on chromosome 2. The moderate negative correlation between increased leptine accumulation and lower Colorado potato beetle field defoliation reported in our study supports a single QTL for both traits. Localization of loci associated with the presence of leptines within the QTL on chromosome 2 suggests that this QTL enables leptine biosynthesis. A lack of recombination within the QTL region on chromosome 2 in Colorado potato beetle individuals with low leptine levels suggests a single gene at this locus associated with the presence of leptines. As a polygenic trait, we propose that a separate regulatory element contributes to the variation observed in leptine accumulation. We identified a putative acetyltransferase within the major QTL on chromosome 2 expressed only 110 in resistant lines which may be responsible for leptine synthesis in this germplasm. Regulation of leptine accumulation may be then accomplished by co-expression of the regulatory elements we identified within the major QTL on chromosome 2 and minor QTL on chromosome 12 (Table S7, S8, Fig. 6). Alternatively, it is possible that glycosylation of acetyl-leptinidine to produce leptines I/II is achieved by a unique UDP-glycosyltransferase(s) that is absent in M6, the non-leptine producer parent. Three distinct UDP-glycosyltransferases are required for the conversion of solanidine to α-solanine and α-chaconine (McCue et al., 2007; McCue et al., 2005; Moehs et al., 1997), but enzymes involved in glycosylation of other aglycones remain undefined. We identified unique expression patterns of uncharacterized UDP-glycosyltransferases on chromosome 2 in 80- 1 and resistant F2 lines that may contribute to leptine I/II production. The complete glycoalkaloid profile of an individual plant may also contribute to the variation in Colorado potato beetle defoliation resistance. There is evidence that the Colorado potato beetle neurosensory response to leptines is modified by the presence of other glycoalkaloids. (Hollister et al., 2001) demonstrated that the Colorado potato beetle neurosensory response to leptine I is reduced in the presence of a-solanine. We observed a modest positive correlation between a-chaconine accumulation and Colorado potato beetle defoliation (Table 2) and negative contribution of the 80-1 genotype in the QTL region on chromosome 2 for a- chaconine accumulation (Table 4). The ratio of acetylated to non-acetylated glycoalkaloids was also the best predictor of field defoliation in this study. Cardeñas et al. (2019) demonstrated that the product of GAME32, responsible for leptinine production in M6, can hydroxylate either the aglycone solanidine or the glycosylated a-solanine and a-chaconine. Taken together, synthesis of leptines by our candidate gene within the QTL on chromosome 2 may involve the acetylation of 111 hydroxylated a-solanine and a-chaconine, resulting in a higher ratio of acetylated to non- acetylated compounds. The QTL identified in this study overlaps with a large region previously associated with leptine synthesis and accumulation in a pseudo F2 population using 80-1 and S. tuberosum Grp. Phureja DH as parents (Manrique-Carpintero et al., 2014). The large size of the QTL region may be explained by the moderate population size and suppression of recombination due to the close proximity of the nucleolar organizer region on the short arm of chromosome 2 (Pijnacker & Ferwerda, 1984), while the presence of two peaks within the region identified by BSA-seq is most likely explained by a potential assembly error in constructing the DM pseudomolecules. Fine mapping using F4 individuals derived from the F2 population to further delineate the region critical to Colorado potato beetle resistance will facilitate efficient introgression of this trait into cultivated backgrounds and contribute to the development of beetle resistant varieties. 112 APPENDICES 113 APPENDIX A: Chapter 3 Tables Table 3.1. Mean, range and standard deviation (SD) of measured glycoalkaloids in the diploid Solanum chacoense USDA8380-1 x M6 F2 population (n = 233) Leptine I (mg/g DW) Leptine II (mg/g DW) a-Solanine (mg/g DW) a-Chaconine (mg/g DW) Total Glycoalkaloids (mg/g DW) Leptine/(a-Solanine & a- Chaconine) Ratioa M6 80-1 F2 Progeny Mean SD Range 0.0 12.1 6.4 6.8 0.0 10.8 8.5 8.5 21.6 5.7 28.6 14.5 19.4 3.0 21.8 13.8 41.0 31.6 65.4 28.2 2.6 0.4 0.4 0.0-25.9 0.0-41.6 6.2-120.1 4.0-115.3 0.1-235.4 0.0-2.3 aThe ratio of acetylated glycoalkaloids to non-acetylated glycoalkaloids was assessed in the subset of the population with the presence of acetylated compounds (n= 162) 114 Table 3.2. Spearman’s rank correlation coefficients among measured traits in the Solanum chacoense USDA8380-1 x M6 F2 population Leptine Ia Leptine IIa Total Leptinea α-Solaninea α-Chaconinea Acetylated/Non-Acetylatedb Total Glycoalkaloidsa Field Defoliationc α-Chaconinea α-Solaninea -0.49*** -0.49*** -0.50*** 0.28** 0.42*** -0.54*** ns -0.40*** -0.49*** -0.45*** 0.92*** ns -0.25** -0.21* Leptine II 0.91*** *** P<0.0001, **P<0.01, *P<0.05, ns not significant, n = 151 a Data represent the mean of three technical replicates mg/g DW b Data represent [mean total leptines (mg/g DW)]/ [mean α-chaconine (mg/g DW) + mean α-solanine (mg/g DW)] c Data represent the mean of three biological replicate plot relative area under the defoliation progression curve (RAUDC x 100) 115 Chromosome No. Mapped SNPs Map Length (cM) Map Length (Mb)a chr01 chr02 chr03 chr04 chr05 chr06 chr07 chr08 chr09 chr10 chr11 chr12 Total 71 66 68 61 68 55 64 61 57 42 90 51 754 136.3 82.9 101.9 100.4 101.3 91.9 94.4 91.4 109.8 93.8 102.2 87.5 1193.8 87.5 39.7 61.5 71.1 51.7 58.4 55.2 55.7 60.1 58.7 44.7 60.7 705 Map Coverage (Mb)a 0.99 0.81 0.99 0.98 0.99 0.98 0.97 0.98 0.98 0.98 0.98 0.99 0.97 Average Interlocus Distance (cM) 1.9 1.3 1.5 1.7 1.5 1.7 1.5 1.5 1.9 2.3 1.1 1.7 1.6 Table 3.3. Summary of single nucleotide polymorphism (SNP) marker information for individual chromosomes of the Solanum chacoense USDA8380-1 x M6 F2 population linkage map aMap length (Mb) and map coverage (Mb) are based on the assembled Solanum tuberosum clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03) 116 Table 3.4. Summary of QTLs detected by MQM in MapQTL® 6 Software in a diploid Solanum chacoense USDA8380-1 x M6 F2 population Trait Defoliationa Leptine Ib Leptine IIb Total Leptineb α-Chaconineb Ratioc Chr 2 7 2 2 2 2 2 Peak Position (cM) 4.1 2.6 0 4.5 0 4.5 0 Nearest Marker PotVar0039036 solcap_snp_c1_10783 solcap_snp_c2_4521 solcap_snp_c2_32460 solcap_snp_c2_4521 solcap_snp_c2_32460 solcap_snp_c2_4521 Physical Position (bp) 22151711 1318666 7676939 22381563 7676939 22381563 7676939 LOD scored 20.52 3.76 7.78 9.50 9.41 8.38 10.86 M6e 39.92 26.28 0.15 0.11 0.72 32.30 0.02 Hete 16.01 19.90 7.70 8.50 16.27 19.30 0.39 80-1e 8.86 31.59 8.31 13.72 22.27 14.40 0.79 Additive effect 15.53 -2.66 -4.08 -6.81 -10.77 8.93 -0.38 Dominance effect -8.37 -9.02 3.48 1.59 4.77 -4.03 -0.02 Variance Explained (%) 49.3 6.2 29.1 34.3 34.1 31.0 38.2 a Data represent the mean of three biological replicate plot relative area under the defoliation progression curve (RAUDC x 100) b Data represent the mean of three technical replicates mg/g DW c Data represent [mean total leptines (mg/g DW)]/ [mean α-chaconine (mg/g DW) + mean α-solanine (mg/g DW)] d LOD threshold at P = 0.05 with MQM for Defoliation, Leptine I, Leptine II, Total Leptines, α-Chaconine and the Ratio of Acetylated/Non-Acetylated Glycoalkaloids was 3.7, 4.0, 4.7, 4.0, 3.8 and 4.2, respectively, based on 1,000x permutations e Mean trait values are given for paternal (Solanum chacoense M6), maternal (Solanum chacoense USDA8380-1), and heterozygous (Het) genotypes 117 APPENDIX B: Chapter 3 Figures Figure 3.1. Colorado potato beetle larval defoliation (%) of Solanum chacoense parental lines M6 and USDA8380-1 (80-1) at three time points post-transplant from tissue culture. Defoliation of detached leaves was scored 9 days after placing neonates on leaves. Each bar is the mean of three replicates ± SEM 118 Figure 3.2. Distribution of Colorado potato beetle resistance in 151 F2 Solanum chacoense progeny and parental lines under field conditions expressed in relative area under the defoliation curve multiplied by 100 (RAUDC x 100). Representative pictures for classes of defoliation are shown below the distribution 119 Figure 3.3. The QTL regions on chromosome 2 associated with Colorado potato beetle resistance under field conditions and foliar concentration of glycoalkaloids identified by bi- parental linkage mapping. QTL are represented by solid bars (1-LOD interval) and extended lines (2-LOD interval) in unique colors for each trait. The genetic positions (cM) are shown on the left and the corresponding physical position of mapped SNPs on the PGSC Version 4.03 Doubled Monoploid pseudomolecule 2 (bp) are shown on the right of the linkage map. The significance (LOD) of SNP association to each trait is plotted against these SNP positions. Figure prepared with MapChart 2.3. (R.E. Voorrips. Plant Research International, Wageningen, The Netherlands) 120 Figure 3.4. Colorado potato beetle resistance phenotypic validation of the F2 phenotypic extremes. a. Percent defoliation by Colorado potato beetle under field conditions of Solanum chacoense parental lines 80-1 and M6 and F2 phenotypic extremes selected from the previous field season. Data represent means of five biological replicates for each parent and for each of the ten F2 individuals in the resistant bulk and ten F2 individuals in the susceptible bulk. b. Foliar leaf concentration of leptines I/II, alpha-solanine and alpha-chaconine in the ten resistant F2 individuals and ten susceptible F2 individuals evaluated in the field. 121 Figure 3.5. Weighted gene co-expression network module associations with three traits: Defoliation (RAUDC), Total Leptines (mg/g DW) and Ratio (the ratio of acetylated compounds to non-acetylated compounds accumulated). Each row corresponds to a module eigengene and each column to a trait. Each cell contains the Pearson correlation value and corresponding Student t test P-value. The color legend indicates the scale for color-coding of the module-trait correlation values 122 Figure 3.6. Interaction of hub genes in the midnight blue module visualized using Cytoscape 3.7 software. The node colors are coded from white to dark blue to indicate differential gene expression. The thickness of connective lines between nodes is representative of connection weight between two nodes. Node labels provide PGSC V4.03 chromosome number (ch) followed by the PGSC numeric gene identifier 123 APPENDIX C: Chapter 3 Supplementary Data Figure S3.1. Analysis of network topology for increasing soft-thresholding powers. Power 22 was selected as the point at which the slope of the curve flattens out. Chart created in R using the WGCNA package. Langfelder P, Horvath S (2008) WGCNA: An R package for weighted correlation network analysis. BMC Bioinformatics 9:559. 124 CH01 CH02 CH03 CH04 CH05 CH06 CH07 CH08 CH09 CH10 CH11 CH12 l e u a v - P M c l e u a v - P M c l e u a v - P M c l e u a v - P M c 20 10 0 160 100 40 20 10 0 160 100 40 20 10 0 160 100 40 20 10 0 160 100 40 50 60 70 80 0 10 20 30 40 50 60 70 80 40 Mb 0 10 20 30 40 50 60 70 80 0 10 20 30 Figure S3.2. Distribution of 754 mapped single nucleotide polymorphisms along the 12 chromosomes. For each chromosome (CH), the upper panel (blue) is the significance of distorted segregation reported as the minus logarithm of chi-square test p-value (P-value), and the lower panel (black) is the Marey map plotting the genetic position (cM) against the physical position in Mb based on the Solanum tuberosum DM genome assemble version 4.03. The 0.1% threshold of significance corresponds to the black line at P-value =3. Charts created in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 125 Figure S3.3. Distribution of segregation rates of maternal (Solanum chacoense USDA8380-1;red), paternal (Solanum chacoense M6; black) and heterozyogous (purple) genotype of 754 mapped single nucleotide polymorphisms along the 12 chromosomes of the genetic map. Loci with distorted segregation at the 0.1% significance level are highlighted with an asterisk (*). Lines (red and blue) represent the confidence interval of segregation for heterozygous and homozygous genotypes, respecitively. Charts created in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 126 Figure S3.4. Means trait values of maternal Solanum chacoense USDA8380-1 alleles (mu_A), paternal Solanum chacoense M6 alleles (mu_B), and heterozygous genotypes (mu_H) plotted along the genetic map (x-axis, cM) of chromosome 2. Charts created with MapQTL® 6 Software (Van Ooijen J, Kyazma B (2009) Mapqtl 6. Software for the mapping of quantitative trait loci in experimental populations of diploid species. :Kyazma BV: Wageningen, Netherlands). 127 Figure S3.5. The minor QTL region on chromosome 7 associated with Colorado potato beetle resistance under field conditions identified by MQM mapping. a. Means trait values of maternal Solanum chacoense USDA8380-1 alleles (mu_A), paternal Solanum chacoense M6 alleles (mu_B), and heterozygous genotypes (mu_H) plotted along the genetic map (x-axis, cM) of chromosome 7. b. Additive (blue) and dominance (majenta) effects are plotted along the genetic positions of chromosome 7. The LOD threshold of 3.7 was determined by 1,000x permutation tests. Charts created with MapQTL® 6 Software (Van Ooijen J, Kyazma B (2009) Mapqtl 6. Software for the mapping of quantitative trait loci in experimental populations of diploid species. :Kyazma BV: Wageningen, Netherlands). 128 Figure S3.6. Distribution of significant QTL along physical positions (Mb) of the 12 chromosomes identified by alignment of whole genome sequence from bulked beetle resistant and bulked beetle susceptible F2 progeny to the Solanum tuberosum clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03), corrected with Solanum chacoense M6 whole genome sequence data, and G’ analysis. The genome wide false discovery rate of 0.01 is shown by a red line. Analyses conducted in QTLseqr package in R (Mansfeld BN, Grumet R for bulk segregant analysis with next-generation sequencing. bioRxiv:208140. https://doi.org/10.1101/208140). (2018) QTLseqr: An R package 129 Figure S3.7. Significant QTL associated with Colorado potato beetle resistance identified by G’ analysis plotted on the physical position (Mb) of the Solanum tuberosum clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03), corrected with Solanum chacoense M6 whole genome sequence reads. Significance is reported as the negative logarithm of the p-value, which is derived from the G’ value. The genome wide false discovery rate of 0.01 is shown by a red line. Analyses conducted in QTLseqr package in R (Mansfeld BN, Grumet R (2018) QTLseqr: An R package for bulk segregant analysis with next-generation sequencing. bioRxiv:208140. https://doi.org/10.1101/208140). 130 Figure S3.8. Genetic map of chromosome 2 and a QTL associated with Colorado potato beetle resistance under field conditions in 96 additional Solanum chacoense F2 individuals. The QTL is represented by the solid green bar (1-LOD interval) and extended lines (2-LOD interval). The genetic positions (cM) of PCR markers are shown on the left and the corresponding physical position on the Solanum chacoense M6 pseudomolecule 2 (bp) are shown on the right of the linkage map. The significance (LOD) of marker association is plotted against these positions. The LOD threshold of 1.9, determined by 1,000x permutation tests, is shown by a dashed, black line. Figure prepared with MapChart 2.3. (R.E. Voorrips. Plant Research International, Wageningen, The Netherlands) 131 Figure S3.9. Principal component analysis of differentially expressed genes between resistant and susceptible lines produced within the R package DESeq2 (Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15:550. https://doi.org/10.1186/s13059-014-0550-8). Parental lines Solanum chacoense USDA8380-1 and M6 are labeled. Unlabeled points correspond to the three biological replicates of resistant and susceptible F2 lines. 132 Figure S3.10. Distribution of significantly (padj < 0.001) down- (left) and up- (right) regulated transcripts in leaf tissue of Solanum chacoense USDA8380-1 and three Colorado potato beetle resistant F2 progeny across the 12 chromosomes. Counts of transcripts determined by FeatureCounts (Liao Y, Smyth GK, Shi W (2013) Featurecounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30:923-930) are given in grey for each chromosome. Differential expression analysis conducted in DESeq2 (Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA- seq data with DESeq2. Genome Biology 15:550. https://doi.org/10.1186/s13059-014-0550-8). Charts created in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 133 Figure S3.11. Distribution of significantly (padj < 0.001) down- (left) and up- (right) regulated transcripts in leaf tissue of Solanum chacoense 80-1 and three Colorado potato beetle resistant F2 progeny across chromosome (Chr) 2 (Solanum tuberosum DM Pseudomolecule PGSC Version 4.03) physical positions (Mb). Differential expression analysis conducted in DESeq2 (Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15:550. https://doi.org/10.1186/s13059-014-0550-8). Charts created in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 134 Figure S3.12. Weighted gene co-expression network module significance values of each of the 29 modules for three traits measured in Colorado potato beetle resistant and susceptible lines. Module significance is represented by the average gene significance in each module. 135 Figure S3.13. Weighted gene co-expression network analysis clustering. a. Cluster dendrogram and module assignment for the 29 modules identified by hierarchical linkage clustering. Branches correspond to modules of highly interconnected groups of genes and each color in the horizontal bar represents one of the color-coded modules. b. Hierarchical clustering dendrogram of module eigengenes. More highly related eigengenes have lower merging heights 136 Figure S3.14. Interaction of 9 hub genes in the darkgrey module visualized using Cytoscape 3.7 software. The node colors are coded from white to grey to indicate differential gene expression. Negative log2FoldChange indicates up-regulation in resistant lines. The thickness of connective lines between nodes is representative of connection weight between two nodes. Node labels provide PGSC V4.03 chromosome number (ch) followed by the PGSC numeric gene identifier. 137 -12.5 1 Figure S3.15. Interaction of 55 hub genes in the red module visualized using Cytoscape 3.7 software. The node colors are coded from rose to red to indicate differential gene expression. Negative log2FoldChange indicates up-regulation in resistant lines. The thickness of connective lines between nodes is representative of connection weight between two nodes. Node labels provide PGSC V4.03 chromosome number (ch) followed by the PGSC numeric gene identifier. 138 -4.0 4.0 Figure S3.16. Interaction of 23 hub genes in the red module visualized using Cytoscape 3.7 software. The node colors are coded from grey to black to indicate differential gene expression. Negative log2FoldChange indicates up-regulation in resistant lines. The thickness of connective lines between nodes is representative of connection weight between two nodes. Node labels provide PGSC V4.03 chromosome number (ch) followed by the PGSC numeric gene identifier. 139 Table S3.1. Phenotypes of the 20 F2 individuals from the Solanum chacoense USDA8380-1 x M6 population used for bulk segregant analysis of Colorado potato beetle resistance. Replicate d Field Trial 2017 (Mean RAUDC x 100) Replicate d Field Trial 2018 (Mean RAUDC x 100) Choice Detached Bioassay (Mean % Defoliatio n) No-Choice Detached Bioassay (Mean % Defoliatio n) Leptin e I (mg/g DW) Leptin e II (mg/g DW) α- Solanin e (mg/g DW) α- Chaconin e (mg/g DW) Total Leptine (mg/g DW) Total Glycoalkaloi ds (mg/g DW) Line EE501F2_00 EE501F2_08 6 9* EE501F2_16 EE501F2_28 EE501F2_29 EE501F2_49 EE501F2_51 6* EE501F2_56 EE501F2_59 EE501F2_64 1* EE501F2_06 1 5 7 9 8 7 3 6 3* 9* 5 Class Resistant Resistant Resistant Resistant Resistant Resistant Resistant Resistant Resistant Resistant Susceptibl e e e e e EE501F2_07 Susceptibl EE501F2_09 Susceptibl EE501F2_09 Susceptibl EE501F2_14 Susceptibl 1.1 0.7 0.7 0.6 0.8 0.5 0.7 0.8 0.8 1.4 56.2 49.2 58.8 49.8 53.8 9.3 2.2 0.9 4.8 7.6 5.8 1.2 1.5 0.5 0.0 63.2 43.3 58.5 57.8 54.3 0 0 0 2 6 0 0.2 6 0.2 0 89 76 55 45 45 37 13.33 6 20 20 14 31.66 10.33 30 0 31.67 55 73.33 63.33 40 2.3 6.5 3.2 4.7 1.3 7.6 6.9 1.3 7.1 17.3 0.0 2.2 0.0 0.0 0.0 140 2.8 7.4 4.6 6.2 2.2 12.0 10.2 1.9 11.6 22.6 0.0 3.3 0.0 0.0 0.0 24.9 13.8 33.9 41.4 32.2 19.7 26.9 49.0 29.8 54.6 38.7 20.6 47.3 55.8 42.6 10.3 11.1 15.2 18.2 14.3 13.8 12.2 27.5 13.5 37.8 40.9 14.5 52.2 52.0 42.3 5.2 13.8 7.9 10.9 3.5 19.6 17.1 3.2 18.8 39.9 0.1 5.5 0.1 0.1 0.1 40.36 38.67 56.93 70.56 49.98 53.20 56.24 79.80 62.13 132.26 79.65 40.58 99.52 107.88 85.01 Ratio Acetylated/No n-acetylated 0.15 0.56 0.16 0.18 0.08 0.59 0.44 0.04 0.43 0.43 0.00 0.16 0.00 0.00 0.00 56.4 50.8 49.5 52.5 23.9 42.9 35.6 69.5 52 61 80 76 35 86.66 88 78.33 0.8 0.0 0.0 0.0 1.3 0.0 0.0 0.0 48.2 32.6 30.9 26.8 22.0 15.5 36.5 25.3 2.1 0.0 0.0 0.1 72.36 48.12 67.47 52.12 0.03 0.00 0.00 0.00 48.3 40.8 89 86.66 4.2 12.3 32.4 25.9 16.5 74.87 0.28 *Lines used for expression profiling Table S3.2. Insertion/deletion (Indel) markers on chromosome 2 designed from Solanum chacoense USDA8380-1 and M6 whole genome sequence data Indel Size Forward Primer (5'-3') Reverse Primer (5'-3') Locus # F2 Progeny screened n=406 n=96 n=96 n=96 n=96 n=96 n=96 n=96 n=96 n=96 AACGGTCACTTATCTCTTAA AGTGGAAGATATTTGAATGG TCTAGGACTGTTGGGTTCT CTTGTATCCACGACCTGAA TCACCTCGTATGAATTAACA GGAATAAGCAGCAATAGCAT CGTGGCTCAATGCTTAGG AGAAGCAGAGATGTTAGTGT AAATGTGAATGCAACAAAGAG TTGATGACTTCTTCCATTGG TCAAAGGTAACAAGGATGTAAAATGT ACATTAATCAGGAGGCAGGACC AACCGTGATCAAGCATAGTC GCGATTCACTAATACATGTAC TGGAGCAAATACAGCCCTACA GCAGCACAAGACATAATTGAGT CTTGGAGAACTTAGTGGAT GCCAATAAGTTGATGACACA TGTGTTCCATGTGAATTGTAT Table S3.1 (cont’d) EE501F2_148 EE501F2_156 EE501F2_462 EE501F2_500 * EE501F2_584 Susceptibl Susceptibl Susceptibl Susceptibl Susceptibl e e e e e ch02_5451852 ch02_6012832 ch02_11283933 ch02_15200067 ch02_19289543 ch02_20766720 ch02_21112606 ch02_21096852 ch02_23527163 ch02_27408064 ch02_29095760 71 45 33 16 74 43 62 38 93 24 36 GTGAAGAACATTCATAGAGTA ATGCTTGTGATGTCCGAAT n=406 CCTACTTTCACCTCTGTATTAC 141 Chromosome Marker Name Linkage Group Genetic Distance Table S3.3. Genetic map of the Solanum chacoense USDA8380-1 x M6 F2 population. Physical Position on PGSC Version 4.03 Pseudomolecules of Solanum tuberosum DM1-3 (bp) solcap_snp_c1_2425 solcap_snp_c2_21100 solcap_snp_c2_19302 solcap_snp_c1_6114 1 PotVar0120099 1 1 PotVar0071966 1 PotVar0119966 1 PotVar0119791 1 PotVar0119976 1 PotVar0044826 1 1 PotVar0045000 1 1 1 PotVar0045435 1 1 1 1 1 1 0 1 1 PotVar0005924 1 solcap_snp_c1_14212 solcap_snp_c2_53842 solcap_snp_c1_8619 solcap_snp_c1_8608 solcap_snp_c2_50013 solcap_snp_c2_43973 solcap_snp_c1_15241 solcap_snp_c1_805 solcap_snp_c1_13810 354295 1028869 1158715 472549 481385 472297 2505120 2591141 2857296 3850533 3693421 4036788 5140602 6789900 6529837 6144618 7447660 13546944 17518620 58567030 61280016 59974768 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 142 0 2.913 4.675 7.823 8.473 8.688 18.61 18.825 22.215 28.345 28.777 30.316 34.682 38.321 39.413 40.728 45.106 49.988 50.858 54.02 56.476 56.908 Table S3.3 (cont’d) solcap_snp_c2_45301 solcap_snp_c2_41338 solcap_snp_c2_40966 solcap_snp_c2_46207 solcap_snp_c2_14616 solcap_snp_c1_6518 1 1 PotVar0049555 1 PotVar0049716 1 1 PotVar0049028 1 1 PotVar0049532 1 1 PotVar0033293 1 1 PotVar0072244 1 1 PotVar0043815 1 PotVar0043608 1 1 1 1 1 1 1 PotVar0041430 1 1 PotVar0110374 1 PotVar0028786 1 1 solcap_snp_c1_3866 solcap_snp_c2_19958 solcap_snp_c2_19975 solcap_snp_c2_20028 solcap_snp_c2_19984 solcap_snp_c2_14350 solcap_snp_c1_5267 solcap_snp_c2_2308 solcap_snp_c2_54547 57.341 60.029 60.461 65.829 66.044 66.259 68.712 72.106 77.466 79.458 83.819 85.583 88.266 89.357 95.991 99.861 100.293 100.943 101.158 104.552 106.317 110.677 112.215 116.082 120.678 123.829 59364003 62154924 61774217 62931118 63365123 63690655 62600331 64094589 65692201 66427942 67603025 67605501 68952770 69664340 71223852 72756308 72835626 73220352 72996962 74628218 74252050 75333653 76290134 77047246 78216532 78922111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 143 Table S3.3 (cont’d) solcap_snp_c2_7344 solcap_snp_c2_7068 solcap_snp_c2_7062 solcap_snp_c2_5076 solcap_snp_c1_3275 1 1 1 1 1 PotVar0061107 1 PotVar0061244 1 1 PotVar0035163 1 PotVar0035721 1 1 PotVar0035437 1 PotVar0124515 1 1 1 PotVar0126949 1 1 PotVar0126587 1 1 PotVar0099782 1 PotVar0099779 1 1 1 PotVar0100004 1 PotVar0122423 2 2 PotVar0039036 solcap_snp_c2_4521 solcap_snp_c2_34490 solcap_snp_c2_14741 solcap_snp_c2_14733 solcap_snp_c2_53075 solcap_snp_c2_46446 solcap_snp_c2_14760 solcap_snp_c1_11288 128.188 130.41 130.842 133.529 137.649 137.864 138.296 142.902 148.25 148.466 150.457 155.553 156.644 156.859 158.85 159.065 159.935 161.249 165.36 166.011 168.001 170.451 170.667 172.204 0 4.107 80003968 80262082 80230736 80598198 81637695 81623184 81750427 83422333 84643858 84972380 84052012 86148266 86602967 86527570 85586812 85902599 85465494 85501690 87245944 87245902 86745999 87558284 87548665 87888814 7676939 22151711 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 144 Table S3.3 (cont’d) solcap_snp_c2_32460 solcap_snp_c2_21759 solcap_snp_c1_13459 solcap_snp_c2_45306 solcap_snp_c1_12329 solcap_snp_c1_14293 2 2 PotVar0029505 2 2 PotVar0117640 2 PotVar0088949 2 2 2 PotVar0123847 2 PotVar0123848 2 2 2 PotVar0062500 2 PotVar0062424 2 PotVar0062099 2 PotVar0062142 2 2 2 2 2 PotVar0082605 2 PotVar0094371 2 PotVar0094218 2 PotVar0094231 2 PotVar0094234 2 2 solcap_snp_c2_46904 solcap_snp_c2_46915 solcap_snp_c2_46890 solcap_snp_c1_13920 solcap_snp_c1_13240 solcap_snp_c2_44777 4.539 6.758 8.522 9.392 10.262 13.657 13.873 14.964 15.835 17.15 22.525 23.175 24.49 24.706 25.138 25.788 26.004 26.436 26.651 27.302 29.068 29.5 29.716 30.148 30.799 32.565 22381563 24387185 25003703 25332805 25895594 27143975 27086486 27602074 27602042 27618678 29163404 29473175 29707398 29763419 29762269 29922863 29955410 30050056 30142847 30394391 31448769 31352137 31352361 31352403 31839875 32670900 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 145 2 PotVar0038096 2 PotVar0038051 2 PotVar0038674 2 2 2 2 PotVar0045976 2 2 PotVar0046549 2 PotVar0046488 2 PotVar0047012 2 PotVar0046764 2 2 2 2 2 PotVar0010684 2 2 2 PotVar0010382 2 PotVar0010429 2 2 PotVar0009997 2 PotVar0009651 2 2 solcap_snp_c2_42169 solcap_snp_c2_55632 solcap_snp_c1_12381 solcap_snp_c1_15466 solcap_snp_c2_53034 solcap_snp_c2_40635 solcap_snp_c2_42128 solcap_snp_c2_25143 solcap_snp_c2_7539 Table S3.3 (cont’d) solcap_snp_c1_14823 solcap_snp_c1_5871 solcap_snp_c2_17931 solcap_snp_c1_16727 35.254 35.47 38.633 41.324 41.975 42.19 42.406 43.946 44.162 44.377 45.028 45.243 45.675 45.891 46.541 48.766 50.76 50.976 52.067 52.718 53.588 54.021 56.244 56.895 57.11 60.031 33962720 33962178 34816049 35675019 36330134 36364959 36420764 36879127 36941026 36931190 37015905 36956584 37438491 37474756 37622394 38386350 38654389 38688454 39073504 39079979 39079305 39369457 39963506 40253455 40435644 41116317 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 146 Table S3.3 (cont’d) solcap_snp_c2_54104 solcap_snp_c2_22842 solcap_snp_c2_43352 solcap_snp_c2_15068 solcap_snp_c2_15251 solcap_snp_c1_7876 solcap_snp_c1_10604 solcap_snp_c2_35687 solcap_snp_c2_36232 solcap_snp_c1_15783 2 2 PotVar0118890 2 2 PotVar0007240 2 PotVar0007181 2 PotVar0006989 2 2 2 2 2 2 3 3 3 PotVar0084666 3 3 3 PotVar0019295 3 3 3 3 PotVar0085747 3 3 3 3 solcap_snp_c1_2051 solcap_snp_c1_12749 solcap_snp_c2_52494 solcap_snp_c2_38068 solcap_snp_c2_51389 solcap_snp_c1_5689 solcap_snp_c2_50372 solcap_snp_c1_12745 solcap_snp_c1_13782 64.397 65.268 65.483 68.403 68.836 69.268 72.185 77.791 78.661 79.312 81.994 82.864 0 4.346 6.563 7.876 10.789 12.327 13.197 20.897 21.112 21.762 23.076 23.508 26.424 27.738 42378785 42737881 42777302 43872417 43871583 43805014 44773069 45695644 46059040 46190640 47177726 47324196 419098 1276863 833199 983008 1956984 2235581 2524231 34492320 33836905 35929359 10654105 34638581 37495967 38758796 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 147 Table S3.3 (cont’d) solcap_snp_c2_45697 solcap_snp_c2_55465 solcap_snp_c1_13506 solcap_snp_c2_20069 solcap_snp_c2_20089 solcap_snp_c2_48507 solcap_snp_c2_45914 solcap_snp_c2_29684 solcap_snp_c2_20135 solcap_snp_c1_6334 3 PotVar0129473 3 3 3 3 PotVar0055003 3 PotVar0042852 3 3 PotVar0120489 3 3 3 3 3 PotVar0070553 3 3 3 PotVar0070385 3 PotVar0056881 3 3 3 3 3 3 PotVar0027580 3 PotVar0029746 3 3 solcap_snp_c2_1567 solcap_snp_c2_1579 solcap_snp_c2_1681 solcap_snp_c2_57260 solcap_snp_c2_17631 solcap_snp_c2_47801 solcap_snp_c2_47802 28.388 35.28 37.269 37.919 42.265 44.945 52.097 52.312 52.528 53.178 55.397 56.488 60.596 61.466 63.229 63.661 64.752 65.843 66.493 71.588 76.438 76.653 77.745 79.737 81.276 81.708 39258117 43326283 44517478 43938298 45612140 46457554 48471111 48467461 48526187 48406270 47769763 47520884 49236778 48764500 49314386 49243176 50226521 50490035 50455611 51506082 53421920 53309062 52979922 53739174 54033139 54033117 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 148 Table S3.3 (cont’d) solcap_snp_c1_14159 solcap_snp_c1_7132 solcap_snp_c2_26402 solcap_snp_c2_18490 solcap_snp_c2_26474 solcap_snp_c2_18502 solcap_snp_c2_18506 3 3 3 PotVar0029964 3 PotVar0029965 3 PotVar0030310 3 3 3 3 3 3 PotVar0013550 3 PotVar0013632 3 PotVar0013816 3 PotVar0014066 3 PotVar0014064 3 3 3 3 PotVar0014106 3 3 3 3 3 3 PotVar0020413 3 PotVar0020402 solcap_snp_c2_9594 solcap_snp_c2_9627 solcap_snp_c2_9580 solcap_snp_c1_131 solcap_snp_c2_9531 solcap_snp_c2_148 solcap_snp_c2_616 solcap_snp_c2_625 82.14 83.455 83.887 84.757 87.916 91.076 92.168 93.039 93.254 93.469 95.694 95.909 97.001 102.883 103.315 104.854 109.717 109.932 112.385 115.539 116.409 117.059 118.373 122.489 124.941 125.156 53970990 54314873 54538909 54538961 55344214 56604264 55901885 56382228 55910650 55911538 57500599 57502252 57568392 58519878 58519761 58295011 58877163 58867880 59163731 60203560 59933404 60168767 59764345 61035987 61494421 61494770 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 149 Table S3.3 (cont’d) solcap_snp_c2_37116 solcap_snp_c2_54378 solcap_snp_c2_54463 solcap_snp_c2_53206 solcap_snp_c1_9084 solcap_snp_c2_21946 solcap_snp_c2_21936 3 3 PotVar0020079 4 4 4 4 4 PotVar0076873 4 PotVar0076875 4 PotVar0076831 4 PotVar0107010 4 4 4 PotVar0100946 4 PotVar0101389 4 4 4 4 4 4 4 4 4 4 4 4 solcap_snp_c2_21914 solcap_snp_c2_51639 solcap_snp_c2_26773 solcap_snp_c2_26838 solcap_snp_c2_53779 solcap_snp_c2_53784 solcap_snp_c2_44609 solcap_snp_c2_53769 solcap_snp_c2_54077 solcap_snp_c2_37325 solcap_snp_c2_56256 solcap_snp_c2_30114 126.247 126.679 0 0.87 4.742 12.448 12.664 12.879 13.094 17.446 18.984 19.854 20.069 21.383 22.474 23.344 34.228 35.993 36.644 36.859 37.951 39.265 39.48 41.245 41.677 41.893 61899146 61796673 856438 1195223 1932918 2680042 2720000 2719958 2720353 3902338 4567755 4595286 4782583 4920111 5192938 5488735 9276426 9733963 9941686 9941194 10132935 10675065 10968547 11355307 12425864 42975226 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 150 Table S3.3 (cont’d) solcap_snp_c2_51220 solcap_snp_c2_53111 solcap_snp_c2_53548 solcap_snp_c2_39463 solcap_snp_c2_39450 solcap_snp_c1_14440 4 PotVar0074745 4 4 PotVar0084444 4 PotVar0116531 4 4 PotVar0100612 4 4 4 PotVar0070707 4 4 4 PotVar0113774 4 4 4 PotVar0000812 4 4 4 4 4 4 PotVar0087064 4 PotVar0087237 4 PotVar0111557 4 PotVar0075331 4 4 PotVar0075681 solcap_snp_c2_43735 solcap_snp_c2_39342 solcap_snp_c2_26758 solcap_snp_c2_55788 solcap_snp_c2_25282 solcap_snp_c2_55796 solcap_snp_c2_55793 solcap_snp_c2_34876 43.657 45.65 47.873 49.187 50.727 50.942 51.592 51.808 57.177 57.827 58.697 59.789 60.004 61.096 63.547 67.906 70.128 70.56 70.775 72.315 74.538 74.97 80.843 81.935 83.026 83.896 52951440 55384468 58263204 58840509 60133081 60124767 60539267 60611728 63040750 63406121 63664170 64087282 64055406 64216808 64801926 65600291 65970724 65867121 65969881 65970096 66147380 66210780 67295666 67822309 67981010 68141339 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 151 solcap_snp_c2_10798 solcap_snp_c1_3499 solcap_snp_c2_10566 solcap_snp_c2_23720 solcap_snp_c2_52276 solcap_snp_c2_33522 solcap_snp_c2_11731 4 PotVar0075882 4 4 4 PotVar0015989 4 PotVar0015856 4 PotVar0017154 4 PotVar0017188 4 PotVar0016517 4 4 4 5 5 5 PotVar0048336 5 5 5 PotVar0114684 5 5 5 5 PotVar0024999 5 PotVar0024652 5 PotVar0024787 5 PotVar0024831 5 5 solcap_snp_c2_57149 solcap_snp_c2_11696 solcap_snp_c2_11685 solcap_snp_c2_11977 solcap_snp_c1_3786 Table S3.3 (cont’d) solcap_snp_c1_13077 solcap_snp_c1_10167 84.111 86.795 92.923 97.037 97.252 100.642 100.857 101.289 101.505 103.268 104.138 0 2.449 2.881 7.719 10.166 10.598 11.03 13.709 14.579 20.677 23.357 24.895 25.545 28.226 30.676 68294006 68865108 70185081 70875788 70791180 71589937 71590759 71336433 71384419 71826344 71954636 303775 561523 496816 1414020 2078804 1956664 1738100 2261080 2288291 3363745 2959035 3358775 3361142 3515956 3960507 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 152 Table S3.3 (cont’d) 5 PotVar0025592 5 PotVar0026296 5 PotVar0079948 5 PotVar0080669 5 PotVar0079935 5 PotVar0079702 5 PotVar0079374 5 PotVar0079479 5 PotVar0079478 5 PotVar0026358 5 PotVar0026313 5 PotVar0080053 5 5 PotVar0116931 5 PotVar0117047 5 PotVar0117259 5 PotVar0117438 5 5 PotVar0089663 5 PotVar0084164 5 PotVar0084074 5 PotVar0083800 5 PotVar0085522 5 PotVar0085401 5 PotVar0091177 5 solcap_snp_c2_5213 solcap_snp_c2_23052 solcap_snp_c2_47610 31.326 33.09 34.403 34.835 35.051 35.483 35.915 36.347 36.997 37.212 37.428 38.742 39.833 41.597 42.467 44.687 45.119 46.657 46.872 54.822 55.254 56.344 60.687 60.902 63.118 68.957 3813315 4252165 4701617 4764411 4701481 4549568 4495794 4507565 4507537 4335381 4252576 4719202 4906728 5364099 5365755 5690795 5693945 5972568 5942512 7670627 7669320 7541185 8807655 8808961 10109724 42895131 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 153 Table S3.3 (cont’d) solcap_snp_c2_53227 solcap_snp_c2_48329 solcap_snp_c2_47393 solcap_snp_c2_47087 solcap_snp_c2_40774 solcap_snp_c1_12008 solcap_snp_c2_46952 solcap_snp_c1_12414 5 PotVar0106493 5 5 5 5 5 PotVar0014376 5 5 5 5 5 PotVar0126201 5 PotVar0081337 5 PotVar0082112 5 PotVar0081615 5 5 PotVar0123209 5 PotVar0123206 5 5 PotVar0128236 5 5 5 5 PotVar0034918 5 5 PotVar0034819 5 solcap_snp_c2_10358 solcap_snp_c2_55240 solcap_snp_c2_8529 solcap_snp_c2_8513 solcap_snp_c2_8521 solcap_snp_c1_1177 solcap_snp_c2_3451 69.826 71.589 72.021 72.453 72.668 73.1 79.718 80.15 84.492 87.168 90.076 92.521 94.737 95.606 95.821 98.498 98.93 100.242 100.892 106.729 107.598 109.135 113.481 117.586 118.898 121.811 43219233 20395121 16884369 42190428 13688447 12237074 45545898 45360490 46691817 47122961 47479039 47989133 48662020 48506716 48541183 49045143 49045164 49467229 49728003 50608349 50584053 50584500 50863665 51501967 51319479 51697156 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 154 solcap_snp_c2_30595 solcap_snp_c2_36456 solcap_snp_c2_36393 solcap_snp_c1_13638 solcap_snp_c1_13641 solcap_snp_c2_57292 5 6 6 6 PotVar0083563 6 PotVar0083440 6 6 6 6 6 6 6 PotVar0026970 6 6 6 PotVar0004038 6 6 6 6 PotVar0104759 6 6 6 PotVar0093231 6 PotVar0133895 6 6 6 solcap_snp_c2_45837 solcap_snp_c2_40242 solcap_snp_c2_33363 solcap_snp_c2_27574 solcap_snp_c2_27564 solcap_snp_c2_33932 solcap_snp_c2_11303 solcap_snp_c2_33933 solcap_snp_c2_27865 solcap_snp_c2_55596 Table S3.3 (cont’d) solcap_snp_c2_3587 solcap_snp_c2_30488 solcap_snp_c2_30501 122.902 0 0.432 0.864 1.079 2.17 4.161 4.811 6.125 6.34 9.965 13.351 14.22 14.436 18.542 19.193 19.625 20.715 22.253 23.79 24.006 24.438 24.653 25.523 25.955 26.17 51988393 368270 257821 103694 106374 764621 1614261 1303853 1832894 1832292 2270608 3214652 3295487 3470384 26013204 6903226 30365962 6903541 37868731 34893352 35077510 35816679 38709550 40905486 40303649 39294201 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 155 Table S3.3 (cont’d) solcap_snp_c2_57014 solcap_snp_c2_16817 solcap_snp_c2_54029 solcap_snp_c2_43127 solcap_snp_c2_52767 solcap_snp_c2_47782 solcap_snp_c1_10838 solcap_snp_c2_36151 solcap_snp_c2_31893 6 6 PotVar0087395 6 PotVar0127327 6 6 6 6 6 6 6 6 PotVar0085941 6 PotVar0086012 6 6 PotVar0090785 6 PotVar0090565 6 PotVar0090474 6 6 PotVar0085062 0 6 PotVar0073914 6 PotVar0073938 6 6 6 PotVar0040289 6 PotVar0041154 6 PotVar0127625 solcap_snp_c2_22239 solcap_snp_c2_22295 solcap_snp_c2_5828 solcap_snp_c2_5793 27.707 27.922 28.354 28.57 29.22 29.652 31.871 35.02 37.7 39.69 44.04 46.03 49.653 50.966 52.955 54.046 61.455 63.217 64.086 67.949 68.165 81.786 83.55 84.641 91.275 92.589 42746310 41506673 42631808 41520176 42877139 43689851 44393449 45267438 45136332 46408984 47270969 47308813 48131679 48429967 48595981 48742965 50620658 50129977 36446498 51270383 51271077 53437178 53544673 53795098 55330787 55908088 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 156 Table S3.3 (cont’d) solcap_snp_c2_9038 solcap_snp_c1_11534 solcap_snp_c2_38867 solcap_snp_c1_10783 solcap_snp_c1_15906 solcap_snp_c2_54652 solcap_snp_c2_26167 solcap_snp_c2_26197 solcap_snp_c2_29169 6 6 PotVar0057119 6 PotVar0056982 6 6 PotVar0065903 7 PotVar0022575 7 7 7 PotVar0022336 7 7 7 7 PotVar0022139 7 7 PotVar0130044 7 7 PotVar0102276 7 PotVar0102536 7 PotVar0102547 7 7 0 7 7 PotVar0132060 7 7 solcap_snp_c2_26248 solcap_snp_c1_16223 solcap_snp_c2_53198 solcap_snp_c2_46749 solcap_snp_c2_47004 solcap_snp_c2_45795 93.459 97.568 98.218 100.209 102.2 0 0.215 0.431 2.423 2.638 3.508 3.724 5.945 12.074 12.29 12.722 15.406 16.057 16.927 17.359 20.99 21.422 26.773 27.205 31.314 31.746 55470036 57245421 56890436 57651092 58462327 795856 583570 783017 1350553 1318666 1002067 1002244 1502809 2610382 2546763 2498400 3172006 3039481 3039313 3118896 3894395 29279410 4834362 4723976 7139104 7584431 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 157 Table S3.3 (cont’d) solcap_snp_c2_56661 solcap_snp_c2_50035 solcap_snp_c2_4453 solcap_snp_c2_23094 solcap_snp_c2_16004 7 7 7 PotVar0069651 7 7 7 7 PotVar0115157 7 PotVar0092990 7 PotVar0115124 7 7 PotVar0093513 7 PotVar0093776 7 7 PotVar0104431 7 7 7 7 7 PotVar0088465 7 7 7 PotVar0134027 7 PotVar0047595 7 7 7 solcap_snp_c2_33019 solcap_snp_c2_28228 solcap_snp_c2_28174 solcap_snp_c1_10001 solcap_snp_c2_25219 solcap_snp_c2_45445 solcap_snp_c2_45180 solcap_snp_c2_38787 solcap_snp_c2_25265 solcap_snp_c2_35078 solcap_snp_c2_35105 31.961 32.177 34.167 34.382 37.532 40.685 43.136 43.568 43.783 45.097 45.529 45.961 50.558 51.208 53.888 55.202 59.799 61.336 67.973 74.343 76.79 77.005 81.594 83.131 86.749 87.181 7056587 6704549 40644313 39087569 42741510 43579421 44218774 43651322 44215750 45239915 45034938 45135821 47348171 47277331 45814156 46813868 47875892 47693809 48794483 49839630 49553646 49455578 50333219 50921700 51606802 51518820 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 158 Table S3.3 (cont’d) solcap_snp_c2_30428 solcap_snp_c1_9215 solcap_snp_c2_18545 solcap_snp_c2_28290 solcap_snp_c1_4029 solcap_snp_c2_16848 solcap_snp_c2_16847 solcap_snp_c1_5452 solcap_snp_c2_12596 7 7 7 PotVar0044278 7 7 7 7 7 PotVar0043855 7 7 7 7 PotVar0037035 7 PotVar0037150 7 PotVar0036741 7 PotVar0036990 7 7 8 PotVar0113740 8 8 8 8 PotVar0063591 8 8 8 8 solcap_snp_c2_28870 solcap_snp_c2_28846 solcap_snp_c2_34179 solcap_snp_c2_52857 solcap_snp_c1_13229 solcap_snp_c2_19639 solcap_snp_c2_19646 solcap_snp_c1_816 solcap_snp_c2_33772 89.398 92.544 93.194 102.816 103.248 105.467 109.818 111.355 118.244 119.114 121.333 125.438 126.308 130.41 132.398 133.711 134.143 0 18.024 23.169 23.385 23.817 24.249 24.682 25.998 26.214 52001357 52693868 52337275 53782889 53782904 53697103 53104327 53252078 54329836 54277246 54856073 55469145 55382464 56006737 55736778 55952533 55887265 1078817 4174544 7445492 6541246 5599267 8195963 8092928 37554546 10832976 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 159 Table S3.3 (cont’d) solcap_snp_c2_45770 solcap_snp_c2_32310 solcap_snp_c1_838 solcap_snp_c2_57589 solcap_snp_c2_19442 solcap_snp_c2_53181 solcap_snp_c1_836 solcap_snp_c2_33766 solcap_snp_c2_54580 solcap_snp_c2_42297 solcap_snp_c2_19431 solcap_snp_c2_29496 solcap_snp_c2_19630 solcap_snp_c2_2179 solcap_snp_c2_19438 solcap_snp_c2_49246 solcap_snp_c2_57300 solcap_snp_c2_18964 solcap_snp_c2_18894 8 8 8 0 8 0 8 8 1 8 8 8 8 8 8 8 8 8 8 8 PotVar0123288 8 PotVar0125199 8 PotVar0077179 8 PotVar0103305 8 8 PotVar0103161 8 solcap_snp_c2_53903 solcap_snp_c2_28568 27.307 27.522 27.954 28.17 28.821 29.692 29.907 30.34 30.772 30.987 31.42 32.071 32.722 33.593 34.244 34.676 40.073 40.289 40.939 42.032 44.489 45.139 47.829 48.48 49.572 52.029 39115651 38687057 37259543 32784424 30149471 18407829 37379264 33597174 24697661 17060690 29758299 26158979 8450151 18393399 30148208 35651364 43848943 43435799 43102244 43897045 45868306 45008776 47376274 47499196 47171793 48333994 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 160 Table S3.3 (cont’d) solcap_snp_c2_28535 solcap_snp_c1_13099 solcap_snp_c1_13094 solcap_snp_c1_15868 solcap_snp_c2_36738 solcap_snp_c2_36760 8 8 8 8 8 8 PotVar0100216 8 PotVar0100132 8 8 PotVar0081171 8 PotVar0081239 8 8 8 8 8 PotVar0119156 8 PotVar0119121 8 PotVar0119089 8 8 8 PotVar0023704 8 PotVar0024071 8 PotVar0097536 8 PotVar0023699 8 PotVar0023850 8 8 solcap_snp_c2_19078 solcap_snp_c2_19079 solcap_snp_c2_19211 solcap_snp_c2_34604 solcap_snp_c1_8297 solcap_snp_c1_8300 solcap_snp_c1_5566 solcap_snp_c2_28433 52.461 54.001 54.433 56.657 59.343 61.565 61.997 63.088 67.447 68.538 70.301 70.516 73.432 73.864 74.296 74.512 77.665 79.429 82.818 85.269 86.139 86.789 87.88 88.312 91.94 100.452 48171790 49061844 49240884 49705404 50913770 51138331 51137032 51192235 52157636 52158230 52491248 52490868 52800147 53138349 53138615 53139206 53457367 53823394 54295146 54777820 54669004 54361711 54777969 54753970 55137894 56382148 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 161 solcap_snp_c1_14393 solcap_snp_c2_39101 solcap_snp_c1_3613 8 8 PotVar0023396 8 9 9 9 9 PotVar0114434 9 PotVar0114460 9 PotVar0114613 9 9 9 9 PotVar0011742 9 PotVar0011891 9 PotVar0011786 9 9 PotVar0012073 9 9 9 9 9 9 0 9 9 solcap_snp_c1_1400 solcap_snp_c1_4271 solcap_snp_c2_13273 solcap_snp_c2_13180 solcap_snp_c2_46413 solcap_snp_c1_13783 solcap_snp_c2_54623 solcap_snp_c2_4400 solcap_snp_c2_56179 solcap_snp_c2_39244 Table S3.3 (cont’d) solcap_snp_c2_28478 solcap_snp_c2_28480 solcap_snp_c1_1059 solcap_snp_c1_1051 solcap_snp_c1_1019 101.989 102.421 103.958 0 0.65 1.741 3.055 3.705 3.92 5.233 6.996 10.619 18.829 19.26 19.476 21.237 21.887 29.568 42.487 43.357 45.574 48.251 50.014 53.159 53.809 54.024 56782299 56590145 56781388 27567 161493 348392 530691 531471 533839 739884 1020724 1556721 2469771 2471523 2470210 2637464 2679615 3920030 6389488 6333199 6722333 8401589 8468741 18258189 13621663 43845142 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 162 Table S3.3 (cont’d) solcap_snp_c2_27666 solcap_snp_c2_21332 solcap_snp_c2_58234 solcap_snp_c1_7530 solcap_snp_c2_54624 solcap_snp_c2_1918 solcap_snp_c2_1917 solcap_snp_c2_27622 solcap_snp_c2_56172 solcap_snp_c2_12760 solcap_snp_c2_26515 9 0 9 9 0 9 9 9 9 9 9 9 PotVar0007613 9 9 9 7 0 9 9 9 9 9 PotVar0051583 9 9 9 9 solcap_snp_c1_219 solcap_snp_c2_53556 solcap_snp_c2_32638 solcap_snp_c2_4393 solcap_snp_c2_21317 solcap_snp_c2_16278 solcap_snp_c2_12758 solcap_snp_c2_44815 solcap_snp_c2_44812 solcap_snp_c1_6192 solcap_snp_c2_44814 solcap_snp_c2_44804 solcap_snp_c1_13612 54.239 54.671 54.886 55.102 55.317 55.967 56.617 56.832 57.264 57.696 58.128 58.778 69.059 79.055 79.271 79.703 80.572 81.004 81.436 81.651 81.867 82.736 83.168 83.6 84.032 84.247 44899486 3499519 11226418 11460409 18258185 19232397 19232247 45471579 43861030 45924103 21264861 30812635 38263840 20711787 45458640 18715095 3811034 31520465 45788396 46687355 46740866 48071305 47753124 46740222 46744528 46837871 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 163 Table S3.3 (cont’d) solcap_snp_c2_21313 0 9 PotVar0129386 9 PotVar0129339 9 PotVar0101834 9 PotVar0103781 9 PotVar0103919 9 PotVar0107747 9 9 9 PotVar0072621 9 PotVar0011128 9 PotVar0105291 9 9 PotVar0108622 9 10 10 PotVar0065466 10 10 PotVar0116620 10 10 PotVar0104083 10 PotVar0129204 10 10 10 10 PotVar0131702 solcap_snp_c2_44249 solcap_snp_c2_24701 solcap_snp_c2_24747 solcap_snp_c2_21992 solcap_snp_c2_20758 solcap_snp_c2_55481 solcap_snp_c2_20879 solcap_snp_c2_1345 solcap_snp_c2_886 solcap_snp_c2_1113 96.686 115.134 115.566 120.165 121.257 124.889 126.203 128.197 138.872 140.867 146.248 150.132 153.293 153.726 154.818 0 3.146 4.683 6.901 15.981 17.745 21.854 22.069 24.984 26.747 30.614 18164321 51665603 51666522 52568992 52769266 53423071 53672529 54060817 56362202 56635134 57422011 58737148 59483431 59586577 60097041 772468 1100487 1372642 1691592 2382807 2646591 3908993 3908588 4398276 4622745 5630858 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 10 10 10 10 10 10 10 164 10 PotVar0007327 10 PotVar0123577 0 10 PotVar0119183 10 10 10 10 PotVar0004836 10 PotVar0005344 10 PotVar0005134 10 PotVar0005097 10 10 10 10 10 10 10 10 PotVar0058242 10 10 PotVar0058165 10 10 10 PotVar0058069 10 PotVar0057955 10 PotVar0058108 solcap_snp_c2_48145 solcap_snp_c2_48091 solcap_snp_c1_9058 solcap_snp_c1_9066 solcap_snp_c2_29749 solcap_snp_c2_29786 solcap_snp_c2_22630 solcap_snp_c1_7187 solcap_snp_c2_22744 solcap_snp_c1_7212 Table S3.3 (cont’d) solcap_snp_c2_57144 solcap_snp_c2_51301 solcap_snp_c2_55819 solcap_snp_c2_15483 33.529 33.961 36.18 38.629 41.545 46.897 56.005 58.688 62.56 62.776 62.991 63.423 64.962 69.572 70.887 71.102 71.972 80.226 80.876 81.746 82.837 83.927 85.465 85.897 86.548 86.98 17628787 44858001 21419947 49584558 51725970 53235977 54996383 55308486 55856581 55842462 55842116 55919521 55898404 56301672 56395440 56366011 56533774 57577812 57479511 57596618 57670512 57468777 57837250 57894655 57896209 57891862 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 165 Table S3.3 (cont’d) solcap_snp_c1_8806 solcap_snp_c2_41693 solcap_snp_c1_12899 solcap_snp_c2_51284 10 PotVar0057839 10 PotVar0057722 10 PotVar0057515 10 10 10 PotVar0122859 11 11 11 PotVar0118043 11 11 11 11 PotVar0008553 11 PotVar0008447 11 11 11 11 PotVar0112395 11 PotVar0008116 11 11 PotVar0112779 11 PotVar0113195 11 PotVar0047373 11 PotVar0047337 11 11 solcap_snp_c2_3686 solcap_snp_c2_54589 solcap_snp_c2_34198 solcap_snp_c2_39890 solcap_snp_c2_31568 solcap_snp_c2_15456 solcap_snp_c2_15364 solcap_snp_c1_4951 solcap_snp_c2_3737 90.133 91.003 91.653 92.744 95.195 97.645 0 0.432 1.082 1.514 7.357 7.789 8.88 13.226 14.539 16.529 16.744 22.081 23.172 26.557 27.207 28.076 31.223 32.985 35.897 43.312 58080606 58185506 58303298 58567056 59177759 59470764 44430287 44620835 45058279 45092064 43294206 43618366 43789547 42601858 42011986 41501023 41448369 40627337 40968037 40397444 40484801 40366995 39786159 39420294 39913375 38405527 10 10 10 10 10 10 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 166 Table S3.3 (cont’d) solcap_snp_c1_1780 solcap_snp_c2_29129 solcap_snp_c2_32341 solcap_snp_c2_29155 solcap_snp_c2_33916 solcap_snp_c2_53685 solcap_snp_c2_49305 solcap_snp_c2_13636 solcap_snp_c2_19458 solcap_snp_c2_54930 11 PotVar0047209 11 PotVar0134713 11 11 PotVar0054059 11 11 11 PotVar0061486 11 11 PotVar0021994 11 PotVar0130709 11 11 11 11 11 PotVar0060032 11 11 PotVar0060017 11 11 PotVar0059185 11 11 11 11 11 11 11 solcap_snp_c2_12258 solcap_snp_c2_21053 solcap_snp_c2_21066 solcap_snp_c2_23915 solcap_snp_c2_21072 solcap_snp_c2_23923 solcap_snp_c2_47382 43.527 45.976 47.74 49.73 50.162 50.812 51.244 51.46 51.675 52.325 71.34 94.297 95.833 96.483 103.357 103.573 103.788 104.22 109.557 111.094 132.193 133.506 133.721 134.153 134.803 137.716 38554619 37384590 36652714 14227194 14221482 13269523 30158470 20362816 19468773 15009820 11265345 10396503 11068645 10773884 9377526 9380064 9377271 9296971 8493529 8841626 5533135 5735381 5977340 5736611 6017871 4250278 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 167 solcap_snp_c2_37660 solcap_snp_c2_20942 solcap_snp_c2_5964 solcap_snp_c2_5960 solcap_snp_c2_5966 solcap_snp_c2_5978 11 11 PotVar0110427 11 PotVar0106251 11 11 11 PotVar0106094 11 PotVar0106023 11 PotVar0105671 11 11 11 11 11 11 11 PotVar0067029 11 PotVar0067381 11 11 11 11 11 PotVar0066480 11 PotVar0067875 11 PotVar0067504 11 11 PotVar0067827 11 solcap_snp_c2_6283 solcap_snp_c2_55972 solcap_snp_c1_2304 solcap_snp_c2_6019 solcap_snp_c2_6249 solcap_snp_c2_33661 Table S3.3 (cont’d) solcap_snp_c2_6303 solcap_snp_c2_47386 solcap_snp_c2_37668 138.148 138.58 139.671 140.321 140.536 140.968 141.4 142.713 142.928 143.578 148.666 148.881 149.313 149.745 150.177 151.047 151.262 151.477 152.128 153.89 154.322 157.472 157.688 158.338 158.988 164.332 3943211 3657926 4227797 4365454 4457195 4346046 4347583 4666149 4777804 5048506 2839006 2854841 2832278 2809210 2774115 3072478 3155246 3182994 3007038 2692043 2705885 3268775 3262580 3252697 3268257 2262006 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 168 Table S3.3 (cont’d) solcap_snp_c2_37189 solcap_snp_c2_37201 solcap_snp_c1_4347 solcap_snp_c1_11105 solcap_snp_c1_10062 solcap_snp_c2_33653 solcap_snp_c1_10069 solcap_snp_c1_2162 solcap_snp_c1_2187 solcap_snp_c2_33709 solcap_snp_c2_13473 11 11 11 11 11 11 PotVar0066311 11 11 11 PotVar0064694 11 11 PotVar0066236 11 11 11 11 PotVar0064473 11 PotVar0064415 11 11 PotVar0064200 11 12 PotVar0098351 12 12 12 PotVar0053570 12 12 PotVar0053629 12 solcap_snp_c2_13419 solcap_snp_c2_13345 solcap_snp_c2_53631 solcap_snp_c2_25327 solcap_snp_c1_8002 solcap_snp_c2_16152 165.202 165.417 165.633 166.946 167.161 169.151 171.6 176.95 177.382 179.373 180.243 180.675 181.545 183.083 185.999 186.65 186.865 187.956 188.172 0 1.539 3.303 6.458 6.673 11.281 14.913 2349165 2289760 2259927 2580985 2562328 2261359 2429279 922248 811667 1748103 2060890 1775392 1163793 1776687 787383 786787 761409 403391 436865 364051 754468 1138029 1509454 1511064 1881848 2209695 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 12 12 12 12 12 12 12 169 Table S3.3 (cont’d) solcap_snp_c2_27959 solcap_snp_c2_28013 solcap_snp_c2_31334 solcap_snp_c2_46303 solcap_snp_c2_46289 solcap_snp_c2_34774 solcap_snp_c2_24611 solcap_snp_c2_34764 solcap_snp_c2_34762 solcap_snp_c2_48902 solcap_snp_c2_27379 12 PotVar0031206 12 PotVar0068584 12 PotVar0069242 12 12 12 12 PotVar0068185 12 12 12 12 12 12 12 12 12 PotVar0109073 12 PotVar0109293 12 12 12 PotVar0110871 12 12 12 12 12 12 solcap_snp_c2_57627 solcap_snp_c1_7495 solcap_snp_c2_23254 solcap_snp_c1_11668 solcap_snp_c2_39410 solcap_snp_c2_48482 solcap_snp_c2_32073 solcap_snp_c2_6432 21.042 25.161 26.031 31.671 36.551 37.422 40.824 43.751 44.844 51.293 51.509 52.825 53.041 54.358 55.9 57.671 58.103 59.873 60.305 60.738 62.054 63.147 63.362 64.678 66.903 69.127 3090598 3549130 3445076 3746825 3928962 4046156 4399051 5258454 5309271 7863490 6936624 8127837 8063612 9119873 10256960 52761637 52756754 53583976 53048469 53314187 53793483 54234293 54234680 54727908 55783322 56226502 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 170 Table S3.3 (cont’d) solcap_snp_c1_2727 solcap_snp_c2_7985 solcap_snp_c2_46213 12 12 12 PotVar0018429 12 PotVar0018250 12 12 PotVar0052908 12 PotVar0052870 12 PotVar0052633 12 PotVar0052528 12 PotVar0052695 12 12 12 PotVar0052374 12 PotVar0052280 12 12 12 12 12 0 0 solcap_snp_c2_5704 solcap_snp_c2_5652 solcap_snp_c2_5507 solcap_snp_c2_5474 solcap_snp_c2_5440 solcap_snp_c2_57485 solcap_snp_c2_57484 solcap_snp_c1_1923 solcap_snp_c1_1924 78.85 79.282 79.498 82.417 87.528 87.743 87.959 91.354 92.005 92.22 92.871 93.303 94.618 95.932 96.148 96.363 97.013 97.446 98.76 98.976 99.408 57468357 57563595 57599073 57952530 59129520 59211572 59271422 59682096 59793754 59680999 59870038 59808631 59957211 59979591 59986990 60129798 60477931 60707179 61086761 32655544 32655517 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 171 Table S3.4. Number of loci with distorted and expected segregation based on a Chi-square test using three thresholds of significance (5%, 1%, and 0.1%) in the 754 mapped SNPs of the Solanum chacoense USDA8380-1 x M6 F2 population P<0.05 P<0.01 P<0.001 Chromosome correct distorted correct distorted correct distorted 1 2 3 4 5 6 7 8 9 10 11 12 Total 32 11 12 40 66 45 2 21 38 28 36 0 331 39 55 56 21 2 10 62 40 19 14 54 51 423 41 25 28 61 68 48 14 24 47 37 58 3 454 30 41 40 0 0 7 50 37 10 5 32 48 300 44 48 37 61 68 51 36 27 54 42 82 5 555 27 18 31 0 0 4 28 34 3 0 8 46 199 172 Table S3.5. Spearman’s rank correlation coefficients among measured traits in 20 F2 individuals from the Solanum chacoense USDA8380-1 × M6 F2 population used for bulk segregant analysis of Colorado potato beetle resistance Replicated Field Trial 2018a Assayb No-Choice Detached Leaf Choice Detached Leaf Replicated Field Trial -0.82*** -0.70** ns 0.52* -0.76*** ns 0.77*** 0.77*** -0.68** -0.62** ns ns -0.61** ns 0.65** 0.78*** Assayc 2017a -0.73** -0.57** ns 0.54* -0.65** ns 0.73** -0.80*** -0.70** ns 0.67** -0.82*** ns Leptine I Leptine II α-Solanine α-Chaconine Acetylated/Non-Acetylated Total Glycoalkaloids Replicated Field Trial 2017a Choice Detached Leaf Assayc 0.77*** No-Choice Detached Leaf Assayb *** P<0.0001, **P<0.01, *P<0.05, ns not significant, n = 20 a Data represent mean relative area under the defoliation progression curve (RAUDC x 100) b Data represent mean percent defoliation after 7 days of larval feeding c Data represent mean percent defoliation after 48 hours of adult beetle feeding 173 (bp) 7217771 13176324 60756417 63452166 54590669 122961 73324 59538016 (bp) 9589234 31699584 62253327 68742551 59454936 2232921 1580137 61152319 Length of QTL (bp) 2371463 18523260 1496910 5290385 4864267 2109960 1506813 1614303 No. SNPs 7880 75062 10932 44558 29450 22167 8500 14813 2 2 4 4 6 7 12 12 Max G' valueb 7.66407 8.99278029 4.00471006 4.94510998 4.94204198 4.62308315 5.9089802 4.33874181 Max G' Position 8363700 25071402 61473854 64856985 59454936 122961 73324 60198332 meanPvalc 2.79E-05 2.60E-05 0.00012416 9.97E-05 2.15E-05 2.76E-05 4.04E-05 4.47E-05 meanQvald 0.00081639 0.00080402 0.00389459 0.00304738 0.00087995 0.00103093 0.00135144 0.00160989 3323 4052 7303 8422 6054 10506 5641 9176 Table S3.6. Significant QTL identified by G' analysis of SNPs generated from alignment to Solanum tuberosum clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03), corrected with Solanum chacoense M6 whole genome sequence data, using the QTLseqr package in R (Mansfeld BN, Grumet R (2018) QTLseqr: An R package for bulk segregant analysis with next-generation sequencing. bioRxiv:208140. https://doi.org/10.1101/208140) Chromosome Start Positiona End Position avgSNPs/Mb aPosition of the first SNP that passes the Benjamini-Hochberg false discovery rate bThe maximum G’ value within the QTL region cThe average p-value in the QTL region. dThe average adjusted p-value in the QTL region 174 Table S3.7. Genes Differentially Expressed between Resistant and Susceptible Individuals using DESeq2 package. Transcripts upregulated in resistant F2 lines and resistant parent 80-1 demonstrate negative logfold change. Solanum tuberosum clone DM1-3 (DM) pseudomolecules (PGSC.Version.4.03) Physical Start Position (bp) Physical End Position (bp) Gene ID Annotated Function baseMean log2FoldChange PGSC0003DMG400000003 PGSC0003DMG400000075 PGSC0003DMG400000078 PGSC0003DMG400000126 PGSC0003DMG400000156 PGSC0003DMG400000177 PGSC0003DMG400000180 PGSC0003DMG400000237 PGSC0003DMG400000288 PGSC0003DMG400000417 PGSC0003DMG400000513 PGSC0003DMG400000541 PGSC0003DMG400000542 PGSC0003DMG400000565 PGSC0003DMG400000584 PGSC0003DMG400000615 PGSC0003DMG400000628 PGSC0003DMG400000652 PGSC0003DMG400000708 PGSC0003DMG400000715 SPX domain-containing membrane protein Conserved gene of unknown function Transferase, transferring glycosyl groups Conserved gene of unknown function Conserved gene of unknown function Ubiquitin carrier protein Zinc finger family protein Conserved gene of unknown function Gamma glutamyl transpeptidases Superoxide dismutase [Cu-Zn] Stachyose synthase Hydrogen peroxide-induced 1 Pentatricopeptide repeat-containing protein Cytochrome P450 Pseudo-response regulator 5 Conserved gene of unknown function Late embryogenesis abundant protein Serine/threonine protein kinase Glycine-rich RNA binding protein Conserved gene of unknown function ch01 ch01 ch01 ch01 ch01 ch01 ch01 ch01 ch12 ch11 ch02 ch03 ch03 ch03 ch03 ch03 ch03 ch12 ch01 ch01 175 1100.1899 71293234 71298560 72810610 72813885 1459.93752 72833577 72842485 1500.82888 73826302 73827911 85.1205581 71906223 71913442 620.778207 72367512 72370872 2032.68722 72397781 72402069 263.523019 73720908 73725220 244.263054 2718.3667 3906126 3910091 39217263 39223656 8498.6044 47301600 47305051 441.534361 46804543 46806857 2949.29413 46787963 46795175 416.501844 46950284 46952292 823.378786 46394146 46399285 498.224052 56543497 56544650 499.897523 56420226 56421126 66.4782319 42294180 42298253 718.107083 85987215 85988480 23134.7808 86111823 86112661 81.4031178 1.62592548 -1.233571013 -0.501405934 -1.692506536 -1.632574488 1.068997506 1.141199171 -0.665878192 -0.940316372 -2.232896282 1.733438021 -1.557300522 -1.406439038 -0.722581602 -2.213874275 1.524641427 4.441708708 -0.377782111 -1.14067678 1.365007283 padj 4.60E-06 0.00019756 0.00090965 9.52E-05 0.00013027 1.92E-06 1.99E-05 0.00099488 7.43E-06 9.05E-05 0.00068514 6.14E-09 8.61E-07 1.98E-09 0.0006206 0.0005988 0.00028909 9.11E-05 7.59E-05 0.00072796 Table S3.7 (cont’d) PGSC0003DMG400000742 PGSC0003DMG400000744 PGSC0003DMG400000745 PGSC0003DMG400000749 PGSC0003DMG400000756 PGSC0003DMG400000861 PGSC0003DMG400000879 PGSC0003DMG400000886 PGSC0003DMG400000963 PGSC0003DMG400000985 PGSC0003DMG400001104 PGSC0003DMG400001131 PGSC0003DMG400001144 PGSC0003DMG400001149 PGSC0003DMG400001221 PGSC0003DMG400001312 PGSC0003DMG400001330 PGSC0003DMG400001338 PGSC0003DMG400001344 PGSC0003DMG400001345 PGSC0003DMG400001363 PGSC0003DMG400001373 PGSC0003DMG400001394 PGSC0003DMG400001413 PGSC0003DMG400001434 Geraniol 10-hydroxylase Geraniol 10-hydroxylase Geraniol 10-hydroxylase Geraniol 10-hydroxylase Salicylic acid-binding protein 2 AMP-activated protein kinase, gamma regulatory subunit White-brown-complex ABC transporter family Cation transport protein chaC Ubiquitin-protein ligase Conserved gene of unknown function DEAD-box ATP-dependent RNA helicase 37 RNA binding Cytochrome P450 92B1 Allene oxide synthase 2 EARLY flowering 4 protein F-box family protein Zinc finger protein Nam 14 Conserved gene of unknown function Pentatricopeptide repeat-containing protein Esterase PIR7B Phospholipase C Mannosidase alpha class 2a FACT complex subunit SSRP1 WRKY transcription factor-c 176 6340483 6341967 ch02 23902305 23902859 42.6081074 ch02 23869995 23872631 1021.31345 ch02 23864174 23865822 9.71994799 ch02 23814454 23816124 3.16429968 ch02 23993315 23994379 370.007163 ch01 13544687 13548706 1262.66989 ch11 11326917 11336464 670.969082 ch11 10998587 11005949 231.252013 ch08 19972788 19974722 380.631407 ch11 393.932089 ch03 14195006 14204658 2842.83828 ch09 55460883 55467663 1238.32998 ch11 41907234 41911479 33.9825574 ch11 41831952 41833781 1997.32173 ch06 26071331 26071919 112.688122 ch02 46407342 46415527 442.095183 ch02 46143998 46147444 92.3195162 ch02 46075362 46076997 266.888361 ch02 45931820 45937635 28.1620412 ch02 45868577 45871118 688.607333 ch02 45480205 45484872 147.769368 ch02 45417362 45419796 3226.18851 ch02 46694810 46700628 978.802485 ch02 46440303 46443585 21.3411939 ch02 45919455 45922325 462.034196 0.00010862 1.308001421 0.00098853 1.313086343 0.00088212 3.715303932 0.00052355 3.330673136 2.46E-05 1.660377435 8.65E-05 0.618215644 2.52E-05 0.975965982 7.32E-08 -1.115506855 2.85E-05 -1.216457911 2.41E-07 1.995864521 -0.319862227 4.33E-05 -0.881843235 0.00099087 7.97E-08 -3.270848385 7.96E-05 -0.992935322 -2.292163829 4.95E-05 2.66E-05 0.817877898 0.00032137 2.160662577 1.531866186 9.10E-05 3.20E-05 2.371381509 2.75E-07 -1.068683177 0.674336935 0.00096402 2.42377065 4.40E-05 -0.544602503 0.00012098 1.772321053 4.66E-05 -1.278752818 0.00025138 PGSC0003DMG40000146 N2,N2-dimethylguanosine tRNA methyltransferase family 4537611 4538583 4555663 4556060 492.15022 1233.6399 1.819013299 5.95E-07 0.302098126 0.0004603 Table S3.7 (cont’d) PGSC0003DMG40000146 0 6 5 9 5 8 0 1 7 2 7 3 3 7 8 9 1 3 4 9 PGSC0003DMG40000152 PGSC0003DMG40000153 PGSC0003DMG40000162 PGSC0003DMG40000163 PGSC0003DMG40000167 PGSC0003DMG40000167 PGSC0003DMG40000167 PGSC0003DMG40000173 PGSC0003DMG40000181 PGSC0003DMG40000182 PGSC0003DMG40000195 PGSC0003DMG40000196 PGSC0003DMG40000201 PGSC0003DMG40000201 PGSC0003DMG40000216 PGSC0003DMG40000217 PGSC0003DMG40000217 PGSC0003DMG40000217 Ninja-family protein Os03g0419100 protein NAD-dependent isocitrate dehydrogenase Conserved gene of unknown function Amidase 39 kDa EF-Hand containing protein Receptor protein kinase Peptide transporter RING finger Kinase Enoyl-CoA-hydratase Ent-kaurenoic acid oxidase AP-1 complex subunit gamma-2 White-brown-complex ABC transporter family Glutathione S-transferase omega Amidase family protein Conserved gene of unknown function Rop guanine nucleotide exchange factor Glutathione S-transferase Glutathione S-transferase 177 0 7 3 3 4 7 2 6 9 0 5 8 7 6 2 3 9 1 1 2 8 7 8 5 2 4 3868798 3869389 566.94236 3880396 3880500 8.7605362 8724058 8724294 46.899458 8712875 8713202 2591.8755 6796299 6797011 5173.5334 6799130 6799600 6823563 6823644 304.59725 202.04945 5 6573632 6780549 6582777 6780815 2304.2052 78.276434 6790815 6791263 492.36386 4709298 4709667 2068.0339 4726459 4726809 980.61881 6933551 6937477 6947555 6951691 1082546 1086035 1075676 1079306 1069048 1070159 2357.2340 274.27593 1248.4675 926.46322 872.81451 6 6 1 2 8 7 4 5 3 2 9 9 8 4 7 ch0 2 ch0 2 ch0 2 ch0 2 ch0 1 ch0 1 ch0 1 ch0 1 ch0 1 ch0 9 ch0 1 ch0 1 ch0 8 ch0 8 ch0 9 ch0 9 ch0 9 ch0 9 ch0 9 ch0 9 - - - - - - 0.499038762 3.209864692 2.741759241 1.149615876 0.616290997 2.030349359 1.077524785 0.533469467 1.386700906 -0.27871155 0.787854062 1.348649887 1.037095395 2.647605551 1.839533305 1.366864116 4.07E-05 0.0007552 6 4.07E-05 1.40E-09 0.0001416 0.0002269 4 6 1.26E-06 1.77E-07 4.66E-05 6.03E-05 0.0008002 6 0.0001525 0.0009678 1 2.70E-07 0.0007288 2 3.53E-05 0.0005123 3 1.033523458 2.05E-05 1044352 1045516 27.886804 1.277187152 Table S3.7 (cont’d) PGSC0003DMG400002203 PGSC0003DMG400002235 PGSC0003DMG400002387 PGSC0003DMG400002517 PGSC0003DMG400002584 PGSC0003DMG400002586 PGSC0003DMG400002601 PGSC0003DMG400002620 PGSC0003DMG400002637 PGSC0003DMG400002720 PGSC0003DMG400002724 PGSC0003DMG400002732 PGSC0003DMG400002781 PGSC0003DMG400002810 PGSC0003DMG400002821 PGSC0003DMG400002967 PGSC0003DMG400002987 PGSC0003DMG400002993 PGSC0003DMG400003013 PGSC0003DMG400003047 PGSC0003DMG400003073 PGSC0003DMG400003090 PGSC0003DMG400003091 PGSC0003DMG400003187 PGSC0003DMG400003229 Conserved gene of unknown function Transcriptional factor TINY O-methyltransferase Conserved gene of unknown function RNA binding protein Harpin-induced 1 IAA-amino acid hydrolase AP2 domain class transcription factor Conserved gene of unknown function Delta14-sterol reductase Conserved gene of unknown function VQ motif-containing protein Conserved gene of unknown function 43895 exonuclease Conserved gene of unknown function Pheophorbide A oxygenase DNAJ protein Regulator of gene silencing Beta-D-glucan exohydrolase GRAS family transcription factor 16S rRNA processing protein RimM family Leucoanthocyanidin dioxygenase Leucoanthocyanidin dioxygenase Temperature-induced lipocalin' Conserved gene of unknown function 178 3927016 3979062 4200806 3919011 3978234 4199867 ch08 36539893 36543930 91.5901384 ch08 39081500 39082508 261.332107 ch04 57239246 57244543 112.251305 ch03 60611416 60612059 49.7653446 ch03 61044550 61054212 397.815581 ch03 61009618 61010762 454.763986 ch03 60667243 60671717 1317.34998 ch03 60259840 60260853 66.0232149 ch03 59900016 59902579 5.22004725 1355.52618 ch09 95.177963 ch09 ch09 172.366573 ch02 24413562 24417481 71.8921504 ch01 77985906 77993484 458.191374 ch04 50.5548669 ch04 25178861 25182625 75.0435164 ch11 43281930 43285433 691.749187 ch11 43231515 43232361 85.4686815 ch11 43124608 43129968 2543.70275 ch08 54102875 54105367 205.986736 ch02 30501190 30511178 1822.44747 ch02 30594268 30595100 18.5950653 ch02 30597160 30600716 635.020532 ch07 6362.32265 ch03 46095581 46097206 642.979519 462000 463320 199873 201504 0.00052355 0.701403394 0.69423568 0.00098354 -1.216597585 0.00013299 1.76E-07 2.193085955 0.00034303 0.457340589 1.177090715 7.32E-05 1.98E-09 -0.787082509 8.54E-05 -1.09513074 -2.092926259 9.84E-05 9.47E-05 -1.818812784 1.71E-08 3.712164209 2.622814984 0.00080254 -3.32210089 6.07E-10 -0.695376403 0.00025316 -4.36975952 9.76E-08 0.00066912 1.542435153 9.48E-06 1.656241393 2.676373332 2.96E-06 -0.898824319 0.00065658 -0.638979829 0.00043637 -0.696036011 3.76E-05 0.00060519 2.802465927 0.00058343 1.889301551 0.856210699 0.00017818 4.98E-09 -3.42157653 Table S3.7 (cont’d) PGSC0003DMG400003270 PGSC0003DMG400003318 PGSC0003DMG400003320 PGSC0003DMG400003322 PGSC0003DMG400003330 PGSC0003DMG400003380 PGSC0003DMG400003399 PGSC0003DMG400003445 PGSC0003DMG400003446 PGSC0003DMG400003465 PGSC0003DMG400003507 PGSC0003DMG400003520 PGSC0003DMG400003576 PGSC0003DMG400003615 PGSC0003DMG400003707 PGSC0003DMG400003709 PGSC0003DMG400003746 PGSC0003DMG400003747 PGSC0003DMG400003765 PGSC0003DMG400003812 PGSC0003DMG400003872 PGSC0003DMG400003941 PGSC0003DMG400004036 PGSC0003DMG400004071 PGSC0003DMG400004081 ARP2/3 complex 21 kDa subunit Adenylyl-sulfate kinase, chloroplastic Conserved gene of unknown function Conserved gene of unknown function Conserved gene of unknown function Late blight resistance protein homolog R1A-4 Conserved gene of unknown function Disease resistance protein Undecaprenyl pyrophosphate synthetase Glycosyl hydrolase family TVLP1 Conserved gene of unknown function Glycosyl hydrolase family 38 protein Thioredoxin H-type 2 Pre-pro-cysteine proteinase Deficiens analogue Transcription factor Taxadien-5-alpha-ol O-acetyltransferase Heat shock protein binding protein Hydrolase ZPT2-11 ADP-sugar diphosphatase ATP binding protein Mismatch repair protein Agamous-like MADS-box protein AGL8 homolog 179 9748792 9753153 5348227 2273832 5352952 2274809 ch02 9.21341481 ch02 22686194 22691274 446.907073 ch02 22625721 22626807 12.5528688 ch02 22613936 22621630 4466.45853 ch02 22644387 22653783 436.965364 ch05 376.381126 ch06 36.9943939 ch00 32642595 32652265 41.0714772 ch00 32653309 32655813 136.544151 ch06 32541630 32547948 2147.54132 ch02 40462161 40470409 245.639023 ch02 40261378 40265879 506.247762 ch02 39213558 39222288 770.635812 ch02 39961471 39963059 925.482788 ch04 70363354 70365693 43030.9742 ch04 70409561 70413264 135.951772 ch04 70095272 70100628 259.539966 ch04 70120175 70123179 12.3776427 ch04 70479254 70484321 2838.31502 ch09 51357208 51360985 746.162425 ch09 47.4606083 ch08 53495210 53501417 467.272407 ch02 43726415 43727792 252.023108 ch06 50967837 50976112 424.883249 ch06 51191112 51198207 608.694087 4829603 4830786 2.98E-05 3.481425463 3.53E-15 -1.629810473 0.00031744 4.983895548 -0.967627371 0.00025032 0.635652756 3.55E-08 -1.293391664 0.00034512 2.74E-11 -2.214066415 1.27E-08 -2.759941479 -7.963849259 8.42E-98 6.32E-06 -0.651632784 0.00012542 1.024406257 2.055024109 3.87E-05 -1.536213797 0.00013218 0.00069618 0.883906188 -1.172710057 1.39E-06 0.00082055 1.00374747 8.25E-06 1.023490148 1.645351612 0.0001638 3.13E-07 -0.709995202 9.53E-09 1.362998307 1.33165105 0.00023493 -0.513132396 0.00027067 1.41E-05 -1.491499591 0.932308624 0.0001884 2.61E-05 1.928155383 Table S3.7 (cont’d) PGSC0003DMG400004151 PGSC0003DMG400004170 PGSC0003DMG400004258 PGSC0003DMG400004275 PGSC0003DMG400004280 PGSC0003DMG400004286 PGSC0003DMG400004287 PGSC0003DMG400004291 PGSC0003DMG400004312 PGSC0003DMG400004332 PGSC0003DMG400004499 PGSC0003DMG400004521 PGSC0003DMG400004547 PGSC0003DMG400004582 PGSC0003DMG400004594 PGSC0003DMG400004607 PGSC0003DMG400004629 PGSC0003DMG400004647 PGSC0003DMG400004651 PGSC0003DMG400004672 PGSC0003DMG400004674 PGSC0003DMG400004698 Thiamine biosynthesis protein ThiC variant L1 Asparagine synthetase [glutamine-hydrolyzing] Cleavage and polyadenylation specificity factor Leucoanthocyanidin dioxygenase Phytocalpain Tap46 Metal tolerance protein Oligopeptide transporter 9-cis-epoxycarotenoid dioxygenase NAC domain protein Homeobox protein Leucine-rich repeat-containing protein Proteinase inhibitor type-2 P303.51 Minor histocompatibility antigen H13 Serine/threonine-protein kinase PBS1 Yth domain-containing protein Epoxide hydrolase 1 Cytochrome P450 CYP82E4v2 nicotine demethylase Polyprotein C2 domain-containing protein Cyclopropyl isomerase Aldehyde dehydrogenase 180 6329642 6332333 3473685 3217033 3467753 3211804 7051.97713 ch06 ch06 8673.67522 ch12 54242591 54246713 783.206276 ch12 61067502 61068832 14.603061 ch12 61145687 61152223 1522.83521 ch12 61035075 61039166 897.195517 ch12 61014021 61016683 54.4897082 ch12 1961.34502 ch08 39213385 39218093 1046.28773 ch03 48030127 48031759 130.936737 ch02 21516771 21518822 21.2791792 ch02 16783207 16784249 19.7230576 ch03 50056094 50056945 7484.42652 ch12 59495645 59504247 1619.0628 ch12 59626798 59635560 1700.86978 ch12 59863894 59872536 2572.45244 ch12 60126521 60130204 1431.68333 ch12 60411417 60419534 44.5273076 ch12 60481184 60490552 1247.2883 ch12 59442421 59444988 200.731068 ch12 59479181 59484632 3698.11212 2618.2686 ch12 60024228 60031694 2.31E-07 -1.209052247 7.32E-05 -3.133456504 2.96E-05 -0.793992218 -3.213089874 0.00093372 6.18E-05 -0.367094109 -3.460928591 8.47E-66 4.00E-24 -5.4517438 0.0005615 1.221182603 -2.505417363 4.93E-05 1.15E-05 1.183497182 1.04E-06 3.034396633 -8.204064918 4.67E-27 7.13E-06 -2.891105241 1.82E-13 0.394978702 -0.322632638 0.0001086 0.00040949 0.261304196 5.02E-11 1.977231184 -1.229676145 9.35E-05 0.0006115 0.336182143 0.512213119 0.00088385 -1.155340762 0.00011613 -1.472007744 0.00064352 Table S3.7 (cont’d) PGSC0003DMG4000047 18 PGSC0003DMG4000047 PGSC0003DMG4000048 22 08 PGSC0003DMG4000048 28 PGSC0003DMG4000048 36 PGSC0003DMG4000048 54 PGSC0003DMG4000048 PGSC0003DMG4000049 PGSC0003DMG4000049 PGSC0003DMG4000050 PGSC0003DMG4000050 PGSC0003DMG4000051 PGSC0003DMG4000051 PGSC0003DMG4000053 76 35 61 30 88 55 60 05 PGSC0003DMG4000053 30 PGSC0003DMG4000053 52 ATP binding / binding / protein kinase/ protein serine/threonine kinase/ protein Cf-2.2 Mitochondrial small heat shock protein Peroxisomal acyl-CoA oxidase 1A BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 Casein kinase Conserved gene of unknown function Conserved gene of unknown function Amino acid transporter Linalool synthase Transcription factor Transporter Sugar transporter Conserved gene of unknown function Protein AFR Protein kinase APK1B, chloroplast 181 ch1 2 ch1 2 ch0 8 ch0 8 ch0 6 ch0 6 ch0 6 ch0 4 ch0 4 ch0 3 ch0 7 ch0 4 ch0 4 ch0 1 ch0 6 ch0 4 1 1 7 8 5 6 4 8 0 5 1 0 4 6 1 8 6039835 9 6040590 4 1324.0608 9 0.38149652 6043389 6043769 8.1600072 2.09840184 5237500 5237626 265.83286 1.79892935 5204164 7 5205046 6 3932.3907 2 4894564 5 4894719 2 6.0178428 4 4862506 0 4863301 0 4676.9776 7 0.56088556 4.68438239 0.82176272 4890759 4890990 40.171731 1.60739150 6614938 6615168 418.39990 1.19240379 9 7 4 3 0.0008680 5 0.0006122 1 7.98E-05 3.04E-07 0.0008221 1 3.09E-08 0.0009186 1.05E-05 0.0003581 2 6607433 6607902 2055007 1492014 3 2062978 1492432 5 6969031 6969498 6961907 6962303 5077427 5078399 1250211 1254852 4137.4659 8.5866233 9 2114.5992 3045.7323 4 839.19408 729.13347 9 294.28353 8 0.5633337 3.47674717 0.0002894 0.41220722 0.0006851 1.35233610 0.0005131 0.34228614 0.49453901 0.0007994 0.0003338 1 4 2 3 1 0.57289106 1.67E-05 1.53E-05 3073424 9 3073712 0 358.96369 0.82094680 4 - 5 6 - 3 - 7 - 4 - 4 2 - 9 4 - 3 3 - 9 - 8 Table S3.7 (cont’d) PGSC0003DMG400005404 PGSC0003DMG400005410 PGSC0003DMG400005464 PGSC0003DMG400005473 PGSC0003DMG400005545 PGSC0003DMG400005572 PGSC0003DMG400005667 PGSC0003DMG400005668 PGSC0003DMG400005677 PGSC0003DMG400005743 PGSC0003DMG400005754 PGSC0003DMG400005903 PGSC0003DMG400005921 PGSC0003DMG400005950 PGSC0003DMG400005974 PGSC0003DMG400006090 PGSC0003DMG400006155 PGSC0003DMG400006170 PGSC0003DMG400006198 PGSC0003DMG400006202 NAD(P)H:quinone oxidoreductase NAD(P)H:quinone oxidoreductase Rhodanese-like domain containing protein Gene of unknown function GAL83 ATP binding protein ch01 74151442 74153806 832.629196 ch01 74148065 74149415 1494.80351 ch06 38707539 38710238 5479.67696 ch06 38709369 38709786 284.309384 ch12 60556129 60558973 105.992979 ch01 86735046 86741007 1714.5106 -1.525278896 0.00028202 -2.455939425 2.69E-05 -1.218623636 0.00012672 2.57E-05 -1.354329521 1.19E-10 -2.528489422 0.874890195 2.62E-06 Conserved gene of unknown function Serine/threonine-protein phosphatase BSL3 Geranylgeranyl transferase type II beta subunit Golgin candidate 2 Aldose-1-epimerase UDP-N-acetylglucosamine--N-acetylmuramyl-(Pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase ch03 58462478 58466113 733.950989 ch03 58456137 58462029 604.240082 ch03 58199197 58207200 883.130704 ch03 58426449 58432853 662.673298 ch03 58296015 58298374 1868.47096 ch06 54567357 54580152 5909.77159 ch06 54172928 54175121 948.208005 ch06 54194363 54198068 8585.66218 ch04 465.993866 ch09 55247742 55252564 722.637932 ch07 44501932 44507039 311.787036 ch01 87577228 87580997 3533.46374 ch01 87361285 87363990 2639.51154 ch01 87300111 87304770 229.917789 Tropinone reductase II F-Box protein Multicystatin Multicystatin 1751233 1753943 Thermal hysteresis protein STHP-64 Gene of unknown function Cucumisin Thaumatin 2.25E-05 -0.556348831 0.0006206 -1.461476947 8.57E-05 0.353484705 3.43E-05 -0.463184069 7.32E-08 -0.58391667 -1.036710384 0.0001802 -2.270713903 0.00065964 -2.358425562 0.00045646 0.00091106 0.637596602 0.00011392 0.964024652 1.053464931 0.00027067 7.86E-06 -1.093357051 5.16E-05 0.557354426 0.982434288 1.10E-05 182 Table S3.7 (cont’d) PGSC0003DMG40000628 PGSC0003DMG40000663 PGSC0003DMG40000670 PGSC0003DMG40000673 PGSC0003DMG40000673 PGSC0003DMG40000677 PGSC0003DMG40000686 PGSC0003DMG40000691 PGSC0003DMG40000695 PGSC0003DMG40000697 PGSC0003DMG40000700 PGSC0003DMG40000713 PGSC0003DMG40000726 PGSC0003DMG40000728 PGSC0003DMG40000732 PGSC0003DMG40000733 PGSC0003DMG40000734 PGSC0003DMG40000735 PGSC0003DMG40000735 PGSC0003DMG40000738 3 2 5 3 5 8 3 4 2 4 6 6 8 6 2 5 7 2 8 9 Multidrug resistance-associated protein 2, 6 (Mrp2, 6), abc- transoprter Conserved gene of unknown function BSD Thioredoxin-like U5 small ribonucleoprotein particle protein DNAJ heat shock N-terminal domain-containing protein Glucosamine-fructose-6-phosphate aminotransferase YA3 LOB domain-containing protein HEAT repeat family protein SET domain-containing protein Arsenical pump-driving atpase SNF7 family protein Plastid isopentenyl diphosphate isomerase CP12 Spliceosome associated protein Apyrase 3 SLD5 Multidrug resistance protein ABC transporter family Aluminum-activated citrate transporter CBL-interacting protein kinase 6 183 ch0 1 ch0 2 ch0 7 ch0 2 ch0 2 ch0 1 ch0 1 ch0 2 ch0 2 ch0 2 ch0 5 ch0 6 ch0 4 ch0 6 ch0 2 ch0 2 ch1 1 ch1 1 ch1 1 ch0 8 6051207 6051886 14455.055 2563818 2564152 14.491033 3970398 3979468 7831403 7837224 7828767 6497728 7830987 6499389 6574069 6574772 2651682 2651999 781.90189 4210.1589 1493.6813 1086.6258 1921.9766 135.57588 2639411 2639691 102.94316 2671710 2672342 2049.7468 4506027 4506512 11.776497 5538974 5539506 1205.3324 5023781 5024248 2773.6348 195064 3762614 195709 3763251 18136.464 1898.4728 3765431 3765866 2353.2704 3820705 3821169 977.49001 3790500 3790747 3809235 3809813 4083228 4083411 37.650013 983.08823 6 8 7 4 8 4 9 0 9 2 1 6 7 4 3 6 1 2 1 4 5 4 0 9 2 9 2 0 5 9 3 8 6 7 9 2 8 1 4 5 3 1 7 1 1 9 8 5 - 0.919155487 4.457603913 0.425573933 0.454783764 2.889874575 0.724968847 1.227317971 2.594593346 2.291106378 0.635801039 3.320850587 0.274514035 -1.11413578 0.708533699 0.579801564 -1.54829615 1.123525848 1.294950134 - - - - - - - - - - - - 1.87E-06 3.14E-10 0.0002427 1 2.86E-05 1.77E-07 0.0004780 0.0003101 0.0002161 5 8 2 6.67E-17 1.13E-05 8.12E-07 0.0009237 6.62E-06 0.0001213 0.0005908 0.0005815 1 9 2 4.20E-07 0.0005749 6 0.879180039 3.79E-05 596.70453 0.974460909 4.73E-06 Table S3.7 (cont’d) PGSC0003DMG400007597 PGSC0003DMG400007624 PGSC0003DMG400007793 PGSC0003DMG400007807 PGSC0003DMG400007898 PGSC0003DMG400008050 PGSC0003DMG400008075 PGSC0003DMG400008101 PGSC0003DMG400008109 PGSC0003DMG400008150 PGSC0003DMG400008343 PGSC0003DMG400008524 PGSC0003DMG400008586 PGSC0003DMG400008640 PGSC0003DMG400008680 PGSC0003DMG400008692 PGSC0003DMG400008805 PGSC0003DMG400009070 PGSC0003DMG400009096 PGSC0003DMG400009108 PGSC0003DMG400009109 PGSC0003DMG400009130 PGSC0003DMG400009173 PGSC0003DMG400009192 Cationic amino acid transporter ERD15 Lipoxygenase 14-3-3 protein Conserved gene of unknown function GcpE Arsenical pump-driving atpase KiTH-2 Beta-galactosidase Nucleic acid binding protein Light harvesting protein 3 F-box family protein Auxin-responsive protein IAA13 Aldose 1-epimerase family protein Transformer-SR ribonucleoprotein Troponin C, skeletal muscle DNA binding protein Kinesin Protein dimerization CAPIP1 Trehalose-6-phosphate synthase ATP-dependent clp protease ATP-binding subunit clpx Small heat stress protein class CIII Mitochondrial deoxynucleotide carrier 184 5686911 5691390 1286913 1508887 2219188 1293220 1511870 2219902 ch11 126.661448 ch04 11354785 11356912 5870.19766 ch12 55.4569804 2681.96417 ch12 ch12 10.8859258 ch11 41712113 41719809 21371.5533 ch11 41741046 41748722 172.334246 ch11 41321245 41324503 15.8477413 ch11 41197787 41206297 7133.60453 ch10 58618604 58622568 2322.57847 60.599226 ch03 36914199 36915581 ch07 7502018 47.8607041 ch09 46204356 46209935 977.946707 ch03 13686731 13688511 1803.98317 ch02 19605690 19610622 2545.24341 ch10 66.858601 ch00 28992772 28997693 333.655719 ch03 44888869 44893015 356.134292 ch03 44794980 44801906 281.123001 ch03 45023262 45026042 1276.47573 ch03 45041922 45043941 75.0249942 ch03 45485675 45495233 2599.75507 ch03 61718867 61719420 109.049148 ch03 62140924 62145910 532.928875 7499230 820159 824485 0.00097168 1.387264272 4.33E-05 -0.99945274 1.98E-06 -2.185576283 6.49E-06 -0.396955798 2.96E-06 -3.752420947 0.00066847 -0.51701646 -0.811851847 0.00061221 8.21E-08 5.798649497 -0.569002745 6.32E-06 1.56E-07 1.253285854 1.40E-09 -4.941705695 1.313310282 1.44E-06 -0.413889592 0.00048875 9.92E-09 -0.993853623 0.460410902 0.00032529 1.008546664 0.00036962 -1.025100359 0.00065757 0.660945815 0.00026985 1.34E-05 0.685021176 2.82E-05 -1.007645837 1.251483756 0.00014525 1.54E-06 -0.819528062 3.194080096 0.00026307 -1.188436413 0.00087649 Table S3.7 (cont’d) PGSC0003DMG40000925 PGSC0003DMG40000932 PGSC0003DMG40000934 PGSC0003DMG40000936 PGSC0003DMG40000937 PGSC0003DMG40000941 PGSC0003DMG40000941 PGSC0003DMG40000945 PGSC0003DMG40000946 PGSC0003DMG40000946 PGSC0003DMG40000949 PGSC0003DMG40000950 PGSC0003DMG40000950 PGSC0003DMG40000951 PGSC0003DMG40000951 PGSC0003DMG40000951 PGSC0003DMG40000967 PGSC0003DMG40000972 PGSC0003DMG40000980 7 6 8 3 6 0 2 6 6 7 7 3 5 1 3 4 3 2 9 6 Neutral/alkaline invertase Conserved gene of unknown function Clathrin coat assembly MADS16 Conserved gene of unknown function UDP-glucosyltransferase UDP-glucosyltransferase F-box family protein Metal-binding isoprenylated protein Metal ion binding protein ABC-type Co2+ transport system, permease component Conserved gene of unknown function Pentatricopeptide repeat-containing protein Aspartic protease inhibitor 8 Aspartic protease inhibitor 5 Kunitz-type protease inhibitor protein Pom14 protein SET domain containing protein Alpha N-terminal protein methyltransferase 1 185 ch1 1 ch0 8 ch0 4 ch0 4 ch0 7 ch0 2 ch0 2 ch1 1 ch0 4 ch0 4 ch0 3 ch0 3 ch0 3 ch0 3 ch0 3 ch0 3 ch0 1 ch0 6 ch0 1 ch0 2 PGSC0003DMG40000961 Uncharacterized plant-specific domain TIGR01589 family 1283624 1284416 1367619 1368338 4874190 4874239 840.91939 124.46632 6490993 6491743 1684.5223 6495966 6496718 2051.9899 4963125 4963390 1337.2008 2694838 2695631 562.85633 2697223 2697560 119.17228 3602038 3602536 2164.9654 6245555 6245679 83.059669 6247968 6248068 67.353503 4414753 4414850 1331.3202 4420869 4421807 1253.0716 4417297 4417855 1305.8365 4403525 4403607 6117.8545 4993005 4993084 16762.690 4994130 4994207 2 3 3 8 3 6 8 5 3 3 6 3 1 6 5 9 9 7 1 0 1 9 5 5 5 2 1 8 9 5 1 8 6 8 109.91531 1617.1098 2098.3312 698.66938 2 5 1 5 2 9 1 3 5 1 6 4 5 4 2 1 1 5248174 5252027 812852 1253262 4 817804 1253671 2 - 0.560024935 0.976917024 0.338028702 0.652511667 0.243510556 1.333865609 1.988650939 0.665897575 2.357829188 2.514794612 0.907670203 0.202663445 0.839509934 1.990889568 2.757280986 4.695005644 0.395383361 1.835103113 0.358530883 - - - - - - - - - - - - 1.00E-04 0.0005406 0.0001047 0.0003996 0.0005471 3 2 1 4 3.13E-12 1.12E-12 6.33E-05 0.0008803 0.0002199 0.0009408 0.0009217 3 9 1 3 4.06E-05 0.0005025 7 6.07E-10 2.00E-12 4.61E-06 9.53E-09 0.0005235 5 249.21998 2.012050084 7.64E-08 Table S3.7 (cont’d) PGSC0003DMG400009850 PGSC0003DMG400009892 PGSC0003DMG400009947 PGSC0003DMG400010000 PGSC0003DMG400010033 PGSC0003DMG400010041 PGSC0003DMG400010129 PGSC0003DMG400010134 PGSC0003DMG400010136 PGSC0003DMG400010137 PGSC0003DMG400010141 PGSC0003DMG400010144 PGSC0003DMG400010145 PGSC0003DMG400010146 PGSC0003DMG400010222 PGSC0003DMG400010232 PGSC0003DMG400010236 PGSC0003DMG400010257 PGSC0003DMG400010263 PGSC0003DMG400010279 PGSC0003DMG400010283 PGSC0003DMG400010332 PGSC0003DMG400010338 PGSC0003DMG400010396 Progesterone 5-beta-reductase Prolyl endopeptidase Subtilisin-type protease Cinnamoyl-CoA reductase Tetratricopeptide repeat-containing protein LIM domain containing protein Aspartic protease inhibitor 10 Cysteine protease inhibitor 1 Stigma expressed protein Cysteine protease inhibitor 1 Stigma expressed protein Stigma expressed protein Cysteine protease inhibitor 9 Kunitz-type tuber invertase inhibitor Phospholipase A1 Cysteine protease Cysteine protease Cp5 Leucine aminopeptidase, chloroplastic MADS-box transcription factor FBP28 Digalactosyldiacylglycerol synthase 2, chloroplastic Class I chitinase Pentatricopeptide repeat-containing protein Leucyl-tRNA synthetase Silencing group A protein ch10 12201791 12210430 638.458208 ch04 71367331 71374544 119.403753 ch04 71120202 71125558 162.467209 ch04 71944169 71948367 2003.62852 ch02 44671383 44675916 1391.13452 ch02 44795348 44800116 1704.46672 ch03 43950496 43951544 26.7446775 ch03 49784928 49785518 414.737191 ch03 49713158 49714012 1103.83505 ch03 49685391 49686264 3916.90971 ch03 49611604 49612275 13735.383 ch03 49514289 49514969 927.656992 ch03 49498051 49498873 1402.75367 ch03 49448372 49449153 793.797088 ch02 32415956 32417689 31.9167054 ch02 32054514 32056959 787.411829 ch02 32074428 32080205 2195.69562 ch05 12727912 12733521 446.324829 ch10 38698247 38706939 120.086433 ch10 38731596 38739735 568.324028 ch10 38265411 38265939 120.590748 ch02 27848945 27854556 209.843613 ch02 28144492 28153257 927.634913 ch02 22154816 22158418 64.2725418 2.01E-06 -1.852907612 9.22E-07 1.48526627 1.54E-06 1.273298672 -0.411686695 0.00015327 0.00093983 0.294247837 0.902886225 0.00051052 4.30E-18 -4.987520962 4.68E-23 -5.222446165 -2.545291713 3.95E-05 3.07E-16 -5.565050124 8.52E-06 -2.770110439 -2.861719027 8.65E-06 8.19E-20 -5.008916486 6.74E-16 -5.788219534 2.691370863 7.22E-08 4.40E-05 -1.704244689 -2.421894752 9.30E-33 -3.021080404 0.00012927 -0.895493161 0.00045584 3.71E-06 0.774109695 -5.835710401 5.74E-07 0.685766479 1.13E-05 -0.666403189 0.00038689 -0.768672205 0.00012385 186 PGSC0003DMG40001074 Leucine-rich repeat family protein / protein kinase family 1141347 1142051 1827.5794 ch0 2 ch0 2 ch0 2 ch0 2 ch1 0 ch0 2 ch0 7 ch0 4 ch0 1 ch0 5 ch0 5 ch1 0 ch0 5 ch1 0 ch1 0 ch1 0 ch0 7 ch0 7 ch0 9 ch1 0 2760996 2761422 1656.8693 2754639 2755032 484.07593 2745715 2746246 268.82921 2746085 2746137 9.3524707 3190467 3197852 8632420 4661004 8635676 4661309 1308.9064 499.93839 45.338284 6691804 6692030 2746.5902 5936126 5936444 5922.4773 1143089 1143394 886.61631 4787552 4788238 538.34059 3518442 3519114 1829.1372 5382871 5383297 2355.5561 5362497 5362695 792.66387 5346788 5347232 257.05881 982266 987735 1141604 5262242 1143559 5262495 360.50266 133.29094 1534.9083 5894659 5895068 246.81986 4 7 3 3 0 9 8 7 8 8 0 0 7 2 0 7 3 3 4 9 6 1 6 2 8 0 8 5 8 0 7 8 8 7 5 3 1 1 4 8 1 7 8 4 4 3 3 1 1 8 8 9 - 1.082016025 4.99E-05 0.755929801 1.16E-12 4.64601322 0.0003915 5.992654698 0.413662331 0.950077609 2.446424854 1.020609891 1.038992086 7.00E-09 0.0008804 4 8.37E-06 0.0002119 2 3.59E-06 0.0002356 9 1.683989469 3.66E-07 0.796160481 0.792294541 1.766135609 1.556374301 0.617323968 0.536901488 0.646369403 0.955134654 2.05E-07 1.90E-05 5.78E-05 2.66E-05 6.49E-06 6.46E-05 3.32E-05 2.79E-05 1.034804335 1.67E-05 0.471580012 4.07E-05 - - - - - - - - Table S3.7 (cont’d) PGSC0003DMG40001042 PGSC0003DMG40001042 PGSC0003DMG40001043 PGSC0003DMG40001045 PGSC0003DMG40001055 PGSC0003DMG40001057 PGSC0003DMG40001058 PGSC0003DMG40001060 PGSC0003DMG40001066 PGSC0003DMG40001073 PGSC0003DMG40001086 PGSC0003DMG40001090 PGSC0003DMG40001102 PGSC0003DMG40001110 PGSC0003DMG40001111 PGSC0003DMG40001114 PGSC0003DMG40001120 PGSC0003DMG40001140 PGSC0003DMG40001141 2 6 0 1 6 9 7 9 4 8 6 9 0 6 6 8 4 1 1 3 Pentatricopeptide repeat-containing protein Conserved gene of unknown function Conserved gene of unknown function Gene of unknown function Trehalose synthase Conserved gene of unknown function Sesquiterpene synthase Transcription factor LIM 1,4-alpha-glucan-maltohydrolase E3 ubiquitin ligase protein Tetracycline transporter Cytosolic acetoacetyl-coenzyme A thiolase Heparanase CBL-interacting protein kinase 8 ATPP2-A13 Hydrophobic protein OSR8 Gene of unknown function Phytochrome A-associated F-box protein Non-structural maintenance of chromosome element 187 Table S3.7 (cont’d) PGSC0003DMG400011420 PGSC0003DMG400011437 PGSC0003DMG400011438 PGSC0003DMG400011544 PGSC0003DMG400011558 PGSC0003DMG400011641 PGSC0003DMG400011650 PGSC0003DMG400011694 PGSC0003DMG400011715 PGSC0003DMG400011716 PGSC0003DMG400011719 PGSC0003DMG400011751 PGSC0003DMG400011780 PGSC0003DMG400011807 PGSC0003DMG400011864 PGSC0003DMG400011865 PGSC0003DMG400011872 PGSC0003DMG400011980 PGSC0003DMG400011997 PGSC0003DMG400012100 PGSC0003DMG400012280 PGSC0003DMG400012315 PGSC0003DMG400012415 PGSC0003DMG400012423 Thioredoxin-like 3-1, chloroplastic LEA D113 homologue type1 Protein LE25 WD-repeat protein Transcription factor LIM CBL-interacting protein kinase 13 Alpha-soluble NSF attachment protein Serpin Sn-2 protein Sn-1 protein 18.5 kDa class I heat shock protein 2-oxoglutarate-dependent dioxygenase Auxilin ARF-GAP domain 13 F-box protein, atfbl3 Transporter Glucosyltransferase Two-component sensor histidine kinase bacteria CPN60A Major latex Conserved gene of unknown function Conserved gene of unknown function Acetylglucosaminyltransferase Conserved gene of unknown function 188 7352367 7348120 888985 7356721 7353590 890624 ch10 58833535 58837062 1193.54972 ch10 58933783 58934530 80.9170812 ch10 58931638 58932735 74.6345185 ch12 56221757 56228576 1721.82755 ch12 56212993 56215429 832.827032 ch05 47506266 47510051 733.171452 ch05 47567094 47573430 1179.2795 ch04 68825985 68830888 12.1545207 ch01 66.8459685 46.3658681 ch01 ch09 5829.33008 ch07 41845573 41847191 68351.1154 ch06 42751074 42757926 903.173362 ch02 13802875 13803354 325.149884 ch10 49465811 49472205 545.676747 ch10 49439604 49446317 193.144316 ch06 43889765 43895421 426.116401 ch05 11541210 11546514 2206.24707 ch06 55568679 55573501 802.978963 ch07 2369.40723 ch08 55997132 55997906 400.957423 ch08 55379410 55380034 248.457051 ch07 53177400 53181191 423.87802 ch07 53285169 53291627 704.669835 6258626 6259653 6.40E-09 -1.535888118 2.75E-05 4.010688955 0.00026985 3.53703817 9.85E-05 -0.521106343 3.60E-06 -1.329203083 2.27E-05 1.582889645 -0.618775344 0.00014483 -2.510229913 0.00019756 -2.600256632 1.25E-05 -1.762097417 0.00033381 2.73E-05 0.753955443 -2.443587353 1.24E-05 2.81E-08 -0.51414899 1.01E-05 -0.615026282 -1.075807942 1.21E-05 -2.548950577 0.00020801 -1.497435031 0.00039961 1.617719929 3.58E-05 4.75E-07 -1.249817864 2.85E-12 -2.947724869 1.004985609 2.38E-05 1.235699052 0.00095046 -0.785414372 0.00012672 -0.620619655 0.00083682 Table S3.7 (cont’d) PGSC0003DMG400012445 PGSC0003DMG400012453 PGSC0003DMG400012503 PGSC0003DMG400012513 PGSC0003DMG400012525 PGSC0003DMG400012605 PGSC0003DMG400012610 PGSC0003DMG400012650 PGSC0003DMG400012664 PGSC0003DMG400012676 PGSC0003DMG400012687 PGSC0003DMG400012704 PGSC0003DMG400012718 PGSC0003DMG400012780 PGSC0003DMG400012800 PGSC0003DMG400012836 PGSC0003DMG400012987 PGSC0003DMG400013019 PGSC0003DMG400013037 PGSC0003DMG400013052 PGSC0003DMG400013055 PGSC0003DMG400013057 PGSC0003DMG400013059 PGSC0003DMG400013094 PGSC0003DMG400013102 Proton pump interactor 1 Nucleolar GTP-binding protein 2 DNA mismatch repair protein mutS Amino acid binding protein DREB1 Cerebral protein Conserved gene of unknown function TMV resistance protein N Multidrug resistance pump ABA induced plasma membrane protein PM 19 Pore protein of 24 kD (OEP24) Conserved gene of unknown function Cytokinin oxidase/dehydrogenase 1 PTAC14 Aminotransferase Conserved gene of unknown function Threonine dehydratase biosynthetic, chloroplastic Prolyl 4-hydroxylase Conserved gene of unknown function TdcA1-ORF1-ORF2 protein Non-LTR retrolelement reverse transcriptase Gene of unknown function TdcA1-ORF2 protein Leucine-rich repeat-containing protein Leucyl-tRNA synthetase 189 580621 338193 110755 ch07 53158312 53163820 6987.1042 ch07 53328483 53330299 115.157422 588125 ch08 55.1033418 596.010181 342214 ch08 12.5176304 ch08 112152 ch02 47452058 47455033 948.92276 ch02 47523088 47527457 1541.96411 ch02 16889189 16891951 130.383187 ch02 16875918 16882893 349.167392 ch02 40981153 40984439 51.0755836 ch02 40770918 40772124 199.870095 ch04 11252614 11257708 678.067616 ch04 11108702 11111248 84.0359251 ch12 45101989 45109764 43.0086176 ch04 52946107 52955379 1549.40284 ch10 13331228 13334391 40.4982921 ch09 11285.7443 ch02 22900205 22905821 550.605578 940.72356 ch06 41433512 41436081 24.5409015 7963430 ch02 ch02 8190275 6.76457436 10.1130588 8182546 ch02 ch02 7967127 4.81145425 ch02 15887638 15891240 58.2780615 ch02 24183991 24188089 4065.92117 7961380 8188698 8181763 7966308 4336457 4342614 9.97E-07 -0.733471288 9.84E-05 0.97524984 0.00027531 0.843274963 1.00E-10 1.906895679 0.00067614 3.881594663 1.44E-05 0.485055769 -0.328728891 0.00064841 2.98E-58 -9.157128794 -1.127746916 1.52E-07 0.0005988 1.929564448 1.94E-08 -0.92519311 -1.486755427 1.98E-06 0.00067868 0.987292801 0.0003855 1.507456159 -0.556061564 1.00E-07 1.89E-16 2.777241752 4.47E-10 -3.303547012 -0.748163477 8.17E-06 2.79E-05 1.27059236 2.09E-05 3.694329181 -4.430940451 4.82E-09 2.00E-12 -4.562792344 3.03E-06 4.499989796 -9.283320424 6.28E-48 3.95E-05 0.463594261 Table S3.7 (cont’d) PGSC0003DMG400013130 PGSC0003DMG400013145 PGSC0003DMG400013178 PGSC0003DMG400013229 PGSC0003DMG400013243 PGSC0003DMG400013248 PGSC0003DMG400013255 PGSC0003DMG400013264 PGSC0003DMG400013282 PGSC0003DMG400013307 PGSC0003DMG400013335 PGSC0003DMG400013378 PGSC0003DMG400013424 PGSC0003DMG400013425 PGSC0003DMG400013426 PGSC0003DMG400013433 PGSC0003DMG400013485 PGSC0003DMG400013490 PGSC0003DMG400013511 PGSC0003DMG400013533 PGSC0003DMG400013627 PGSC0003DMG400013629 PGSC0003DMG400013637 PGSC0003DMG400013645 PGSC0003DMG400013666 Conserved gene of unknown function Conserved gene of unknown function DNA binding protein Gene of unknown function Potassium channel TORK1 Lipoxygenase ERF transcription factor 5 Extracellular ligand-gated ion channel Tonoplast dicarboxylate transporter Aspartic proteinase nepenthesin-1 Alternative oxidase Metalloendopeptidase Conserved gene of unknown function Lipase Conserved gene of unknown function Conserved gene of unknown function ATP-citrate synthase Prf Pantothenate kinase family protein Flavonol 4'-sulfotransferase Tir-nbs-lrr resistance protein Cytochrome P450 hydroxylase Conserved gene of unknown function EMB1624 RING-H2 finger protein ATL80 190 6314231 3820243 6714370 6316028 3822055 6714828 1149991 1060172 1002200 775084 489114 1233766 785527 185575 189146 197809 204603 294714 8974024 9139914 8807178 9065839 246.220586 ch01 620.703012 ch10 132.175755 ch12 ch00 32683427 32690771 20.6363721 289.944287 ch11 ch11 142.117554 58.1662118 ch11 511.967169 ch11 ch11 9443.0132 4554.35637 ch11 645.832774 ch11 ch03 2198.63732 875.635333 ch03 74.7775708 ch03 ch03 4055.58296 187.49429 ch03 2467.49637 ch05 ch05 1618.28778 620.004911 ch05 ch05 4544.02033 ch02 37802516 37808556 495.084558 ch02 37830842 37832400 399.089918 ch02 38004049 38004778 24.0107907 ch02 38127585 38130411 6.22659222 ch02 38184210 38185499 502.368587 1156528 1064952 1002736 778244 493276 1235285 790528 188815 197051 200663 207670 295402 8980534 9148796 8817341 9067059 0.00027457 1.709972855 0.00097898 0.379978383 6.93E-08 3.193669549 0.00095416 1.863739956 6.45E-05 1.515407695 0.00086786 -1.1018486 9.84E-05 1.442868277 0.00059646 0.946494131 6.45E-05 1.453291933 1.829140245 9.85E-05 -1.355701894 0.00059822 -1.036699376 7.70E-10 2.89E-05 -1.279455603 4.40E-12 -2.779337021 -0.806706139 3.57E-05 0.00068894 0.807351097 -2.095261094 7.98E-05 -0.798566017 0.00068172 4.54E-05 -0.503260479 5.74E-07 -2.094248146 2.117880851 9.88E-10 4.65E-24 6.086354248 0.00058475 1.261567272 3.58368411 0.00023721 1.88E-07 0.745810142 Table S3.7 (cont’d) PGSC0003DMG400013676 PGSC0003DMG400013686 PGSC0003DMG400013821 PGSC0003DMG400013875 PGSC0003DMG400013882 PGSC0003DMG400013884 PGSC0003DMG400013895 PGSC0003DMG400013929 PGSC0003DMG400013977 PGSC0003DMG400014029 PGSC0003DMG400014083 PGSC0003DMG400014132 PGSC0003DMG400014182 PGSC0003DMG400014187 PGSC0003DMG400014198 PGSC0003DMG400014200 PGSC0003DMG400014210 PGSC0003DMG400014212 PGSC0003DMG400014272 PGSC0003DMG400014284 PGSC0003DMG400014298 PGSC0003DMG400014314 PGSC0003DMG400014325 PGSC0003DMG400014372 CLB1 Auxin response factor 19 Potassium transporter Transcription factor Negative regulator of the PHO system Conserved gene of unknown function RabGAP/TBC domain-containing protein Cytochrome P450 92B1 Actin binding protein Abhydrolase domain containing Ninja-family protein AFP2 Conserved gene of unknown function Nuclear transcription factor, X-box binding Conserved gene of unknown function Conserved gene of unknown function Flotillin-1 DnaJ protein Heat shock protein (S)-N-methylcoclaurine 3'-hydroxylase isozyme Conserved gene of unknown function Conserved gene of unknown function F-box family protein Sugar isomerase domain-containing protein Uridine kinase 191 6466768 ch05 43392201 43396090 98.9732476 ch05 43160552 43167160 2620.47225 ch04 22319412 22327197 2490.84387 ch02 35441558 35444347 1013.73542 ch02 35389834 35394515 872.993181 ch02 35366764 35368077 91.7002502 844.63379 ch12 6472832 ch11 13175386 13180398 59.000393 ch05 42833027 42839003 40.2026768 ch03 61598488 61600867 1042.33611 ch02 25185566 25186519 20.4456738 ch03 57117491 57118750 6.86744301 ch03 58055767 58059980 1397.85957 ch03 58153239 58156352 197.939501 ch03 57078465 57084441 378.671785 ch03 57113395 57115435 408.327105 ch03 57385089 57386521 1400.12849 ch03 57412216 57414401 44.3572807 ch03 42351046 42356188 101.652092 ch03 42549837 42551918 98.3476549 ch03 43043166 43043951 103.099793 ch03 42263976 42268076 664.112672 248.16683 ch03 42558202 42558810 ch10 922546 580.273196 912734 -0.827645376 0.00048875 2.35E-05 0.68193804 -0.684918912 1.98E-12 9.05E-05 1.109622441 0.00011191 0.77083388 2.320829509 1.45E-13 8.92E-07 -0.946878278 2.03E-05 -3.698797006 1.490807955 0.00028202 4.99E-05 -2.528367769 2.550683865 3.68E-07 -3.213608275 0.00043555 -0.257845094 0.00057496 1.83E-05 0.551348466 1.036543724 0.00017188 -2.251837455 0.00091093 2.25E-06 -1.934531808 2.522267778 0.00022091 0.0009769 -1.829809316 -4.999221339 4.22E-08 -1.267241362 0.00068061 4.26E-11 -0.984838429 7.87E-11 -4.57809867 -1.125302946 1.34E-13 Table S3.7 (cont’d) PGSC0003DMG400014470 PGSC0003DMG400014492 PGSC0003DMG400014566 PGSC0003DMG400014673 PGSC0003DMG400014760 PGSC0003DMG400014763 PGSC0003DMG400014819 PGSC0003DMG400014863 PGSC0003DMG400014867 PGSC0003DMG400014900 PGSC0003DMG400014931 PGSC0003DMG400014936 PGSC0003DMG400015093 PGSC0003DMG400015105 PGSC0003DMG400015119 PGSC0003DMG400015153 PGSC0003DMG400015158 PGSC0003DMG400015174 PGSC0003DMG400015191 PGSC0003DMG400015229 PGSC0003DMG400015275 PGSC0003DMG400015289 PGSC0003DMG400015385 PGSC0003DMG400015420 MtN19 Truncated hemoglobin Transcription factor Dimethylaniline monooxygenase Taxane 13-alpha-hydroxylase cytochrome P450 Gene of unknown function Nitric oxide synthase-associated protein I Phenylacetaldehyde synthase Peroxidase Chitin-inducible gibberellin-responsive protein 50S ribosomal protein L7/L12 Zinc/iron transporter Gene of unknown function Alpha N-terminal protein methyltransferase 1 Progesterone 5 beta-reductase Tryptophan biosynthesis protein, trpc Protein bem46 DUF26 domain-containing protein 2 Conserved gene of unknown function BTB/POZ domain-containing protein Aquaporin, MIP family, TIP subfamily Proteinase inhibitor type-2 CEVI57 Phosphoenolpyruvate carboxylase Conserved gene of unknown function 192 2941260 2943570 7206788 7132680 7208616 7135798 ch12 49139008 49141127 26.1132253 ch08 42839490 42844914 451.046811 ch05 472.204787 ch01 88291988 88294566 71.5609355 ch01 17681887 17683177 6.22218096 ch08 44374559 44377460 39.4186028 ch03 38629353 38632389 736.290499 322.368564 ch03 ch03 940.769324 ch02 40546545 40549653 1365.71087 ch02 15517635 15522570 682.862666 ch02 15695173 15697155 127.55616 ch08 41308063 41309028 1.59088123 ch02 12519201 12524409 111.232352 ch00 31823679 31827925 11.9993285 ch03 51601379 51606789 3072.70671 ch03 51505704 51510446 879.382214 ch03 51177387 51177785 17.5242151 ch03 50849874 50855089 410.845486 ch03 51465624 51468593 1371.17193 ch03 50367048 50368315 39.8782477 ch03 50143509 50144247 16.0404107 ch12 2864.23602 ch12 56695532 56697912 59.3033574 239059 245128 -1.473134526 0.0001218 -0.579209994 0.00060887 -3.053086992 7.98E-05 -1.839989517 0.00013335 0.0005945 2.976951298 3.03722548 4.37E-12 3.60E-05 -0.839523653 0.00076672 2.11897488 1.8534164 0.00075526 1.61E-05 -0.422856465 6.85E-07 -0.505914735 1.642463659 4.05E-06 -4.059085587 0.00066813 6.56E-11 2.168718299 -2.256317025 6.76E-05 -0.582037033 0.00028971 0.00068172 0.802059188 1.502927459 0.0003915 0.709047624 7.26E-07 -1.920281144 0.00077144 4.812415648 1.74E-08 2.89E-07 -3.351494167 2.23E-22 -1.17553151 1.042264631 1.04E-06 Table S3.7 (cont’d) PGSC0003DMG40001542 PGSC0003DMG40001548 PGSC0003DMG40001550 PGSC0003DMG40001551 PGSC0003DMG40001557 PGSC0003DMG40001562 PGSC0003DMG40001566 PGSC0003DMG40001572 PGSC0003DMG40001577 PGSC0003DMG40001581 PGSC0003DMG40001586 PGSC0003DMG40001600 PGSC0003DMG40001601 PGSC0003DMG40001601 PGSC0003DMG40001616 PGSC0003DMG40001618 PGSC0003DMG40001619 PGSC0003DMG40001622 PGSC0003DMG40001627 2 4 5 3 7 5 1 8 0 7 0 1 0 8 6 5 7 0 0 3 ATP-dependent RNA helicase Laccase Anthranilate N-benzoyltransferase protein Gene of unknown function RNase H domain-containing protein Conserved gene of unknown function RING-H2 finger protein ATL2N HIPL1 protein Cyclin-dependent kinase inhibitor UDP-glucose:sterol 3-O-glucosyltransferase Molybdopterin cofactor sulfurase Glutamate dehydrogenase A Amino acid binding protein Conserved gene of unknown function HVA22 e Anthocyanidine rhamnosyl-transferase Pentatricopeptide repeat-containing protein Conserved gene of unknown function 193 ch1 2 ch0 2 ch0 2 ch0 2 ch0 5 ch0 8 ch0 6 ch1 2 ch0 2 ch0 9 ch0 1 ch0 5 ch0 0 ch0 4 ch1 1 ch1 1 ch1 1 ch1 1 ch1 1 ch0 6 PGSC0003DMG40001612 BRASSINOSTEROID INSENSITIVE 1-associated receptor Alpha-glucosidase kinase 1 5678518 5679127 390.16862 2093867 2094136 35.353663 2100355 2100514 306.46328 2079407 2079650 93.451362 7219550 2156601 7222918 2157345 92.015894 471.42422 2865856 2865933 136.07837 3710693 3711087 189.52258 4397631 4397804 374.44909 4498467 4499754 1481.7618 1694367 1695065 1407.9432 4 0 7 2 2 0 5 7 6 8 3 2 9 7 3 4 6 5 3 2 2 1 7 9 0.786055011 1.972309186 -9.49936276 1.777847938 0.684236167 0.534558984 1.337066049 -1.14482245 1.253082139 1.354934289 0.571937975 1.01E-05 1.66E-07 2.16E-79 0.0009311 0.0007034 0.0004389 5 6 8 1.03E-07 0.0007542 9 4.66E-05 0.0001975 6 1.10E-05 4734811 4735399 2335149 2335532 5258251 5264186 2189365 2198498 2923502 2929339 2060384 2062711 2129563 2131267 2638392 4014228 7 2641771 4014447 7 124.99378 0.926507717 4.18E-06 1003.2441 1634.5265 165.94432 42.372840 961.37080 157.07873 57.092234 2878.5115 1.175778388 1.131644986 0.835722681 1.58E-05 0.0001476 0.0003165 7 1 1.726270981 8.23E-05 1.830812853 0.918969673 0.730073609 2.27E-05 0.0001732 0.0004420 5 1.001280698 3.61E-05 - - - - - - - - - - - - 1 4 8 2 7 2 5 5 2 9 1 7 4 3 1 5 5 2 Table S3.7 (cont’d) PGSC0003DMG400016339 PGSC0003DMG400016383 PGSC0003DMG400016411 PGSC0003DMG400016437 PGSC0003DMG400016442 PGSC0003DMG400016460 PGSC0003DMG400016462 PGSC0003DMG400016480 PGSC0003DMG400016729 PGSC0003DMG400016733 PGSC0003DMG400016765 PGSC0003DMG400016776 PGSC0003DMG400016930 PGSC0003DMG400016932 PGSC0003DMG400016991 PGSC0003DMG400016993 PGSC0003DMG400017020 PGSC0003DMG400017022 PGSC0003DMG400017069 PGSC0003DMG400017116 PGSC0003DMG400017123 PGSC0003DMG400017141 PGSC0003DMG400017227 PGSC0003DMG400017246 PGSC0003DMG400017287 Lipid binding protein Ccr4-associated factor Isochorismatase Conserved gene of unknown function Aspartic proteinase 15.4 kDa class V heat shock protein Conserved gene of unknown function Conserved gene of unknown function Conserved gene of unknown function Acyl:coa ligase acetate-coa synthetase Conserved gene of unknown function Cytochrome P450 Conserved gene of unknown function Nucleolar GTP-binding protein 2 LescPth2 Pto-like serine/threonine kinase Pentatricopeptide repeat-containing protein Conserved gene of unknown function Beta-amyrin synthase Nodulin family protein Cytochrome P450 Transposon protein, CACTA, En/Spm sub-class Serine/threonine-protein phosphatase 1-aminocyclopropane-1-carboxylate oxidase homolog Conserved gene of unknown function 194 3413078 3558766 3411973 3555785 ch06 39105067 39106017 158.493823 389.683116 ch01 ch01 749.660538 ch02 36648396 36655649 388.347237 ch02 36582089 36587773 955.22491 ch02 36223235 36224237 1072.88946 ch02 36727523 36735334 227.6241 ch02 36385262 36386136 55.1704589 ch02 36879763 36884526 95.9824082 ch02 36961118 36964883 196.981631 ch06 45266675 45268111 95.8210255 ch06 45321291 45323254 251.442283 ch04 48952514 48954196 17.8048269 11.575201 ch04 48765725 48766123 ch05 9647499 28.9140896 ch05 30.5989382 9684276 ch02 11031511 11036203 40.3192863 ch02 11086314 11088387 10.8729445 ch12 37815090 37824497 1050.49934 ch03 53307178 53312264 194.439981 ch00 36184650 36186778 31.7144734 ch01 21.5678341 ch09 56359020 56364320 780.892959 ch09 56079326 56081254 523.189218 ch07 50746949 50748052 767.041966 9646117 9683191 5767436 5770198 -1.33367907 2.84E-05 -0.558724542 0.00062909 -0.638029075 5.84E-05 0.00074288 0.310002737 1.54E-05 1.352368296 -1.326211868 1.06E-05 6.38E-11 -4.118600533 1.53E-05 1.509854084 -1.063926165 6.25E-06 -1.331231915 0.00043565 4.42E-05 3.110485684 -2.944166617 1.51E-05 9.19E-05 -1.978566738 4.08E-05 2.031989353 -1.568127464 8.65E-05 6.46E-05 -0.94419313 0.00017377 1.217692581 -4.009243661 7.74E-12 0.00035614 1.018193132 0.00060887 0.614043636 -2.338925824 0.0006967 1.98E-06 -2.607637544 3.24E-05 0.51111164 -1.726055975 1.81E-12 5.37E-05 -0.733632956 Table S3.7 (cont’d) PGSC0003DMG400017400 PGSC0003DMG400017413 PGSC0003DMG400017465 PGSC0003DMG400017473 PGSC0003DMG400017491 PGSC0003DMG400017513 PGSC0003DMG400017602 PGSC0003DMG400017610 PGSC0003DMG400017728 PGSC0003DMG400017730 PGSC0003DMG400017739 PGSC0003DMG400017873 PGSC0003DMG400017876 PGSC0003DMG400017967 PGSC0003DMG400017999 PGSC0003DMG400018012 PGSC0003DMG400018062 PGSC0003DMG400018063 PGSC0003DMG400018073 PGSC0003DMG400018134 PGSC0003DMG400018157 PGSC0003DMG400018160 PGSC0003DMG400018178 PGSC0003DMG400018206 PGSC0003DMG400018332 Mutt domain protein Phosphofructokinase Lipoxygenase Amino acid transporter Plant ubiquilin Acetylornithine deacetylase Chloroplast-targeted copper chaperone Protein ALUMINUM SENSITIVE 3 Conserved gene of unknown function FtsH protease Nibrin Tetratricopeptide repeat protein Tetratricopeptide repeat protein Hcr2-0B Cytochrome P450 hydroxylase Conserved gene of unknown function UDP-glucuronosyltransferase UDP-glucuronosyltransferase Kelch repeat-containing protein Cytochrome P450 71A4 Glutaredoxin Conserved gene of unknown function Histone deacetylase Conserved gene of unknown function DNA binding protein 195 ch07 43459160 43462088 35.5573596 ch07 43121565 43126165 1822.03879 ch05 12305546 12312297 194.278989 ch02 19129779 19132621 14.4863439 ch02 19399496 19406825 86.8164023 ch08 49666212 49670996 3143.76344 ch10 52417462 52421546 1205.03273 ch10 52265066 52269176 42.6152142 ch02 37321840 37323602 36.8964391 ch02 37358682 37361593 154.820814 ch02 37334430 37343856 428.019421 ch02 18356198 18362279 81.1761953 ch02 18448855 18454560 2.53688822 ch05 10969744 10971986 3.6592988 ch00 25480003 25482012 107.901702 ch07 46.7373794 ch03 32418043 32419236 69.9302043 ch03 32424153 32424990 111.028626 ch05 34469019 34469360 287.836368 ch03 51885505 51886302 86.7817674 ch03 52659498 52662549 941.848515 ch03 52611505 52613549 88.1647163 ch03 52277098 52280098 194.623074 ch03 52635181 52637606 40.6657927 ch01 74024336 74026082 1438.80234 5054362 5044406 -2.556099578 0.00056274 9.85E-05 2.024471068 -1.463960895 2.35E-05 -1.480598982 0.00027806 0.00067614 1.056891068 -3.478265316 1.69E-12 0.399882921 9.78E-05 -1.418669762 0.00055174 1.888497002 6.32E-06 4.08E-05 4.193957025 1.87E-06 0.766583345 -9.043092808 9.62E-46 5.88E-08 -5.273363555 4.73E-09 -5.283063455 1.828114107 4.12E-06 1.73E-11 2.629794535 0.0005971 -1.338576885 -1.526800361 9.03E-07 -0.780798985 0.00032866 0.00030961 0.949864612 0.610555802 0.00013946 2.11E-07 -0.729883913 0.752117767 0.00034128 -0.973488293 0.00015594 -0.912108514 0.00078342 Table S3.7 (cont’d) PGSC0003DMG400018334 PGSC0003DMG400018381 PGSC0003DMG400018401 PGSC0003DMG400018408 PGSC0003DMG400018423 PGSC0003DMG400018442 PGSC0003DMG400018443 PGSC0003DMG400018492 PGSC0003DMG400018510 PGSC0003DMG400018514 PGSC0003DMG400018535 PGSC0003DMG400018540 PGSC0003DMG400018574 PGSC0003DMG400018578 PGSC0003DMG400018584 PGSC0003DMG400018589 PGSC0003DMG400018605 PGSC0003DMG400018751 PGSC0003DMG400018774 PGSC0003DMG400018791 PGSC0003DMG400018852 PGSC0003DMG400018857 PGSC0003DMG400018894 PGSC0003DMG400018900 PGSC0003DMG400018901 PGSC0003DMG400018903 Conserved gene of unknown function Conserved gene of unknown function Gene of unknown function Zinc finger protein Phytanoyl-CoA dioxygenase domain containing Late blight resistance protein UDP-glucuronosyltransferase Cell division control 20 Aldo/keto reductase ATP binding protein Yellow stripe Nucleic acid binding protein Disease resistance protein R3a Clathrin interactor EPSIN 1 Beta-1,3-glucuronyltransferase Conserved gene of unknown function Binding protein Protein kinase Cell division protease ftsH Protein GIGANTEA Rho GTPase activator Conserved gene of unknown function Gene of unknown function Cc-nbs-lrr resistance protein Phosphoprotein phosphatase Cc-nbs-lrr resistance protein 196 3841902 3844819 4541736 4435486 4843837 4538880 4431043 4839966 ch01 74077032 74083545 710.715467 ch01 74036357 74037420 11.8154871 ch07 48345195 48345749 63.9283543 1813.88984 ch05 1734.29413 ch05 ch05 63.2905176 ch03 32305121 32309574 15.1247975 ch08 255.198765 ch03 46006400 46010878 1595.17538 ch03 45936235 45940161 16.6696337 ch03 45963294 45967167 2768.21932 ch03 45850440 45854076 442.983747 ch11 42735484 42737313 65.7727659 ch11 42423771 42432321 379.515977 ch11 42573210 42578702 504.297233 ch11 42657058 42660736 6.20206367 ch05 10268787 10274376 3437.56849 ch07 2193.69656 ch03 10643807 10646914 913.650072 ch12 54722764 54731694 3409.30058 ch03 38314819 38321395 929.154757 ch03 38472320 38476345 598.704028 ch00 201.360211 ch12 10577577 10582411 305.456885 ch12 10592460 10593287 73.6681995 ch12 10614834 10622662 390.225664 2494232 2501308 1604816 1607351 0.00094325 -0.87765235 1.36E-06 3.766971651 0.00020973 1.229249402 0.0007982 1.9681799 2.43E-08 -0.412453675 2.48E-05 -2.741309142 -1.708543136 0.00018765 -0.797739669 1.74E-09 -0.548215452 0.00025383 3.58E-05 1.1937214 2.71E-07 1.370753786 -1.185545254 7.60E-06 -1.20220278 0.00077144 -0.365889338 0.00040392 -0.903371503 7.72E-06 2.366747931 0.00051052 -0.900534015 0.00012013 0.81223421 0.0009217 -0.785786275 0.00072203 -1.478036887 0.00048179 0.707484605 9.39E-05 1.87E-06 -1.18065591 1.01E-05 1.334847202 -0.888672604 3.44E-12 3.88E-16 -1.059964587 -0.894238912 5.61E-06 Table S3.7 (cont’d) PGSC0003DMG400018925 PGSC0003DMG400019082 PGSC0003DMG400019103 PGSC0003DMG400019157 PGSC0003DMG400019196 PGSC0003DMG400019217 PGSC0003DMG400019232 PGSC0003DMG400019257 PGSC0003DMG400019287 PGSC0003DMG400019337 PGSC0003DMG400019394 PGSC0003DMG400019395 PGSC0003DMG400019408 PGSC0003DMG400019518 PGSC0003DMG400019536 PGSC0003DMG400019634 PGSC0003DMG400019697 PGSC0003DMG400019714 PGSC0003DMG400019813 PGSC0003DMG400019836 PGSC0003DMG400019852 PGSC0003DMG400019881 PGSC0003DMG400019971 PGSC0003DMG400020078 PGSC0003DMG400020118 PGSC0003DMG400020316 Polyphenol oxidase B, chloroplastic Mutt domain protein Zinc binding dehydrogenase SLT1 protein Tropinone reductase AN2 GRAS2 Chloroplast thiazole biosynthetic protein Conserved gene of unknown function Conserved gene of unknown function DNA binding protein Ubiquitin carrier protein WRKY transcription factor Pseudo response regulator Conserved gene of unknown function Autophagy protein Organic anion transporter Tubulin gamma complex-associated protein Gene of unknown function Pentatricopeptide repeat-containing protein Translocon-associated protein, beta subunit JAR1 Circadian clock-associated FKF1 DUF26 domain-containing protein 2 Oxidoreductase Ribosomal RNA methyltransferase 197 6702171 6731003 6282129 6707762 6735863 6284276 ch08 45840965 45842954 223.645008 ch00 34803261 34806222 102.676072 ch00 34332094 34339804 474.261291 ch10 51356007 51358493 6441.21665 ch10 50824924 50827881 72.7586398 ch10 51469785 51470105 29.8782049 ch07 54095486 54098718 510.30254 ch07 54327915 54330135 56233.6923 ch07 53682654 53687336 785.073867 ch09 48639777 48642730 721.511693 98.1840304 ch10 ch10 864.364089 ch10 14.9972615 ch03 55734633 55741670 181.662541 ch03 55998988 56003542 1377.73422 ch11 40758920 40764299 752.229405 ch04 55405970 55410341 1332.11922 ch02 37047957 37057117 1113.57285 ch05 15153263 15156511 171.887227 502.476082 ch02 7673272 ch03 48335188 48338656 68.185689 36.6524245 5782434 ch10 ch01 536380 1817.24922 ch06 58640829 58643258 219.419418 ch06 59193490 59197152 492.987835 ch09 671.622463 317379 325075 7667933 5779667 531784 -2.014810442 0.00059615 1.70E-06 -2.578117625 1.061302093 4.19E-13 0.00081464 0.643602614 9.82E-07 0.895577416 2.506857505 0.00069868 0.90172526 0.00017188 -0.797948523 0.00051665 -0.921307012 1.37E-07 3.26E-06 1.702073195 2.85E-05 -0.920754284 -0.866802801 3.70E-10 2.04E-07 1.973987669 1.333264378 0.00017004 -0.796273049 0.00019225 8.34E-05 -0.272237116 1.06E-05 0.416782495 0.419244964 0.00025767 0.00018717 3.34743554 5.52E-08 -1.108137958 0.923844586 2.35E-05 -1.656738338 1.90E-05 -2.579914643 0.00034176 1.205908837 2.06E-05 6.41E-05 1.258848831 0.306593796 0.00042188 Table S3.7 (cont’d) PGSC0003DMG400020352 PGSC0003DMG400020443 PGSC0003DMG400020502 PGSC0003DMG400020582 PGSC0003DMG400020760 PGSC0003DMG400020844 PGSC0003DMG400020895 PGSC0003DMG400020968 PGSC0003DMG400021079 PGSC0003DMG400021099 PGSC0003DMG400021116 PGSC0003DMG400021131 PGSC0003DMG400021136 PGSC0003DMG400021171 PGSC0003DMG400021194 PGSC0003DMG400021209 PGSC0003DMG400021214 PGSC0003DMG400021216 PGSC0003DMG400021322 PGSC0003DMG400021323 PGSC0003DMG400021344 PGSC0003DMG400021346 PGSC0003DMG400021400 PGSC0003DMG400021459 PGSC0003DMG400021477 PGSC0003DMG400021534 OTU-like cysteine protease family protein Conserved gene of unknown function AP47/50p Selenium-binding protein Ankyrin repeat-containing protein Conserved gene of unknown function UDP-glucose:glucosyltransferase Flavonol 4'-sulfotransferase Potassium channel KLT1 Ubiquitin-protein ligase AAA ATPase Cytochrome P450 Protection of telomeres 1 protein Jumonji domain protein RNA 3' terminal phosphate cyclase Zinc finger protein Nectarin 5 Digalactosyldiacylglycerol synthase 1 BEL29 protein Conserved gene of unknown function Transcription factor Nuclear matrix constituent protein 2 (Fragment) Cc-nbs-lrr resistance protein Cytochrome B5 198 UDP-glucoronosyl/UDP-glucosyl transferase family protein ch02 29135616 29137448 530239 534805 1278048 1286091 3824192 3828008 8751815 8753374 ch09 1072.21877 ch07 50144615 50149480 2306.30577 ch08 914.019667 ch09 59731575 59736411 4604.91646 ch05 33183314 33188813 21.3056712 ch06 783.655231 ch00 24414183 24417600 38.8588669 ch09 345.950383 ch00 35205231 35206661 18.9139506 ch02 29513928 29520655 114.455824 21.275797 ch02 28863321 28865120 1786.75088 ch02 28711736 28714333 133.357644 ch02 29500348 29509451 316.499417 ch02 28850164 28855771 223.99229 ch02 28475473 28481140 1043.83337 ch02 28361993 28368245 2055.94473 ch02 29384162 29385406 68.0767199 4009.07889 ch01 1723.25465 ch01 ch01 718.728642 ch01 169.441583 ch02 42366746 42372993 1524.09761 ch02 17758722 17762087 533.606788 ch02 17659025 17664191 633.253903 ch04 67310831 67318245 267.912694 3061072 2955443 2528264 2499796 3066062 2959120 2537150 2505473 -0.507714385 0.00031623 3.42E-08 -1.251317506 -0.342214057 7.88E-05 1.79E-05 -1.514531165 3.46E-05 1.67434718 -3.90360364 1.32E-13 1.10E-05 -3.979882775 4.91E-05 -1.300063477 1.865725771 3.72E-08 0.00076657 0.564868751 0.00026443 1.792143602 1.75531573 2.25E-05 3.62E-10 1.97862468 4.52E-15 1.197553941 1.719313609 5.71E-07 0.00058193 0.388296103 0.00072203 0.352356771 1.130073288 5.32E-05 0.00075388 0.449806699 3.72E-07 0.938805747 0.598775002 0.00012385 -2.321878354 0.00021729 0.00018355 0.476465346 2.081104602 1.01E-07 1.79E-05 0.820742591 -2.706666347 0.0003091 Table S3.7 (cont’d) PGSC0003DMG400021560 PGSC0003DMG400021636 PGSC0003DMG400021649 PGSC0003DMG400021650 PGSC0003DMG400021680 PGSC0003DMG400021683 PGSC0003DMG400021728 PGSC0003DMG400021787 PGSC0003DMG400021797 PGSC0003DMG400022012 PGSC0003DMG400022062 PGSC0003DMG400022135 PGSC0003DMG400022161 PGSC0003DMG400022172 PGSC0003DMG400022179 PGSC0003DMG400022187 PGSC0003DMG400022206 PGSC0003DMG400022217 PGSC0003DMG400022220 PGSC0003DMG400022239 PGSC0003DMG400022311 PGSC0003DMG400022358 PGSC0003DMG400022371 PGSC0003DMG400022387 PGSC0003DMG400022436 Auxin response factor ARF16 Phosphomannomutase Mitogen-activated protein kinase 4 Conserved gene of unknown function ELMO domain-containing protein 2 E3 ubiquitin-protein ligase RMA1H1 Conserved gene of unknown function UDP-glucuronosyltransferase Conserved gene of unknown function Electron transporter Aspartic proteinase Replicase Conserved gene of unknown function Ccaat-binding transcription factor subunit A Zinc transporter GRAS7 Thioredoxin HCF164, chloroplastic Inducible plastid-lipid associated protein RNA Binding Protein 45 Seed maturation protein Conserved gene of unknown function Conserved gene of unknown function Nucleic acid binding protein DNA mismatch repair protein muts2 Serine/threonine-protein kinase PBS1 199 5682392 5686326 3978738 3936652 3982635 3938256 ch09 1165.89982 ch05 43715384 43719542 1541.94359 ch05 44000845 44007763 1307.01079 ch05 44048984 44051638 646.336054 76.8935067 ch10 ch10 900.402629 ch02 35678544 35681073 210.852562 ch03 32712270 32713950 21.9215023 ch03 32826904 32833426 950.487603 ch02 24525986 24529953 20.1496063 ch02 17231316 17236548 476.270045 ch07 56366456 56370688 695.356621 ch07 55775632 55778464 263.7554 ch07 55488235 55489682 2511.77192 ch07 55406651 55409430 1863.81134 ch07 55324106 55327585 1952.02523 ch07 55108225 55111426 3140.2939 ch07 54846232 54850298 1128.94716 ch07 54800999 54804937 5045.08484 ch07 56446593 56447148 3.86797506 ch07 54953135 54962245 436.044339 ch02 34835090 34840045 493.265606 ch02 34515823 34516551 445.983232 ch02 34080115 34084138 726.513118 ch02 34208173 34212424 54.4104623 0.00064352 0.587341957 9.59E-06 -0.484908362 5.37E-05 0.659754855 1.91E-05 1.632927212 -1.275059862 1.67E-09 -2.283366676 0.00045987 -1.152555756 1.19E-08 -1.564092567 0.00080609 -1.232757189 0.00095478 1.25E-13 -5.480239692 5.75E-06 0.726069304 -1.560193398 7.07E-11 -0.877918941 0.00035692 0.0007278 -1.136673605 0.959624203 1.31E-05 2.92E-06 -0.894161714 3.39E-05 -0.540561771 -2.817588626 1.01E-07 -0.286054026 0.00076657 0.00026928 4.716532779 -0.40679395 0.00075526 1.11E-05 -0.482660538 3.34E-05 -1.321560021 -0.531768752 6.04E-05 6.81E-05 1.371262005 Table S3.7 (cont’d) PGSC0003DMG400022440 PGSC0003DMG400022466 PGSC0003DMG400022469 PGSC0003DMG400022507 PGSC0003DMG400022509 PGSC0003DMG400022740 PGSC0003DMG400022742 PGSC0003DMG400022743 PGSC0003DMG400022744 PGSC0003DMG400022902 PGSC0003DMG400022932 PGSC0003DMG400023164 PGSC0003DMG400023195 PGSC0003DMG400023198 PGSC0003DMG400023365 PGSC0003DMG400023374 PGSC0003DMG400023375 PGSC0003DMG400023391 PGSC0003DMG400023428 PGSC0003DMG400023442 PGSC0003DMG400023465 PGSC0003DMG400023570 PGSC0003DMG400023738 Conserved gene of unknown function S-adenosylmethionine-dependent methyltransferase N-acetyltransferase With no lysine kinase DNA-damage-repair/toleration protein DRT102 APO protein 1, chloroplastic Acetylornithine aminotransferase, mitochondrial Cytochrome P450 Cytochrome P450 Pre-mRNA-splicing factor cwc-22 Na+/h+ antitransporter Translation initiation factor DEAD-box ATP-dependent RNA helicase 39 Leucine-rich repeat-containing protein Flowering locus T protein Glycine-rich cell wall structural protein 1 Histone H2B Ferredoxin-3, chloroplast TSC13 protein DDT domain-containing protein Phospholipase Conserved gene of unknown function Conserved gene of unknown function 200 ch02 34120454 34122690 1463.53502 ch01 75661708 75664683 617.430777 ch01 75643341 75644021 54.4888209 ch01 75508655 75512302 399.649718 ch01 75474528 75477100 1241.5181 ch08 53933044 53937976 694.275282 ch08 54049949 54053895 1983.99371 ch08 54054249 54056429 549.110676 ch08 54066816 54068358 113.092433 ch03 61394725 61396307 31.8178467 ch11 30761643 30770791 1275.75728 ch02 18878488 18883269 5325.98484 ch12 54853693 54861561 2259.86616 ch00 21610499 21613702 651.15574 ch05 51319128 51320774 81.7220532 ch05 51177306 51178496 37.2879556 ch05 51163436 51164074 3782.02568 ch05 50956295 50959091 1070.17984 ch05 50250125 50254609 2307.93298 ch05 51967262 51976469 424.48641 ch05 51516702 51518689 2238.61575 ch04 33.557655 ch10 57362614 57363393 480.392004 8343130 8345629 5.17E-09 -1.192526687 -0.584911965 2.66E-05 -1.138540567 0.00028226 -0.407061246 0.00022091 -0.856484687 0.00021434 -0.753123464 1.03E-05 0.0001525 -1.056049762 5.52E-07 -2.551116612 -2.458015261 2.93E-06 -0.953492337 0.00052361 -0.626490059 0.00032866 -0.809584112 0.00068394 2.85E-09 -0.603033772 4.94E-06 0.872140879 2.431237133 0.00066895 0.0001268 2.825514793 -0.879029544 2.23E-05 -0.363454257 0.00089152 5.88E-07 -0.908682808 0.00022224 0.432480553 -0.441385458 2.62E-06 0.00035614 1.744989621 -1.1922751 7.45E-05 Table S3.7 (cont’d) PGSC0003DMG4000238 PGSC0003DMG4000238 PGSC0003DMG4000238 PGSC0003DMG4000238 PGSC0003DMG4000238 PGSC0003DMG4000239 PGSC0003DMG4000239 PGSC0003DMG4000241 PGSC0003DMG4000241 PGSC0003DMG4000241 PGSC0003DMG4000241 PGSC0003DMG4000242 PGSC0003DMG4000242 PGSC0003DMG4000242 PGSC0003DMG4000243 PGSC0003DMG4000243 PGSC0003DMG4000244 PGSC0003DMG4000244 PGSC0003DMG4000244 PGSC0003DMG4000245 36 47 50 64 69 42 78 02 76 81 92 61 74 81 17 36 78 88 94 60 SAM (And some other nucleotide) binding motif Methyltransferase small 2,4-dienoyl-CoA reductase 2-oxoglutarate-dependent dioxygenase Hcr2-0B Cf-2.2 Sorting nexin-4 Mevalonate disphosphate decarboxylase F-box family protein Carbohydrate transporter CER6 MurB reductase Conserved gene of unknown function Cytochrome P450 monooxygenase Gamma aminobutyrate transaminase isoform2 Storage protein Conserved gene of unknown function Calmodulin-binding protein IS10 transposase Phosphoglycerate mutase Formin 20 201 ch1 2 ch1 2 ch1 2 ch1 2 ch1 2 ch0 6 ch1 1 ch0 4 ch0 2 ch0 2 ch0 2 ch0 9 ch1 2 ch1 2 ch0 6 ch0 3 ch0 3 ch0 3 ch0 3 ch0 3 6071678 6071962 275.39435 6083884 6083992 50.936565 6078395 6078637 39.885359 6069448 6069746 86.474723 6065680 6065810 3043597 3044343 5283304 5999627 5290904 6000067 67.708723 2716.2916 2084.9529 1408.2795 2167015 2167324 505.06022 2189281 2189547 1235.3185 2195447 2195883 313.23893 1227226 1233877 5853429 5858832 5776516 5577723 5780741 5578414 2119237 2119430 1243.5201 4831.9921 31825.987 902.42892 266.49730 5389729 5390116 866.14015 5407251 5407349 369.73965 5426659 5426878 762.58964 5561506 5562439 1707.8804 0 1 6 9 7 7 0 8 0 1 4 0 8 6 1 5 0 5 4 0 5 9 0 1 8 4 1 3 3 1 0 1 6 3 3 2 7 7 2 7 1 1 3 8 8 2 7 8 6 9 - 0.712928208 1.61E-10 2.147480792 0.0001217 1.861270227 2.970185815 4.981025488 0.498207211 1.244143747 0.273291011 2.05E-06 7.65E-19 4.48E-31 0.0001807 0.0003151 0.0006019 3 6 9 -1.37003316 3.38E-13 1.631333881 3.72E-08 0.883476654 -0.53523602 1.652512078 1.559959321 0.288379637 0.948482879 1.323941609 1.612511472 -2.22706933 0.295521762 7.22E-07 0.0002947 0.0001555 2 5 3.62E-09 4.99E-05 0.0006761 4 8.72E-05 0.0001341 3 1.03E-10 0.0004218 8 - - - - - - - - - Table S3.7 (cont’d) PGSC0003DMG400024612 PGSC0003DMG400024631 PGSC0003DMG400024764 PGSC0003DMG400024784 PGSC0003DMG400024843 PGSC0003DMG400024942 PGSC0003DMG400025009 PGSC0003DMG400025033 PGSC0003DMG400025112 PGSC0003DMG400025122 PGSC0003DMG400025144 PGSC0003DMG400025149 PGSC0003DMG400025228 PGSC0003DMG400025299 PGSC0003DMG400025328 PGSC0003DMG400025342 PGSC0003DMG400025407 PGSC0003DMG400025414 PGSC0003DMG400025433 PGSC0003DMG400025473 PGSC0003DMG400025480 PGSC0003DMG400025499 PGSC0003DMG400025595 PGSC0003DMG400025610 PGSC0003DMG400025612 Uridine cytidine kinase I MtN3/saliva family protein Conserved gene of unknown function F-box/kelch-repeat protein LEA-18 Rnf5 Conserved gene of unknown function SP1L DnaJ 1895871 1900303 1737542 1443538 1897945 1903287 1738700 1454045 Rubisco subunit binding-protein beta subunit, rubb Conserved gene of unknown function Conserved gene of unknown function ch03 54627728 54639244 1744.19397 ch03 55057180 55060002 1804.11373 ch01 77612333 77615421 568.315425 ch01 77931865 77933839 1493.07991 ch04 58561673 58563662 3184.28907 ch02 43123839 43124291 76.6442304 500.664606 ch10 176.933733 ch10 ch05 153.558196 ch05 1115.40383 ch02 25351803 25352365 203.553456 ch02 25477991 25487584 731.047659 957.576319 ch08 Alpha-galactosidase/alpha-n-acetylgalactosaminidase ch04 1973.60997 ch03 53276654 53283732 7339.40338 ch03 53203041 53205307 9268.10512 ch05 16698981 16707877 975.6162 ch05 16834679 16838305 120.449769 ch04 411.614084 ch02 23419179 23426233 1581.74039 ch02 23414636 23415103 2819.76377 ch07 47049000 47054125 878.12785 ch02 37305234 37305545 7.16641295 ch05 311.980796 217.478818 ch05 Receptor protein kinase Glutamate receptor 3 plant 8057030 3887194 8065286 3891481 5692338 5765092 5694725 5768126 Conserved gene of unknown function CBS domain containing protein Kinase Sucrose transporter Gamma aminobutyrate transaminase isoform2 Zinc finger protein CONSTANS-LIKE 15 Plasma membrane ATPase 1 Homeobox Pyruvate dehydrogenase 5511964 5516399 Myb-like DNA-binding protein 202 0.0001301 -0.879228497 4.84E-06 -1.40987244 0.00012672 1.52562084 -1.847508206 4.46E-07 -2.396846116 0.00059615 2.454164595 0.0008286 1.84E-06 0.922049401 2.41E-05 1.07575989 0.894934833 2.06E-05 -1.456484639 0.00010862 -1.255251399 0.00035035 -0.499382016 0.00063344 1.33E-06 -1.323713457 -1.667588291 2.41E-07 -0.447098959 0.00017686 0.00016363 0.891647963 2.11E-05 1.328022671 -2.120757437 1.02E-05 7.95E-11 -4.698470083 -1.532319541 3.29E-06 -0.761625793 0.00034176 1.22E-06 -0.901999791 1.71E-10 5.120076953 0.358970515 0.00020842 0.00051665 0.794464785 Table S3.7 (cont’d) PGSC0003DMG400025655 PGSC0003DMG400025656 PGSC0003DMG400025722 PGSC0003DMG400025843 PGSC0003DMG400025887 PGSC0003DMG400025890 PGSC0003DMG400025927 PGSC0003DMG400025957 PGSC0003DMG400025980 PGSC0003DMG400025988 PGSC0003DMG400026112 PGSC0003DMG400026169 PGSC0003DMG400026181 PGSC0003DMG400026221 PGSC0003DMG400026241 PGSC0003DMG400026267 PGSC0003DMG400026275 PGSC0003DMG400026303 PGSC0003DMG400026354 PGSC0003DMG400026375 PGSC0003DMG400026417 PGSC0003DMG400026502 PGSC0003DMG400026554 PGSC0003DMG400026566 PGSC0003DMG400026569 PGSC0003DMG400026631 Ankyrin repeat-containing protein Conserved gene of unknown function Oligouridylate binding protein Anthranilate N-benzoyltransferase protein Thioredoxin M3, chloroplastic NADPH oxidoreductase Conserved gene of unknown function Conserved gene of unknown function Conserved gene of unknown function Non-specific lipid-transfer protein ReMembR-H2 protein JR702 DNA binding protein Transcription factor Major pollen allergen Ory s 1 MybSt1 Gene of unknown function O-methyltransferase Transporter Conserved gene of unknown function Electron transporter UPA22 Vacuolar H+-ATPase A2 subunit isoform Conserved gene of unknown function Gene of unknown function Conserved gene of unknown function Malonyltransferase 203 ch02 27701384 27703638 24.2519504 ch02 27695689 27696882 22.6578078 ch07 39571078 39578237 2654.83959 ch01 83622408 83625445 13.7292094 ch01 84401258 84405639 1711.91061 ch01 84539605 84558799 1681.0612 ch01 85243257 85245511 2613.88974 ch01 70563912 70566970 278.597693 ch01 69887660 69890149 1137.50102 ch01 69558764 69559120 2.23864579 ch06 46552396 46558438 781.810294 ch07 47281684 47282147 258.83303 ch07 47790598 47791812 2326.0825 ch08 51064955 51067304 343.923931 ch08 51486596 51489777 1764.2392 ch08 51489971 51491072 95.9686721 84.0833438 ch08 ch08 217.658185 ch11 40455628 40457217 1283.77092 ch10 24275922 24281820 614.061944 ch09 59174648 59175464 13883.864 ch06 48043865 48051749 618.107536 ch02 24825722 24828991 39.6361398 ch02 25020908 25021558 602.238922 ch02 25068355 25072758 33.3846767 ch10 827.524729 1794626 1920720 1796740 1927912 4252547 4254216 3.74E-08 2.86606308 1.998814706 0.00096312 -0.400654992 0.00067163 6.07E-10 -3.472855132 1.47E-22 -1.663881746 -0.338777367 8.69E-05 3.75E-06 -2.84114339 6.60E-05 1.05492457 -1.071865412 5.06E-07 -3.798827428 0.00074288 -0.596659432 0.00077607 2.555914166 1.44E-07 4.12E-06 1.419184586 1.75E-05 5.466043041 -0.326611997 0.0006206 -0.813153718 0.0008168 -8.849481964 0.00065414 -0.493635851 9.84E-05 1.259946435 4.73E-06 -0.532841568 0.00078759 -3.322066396 4.17E-09 -0.90939456 0.0001268 -1.461473316 0.00047662 1.10356933 4.20E-09 1.13E-08 4.199772413 1.056604243 0.00072115 Table S3.7 (cont’d) PGSC0003DMG400026779 PGSC0003DMG400026784 PGSC0003DMG400026867 PGSC0003DMG400026920 PGSC0003DMG400026995 PGSC0003DMG400027005 PGSC0003DMG400027017 PGSC0003DMG400027062 PGSC0003DMG400027119 PGSC0003DMG400027120 PGSC0003DMG400027130 PGSC0003DMG400027192 PGSC0003DMG400027207 PGSC0003DMG400027234 PGSC0003DMG400027253 PGSC0003DMG400027345 PGSC0003DMG400027392 PGSC0003DMG400027403 PGSC0003DMG400027423 PGSC0003DMG400027428 PGSC0003DMG400027462 PGSC0003DMG400027463 PGSC0003DMG400027477 PGSC0003DMG400027519 PAS/LOV protein A Inorganic pyrophosphatase LescPth4 ProFAR isomerase associated family protein ATM (ATAXIA-TELANGIECTASIA MUTATED) Phototropic-responsive NPH3 family protein Fructokinase 2 Conserved gene of unknown function Ferritin Multiple inositol polyphosphate phosphatase 1 Phosphoinositide-specific phospholipase C Zinc ion binding protein Conserved gene of unknown function Conserved gene of unknown function Gene of unknown function Gene of unknown function 2-oxoglutarate-dependent dioxygenase ThiF family protein Hyoscyamine 6-dioxygenase Sigma factor sigb regulation protein rsbq Anthranilate N-benzoyltransferase protein Anthranilate N-benzoyltransferase protein Xylose isomerase Conserved gene of unknown function 204 9560447 9567505 ch01 43192763 43196447 5257.454 ch10 31204939 31211655 1398.69093 ch05 201.27484 ch06 53694852 53700000 170.806348 ch06 52340043 52344326 1063.41914 ch06 53994547 53996424 9.10710248 ch06 53746105 53749420 16340.4475 ch06 52907944 52913908 1154.78941 ch05 47747602 47751071 1673.51073 ch05 47752726 47761321 823.090941 ch05 48052466 48057779 3752.24928 ch05 48192235 48199860 669.842056 ch05 48629307 48632235 782.168699 ch04 1243.99931 ch04 10348636 10351120 81.1990741 ch11 127.216421 ch11 43707201 43714692 1197.72768 ch11 44069117 44084483 516.48439 ch05 18608237 18617613 860.272314 ch05 19127293 19129332 156.953395 ch07 311.433778 3.4142854 ch07 ch07 3279.67178 ch01 74807891 74812615 1078.34374 2312501 2324114 2306278 2311098 2322903 2301043 4658595 4658971 9776232 9783559 -1.490634999 5.69E-05 -2.172234177 0.00079271 -1.079550568 2.97E-06 -0.777907662 0.00054969 -0.567423778 0.00048358 2.945094132 0.00011637 1.017754001 0.0001884 -0.373837573 0.00019313 -0.938791722 0.00065907 0.688510386 5.88E-07 -0.718927109 0.00098354 0.671571812 0.00015419 -1.341509377 0.00061221 -1.269782328 5.53E-06 -1.048780431 0.00036137 0.00091477 1.710799726 9.73E-05 0.850610943 -0.296838731 0.0006206 -0.575074708 0.00072168 1.18E-05 1.573347381 -3.326633761 2.07E-09 -3.445493224 0.00020309 -1.630083101 0.00043191 0.693514319 4.97E-06 Table S3.7 (cont’d) PGSC0003DMG400027520 PGSC0003DMG400027660 PGSC0003DMG400027684 PGSC0003DMG400027750 PGSC0003DMG400027766 PGSC0003DMG400027774 PGSC0003DMG400027779 PGSC0003DMG400027809 PGSC0003DMG400028009 PGSC0003DMG400028046 PGSC0003DMG400028097 PGSC0003DMG400028160 PGSC0003DMG400028175 PGSC0003DMG400028182 PGSC0003DMG400028222 PGSC0003DMG400028260 PGSC0003DMG400028331 PGSC0003DMG400028415 PGSC0003DMG400028421 PGSC0003DMG400028438 PGSC0003DMG400028461 PGSC0003DMG400028541 PGSC0003DMG400028543 PGSC0003DMG400028663 PGSC0003DMG400028710 PGSC0003DMG400028789 Periplasmic beta-glucosidase Gene of unknown function C-8,7 sterol isomerase Hsc70 Generic methyltransferase F-box family protein Flavonol 3-sulfotransferase Electron transporter Conserved gene of unknown function Conserved gene of unknown function Short-chain type alcohol dehydrogenase F-box protein Cytochrome P450 76A2 Aquaporin TIP1 3 KED Conserved gene of unknown function Zeatin O-glucosyltransferase Conserved gene of unknown function Ubiquitin-protein ligase Conserved gene of unknown function Serine-threonine protein kinase, plant-type Polygalacturonase Gene of unknown function Conserved gene of unknown function Ammonium transporter 1 member 2 Conserved gene of unknown function 205 7034956 7037871 ch01 74764148 74770470 462.699424 ch07 51856695 51857749 50.6003098 ch06 57946272 57948790 4515.87505 ch04 1913.81443 ch01 71142591 71147054 138.785846 ch01 71181237 71185583 402.675583 ch01 71113182 71114219 133.52673 ch11 24838193 24847707 1007.86406 ch09 44401803 44408744 2389.96759 ch03 39592411 39594799 13.1914112 105.89566 ch10 57044476 57051061 ch10 55890926 55892317 39.376409 ch10 55479295 55481138 388.936189 ch10 55272336 55274167 21.8550592 ch10 56404784 56410132 1578.94556 ch10 55659728 55664320 75.9233872 1844.33557 ch05 239420 ch02 31431149 31434858 17.484824 ch02 31596041 31597580 167.476742 ch02 31830269 31834155 11.2342876 ch02 31441857 31444742 8.76822504 ch06 49751327 49751976 20.5800516 619.64606 ch06 49714457 49715456 ch11 6739100 133.449799 ch04 47052454 47055928 1136.9675 ch06 50850881 50851914 76.8169094 237549 6737028 -1.214431998 0.00019146 1.96E-07 3.953234628 -1.39792448 5.53E-09 0.00028367 1.969141904 3.23E-05 1.469204575 -0.5309001 0.00089152 -2.392468566 0.00029074 -0.590373341 2.06E-12 -0.499484617 0.00058193 0.0005988 -2.242987128 0.604231679 0.00012402 -1.381469934 0.00095416 1.118493171 1.01E-05 -3.866547623 0.00019541 0.952924407 0.00053823 0.00076926 0.824354768 -2.31614831 0.00053823 -2.013213421 0.00032866 7.72E-06 1.816314081 5.265920735 3.63E-07 -1.919185953 0.00020309 -2.691162024 0.00021434 2.12E-07 4.727315868 2.426027088 9.34E-06 0.00089152 1.842087077 -3.237023209 3.38E-13 Table S3.7 (cont’d) PGSC0003DMG400028815 PGSC0003DMG400028818 PGSC0003DMG400028851 PGSC0003DMG400028886 PGSC0003DMG400028985 PGSC0003DMG400028992 PGSC0003DMG400029195 PGSC0003DMG400029201 PGSC0003DMG400029269 PGSC0003DMG400029313 PGSC0003DMG400029390 PGSC0003DMG400029405 PGSC0003DMG400029522 PGSC0003DMG400029576 PGSC0003DMG400029618 PGSC0003DMG400029637 PGSC0003DMG400029664 PGSC0003DMG400029724 PGSC0003DMG400029748 PGSC0003DMG400029756 PGSC0003DMG400029857 PGSC0003DMG400029895 PGSC0003DMG400029934 PGSC0003DMG400029937 Reticulon family protein Transcription factor Translation initiation factor eif-2b alpha subunit Gene of unknown function Calcium ion binding protein Adenylate kinase 1 chloroplast Ferritin Sesquiterpene synthase 2 Ankyrin repeat-containing protein R2 protein Cysteine protease Disease resistance protein RPM1 Clathrin assembly protein Polyphenol oxidase 3-ketoacyl CoA thiolase 2 Peptidoglycan binding domain containing protein Non-lysosomal glucosylceramidase PHO2 Auxin-induced protein 5NG4 Conserved gene of unknown function Strictosidine synthase Importin alpha Sphingolipid delta-8 desaturase ZIP family metal transporter 206 6028478 6077490 6033927 6085194 2413.62975 ch12 ch12 339.675069 ch12 53051863 53056164 1118.20379 ch09 26418762 26419454 14.5088694 ch01 67013023 67013750 418.173944 ch04 32935520 32945039 546.83085 ch09 10548979 10552001 112.797127 ch00 23638976 23642120 95.7570723 ch12 58261858 58266713 2403.22623 ch12 57509849 57513256 192.392004 ch12 57732653 57738359 6553.11522 ch12 57474535 57477976 386.792251 ch04 2276.77063 ch08 45676042 45677832 569.174384 ch09 58286615 58291622 5732.90401 ch09 58731114 58739212 979.834172 ch09 58747188 58756210 700.113447 ch02 33974644 33983840 7367.37451 ch01 81522631 81525449 67.2492802 ch01 81463598 81465776 579.900852 ch10 58200469 58203988 2951.91651 ch08 20459521 20466178 3253.05798 ch08 35730232 35732357 455.207409 ch08 35990888 35994505 787.180556 2500034 2506737 4.60E-06 -0.846995162 2.53E-05 -2.856851577 0.00037317 0.396958254 6.22E-10 2.818512274 1.089665597 0.00033475 -0.331113331 0.00088033 -2.015847705 4.49E-06 -4.514680887 0.00032682 -0.78249611 2.54E-05 6.04E-05 -1.098334869 -1.189294063 5.58E-19 -1.223037594 0.00050557 0.486746068 0.00014031 -3.892567121 0.00022957 -0.628282007 0.00025125 3.20E-05 -0.711904718 0.00023506 0.43871909 1.079733756 9.11E-06 0.00012501 0.907966959 6.37E-05 0.554443444 0.682937009 0.00074288 -0.262321985 0.00085671 2.70E-07 -2.304361157 0.84575045 2.06E-07 Table S3.7 (cont’d) PGSC0003DMG400029940 PGSC0003DMG400029964 PGSC0003DMG400030008 PGSC0003DMG400030017 PGSC0003DMG400030082 PGSC0003DMG400030097 PGSC0003DMG400030170 PGSC0003DMG400030172 PGSC0003DMG400030232 PGSC0003DMG400030234 PGSC0003DMG400030336 PGSC0003DMG400030352 PGSC0003DMG400030369 PGSC0003DMG400030426 PGSC0003DMG400030454 PGSC0003DMG400030460 PGSC0003DMG400030473 PGSC0003DMG400030574 PGSC0003DMG400030575 PGSC0003DMG400030590 PGSC0003DMG400030635 PGSC0003DMG400030640 PGSC0003DMG400030662 PGSC0003DMG400030731 PGSC0003DMG400030804 Conserved gene of unknown function Phospholipase A2 Low-temperature-induced 65 kDa protein Vacuolar sorting receptor protein PV72 Primary amine oxidase Gamma-glutamyl transferase Stem-specific protein TSJT1 Aspartic proteinase oryzasin-1 Conserved gene of unknown function SAUR family protein IMP dehydrogenase/GMP reductase Flavoprotein wrbA Pyruvate decarboxylase isozyme 2 17.6 kD class I small heat shock protein UDP-glucosyltransferase Conserved gene of unknown function Ubiquitin-conjugating enzyme E2-17 kDa Fiber protein Fb34 Conserved gene of unknown function Phosphatidic acid phosphatase alpha DNA ligase Conserved gene of unknown function Glycosyl hydrolase family 3 protein Metallocarboxypeptidase inhibitor UDP-glucose:glucosyltransferase 207 8357046 7837855 8361081 7847421 ch08 36094738 36100762 448.571574 71.5013573 ch07 ch01 51.4352988 ch09 18598486 18607646 5295.09199 ch09 53558075 53561051 220.538932 ch05 46888513 46893536 986.927599 ch09 54527563 54530999 3595.72217 ch09 54513083 54516979 635.637138 ch06 38882037 38884217 1402.15219 ch06 38928224 38928996 257.872866 ch06 56873853 56879282 1065.03734 ch06 57148354 57152102 1027.99501 6.2015224 ch06 57330850 57333771 ch06 56893292 56894077 450.8051 ch06 57477053 57479399 139.516893 ch06 57532764 57535052 268.698065 ch06 57684685 57689801 3183.61799 ch05 660.305344 ch05 2903.42071 ch02 41537051 41541585 65.9496169 ch02 41644298 41653900 806.316412 ch02 41735967 41736807 28.0395224 ch02 42025809 42030051 735.149971 ch07 4053.83153 ch04 63830969 63832893 312.610202 3198624 3200152 3446147 3430984 3452767 3438165 2.34E-08 -1.826319942 6.21E-08 -4.453450602 3.48E-07 3.152584254 -1.116628801 2.27E-05 -1.660490618 0.00078759 -0.453271134 0.00014946 3.52E-06 -1.093578141 0.0006062 -2.436736354 0.46947494 1.79E-05 0.00021434 0.839963076 0.00015173 0.950860666 1.462506829 1.98E-06 0.00058193 3.872334741 1.29216275 7.37E-06 -0.939224099 0.00092072 -0.65978974 0.00091477 -0.487737013 0.00045646 0.50106068 0.00043191 0.00072606 0.480270262 2.01286097 9.85E-05 -0.753044741 0.00023506 3.733210812 7.11E-10 -0.799101332 0.00027289 -1.858222196 1.00E-07 1.24E-05 -2.465573198 Table S3.7 (cont’d) PGSC0003DMG400030823 PGSC0003DMG400030891 PGSC0003DMG400030902 PGSC0003DMG400030918 PGSC0003DMG400031041 PGSC0003DMG400031053 PGSC0003DMG400031087 PGSC0003DMG400031169 PGSC0003DMG400031201 PGSC0003DMG400031212 PGSC0003DMG400031311 PGSC0003DMG400031342 PGSC0003DMG400031399 PGSC0003DMG400031743 PGSC0003DMG400031753 PGSC0003DMG400031772 PGSC0003DMG400031818 PGSC0003DMG400031834 PGSC0003DMG400032131 PGSC0003DMG400032137 PGSC0003DMG400032151 PGSC0003DMG400032155 PGSC0003DMG400032157 PGSC0003DMG400032159 PGSC0003DMG400032181 Glycosyltransferase Hydrolase, hydrolyzing O-glycosyl compounds Dicyanin Dicyanin D-cysteine desulfhydrase Tubulin-specific chaperone B UPF0497 membrane protein 11 Conserved gene of unknown function Chloroplast lumen common family protein Nucellin F-Box protein Subtilase UDP-arabinose 4-epimerase 1 Conserved gene of unknown function NAC domain protein Gene of unknown function TPR Domain containing protein Protein SSM1 Maintenance of killer 16 (Mak16) protein Dead box ATP-dependent RNA helicase U box Lipoxygenase Oxidoreductase, 2OG-Fe(II) oxygenase family protein Dynein light chain type 1 family protein Conserved gene of unknown function 208 100.90027 ch04 63872817 63874618 960.412062 4101254 ch07 13.3907014 ch07 4397393 ch07 52.1437623 4387700 ch03 48900767 48907402 659.172622 ch03 48785885 48793264 528.087347 ch07 51525374 51527164 736.488714 ch01 68949977 68950807 305.252039 ch04 54551048 54555727 59.61239 ch06 17390768 17394217 1640.42751 ch03 37384487 37387419 57.6932696 ch03 31730939 31736141 119.038483 ch02 14373865 14379239 725.671254 ch03 47632362 47632668 2.67959152 ch02 33210830 33214813 84.6655523 ch02 33109327 33113214 99.2549162 ch09 52902467 52905497 10.928236 ch09 53402615 53405289 554.256908 1580.11757 ch01 1222.42132 ch01 ch01 403.512785 1761.92268 ch01 165.078459 ch01 ch01 168.541799 337.064884 ch01 1404111 1493275 1935765 2146062 2263019 1024380 1513966 4096440 4395490 4385448 1399692 1486493 1929493 2132539 2261123 1021496 1510329 -1.556150025 0.0006206 -1.295804958 0.00016412 3.164623983 4.91E-05 1.65E-05 2.623428062 -1.206701824 3.75E-06 -0.561484904 0.00044205 0.699417704 2.05E-05 -0.514729781 0.00049543 -0.981947522 5.15E-05 1.80E-05 -0.758759853 2.84E-05 -2.286980974 -4.1267085 1.24E-06 -0.679085304 0.00013338 1.16E-06 4.20506577 2.787300913 7.32E-05 -1.910496269 0.00022091 -3.385689347 0.00019313 -1.34926236 9.35E-05 -0.674964096 0.00052205 7.72E-06 -0.344731203 -1.141459837 8.58E-09 3.30E-15 -4.092984375 -5.080966669 1.64E-11 -1.094773503 0.00030241 2.15E-12 -1.205497813 Table S3.7 (cont’d) PGSC0003DMG400032183 PGSC0003DMG400032246 PGSC0003DMG400032793 PGSC0003DMG400033065 PGSC0003DMG400033116 PGSC0003DMG400033171 PGSC0003DMG400033357 PGSC0003DMG400033569 PGSC0003DMG400033589 PGSC0003DMG400033688 PGSC0003DMG400033690 PGSC0003DMG400033903 PGSC0003DMG400033905 PGSC0003DMG400033906 PGSC0003DMG400033907 PGSC0003DMG400034106 PGSC0003DMG400034672 PGSC0003DMG400035253 PGSC0003DMG400035666 PGSC0003DMG400035689 PGSC0003DMG400037262 PGSC0003DMG400037286 PGSC0003DMG400037729 PGSC0003DMG400039599 PGSC0003DMG400040038 PGSC0003DMG400040145 Conserved gene of unknown function Endonuclease/exonuclease/phosphatase family protein Heat stress transcription factor HSFA9 HIPL1 protein Adenylate isopentenyltransferase Cytochrome P450 Gene of unknown function NPH3 (NON-PHOTOTROPIC HYPOCOTYL 3) Periaxin Tartrate-resistant acid phosphatase type 5 Conserved gene of unknown function SGRP-1 protein Isoflavone reductase homolog Isoflavone reductase homolog Isoflavone reductase homolog Conserved gene of unknown function Glucosyl/glucuronosyl transferases Gene of unknown function UDP-glucosyltransferase Flavonoid glucoyltransferase UGT73N1 Mitochondrial deoxynucleotide carrier Ferredoxin II 2,4-dienoyl-CoA reductase Gene of unknown function Gene of unknown function Gene of unknown function 209 1546146 1542712 ch01 2409.71682 ch09 54023125 54028439 367.540073 ch07 36269190 36271391 126.28958 ch06 51998976 52003557 28.7635821 ch01 61389336 61393183 154.376398 ch10 57142011 57143646 55.8041964 ch00 36044744 36045905 15.2118211 ch01 82314789 82318067 2385.58328 ch01 82360337 82361579 38.8162394 ch03 48558976 48561647 265.975785 ch03 48533468 48536913 1224.82177 ch10 44144168 44145232 19598.9747 ch10 44261999 44265327 5.79736691 ch10 44285130 44292822 78.1581048 ch10 43998825 44000679 2.19089288 9.1261221 ch12 39706880 39707286 ch11 4865229 14.2425847 ch10 46075172 46075672 7.23712395 ch07 41165973 41167403 1694.69725 ch06 53529488 53530984 779.04553 ch12 60263725 60265664 107.211766 ch00 22126282 22126596 918.094542 ch12 60926766 60930734 20.3131423 ch00 27.8217381 ch10 53718625 53719008 3.31498074 ch05 16430600 16432721 7.68996598 4864096 5180389 5181182 6.18E-05 -0.930384806 2.89E-07 0.770221356 7.94E-05 2.325500209 0.0006992 -1.536058364 0.0004603 1.249177717 2.98E-05 1.402902725 2.90E-05 -1.74605401 3.24E-07 -0.762350045 0.00031339 2.010763354 6.60E-05 2.189761658 9.07E-07 -0.715568069 5.61E-05 -1.022623046 2.85E-05 -3.565717661 2.60E-06 -2.76447457 1.33E-06 -4.798087181 0.00067868 1.708240079 3.39E-05 -3.942323296 0.00087183 1.964409932 -0.527244791 0.00068172 9.67E-08 -1.994055619 -0.562114507 0.0008842 -1.842669717 0.00098853 1.73E-21 -4.946861276 4.096809685 2.31E-09 6.97E-06 4.215409264 2.771703032 1.90E-05 Table S3.7 (cont’d) PGSC0003DMG400041 007 PGSC0003DMG400042 385 PGSC0003DMG400042 PGSC0003DMG400045 914 090 Poly(A)-specific ribonuclease LINE-type retrotransposon LIb DNA, complete sequence, Insertion at the S11 site Gene of unknown function Pentatricopeptide repeat-containing protein PGSC0003DMG400045 350 Galactose-binding like PGSC0003DMG400046 PGSC0003DMG400046 107 334 PGSC0003DMG401001 521 PGSC0003DMG401002 119 PGSC0003DMG401002 PGSC0003DMG401002 122 164 PGSC0003DMG401002 270 PGSC0003DMG401002 683 PGSC0003DMG401003 142 2,4-dienoyl-CoA reductase Conserved gene of unknown function Rab escort protein Conserved gene of unknown function Multidrug resistance pump Glutathione S-transferase Mutt domain protein Heat stress transcription factor A3 Cytochrome P450 210 ch1 2 ch0 1 ch0 2 ch1 1 ch0 2 ch1 2 ch0 3 ch0 2 ch0 4 ch0 4 ch0 9 ch0 8 ch0 9 ch0 3 4079219 8 4079271 9 7.3947492 2 7383523 3 7383753 6 10.829226 4 2.34161622 23.4323105 2103273 2103336 77.384691 9.20577499 3981238 3981413 272.30153 2804233 3 2804310 3 104.33598 7 6093218 6093327 6.8838250 5.48859145 5715534 5715595 97.145486 0 5 0 6 2 3 3 1 1 1 3 8 - 7 - 5 - 5 - - 2 - 5 - 4 - 7 - 6 - 9 - 2 - 1 0.91855638 1.06918544 0.84303885 0.60407896 0.54115114 2.30624149 2.78970498 1.42800790 0.64048172 0.0001864 5 1.12E-12 1.07E-41 6.33E-05 2.41E-07 3.31E-11 1.07E-06 0.0001837 9 0.0003569 2 0.0005166 3 5 1.97E-07 0.0002849 1 2.59E-08 0.64461098 0.0009635 3853673 5 3854433 6 1093.285 6450002 9 6450439 6 254.32850 2 6444862 2 6445343 2 1035276 1036990 872473 876174 3849902 3856182 335.42447 126.77295 9 51.459622 3 519.43615 9 4704364 8 4704871 9 1068.6087 Table S3.7 (cont’d) PGSC0003DMG401003592 PGSC0003DMG401003606 PGSC0003DMG401004726 PGSC0003DMG401004862 PGSC0003DMG401005047 PGSC0003DMG401006174 PGSC0003DMG401006926 PGSC0003DMG401007970 PGSC0003DMG401010918 PGSC0003DMG401012406 PGSC0003DMG401012961 PGSC0003DMG401013152 PGSC0003DMG401014186 PGSC0003DMG401015244 PGSC0003DMG401015669 PGSC0003DMG401016370 PGSC0003DMG401016434 PGSC0003DMG401017626 PGSC0003DMG401017944 PGSC0003DMG401020453 PGSC0003DMG401020557 PGSC0003DMG401021786 PGSC0003DMG401021988 PGSC0003DMG401021989 PGSC0003DMG401023866 PGSC0003DMG401023870 Phospholipase D delta isoform 1a Conserved gene of unknown function Hcr2-0B Gene of unknown function Receptor protein kinase Plant synaptotagmin E2 protein isoform 5 Serine/threonine-protein kinase Conserved gene of unknown function Protein yippee Aspartic proteinase nepenthesin-1 Conserved gene of unknown function Chaperone protein DNAj Conserved gene of unknown function Glucan endo-1,3-beta-glucosidase Lipase Conserved gene of unknown function Alpha-amylase Gene of unknown function Cytochrome P450 TatD DNase domain-containing deoxyribonuclease Gene of unknown function Sodium/hydrogen exchanger Pax transcription activation domain interacting protein Hcr2-p4.1 Cf-2.1 211 2325647 2333359 ch02 38969569 38972702 1237.29577 ch02 40234806 40235963 27.7350309 ch12 60512756 60515575 30.436269 ch06 48494672 48495794 70.5157778 ch03 22.4901103 ch01 87454143 87463868 3611.29476 ch02 26773990 26778122 698.087089 ch04 68192362 68199138 888.055696 ch11 37373567 37377091 13.5195081 549.06994 ch07 53003962 53007535 1046.31059 ch05 2151041 ch07 34590918 34595091 2086.8869 ch03 58100739 58107097 692.293953 ch03 50954189 50957371 1025.80429 ch11 180.418204 ch01 68.0374159 ch02 36696924 36705272 752.298452 ch05 1797.43929 ch02 54.4430873 ch06 45464851 45466905 65.2726688 3045662 ch06 662.180427 150.4013 ch03 32668144 32673314 136.145949 825564 ch06 ch06 806738 86.7029361 ch12 60664839 60672113 6.40529278 ch12 60641248 60645994 4.37992193 1793557 3599829 1798576 3605009 5918681 7340226 5923232 7348506 2149023 3036160 818762 801621 8.65E-05 -0.764291061 2.40E-07 -2.165773016 9.11E-09 -3.289805847 -1.390666554 0.00063598 -1.343159467 0.00067521 -0.463775819 0.0001842 -0.47009761 4.93E-05 -0.748108014 0.00029006 -1.438377685 6.33E-05 3.01E-05 0.390568539 0.00021612 0.593510864 -0.616892849 1.10E-05 -0.369845159 0.00019672 -1.021399302 0.00053651 -1.168563568 0.00057496 -1.487036212 0.00070535 -0.951570457 0.00011066 -1.356651241 1.40E-06 4.67E-11 -2.293650763 -3.065447166 7.73E-06 -0.314374636 0.00014137 0.00088044 0.783897178 0.00024459 0.939620075 1.889874996 0.00030241 8.36E-08 -3.909238859 -4.082840247 5.07E-06 Table S3.7 (cont’d) PGSC0003DMG401024074 PGSC0003DMG401025788 PGSC0003DMG401025892 PGSC0003DMG401026387 PGSC0003DMG401026931 PGSC0003DMG401027620 PGSC0003DMG401029906 PGSC0003DMG401031052 PGSC0003DMG402000097 PGSC0003DMG402000528 PGSC0003DMG402000923 PGSC0003DMG402001341 PGSC0003DMG402001978 PGSC0003DMG402002270 PGSC0003DMG402002635 PGSC0003DMG402002661 PGSC0003DMG402002794 PGSC0003DMG402003606 PGSC0003DMG402003745 PGSC0003DMG402004500 PGSC0003DMG402004978 PGSC0003DMG402005679 PGSC0003DMG402006194 PGSC0003DMG402006783 PGSC0003DMG402007138 Amine oxidase 2763075 2768867 Conserved gene of unknown function CCHC-type integrase Cgi67 serine protease UDP-glucosyltransferase Conserved gene of unknown function Conserved gene of unknown function Conserved gene of unknown function Conserved gene of unknown function Conserved gene of unknown function Conserved gene of unknown function ch03 146.450874 ch01 84482429 84488047 41.0027226 ch01 84604437 84605674 179.018747 ch02 41445448 41451320 1687.69434 ch06 53541806 53543434 6.27397168 ch11 12619426 12627140 343.397793 ch01 54580019 54581314 372.658856 ch03 48745281 48746801 558.431751 ch01 73196771 73200269 457.439197 ch02 46920438 46926955 365.065763 ch07 42736036 42739222 658.113673 ch02 45976570 45979430 58.4571046 496.105893 Pyridine nucleotide-disulfide oxidoreductase family protein ch11 ch08 144.972378 ch03 59919277 59920939 510.055676 ch09 496.155408 ch01 78353666 78354844 333.717086 ch02 40233987 40241246 207.791278 ch04 70087398 70091269 5245.01727 ch02 21647631 21649330 941.422369 ch12 53989476 53993626 1440.40239 ch03 58171876 58173895 198.287161 ch01 87390883 87395297 1760.39485 ch01 65130372 65132031 563.773957 ch06 55417034 55421895 1421.52961 Early-responsive to dehydration 7 RNA polymerase sigma subunit SigD Conserved gene of unknown function Conserved gene of unknown function Conserved gene of unknown function Glycosyltransferase 1 Glutathione peroxidase Cytochrome P450 Mutt domain protein Auxin-responsive protein IAA1 Nicotiana tabacum wound inducive mRNA , complete cds Aldehyde dehydrogenase 6015170 844679 6018361 848394 FtsZ 3268985 3273075 -0.949530042 0.00031623 1.51E-07 -2.653623454 -0.771732833 8.08E-06 0.00052791 0.516325086 0.00072796 -2.37763374 -0.467789678 4.54E-05 -1.761088899 0.00047225 -0.54018853 6.11E-06 -0.880625475 0.00013946 -2.371041275 0.00015259 9.59E-06 -1.966774859 -2.458070303 4.49E-06 5.75E-05 0.958924642 7.35E-07 -2.303437777 1.638132307 0.00051233 6.49E-05 -0.755447713 3.43E-05 -1.216681131 -1.417884061 9.12E-05 -1.500374317 0.00023197 0.0002405 2.890439329 -0.551951693 8.65E-05 1.13E-08 -2.105332123 0.762424346 1.28E-05 -0.638190061 0.00061221 2.27E-05 -0.711739148 212 Table S3.7 (cont’d) PGSC0003DMG402007 PGSC0003DMG402009 PGSC0003DMG402010 970 791 991 PGSC0003DMG402011 202 PGSC0003DMG402012 386 PGSC0003DMG402013 020 PGSC0003DMG402014 450 PGSC0003DMG402015 792 PGSC0003DMG402016 PGSC0003DMG402016 PGSC0003DMG402018 244 574 552 Conserved gene of unknown function Cytochrome P450 hydroxylase Cytochrome P450 hydroxylase Kinase pac.W.Ch.162 CYP72A58 Heat shock protein binding protein ADP-ribosylation factor GTPase-activating protein AGD3 Short-chain dehydrogenase Conserved gene of unknown function Nucleotide pyrophosphatase/phosphodiesterase Soluble starch synthase 1, chloroplastic/amyloplastic PGSC0003DMG402020 126 Armadillo/beta-catenin repeat family protein / BTB/POZ domain- containing protein PGSC0003DMG402021 PGSC0003DMG402021 921 988 Ribose-phosphate pyrophosphokinase Sodium/hydrogen exchanger PGSC0003DMG402022 215 Aconitate hydratase 3 213 ch0 4 ch0 0 ch1 0 ch0 7 ch0 7 ch0 2 ch1 2 ch0 3 ch1 1 ch0 4 ch0 3 ch0 6 ch1 0 ch0 6 ch0 7 6820144 6820420 106.3523 3.70966047 2650457 2650597 45.35103 1.91953043 5453850 5454060 132.4416 7 1 9 1 7 6 1143710 1145517 141.9104 5265109 9 5265977 6 1457.064 39 2288629 8 2289194 8 1909.213 06 4879881 0 4880447 7 112.2386 12 4349281 9 4349876 9 1992.758 98 3144226 5448920 3151007 5449437 131.9568 4560329 4561230 4819.495 2 2 8 7 5909833 8 5910110 2 3727475 2 3728068 0 815313 818676 12.39322 2856.047 0.98961480 330.2144 0.81219797 5487574 2 5488345 2 34.48723 71 1.84221772 25 37 3 4 73 18 21 79 1.31644207 0.71543757 1.29479122 0.67631957 0.77394401 2.03577216 1.22861379 1.02630486 1.41190886 - 7 8 - - 1 - 5 - 3 - 4 - 4 - 3 - - - 4 - 2 6 - 8 4.40E-05 5.61E-06 3.61E-05 0.000111 56 4.68E-23 0.000216 12 3.71E-05 0.000377 96 0.000459 96 64 2.13E-11 2.64E-05 4.94E-09 2.96E-06 0.000165 72 275.8283 0.43186119 0.000574 PGSC0003DMG40202862 Chloroplast post-illumination chlorophyll fluorescence increase 4975009 4975315 5954.6239 3 0 2 2 1 2 3 6 8 7 9 1 9 1 9 5 4 7 6 8 0 9 3 7 3 4 7 6 1 0 0 9 5602033 5602359 861.56974 5005386 5006108 1054.2486 6067219 6067315 2.0642147 3167463 6115343 3169938 6115566 901.08695 1764.4889 1570768 1571019 290.30137 7017424 7017678 449.17376 5390436 5390866 175.98338 5363195 5363599 2356.3435 5798470 5798870 1858.9403 3257664 6384166 3260199 6384211 1976.1683 13.488343 6009456 6009651 714.89862 5634257 5634342 97.204849 4596459 4596816 7434874 7435583 468.62562 3421.1733 5816728 5817389 56.877590 2495782 4552993 0 2500630 4553055 3 98.719197 44.848808 6 1 1 2 5 9 3 2 5 2 2 4 3 2 6 7 5 4 1 0.639779147 1.773505081 4.962779612 1.416613518 1.200141416 1.606259344 0.446075945 0.594731966 1.092522043 3.483709726 -1.13130859 -0.36092917 2.481846877 0.610487091 1.616110849 0.471609062 -0.55727811 1.481083841 1.856126658 - - - - - - - - - - - - 0.0007705 4 3.88E-13 0.0001403 0.0004047 0.0006290 1 3 9 5.22E-11 0.0007919 3 1.32E-09 0.0004256 8 6.12E-10 1.66E-05 0.0007971 0.0007670 0.0007714 9 5 4 7.79E-05 0.0003417 6 3.74E-08 0.0001810 0.0001668 9 8 -2.96423218 9.00E-06 Table S3.7 (cont’d) PGSC0003DMG40202225 PGSC0003DMG40202343 PGSC0003DMG40202386 PGSC0003DMG40202414 PGSC0003DMG40202506 PGSC0003DMG40202564 PGSC0003DMG40202601 PGSC0003DMG40202690 PGSC0003DMG40202702 PGSC0003DMG40202768 PGSC0003DMG40202944 PGSC0003DMG40203080 PGSC0003DMG40203152 PGSC0003DMG40300059 PGSC0003DMG40300134 PGSC0003DMG40300328 PGSC0003DMG40300567 PGSC0003DMG40300783 PGSC0003DMG40302045 3 8 6 0 6 7 8 6 3 7 6 4 5 0 4 1 5 9 8 3 Ubiquitin carrier protein Epoxide hydrolase Hcr2-0B PAE Short-chain dehydrogenase/reductase Hydrolase Gene of unknown function Endonuclease III Nam 1 Wound-inducible carboxypeptidase protein GAGA-binding transcriptional activator BBR/BPC1 UDP-glucose:glucosyltransferase Cytochrome P450 Flavonol synthase/flavanone 3-hydroxylase Conserved gene of unknown function Histone mRNA exonuclease 1 Cytochrome P450 Purple acid phosphatase 3 Cytochrome P450 71D7 214 ch0 7 ch0 5 ch1 2 ch0 8 ch0 4 ch1 1 ch0 1 ch0 6 ch0 6 ch0 6 ch1 2 ch0 4 ch0 4 ch0 9 ch0 3 ch0 2 ch0 1 ch0 3 ch1 2 ch0 6 493110 489323 ch01 2041.73162 ch01 82642611 82643869 1491.32144 ch03 56330296 56339009 38.6320699 0.690802007 -0.44581783 -1.532266264 7.32E-05 1.11E-06 1.14E-05 Table S3.7 (cont’d) PGSC0003DMG403022797 PGSC0003DMG403033574 PGSC0003DMG404000594 Aspartate semialdehyde dehydrogenase EXECUTER1 protein, chloroplast Flavonol synthase/flavanone 3-hydroxylase 215 Table S3.8. Number of genes in each of the 29 modules detected by weighted gene co-expression network analysis. Number of Module skyblue white genes 45 56 59 60 67 85 111 114 115 133 150 152 156 213 225 302 310 353 363 372 406 581 591 698 917 1121 1292 4449 8499 21996 darkorange orange darkgrey darkturquoise darkgreen darkred royalblue lightyellow lightgreen grey60 lightcyan midnightblue cyan salmon tan greenyellow purple magenta pink black red green yellow brown blue turquoise grey Total 216 Table S3.9. Hub genes in the darkgrey, grey60, midnightblue and red modules detected by weighted gene co-expression analysis. PGSC ID Figure ID Chromsome PGSC0003DMG400010136 PGSC0003DMG400010141 PGSC0003DMG400010144 PGSC0003DMG400005921 PGSC0003DMG400005950 PGSC0003DMG400022217 PGSC0003DMG400017513 PGSC0003DMG400029576 PGSC0003DMG400012987 PGSC0003DMG400018930 PGSC0003DMG400003626 PGSC0003DMG400014894 PGSC0003DMG400016711 PGSC0003DMG400000576 PGSC0003DMG400013439 PGSC0003DMG400014200 PGSC0003DMG400022535 PGSC0003DMG400022541 PGSC0003DMG400025346 PGSC0003DMG400011497 PGSC0003DMG400004158 PGSC0003DMG400027047 PGSC0003DMG400028541 PGSC0003DMG400019243 ch03:400010136 ch03:400010141 ch03:400010144 ch06:400005921 ch06:400005950 ch07:400022217 ch08:400017513 ch08:400029576 ch09:400012987 ch01:400018930 ch02:400003626 ch02:400014894 ch02:400016711 ch03:400000576 ch03:400013439 ch03:400014200 ch03:400022535 ch03:400022541 ch03:400025346 ch04:400011497 ch06:400004158 ch06:400027047 ch06:400028541 ch07:400019243 -2.545291713 -2.770110439 -2.861719027 -2.270713903 -2.358425562 -2.817588626 -3.478265316 -3.892567121 -3.303547012 -1.82533385 -2.382564762 -2.115355798 -2.464063862 -3.000026044 -2.621561723 -2.251837455 -1.475352538 -2.542073151 -2.150255316 -2.124029094 -2.398298384 -2.412609998 -2.691162024 -2.083926935 ch03 ch03 ch03 ch06 ch06 ch07 ch08 ch08 ch09 ch01 ch02 ch02 ch02 ch03 ch03 ch03 ch03 ch03 ch03 ch04 ch06 ch06 ch06 ch07 End Start Module 49713158 49714012 darkgrey 49611604 49612275 darkgrey 49514289 49514969 darkgrey 54172928 54175121 darkgrey 54194363 54198068 darkgrey 54846232 54850298 darkgrey 49666212 49670996 darkgrey 45676042 45677832 darkgrey darkgrey 4336457 4342614 grey60 42196658 42198625 39706489 39707596 grey60 grey60 40639078 40642512 grey60 36798230 36803111 46614171 46615243 grey60 grey60 343737 347232 grey60 57113395 57115435 3739714 3741448 grey60 grey60 3883801 3886618 grey60 53160568 53162079 6230160 6232158 grey60 grey60 3353777 3362834 grey60 53102742 53104170 49751327 49751976 grey60 grey60 53789533 53790113 Annotation Stigma expressed protein Stigma expressed protein Stigma expressed protein Multicystatin Multicystatin Inducible plastid-lipid associated protein Acetylornithine deacetylase Polyphenol oxidase Threonine dehydratase biosynthetic, chloroplastic Proteinase inhibitor I4, serpin Lactoylglutathione lyase Membrane protein Homeobox protein knotted-1-like LET6 PGPS/D3 Aspartic proteinase oryzasin-1 Flotillin-1 Lactoylglutathione lyase Peroxidase 72 Voltage-dependent anion channel Light-dependent short hypocotyls 1 Ca2+ antiporter/cation exchanger UPF0497 membrane protein Polygalacturonase Conserved gene of unknown function 217 Table S3.9 (cont’d) PGSC0003DMG400004736 PGSC0003DMG400009300 PGSC0003DMG400012183 PGSC0003DMG400022756 PGSC0003DMG400024286 PGSC0003DMG400030172 PGSC0003DMG400008276 PGSC0003DMG400021814 PGSC0003DMG400003446 PGSC0003DMG400025887 PGSC0003DMG400015505 PGSC0003DMG400013094 PGSC0003DMG400042914 PGSC0003DMG400012650 PGSC0003DMG400017873 PGSC0003DMG400004521 PGSC0003DMG400013057 PGSC0003DMG400010236 PGSC0003DMG400010426 PGSC0003DMG400021171 PGSC0003DMG400009806 PGSC0003DMG400015105 PGSC0003DMG400014906 PGSC0003DMG400025655 PGSC0003DMG400003270 PGSC0003DMG400013052 ch08:400004736 ch08:400009300 ch08:400012183 ch08:400022756 ch09:400024286 ch09:400030172 ch10:400008276 ch12:400021814 ch00:400003446 ch01:400025887 ch02:400015505 ch02:400013094 ch02:400042914 ch02:400012650 ch02:400017873 ch02:400004521 ch02:400013057 ch02:400010236 ch02:400010426 ch02:400021171 ch02:400009806 ch02:400015105 ch02:400014906 ch02:400025655 ch02:400003270 ch02:400013052 -2.211277943 -1.648963506 -2.332438315 -1.600932407 -2.318151846 -2.436736354 1.078989436 -2.338898406 -7.963849259 -1.663881746 -9.49936276 -9.283320424 -9.205774995 -9.157128794 -9.043092808 -8.204064918 -4.562792344 -2.421894752 0.755929801 1.197553941 2.012050084 2.168718299 2.865049442 2.86606308 3.481425463 3.694329181 8091264 grey60 grey60 grey60 grey60 grey60 grey60 grey60 grey60 ch08 52816522 52817143 ch08 48660644 48666483 ch08 55277792 55281642 ch08 53947780 53951150 ch09 50459496 50461280 ch09 54513083 54516979 ch10 43622511 43623419 ch12 8092695 ch00 32653309 32655813 Midnightblue ch01 84401258 84405639 Midnightblue ch02 21003553 21005147 Midnightblue ch02 15887638 15891240 Midnightblue ch02 21032730 21033362 Midnightblue ch02 16889189 16891951 Midnightblue ch02 18356198 18362279 Midnightblue ch02 16783207 16784249 Midnightblue ch02 8182546 Midnightblue ch02 32074428 32080205 Midnightblue ch02 27546393 27550327 Midnightblue ch02 29500348 29509451 Midnightblue ch02 12532624 12536712 Midnightblue ch02 12519201 12524409 Midnightblue ch02 40669679 40670305 Midnightblue ch02 27701384 27703638 Midnightblue 9753153 Midnightblue ch02 ch02 7963430 Midnightblue 8181763 9748792 7961380 P-rich protein EIG-I30 Xyloglucan endotransglucosylase/hydrolase protein 2 Endo-1,4-beta-glucanase Adenosine 3'-phospho 5'-phosphosulfate transporter Ca2+ antiporter/cation exchanger Aspartic proteinase oryzasin-1 Gene of unknown function Chlorophyllase 1 Undecaprenyl pyrophosphate synthetase Thioredoxin M3, chloroplastic Anthranilate N-benzoyltransferase protein Leucine-rich repeat-containing protein Gene of unknown function TMV resistance protein N Tetratricopeptide repeat protein Leucine-rich repeat-containing protein Gene of unknown function Cysteine protease Cp5 Conserved gene of unknown function Protection of telomeres 1 protein Alpha N-terminal protein methyltransferase 1 Alpha N-terminal protein methyltransferase 1 Conserved gene of unknown function Ankyrin repeat-containing protein ARP2/3 complex 21 kDa subunit TdcA1-ORF1-ORF2 protein 218 Table S3.9 (cont’d) PGSC0003DMG400037923 PGSC0003DMG400010430 PGSC0003DMG400013629 PGSC0003DMG400010146 PGSC0003DMG400013821 PGSC0003DMG400014763 PGSC0003DMG400023869 PGSC0003DMG400029390 PGSC0003DMG400004286 PGSC0003DMG400043070 PGSC0003DMG400020035 PGSC0003DMG400020032 PGSC0003DMG400024450 PGSC0003DMG400001017 PGSC0003DMG400021348 PGSC0003DMG400025795 PGSC0003DMG400025600 PGSC0003DMG400003642 PGSC0003DMG400019712 PGSC0003DMG400003641 PGSC0003DMG401005669 PGSC0003DMG400034325 PGSC0003DMG400010921 PGSC0003DMG400029452 PGSC0003DMG400029493 ch02:400037923 ch02:400010430 ch02:400013629 ch03:400010146 ch04:400013821 ch08:400014763 ch12:400023869 ch12:400029390 ch12:400004286 ch00:400043070 ch00:400020035 ch00:400020032 ch01:400024450 ch01:400001017 ch01:400021348 ch01:400025795 ch02:400025600 ch02:400003642 ch02:400019712 ch02:400003641 ch03:401005669 ch04:400034325 ch04:400010921 ch04:400029452 ch04:400029493 3.7712659 4.64601322 6.086354248 -5.788219534 -0.684918912 3.03722548 -4.981025488 -1.189294063 -3.460928591 -4.736989332 -2.323862356 -2.197445682 -6.01146723 -5.177311642 -2.467549852 -1.823560779 -5.538888064 -2.761187265 -1.925489697 -0.920184323 -0.079970427 -4.075375115 -3.872439934 -2.903024693 -1.61998836 ch02 ch02 ch02 ch03 ch04 ch08 ch12 ch12 ch12 ch00 ch00 ch00 ch01 ch01 ch01 ch01 ch02 ch02 ch02 ch02 ch03 ch04 ch04 ch04 ch04 18102072 Midnightblue 18101449 27462463 Midnightblue 27457154 37832400 Midnightblue 37830842 49449153 Midnightblue 49448372 22327197 Midnightblue 22319412 44377460 Midnightblue 44374559 60658107 Midnightblue 60656805 57732653 57738359 Midnightblue 61035075 61,039,166 Midnightblue 3241084 35418095 27746206 43084648 13951129 2466455 84631734 37200576 39375243 37092937 39380731 58440542 35444028 4403170 3006111 3193641 3245717 35422186 27749094 43085217 13954241 2476089 84636755 37201418 39375935 37095751 39381418 58445893 35449144 4404009 3006536 3194489 red red red red red red red red red red red red red red red red 219 Conserved gene of unknown function Conserved gene of unknown function Cytochrome P450 hydroxylase Kunitz-type tuber invertase inhibitor Potassium transporter Gene of unknown function Cf-2.2 Cysteine protease Tap46 Gene of unknown function Histidine decarboxylase Conserved gene of unknown function Short-chain type alcohol dehydrogenase Conserved gene of unknown function Subtilisin-type protease Cytochrome P450 Conserved gene of unknown function Gast1 Conserved gene of unknown function Transposon MuDR mudrA Conserved gene of unknown function Gene of unknown function Conserved gene of unknown function Disease resistance protein Glucosyltransferase Table S3.9 (cont’d) PGSC0003DMG400029505 PGSC0003DMG400006523 PGSC0003DMG400013210 PGSC0003DMG400012703 PGSC0003DMG400013701 PGSC0003DMG400034515 PGSC0003DMG400031002 PGSC0003DMG400027020 PGSC0003DMG400030390 PGSC0003DMG400031356 PGSC0003DMG400027473 PGSC0003DMG401011202 PGSC0003DMG400027465 PGSC0003DMG400026275 PGSC0003DMG402029949 PGSC0003DMG400003938 PGSC0003DMG400018476 PGSC0003DMG400022070 PGSC0003DMG400013721 PGSC0003DMG400003847 PGSC0003DMG400020332 PGSC0003DMG402028118 PGSC0003DMG400003236 PGSC0003DMG402010281 PGSC0003DMG400046473 ch04:400029505 ch04:400006523 ch04:400013210 ch04:400012703 ch05:400013701 ch05:400034515 ch05:400031002 ch06:400027020 ch06:400030390 ch06:400031356 ch07:400027473 ch07:401011202 ch07:400027465 ch08:400026275 ch08:402029949 ch08:400003938 ch08:400018476 ch08:400022070 ch08:400013721 ch09:400003847 ch09:400020332 ch10:402028118 ch10:400003236 ch10:402010281 ch10:400046473 -1.615027328 -1.235240837 -0.116421678 -0.043161105 -5.723530296 -3.229704084 -2.465796727 -2.918700842 -1.799734014 -0.417478158 -5.828853422 -5.237188117 -0.624957467 -8.849481964 -3.639224973 -3.156745263 -2.937419599 -0.176836427 -0.115033172 -5.649385934 -3.054141908 -6.175712283 -5.817882181 -4.640331467 0.005759836 8291731 2981078 4326938 2134760 1148861 2382858 1794626 2987474 ch04 ch04 4334896 ch04 35777054 35778091 ch04 11189908 11195283 ch05 43080013 43080872 ch05 34394716 34396093 ch05 8292138 ch06 53666248 53668276 ch06 56238355 56242764 ch06 20312334 20318431 2138926 ch07 ch07 1152310 2386771 ch07 ch08 1796740 ch08 35868617 35872897 ch08 53555111 53556281 ch08 3585269 ch08 13799343 13802555 ch08 8469048 ch09 51322791 51326087 ch09 660041 ch10 56663717 56670436 ch10 13888658 13892430 ch10 38912954 38919046 ch10 23948850 23952707 8465002 3584114 658164 220 NRC1 Nodulin family protein Proteasome subunit beta type Ubiquitin NOX1 Wall-associated kinase Gene of unknown function Alpha-L-fucosidase 2 Conserved gene of unknown function Conserved gene of unknown function Hypersensitive response assisting protein red red Multidomain cyclophilin type peptidyl-prolyl cis-trans isomerase red red red red red red red red red red red red red red red red red red red red red red red Gene of unknown function Gene of unknown function Gene of unknown function Gene of unknown function Kunitz trypsin inhibitor FK506-binding protein Gene of unknown function TRANSPARENT TESTA 12 protein Conserved gene of unknown function Integrin-linked protein kinase Gene of unknown function O-methyltransferase Gast1 PGSC0003DMG4000154 ch12:4000154 5692075 5692243 LINE-type retrotransposon LIb DNA, complete sequence, Insertion at the 0.509017191 0.601029354 -5.867607566 -3.116158053 -2.504348638 -0.878009772 -0.367388438 -0.239293091 0.012683031 0.034656763 0.099479855 0.187713799 0.189167164 0.276832434 ch 10 ch 10 ch 12 ch 12 ch 12 ch 12 ch 12 ch 12 ch 12 ch 12 ch 12 ch 12 ch 12 ch 12 5450375 5450432 4690065 4690426 5403891 5404533 4521181 4522022 4957311 4957451 5203973 5204186 2462173 2462739 5256522 5257495 5287128 5287424 4042888 4043063 1 6 5 7 8 7 9 7 4 5 5 8 5 7 6 3 1 8 5 3 8 7 5 4 6415673 2796385 0 6418862 2796713 3 red red red red red red red red red red red red red red Ribosomal protein L23 Developmentally regulated GTP-binding protein S14 site C-terminal zinc-finger U4/U6 small nuclear ribonucleoprotein Prp4 Fatty acid desaturase Resistance protein PSH-RGH6 Gene of unknown function Conserved gene of unknown function P69E protein ERF transcription factor 5 Histidine acid phosphatase Plant disease resistant protein Gene of unknown function PGSC0003DMG4000159 ch12:4000159 2864619 2865185 Table S3.9 (cont’d) PGSC0003DMG4000403 ch10:4000403 PGSC0003DMG4000120 ch10:4000120 PGSC0003DMG4020049 ch12:4020049 PGSC0003DMG4000127 ch12:4000127 PGSC0003DMG4000286 ch12:4000286 PGSC0003DMG4000140 ch12:4000140 PGSC0003DMG4000256 ch12:4000256 PGSC0003DMG4000115 ch12:4000115 PGSC0003DMG4000043 ch12:4000043 PGSC0003DMG4000158 ch12:4000158 PGSC0003DMG4000138 ch12:4000138 PGSC0003DMG4000279 ch12:4000279 32 43 61 73 85 50 15 08 97 69 28 13 91 30 32 43 61 73 85 50 15 08 97 69 28 13 91 30 221 APPENDIX D: Chapter 3 Copyright Permission RightsLink Printable License https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=6ae802da-7cae... SPRINGER NATURE LICENSE TERMS AND CONDITIONS Feb 16, 2021 This Agreement between Dr. Natalie Kaiser ("You") and Springer Nature ("Springer Nature") consists of your license details and the terms and conditions provided by Springer Nature and Copyright Clearance Center. License Number 5003210613352 License date Feb 06, 2021 Licensed Content Publisher Licensed Content Publication Springer Nature Theoretical and Applied Genetics Licensed Content Title Mapping Solanum chacoense mediated Colorado potato beetle (Leptinotarsa decemlineata) resistance in a self-compatible F2 diploid population Licensed Content Author Natalie Kaiser et al Licensed Content Date May 30, 2020 Type of Use Thesis/Dissertation Requestor type academic/university or research institute Format Portion print and electronic full article/chapter 1 of 6 Figure S3.10 Springer Copyright Permission for inclusion of Chapter 3 in this dissertation. 2/16/21, 12:30 PM 222 APPENDIX E: Tuber Yield Table S3.10 Tuber yield of greenhouse grown plants in the Solanum chacoense F2 mapping population. Individual Number of Total Tuber Weight (g) Mean Tuber Weight (g) Tubers EE501F2_002 EE501F2_003 EE501F2_006 EE501F2_007 EE501F2_010 EE501F2_011 EE501F2_015 EE501F2_019 EE501F2_021 EE501F2_022 EE501F2_026 EE501F2_028 EE501F2_029 EE501F2_036 EE501F2_037 EE501F2_038 EE501F2_041 EE501F2_044 EE501F2_045 EE501F2_047 EE501F2_050 EE501F2_051 EE501F2_054 EE501F2_056 EE501F2_061 EE501F2_062 EE501F2_063 EE501F2_064 EE501F2_066 EE501F2_069 EE501F2_071 EE501F2_075 EE501F2_076 EE501F2_080 EE501F2_081 EE501F2_082 19 0 21 0 14 4 0 4 13 15 5 12 0 6 28 10 5 29 22 0 21 15 13 20 29 4 10 0 0 21 6 5 14 15 16 16 57.0 44.0 19.0 8.0 2.0 34.0 35.0 20.0 22.0 8.0 40.0 17.0 11.0 94.0 50.0 65.0 18.0 32.0 83.0 44.0 4.0 67.0 104.0 4.0 4.0 75.0 41.0 44.0 16.0 3.0 2.1 1.4 2.0 0.5 2.6 2.3 4.0 1.8 1.3 1.4 1.7 2.2 3.2 2.3 3.1 1.2 2.5 4.2 1.5 1.0 6.7 5.0 0.7 0.8 5.4 2.7 2.8 1.0 223 Table S3.10 (cont’d) EE501F2_084 EE501F2_089 EE501F2_092 EE501F2_093 EE501F2_095 EE501F2_098 EE501F2_099 EE501F2_100 EE501F2_105 EE501F2_106 EE501F2_113 EE501F2_121 EE501F2_123 EE501F2_124 EE501F2_125 EE501F2_126 EE501F2_128 EE501F2_131 EE501F2_135 EE501F2_137 EE501F2_139 EE501F2_140 EE501F2_142 EE501F2_143 EE501F2_145 EE501F2_148 EE501F2_151 EE501F2_154 EE501F2_155 EE501F2_156 EE501F2_157 EE501F2_160 EE501F2_161 EE501F2_162 EE501F2_163 EE501F2_164 EE501F2_172 EE501F2_173 EE501F2_176 EE501F2_179 EE501F2_180 EE501F2_182 2 44 21 19 7 11 17 14 28 4 20 0 1 0 19 9 5 47 0 3 0 0 15 8 7 11 7 21 19 15 24 18 19 5 16 7 14 4 7 14 10 20 1.0 116.0 40.0 31.0 4.0 9.0 30.0 24.0 64.0 23.0 41.0 1.0 38.0 6.0 14.0 106.0 3.0 22.0 28.0 18.0 27.0 5.0 36.0 53.0 32.0 65.0 30.0 57.0 7.0 29.0 12.0 70.0 3.0 11.0 68.0 27.0 39.0 0.5 2.6 1.9 1.6 0.6 0.8 1.8 1.7 2.3 5.8 2.1 1.0 2.0 0.7 2.8 2.3 1.0 1.5 3.5 2.6 2.5 0.7 1.7 2.8 2.1 2.7 1.7 3.0 1.4 1.8 1.7 5.0 0.8 1.6 4.9 2.7 2.0 224 Table S3.10 (cont’d) EE501F2_183 EE501F2_188 EE501F2_193 EE501F2_195 EE501F2_196 EE501F2_199 EE501F2_200 EE501F2_202 EE501F2_214 EE501F2_215 EE501F2_217 EE501F2_220 EE501F2_221 EE501F2_222 EE501F2_223 EE501F2_226 EE501F2_228 EE501F2_230 EE501F2_231 EE501F2_233 EE501F2_234 EE501F2_236 EE501F2_237 EE501F2_242 EE501F2_247 EE501F2_250 EE501F2_254 EE501F2_259 EE501F2_262 EE501F2_268 EE501F2_270 EE501F2_271 EE501F2_275 EE501F2_277 EE501F2_278 EE501F2_285 EE501F2_288 EE501F2_291 EE501F2_294 EE501F2_296 EE501F2_297 EE501F2_299 12 27 12 0 15 9 22 9 18 0 29 31 0 18 12 20 0 14 0 14 14 0 0 12 12 14 10 12 17 16 21 17 19 22 22 30 38 5 12 7 6 0 10.0 30.0 24.0 18.0 56.0 57.0 22.0 47.0 100.0 56.0 48.0 56.0 32.0 30.0 20.0 45.0 24.0 13.0 14.0 75.0 40.0 16.0 36.0 24.0 25.0 64.0 31.0 35.0 64.0 57.0 13.0 15.0 17.0 16.0 0.8 1.1 2.0 1.2 6.2 2.6 2.4 2.6 3.4 1.8 2.7 4.7 1.6 2.1 1.4 3.2 2.0 1.1 1.0 7.5 3.3 0.9 2.3 1.1 1.5 3.4 1.4 1.6 2.1 1.5 2.6 1.3 2.4 2.7 225 Table S3.10 (cont’d) EE501F2_300 EE501F2_301 EE501F2_305 EE501F2_311 EE501F2_312 EE501F2_313 EE501F2_316 EE501F2_318 EE501F2_319 EE501F2_320 EE501F2_321 EE501F2_322 EE501F2_324 EE501F2_325 EE501F2_326 EE501F2_334 EE501F2_335 EE501F2_336 EE501F2_337 EE501F2_338 EE501F2_339 EE501F2_341 EE501F2_343 EE501F2_348 EE501F2_349 EE501F2_354 EE501F2_358 EE501F2_361 EE501F2_362 EE501F2_363 EE501F2_365 EE501F2_370 EE501F2_371 EE501F2_375 EE501F2_377 EE501F2_378 EE501F2_380 EE501F2_382 EE501F2_383 EE501F2_386 EE501F2_387 EE501F2_388 0 0 0 18 7 17 4 21 21 18 2 3 15 1 21 0 0 14 14 13 0 4 0 13 9 0 26 20 18 20 31 0 20 13 0 0 23 10 26 17 0 0 19.0 9.0 42.0 3.0 16.0 27.0 10.0 2.0 52.0 26.0 1.0 30.0 27.0 33.0 13.0 5.0 17.0 6.0 23.0 52.0 37.0 39.0 70.0 35.0 21.0 47.0 14.0 20.0 38.0 1.1 1.3 2.5 0.8 0.8 1.3 0.6 1.0 17.3 1.7 1.0 1.4 1.9 2.4 1.0 1.3 1.3 0.7 0.9 2.6 2.1 2.0 2.3 1.8 1.6 2.0 1.4 0.8 2.2 226 Table S3.10 (cont’d) EE501F2_390 EE501F2_391 EE501F2_393 EE501F2_394 EE501F2_397 EE501F2_398 EE501F2_401 EE501F2_402 EE501F2_403 EE501F2_404 EE501F2_405 EE501F2_407 EE501F2_408 EE501F2_410 EE501F2_411 EE501F2_413 EE501F2_417 EE501F2_418 EE501F2_419 EE501F2_421 EE501F2_422 EE501F2_423 EE501F2_426 EE501F2_427 EE501F2_431 EE501F2_433 EE501F2_438 EE501F2_446 EE501F2_447 EE501F2_448 EE501F2_449 EE501F2_451 EE501F2_454 EE501F2_455 EE501F2_456 EE501F2_457 EE501F2_458 EE501F2_460 EE501F2_462 EE501F2_465 EE501F2_466 EE501F2_468 3 20 18 18 15 12 17 7 12 12 15 15 18 20 4 0 13 14 0 18 21 21 26 24 17 19 6 13 10 10 16 8 20 10 18 0 9 8 20 12 12 11 6.0 40.0 29.0 44.0 40.0 48.0 20.0 56.0 23.0 36.0 34.0 40.0 29.0 56.0 2.0 17.0 31.0 53.0 68.0 45.0 25.0 44.0 67.0 28.0 40.0 25.0 25.0 50.0 65.0 14.0 66.0 43.0 58.0 33.0 5.0 41.0 21.0 20.0 47.0 2.0 2.0 1.6 2.4 2.7 4.0 1.2 8.0 1.9 3.0 2.3 2.7 1.6 2.8 0.5 1.3 2.2 2.9 3.2 2.1 1.0 1.8 3.9 1.5 6.7 1.9 2.5 5.0 4.1 1.8 3.3 4.3 3.2 3.7 0.6 2.1 1.8 1.7 4.3 227 Table S3.10 (cont’d) EE501F2_470 EE501F2_471 EE501F2_474 EE501F2_475 EE501F2_477 EE501F2_478 EE501F2_480 EE501F2_481 EE501F2_483 EE501F2_484 EE501F2_488 EE501F2_491 EE501F2_492 EE501F2_493 EE501F2_494 EE501F2_495 EE501F2_496 EE501F2_499 EE501F2_500 EE501F2_501 EE501F2_503 EE501F2_504 EE501F2_505 EE501F2_506 EE501F2_507 EE501F2_508 EE501F2_511 EE501F2_513 EE501F2_514 EE501F2_516 EE501F2_519 EE501F2_520 EE501F2_521 EE501F2_523 EE501F2_524 EE501F2_525 EE501F2_526 EE501F2_527 EE501F2_528 EE501F2_532 EE501F2_533 EE501F2_534 5 10 18 19 18 8 7 3 1 18 7 16 38 4 0 7 12 14 14 10 10 20 0 14 1 6 14 9 9 17 8 0 0 5 0 14 0 0 18 12 18 17 2.0 47.0 32.0 53.0 31.0 18.0 33.0 5.0 1.0 56.0 25.0 66.0 96.0 4.0 8.0 16.0 72.0 55.0 22.0 54.0 51.0 11.0 1.0 3.0 41.0 16.0 18.0 41.0 8.0 1.0 46.0 66.0 5.0 26.0 92.0 0.4 4.7 1.8 2.8 1.7 2.3 4.7 1.7 1.0 3.1 3.6 4.1 2.5 1.0 1.1 1.3 5.1 3.9 2.2 5.4 2.6 0.8 1.0 0.5 2.9 1.8 2.0 2.4 1.0 0.2 3.3 3.7 0.4 1.4 5.4 228 Table S3.10 (cont’d) EE501F2_537 EE501F2_538 EE501F2_540 EE501F2_543 EE501F2_544 EE501F2_545 EE501F2_549 EE501F2_551 EE501F2_553 EE501F2_554 EE501F2_557 EE501F2_558 EE501F2_562 EE501F2_563 EE501F2_565 EE501F2_568 EE501F2_570 EE501F2_572 EE501F2_576 EE501F2_582 EE501F2_584 EE501F2_586 EE501F2_587 EE501F2_588 EE501F2_591 EE501F2_597 EE501F2_599 EE501F2_603 EE501F2_604 EE501F2_605 EE501F2_608 EE501F2_610 EE501F2_611 EE501F2_615 EE501F2_621 EE501F2_622 EE501F2_623 EE501F2_625 EE501F2_636 EE501F2_637 EE501F2_640 EE501F2_641 8 2 17 9 7 19 0 17 5 20 10 12 33 3 10 23 4 7 0 8 11 12 10 24 13 14 6 14 14 14 24 16 14 9 15 23 10 21 22 3 0 23 5.0 19.0 47.0 18.0 9.0 44.0 65.0 4.0 21.0 34.0 50.0 57.0 21.0 55.0 52.0 12.0 12.0 14.0 15.0 35.0 74.0 57.0 26.0 47.0 11.0 50.0 66.0 18.0 79.0 55.0 19.0 14.0 101.0 42.0 8.0 24.0 113.0 5.0 98.0 0.6 9.5 2.8 2.0 1.3 2.3 3.8 0.8 1.1 3.4 4.2 1.7 7.0 5.5 2.3 3.0 1.7 1.8 1.4 2.9 7.4 2.4 2.0 3.4 1.8 3.6 4.7 1.3 3.3 3.4 1.4 1.6 6.7 1.8 0.8 1.1 5.1 1.7 4.3 229 Table S3.10 (cont’d) EE501F2_642 EE501F2_646 EE501F2_647 EE501F2_648 EE501F2_649 EE501F2_652 EE501F2_653 EE501F2_654 EE501F2_656 EE501F2_658 EE501F2_660 EE501F2_665 EE501F2_670 EE501F2_672 EE501F2_673 EE501F2_701 EE501F2_703 EE501F2_705 EE501F2_706 EE501F2_708 12 9 6 9 5 5 24 24 4 6 25 13 20 1 14 18 14 1 16 18 19.0 19.0 4.0 19.0 8.0 47.0 110.0 58.0 3.0 10.0 53.0 21.0 106.0 5.0 119.0 43.0 22.0 2.0 37.0 23.0 1.6 2.1 0.7 2.1 1.6 9.4 4.6 2.4 0.8 1.7 2.1 1.6 5.3 5.0 8.5 2.4 1.6 2.0 2.3 1.3 230 REFERENCES 231 REFERENCES Agelopoulos, N., Chamberlain, K., & Pickett, J. 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R., Shang, Y., Li, C., & Huang, S. (2019). The genetic basis of inbreeding depression in potato. Nature Genetics, 51(3), 374-378. 243 CHAPTER 4 ASSESSING THE CONTRIBUTION OF SLI TO SELF-COMPATIBILITY IN NORTH AMERICAN DIPLOID POTATO GERMPLASM USING KASPTM MARKERS This chapter is a published journal article (Kaiser, et al., 2021). The article is reproduced here with permission from the publisher (Appendix D). 244 Abstract Diploid hybrid potato variety development requires the introduction of reliably transmitted self-compatibility to largely self-incompatible (SI) elite diploid germplasm. The diploid Solanum chacoense clone M6 has been widely used to introgress self-compatibility into North American potato diploid breeding programs. We determined that M6 is homozygous for six DNA Kompetitive Allele Specific PCR (KASP)TM markers spanning a 224 kb region, linked to Sli in Dutch germplasm. Self-compatible (SC) Sli alleles were identified in dihaploids of the cultivars ‘Atlantic’ and ‘Superior’ and breeding clone NY148. This finding demonstrates the potential of Sli genotyping to select S. tuberosum dihaploids that will contribute to self-compatibility. We appraised the transmission of Sli in a diploid recurrent selection population and in a diploid backcross population, each designed to introgress self-compatibility while improving agronomic traits. The frequency of the homozygous SC Sli genotype at the six marker loci increased over the course of four cycles of recurrent selection. The homozygous Sli SC genotype at any one of five marker loci within an 80.8kb region on chromosome 12 (58,960,090-59,040,898 bp) perfectly predicted a SC phenotype in the recurrent selection population. The heterozygous Sli genotype was found in SC and SI individuals. The discrepancy between phenotype and marker genotype can be attributed in part to the difficulty of accurately phenotyping self-compatibility. We also identified SC individuals with the homozygous SI Sli genotype at all tested loci. The presence of the homozygous SI Sli haplotype in SC clones 1S1 and DMRH-89 and a SC individual from the S. chacoense PI 133664 further suggests that other genetic components contribute to self- compatibility. This work illustrates the ability of Sli markers to predict self-compatibility in some germplasm, but it also underscores the need to identify other genomic regions critical to self- 245 compatibility and the role of the environment in expression of genes involved in the self- compatibility reaction. Introduction Conducting genetic improvement of potato at the diploid level offers the advantage of a more efficient genetic system for mapping and stacking valuable traits. In addition, inbreeding diploids exposes genetic load more rapidly, allowing a breeder to select against undesirable alleles. However, the common occurrence of gametophytic self-incompatibility in diploid potato (Carson & Howard, 1942; Fujii, et al., 2016) impedes inbred line development. Gametophytic self- incompatibility in the Solanaceae is controlled by the multiallelic S-locus on chromosome 1 that contains tightly linked genes encoding S-RNase and multiple S-locus F-box (SLF) genes (Enciso- Rodriguez, et al., 2019; Kubo, et al., 2015; McClure, et al., 1989; Takayama & Isogai, 2005). Pollen is rejected if the pollen S-haplotype matches either of the S-haplotypes in the diploid pistil. In self-incompatible (SI) individuals, the pollen-expressed SLF genes fail to recognize and ubiquitinate the native pistil-expressed S-RNase, leading to the degradation of self-pollen RNA and inhibition of self-pollen tube growth (Hua, et al., 2008). The self-compatible (SC) diploid Solanum chacoense Bitter clone M6 has been widely used to introgress SC into North American diploid potato breeding programs. The presence of the dominant self-incompatibility inhibitor gene Sli on chromosome 12 (Hosaka & Hanneman, 1998a, 1998b) is hypothesized to confer SC in M6 (Jansky, et al., 2014). There are several drawbacks to relying upon Sli donors to improve self-compatibility in diploid breeding programs. The use of unadapted SC S. chacoense clones, including M6, introduces unfavorable traits such as prolific stolon production, high tuber glycoalkaloid content and late maturity into the germplasm pool. Selection against these phenotypes is possible but may require several generations of 246 recombination and selection. The use of SC sources with a S. tuberosum background is an attractive method to retain desirable adapted alleles while improving self-compatibility. The diploid S. tuberosum clones US-W4 (De Jong & Rowe, 1971) and RH89‐039‐16 (RH) (Park, et al., 2005; van der Voort J., et al., 1998) have been used for this purpose but the genetic basis of self-compatibility in these clones remains unknown. Clot et al. (2020) report Sli SC alleles within North American tetraploid varieties. Dihaploids extracted from these varieties could be a valuable source of self-compatibility. Because dihaploid extraction is a very time and labor-intensive process, determination of the presence and dosage of Sli SC alleles in tetraploid varieties would enhance dihaploid characterization. Successful implementation of self-compatibility in breeding programs also requires reliable transmission to offspring. SC clones occurring at lower frequencies than expected has been reported in selfed populations derived from Sli donors (Birhman & Hosaka, 2000; Phumichai, et al., 2005). These results suggest that in addition to Sli, other loci may be involved in modulating the self-incompatibility response in Solanum species (Goldraij, et al., 2006; McClure, et al., 1999; O’Brien, et al., 2002). Recently, Clot et al. (2020) proposed a 333kb candidate Sli region on chromosome 12 in M6 and describe 18 Kompetitive Allele Specific PCR (KASP)TM markers across the interval associated with self-compatibility in two diploid S. tuberosum mapping populations. Using a subset of these Sli KASPTM markers we assessed the contribution of Sli to self-compatibility in Michigan State University diploid germplasm which represents diverse clones derived from multiple North American breeding programs. 247 Materials and Methods Plant Material Self-compatible diploid clones Diploid breeding clones were selected to compare self-compatibility phenotypes with KASPTM marker genotypes in the candidate Sli region. Two S7 S. chacoense clones were used in this study: M6 (Jansky, et al., 2014) and clone ‘524-8’. The S. chacoense clone M6 is highly male fertile and has been widely used in North American potato breeding programs as a male parent to introgress SC. ‘524-8’ is another inbred S. chacoense clone developed in the same breeding program that created M6. A SC individual selected from the S. chacoense PI 133664 (PI 133664- 40) was also assessed, because individuals in this PI were previously determined to segregate for self-compatibility in a 1:1 ratio (data not shown). To examine if Sli plays a role in the S. tuberosum self-compatibility sources, we selected the dihaploid clone US-W4 produced by parthenogenesis from the Minnesota breeding clone ‘20-20-34’ (De Jong, et al., 1971) and clone XD3, derived from a cross between US-W4 and the SC S. chacoense clone ‘39-7’ (PI 275138). Other cultivated potato self-compatibility sources used in this study include the S. tuberosum Group Tuberosum clone RH89‐039‐16 (RH) (pedigree can be found in (PGSC, 2011) supplemental data), a SC F1 progeny (DMRH-89) produced from a cross between RH and S. tuberosum Group Phureja DM 1‐ 3 516 R44 (DM) (Peterson, et al., 2016) and S. tuberosum Group Phureja clone 1S1 acquired from Richard Veilleux at Virginia Tech University. The expression of self-fertility exhibited by clone 1S1 has varied under our greenhouse conditions in Michigan (data not shown). Recurrent Selection and Backcross Populations We evaluated the transmission of markers linked to Sli in a diploid recurrent selection population and a diploid backcross population created at Michigan State University. The diploid 248 recurrent selection population was founded by introgressing self-compatibility from six self- compatibility donors into seven self-incompatible (SI) clones to create a multi-species potato germplasm pool (Alsahlany, et al., 2020). Four cycles of recurrent selection were conducted to improve self-fertility, photoperiod adaptation and tuber quality traits while maintaining genetic diversity (Alsahlany, et al., 2020). In this study, we assessed the genotype of five SC and eight SI founder clones, 39 cycle 0 clones, 25 cycle 1 clones, 50 cycle 2 clones, 85 cycle 3 clones and 29 cycle 4 clones at six KASPTM marker loci (Table S1). The clones used for analysis in cycle 0-4 were randomly selected. The backcross germplasm was previously developed to introduce self-compatibility into S. tuberosum diploid germplasm (Alsahlany, 2019). This backcross population differs from a traditional backcross scheme in that different dihaploid, recurrent parents were used at each generation to maintain vigor and fertility traits. Briefly, the backcross population recurrent parents were dihaploids generated from cultivated S. tuberosum tetraploid varieties (‘Atlantic’, ‘Superior’, ‘Kalkaska’) and advanced breeding clones (MSR127-2 and NY148) by crossing with the haploid inducer IVP101 (S. tuberosum Gp. Phureja) (Manrique-Carpintero, et al., 2018). Self-compatible F1 families were created by crossing these dihaploids to three diploid self-compatibility donors: S. chacoense clone M6, a cycle 0 recurrent selection clone MSBB912-B (MCD205 (MSA133-57 x TF75.5) x M6), and bulk pollen (Bulk3) created by mixing equal pollen from four SC cycle 0 recurrent selection clones (MSBB912-B, MSBB920-A, MSBB930-A, and MSBB932-A) (Alsahlany, 2019). The F1 hybrids were then backcrossed to various dihaploids to form the BC1 generation and the subsequent BC2 generation. In this study we surveyed the genotype of the four clones comprising the SC Bulk3 pollen, 17 dihaploids (4 ‘Superior’ dihaploids, 11 ‘Atlantic’ 249 dihaploids, 1 NY148 dihaploid and 1 MSR127-2 dihaploid), 36 F1 hybrids, and 24 BC1 clones. These clones and their pedigree information can be found in Table S2. DNA Isolation and KASPTM Genotyping DNA was isolated from young leaf tissue using the QIAGEN DNeasy Plant Mini Kit (QIAGEN, Germantown, MD). Using ThermonucleotideBLAST v2.04 (Gans & Wolinsky, 2008), we determined that nine of 18 Kompetitive Allele Specific PCR (KASP)TM markers designed by Clot et al. (2020) within a candidate Sli region on chromosome 12 were predicted to amplify a single product in S. chacoense M6 (Leisner, et al., 2018). These nine KASPTM markers were ordered from LGC Genomics. In this study, each marker name contains ‘Sli’ followed by the last three digits of the marker physical position on chromosome 12 of the potato doubled monoploid S. phureja clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03) (Table S3). Each KASPTM assay included a SC and SI allele-specific forward primer as well as a common reverse primer (Table S3). KASPTM assays were run with 3.25 μL reaction system including 1.5 μL v4.0 2x Low ROX KASPTM Mastermix (LGC Genomics, Beverly, MA), 0.05 μL of KASPTM Assay by Design primer and 1.7 μL of 15-30 ng/μl genomic DNA. The PCR conditions for KASPTM marker assay was 95 °C for 10 min, followed by 10 cycles of touch down PCR from 65 °C to 57 °C with 0.8 °C decrease per cycle, then followed by 37 cycles of 95 °C for 20 s and 58 °C for 1 min. PCR fluorescent endpoint readings were performed using the Light Cycler® 480 Real-Time PCR System (Roche, Germany). SNP genotyping SNP genotyping of the recurrent selection population was performed previously by Alsahlany et al. 2020. Briefly, DNA was quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA) and normalized to a concentration of 50 ng μl-1. The Infinium 8.3K 250 Illumina Array (V1) was used to genotype founder clones and individuals from the recurrent selection population cycles 0-3 using an Illumina iScan Reader (Illumina, San Diego, CA) according to the manufacturer’s suggested protocol. Individuals from cycle 4 of the recurrent selection population were genotyped on the Infinium 22K Illumina Array (V3). GenomeStudio software was used for calling SNP genotypes (Illumina, San Diego, CA). In this study, the physical position of mapped SNPs from the Illumina Array on the DM pseudomolecules (PGSC Version 4.03) was used to select only SNPs on chromosome 12 between 58,047,342- 59,205,539 bp that were polymorphic in the sampled individuals. Self-compatibility Phenotyping Clones from the recurrent selection population were phenotyped for self-compatibility in the greenhouse between 2013-2018. One tuber for each clone was planted in a 18.9 L (5 gallon) pot and grown in the greenhouse under 16h-day/8h-night photoperiod conditions at 20-25 °C during both day and night (Alsahlany, 2019). Self-pollinations were made using fresh pollen collected from open flowers and self-compatibility was determined by fruit set (Alsahlany, 2019). Plants lacking pollen production (NP) and non-flowering (NF) plants were recorded. Self- compatibility phenotyping data for the recurrent selection population clones can be found in Table S1. For this study, clones with ≥ 10 pollinations that did not set fruit were considered SI while clones that did not set any fruit upon fewer than 10 pollinations were not considered for analysis. Non-flowering plants and plants that did not produce pollen were excluded from analysis. Data analysis Three Sli KASPTM markers resulting in ambiguous or negative calls in greater than 15% of tested clones were removed for future analysis (Table S3). Ambiguous calls refer to those samples where one of three genotype clusters could not be assigned. Marker data were filtered to remove 251 individuals with missing data at >2 marker loci (n=13). The allele amplified by the SC Sli allele- specific primer was coded as ‘A’ and the allele amplified by the SI Sli allele-specific primer was coded as ‘B.’ It is important to note that the terms SC Sli allele and SI Sli allele are derived from Clot et al. (2020) and refer to the marker genotype rather than the phenotypic self-compatibility characterization conducted in this study. Genotype frequencies of the homozygous SC Sli genotype (AA), heterozygous Sli genotype (AB), and SI Sli homozygous genotype (BB) were calculated for each of the six markers in each cycle of the recurrent selection population as well as the parental, F1, and BC1 generations of the backcross population. The association of each Sli KASPTM marker genotype with the self-compatibility phenotype was evaluated using Chi-squared likelihood ratio testing with contingency tables containing marker genotype (AA, AB or BB) and phenotype (SC or SI) data in JMP® software (Version Pro 13. SAS Institute Inc., Cary, NC). The R2 (U) reported is the -Loglikelihood of the model divided by the total -Loglikelihood. The Chi-squared test was used to test the association between select SNPs from the Illumina Array and the six Sli KASPTM marker genotypes as well as between the SNPs and the SC phenotype. Results KASPTM Haplotypes of Eight SC diploids We selected nine Sli KASPTM markers predicted to amplify a singular product in S. chacoense M6. These nine markers spanned a region of 224 kb on DM v4.03 chromosome 12 from 58,960,090-59,184,424 bp within the 333 kb Sli region reported by Clot et al. (2020) (58,945,000- 59,278,000 bp) (Figure 1). Of the nine, only six markers (Sli_090, Sli_561, Sli_304, Sli_626, Sli_898, and Sli_424) produced non-ambiguous genotype calls in ≥ 85% of the diploid germplasm tested. M6 is homozygous for the SC Sli genotype at all six loci while the SC clones 1S1, DMRH- 89, and S. chacoense PI 133664-40 have the SI Sli homozygous genotype at these six loci (Table 252 1). US-W4 and XD3 share a heterozygous Sli haplotype between markers Sli_090 (58,960,090 bp) and Sli_898 (59,040,898 bp) and RH is heterozygous for the six Sli marker loci (Table 1). KASPTM Analysis of a Diploid Recurrent Selection Population To investigate the effect of recurrent selection for self-compatibility on the Sli haplotype, we examined the mean genotype frequency at each of the six Sli KASPTM marker loci in each generation of recurrent selection (N=228). The frequency of the homozygous SC Sli genotype (AA) at each of the six Sli marker loci increased progressively from between 0.03-0.06 to between 0.79-0.88 over the course of five generations (Figure 2). By cycle 4, the frequency of the homozygous SI Sli genotype (BB) was zero at five of the six marker loci (Figure 2). Only the most distal marker, Sli_424, presented BB genotypes in 10% of cycle 4 clones (Figure 2). The frequency of heterozygous Sli genotypes also decreased in cycle 4 compared to cycle 0 at all six marker loci (Figure 2). The divergence between genotype frequency at linked markers Sli_626 and Sli_898 between cycle 1 and cycle 2 is an artifact of missing data for individuals in cycle 2 (Figure 2, Table S1). We tested the association between Sli KASPTM marker genotype and self-compatibility in 178 individuals with unambiguous phenotypes. Five contiguous Sli markers (Sli_090, Sli_561, Sli_304, Sli_626, and Sli_898) were each significantly associated with the SC phenotype (p < 0.0001) and demonstrate moderate prediction accuracy, with R2 (U) ranging from 0.27-0.35 (Table 2). For each of these five Sli markers, the homozygous SC Sli genotype (AA) was only found in SC individuals. The heterozygous Sli genotype (AB) was present in both SC and SI individuals. The homozygous SI Sli genotype (BB) was primarily present in SI individuals. However, three SC individuals (MSBB946-B, MSCC811-04, and MSDD807-05) were homozygous for genotype BB across these five Sli marker loci. The proportion of flowers that set fruit upon selfing in these three 253 SC clones varied (1.00, 0.10, and 0.27, respectively). Marker Sli_561 (58,962,561 bp) was the best predictor, while the most distal marker in the region (Sli_424) was least predictive of self- compatibility (Table 2). In the 178 individuals with unambiguous SC phenotypes, we observed a greater proportion of flowers setting fruit upon selfing under greenhouse conditions conferred by the homozygous SC Sli and heterozygous Sli genotype between markers Sli_090 – Sli_898 in cycles 1-4 (Figure S1). Marker Sli_424 did not follow this trend (Figure S1). Among the 178 individuals, none in cycle 0 had the homozygous SC Sli genotype. The heterozygous Sli genotype between markers Sli_090 – Sli_898 resulted in a higher proportion of fruit set in cycle 0 (Figure S1). Connecting SNP genotyping to self-compatibility phenotyping We compared the SC phenotype of 164 recurrent selection clones (13 founders, 11 cycle 0 clones, 15 cycle 1 clones, 42 cycle 2 clones, 56 cycle 3 clones, and 27 cycle 4 clones) with four SNP markers from the Illumina Potato Array 8.3K array residing near the Sli KASPTM markers used in this study (Table S4). The SNP solcap_snp_c1_13698 located at 58,983,259 bp on chromosome 12 was significantly associated with SC phenotype (R2 (U) = 0.23; p<0.0001). We then interrogated the SNP genotype of SI founder clones 84SD22 and Ber83, SC founder clones RH and M6, as well as 26 SC and 1 SI cycle 4 clones at 11 SNP markers from the 22K V3 Illumina Potato Array (Table S5). There was a significant association of SNP PotVar0053460, positioned 315 bp upstream of SNP solcap_snp_c1_13698 at 58,983,574 bp, with the SC phenotype (R2 (U) = 0.69; p<0.0001). Both SNPs solcap_snp_c1_13698 and PotVar0053460 lay between Sli KASPTM markers Sli_561 and Sli_304 (Figure 1) and are within a gene annotated as a potassium channel beta subunit protein (PGSC0003DMG400016870). 254 Sli Alleles in S. tuberosum Dihaploids Examining the marker genotype of dihaploid clones derived from S. tuberosum tetraploid varieties ‘Atlantic’ and ‘Superior’ revealed heterozygosity at the candidate Sli region. In the 11 ‘Atlantic’ dihaploids, both homozygous and the heterozygous haplotypes were observed (Table S2). Notably, the homozygous SC Sli haplotype was present in the ‘Atlantic’ dihaploid clone ATL.M.170. The SI Sli homozygous haplotype between markers Sli_090 and Sli_898 was most prevalent, found in seven ‘Atlantic’ dihaploids. The homozygous SC Sli haplotype was not observed in the four ‘Superior’ dihaploids analyzed (Table S2). The dihaploid derived from breeding clone MSR127-2 contains the homozygous SI Sli haplotype while the dihaploid generated from breeding clone NY148 has the heterozygous Sli genotype at five contiguous loci (with the exception of a no-call observed in marker Sli_424) (Table S2). Overall, the frequency of the SI Sli homozygous genotype was ≥ 0.50 at each marker in the dihaploid parental clones (Figure S2). Male sterility in the dihaploid clones prevented determination of a SC phenotype. KASPTM Analysis of a Diploid Backcross Population The frequency of the SI Sli homozygous genotype between markers Sli_090 – Sli_898 was low (0.20) in the SC sources used in the diploid backcross population (Figure S2). Only clone MSBB920-A contained the SI Sli homozygous haplotype at all six marker loci. In the 36 F1 clones used in this study, the heterozygous Sli (AB) and SI Sli homozygous (BB) genotypes were present in nearly equivalent frequency between markers Sli_090 – Sli_898 (Figure S2). A single F1 clone CC849-04 had the SC homozygous (AA) genotype between markers Sli_090 – Sli_898 (Table S2). This clone was derived from a cross between ‘Atlantic’ dihaploid ATL.M.198 and self- compatibility donor MSBB912-B, both of which were heterozygous for these five Sli marker loci (Table S2). The frequency of the SC Sli homozygous genotype increased in the 24 BC1 clones 255 compared to the F1 clones at all marker loci (Figure S2, 3). However, the BC1 individuals were predominantly heterozygous at all marker loci (Figure S2, 3). Discussion Self-compatibility Donors without Sli SC haplotypes Three SC clones lacked SC alleles across the Sli region analyzed in this study, suggesting that other genetic factors underlay self-compatibility in S. chacoense PI 133664-40, DMRH-89 and 1S1. The action of genetic factors besides Sli in SC DMRH progeny was previously hypothesized by Peterson et al. (2016). Independent mutation of S-RNase has been reported in several SC tomato species (Kondo, et al., 2002). Although relatively few S-RNase sequences have been cloned in potato, considerable effort to characterize S-RNase alleles has been undertaken in S. chacoense (Despres, et al., 1994; Marcellan, et al., 2006; Qi, et al., 2001; Saba-el-Leil, et al., 1994; Xu, et al., 1990). A survey of 16 S. phureja, S. stenotomum and S. okadae clones (Dzidzienyo, et al., 2016) suggests a wide allelic S-RNase diversity in potato. Further characterization of the S-RNase alleles in potato would be useful in designing crosses and selecting SC individuals. In Lycopersicon and Petunia, additional modifier loci are necessary for proper functioning of the S-locus in the SI reaction (Ai, et al., 1991; Bernatzky, et al., 1995; Clark & Kao, 1994; Martin, 1968). Several candidate modifier genes with unique function in the SI reaction of the pistil and pollen have been identified (reviewed in (McClure, et al., 2011)). The SC clones S. chacoense PI 133664-40, DMRH-89 and 1S1 lacking SC Sli alleles may instead contain self- compatibility alleles of modifier genes. Identification of these alternate sources of self- compatibility expands the genetic base available to breeders seeking to improve self-compatibility in breeding populations. 256 Sli Linkage to a Deleterious Allele A recessive lethal allele la3 overlapping Sli was localized to a 1.8 Mb region (58,479,322- 60,283,649 bp) on chromosome 12 in clone RH (Zhang, et al., 2019). We observed a heterozygous Sli haplotype in RH. There is also prior evidence for a recessive lethal allele on chromosome 12 in SC S. chacoense. In a population created by selfing XD3, Endelman, et al. (2019) identified distorted segregation between 57.6-59.2 Mb on chromosome 12 and conclude that XD3 maternal parent SC S. chacoense clone ’39-7’ harbors a recessive lethal allele in this region. Our study confirms the homozygous presence of Sli in S. chacoense M6 between 58,960,090-59,184,424 bp on chromosome 12 (Table 1) and suggests that Sli in M6 may be linked to a non-lethal allele of la3. In contrast, we report a heterozygous Sli haplotype in the inbred S. chacoense clone ‘524-8’. Retention of heterozygosity at Sli in this clone that has been inbred for seven generations could be a function of reduced recombination in the region. However, unlike in heterochromatic regions, residual heterozygosity in this gene-dense region most likely signals the presence of a recessive deleterious allele. Sli Contributes to Self-compatibility in a Diploid Recurrent Selection Population It is clear from this work that recurrent selection is an effective strategy in diploid potato to rapidly drive a desirable haplotype to fixation. Self-compatibility and the SC Sli haplotype were concurrently enriched over the course of five generations in a recurrent selection population. Five contiguous Sli KASPTM markers (Sli_090-Sli_898) spanning an 80.8 kb interval (58,960,090- 59,040,898 bp) were positively associated with self-compatibility in the recurrent selection population. The genotype at the sixth and most distal position within the region we surveyed, marker Sli_424, was not significantly associated with self-compatibility (∝ = 0.001). The uninformative marker Sli_424 is located 144 kb downstream from the closest neighbor marker 257 Sli_898, which could permit recombination between the markers (Figure 1). Several genes annotated as F-box proteins reside within the 80.8 kb interval linked to SC, including StSCI (PGSC0003DMG400016861), which was recently shown to be necessary for self-compatibility in clone RH (Sanwen, et al., 2019). The discrepancies we observed between Sli KASPTM marker genotype and SC phenotype can be attributed in part to the difficulty of accurately phenotyping self-compatibility and the inheritance of diverse sources of self-compatibility in the recurrent selection population. The homozygous SC Sli genotype (AA) at the five informative markers (Sli_090-Sli_898) was only present in M6 and SC recurrent selection individuals. The heterozygous Sli genotype (AB) at these markers was less reliable in predicting a SC phenotype. In other words, both SC and SI individuals from the recurrent selection population carried the heterozygous Sli genotype at these marker loci. The minimum self-pollinations required to determine SC phenotype in this study was relatively low (≥ 10 pollinations). Consequently, clones we classified as SI may have set fruit with additional self-pollinations attempts over a longer time period. (Haynes & Guedes, 2018) observed significant genotype x environment interactions for selfed fruit/seed set over two years of selfing a diploid hybrid Solanum phureja – S. stenotomum population. In addition, we could not phenotypically classify non-flowering and male sterile clones as SC or SI and their removal from analysis reduced our power to draw correlations with marker genotype. We also identified three SC individuals with the homozygous SI Sli genotype (BB) at the tested Sli marker loci. One of these individuals, MSBB946-B was the product of a cross with DMRH-89 while self-compatibility in the other two individuals may stem from genetic factors contributed by the bulked pollen of SC selections in their pedigree (Table S1). Introduction of non-Sli based self-compatibility to the recurrent selection population from clone DMRH-89 and the segregation of other genes involved 258 in self-compatibility could confound the ability of Sli markers to reliably predict self-compatibility in this population. Despite these challenges, our findings inform breeders seeking to improve Sli-derived self- compatibility in breeding populations and to more generally understand the genetic landscape of SC. When using Sli-derived SC, the laborious burden of phenotyping self-compatibility can be circumnavigated by selecting only clones with the homozygous SC Sli genotype (AA) at any one of the five informative Sli KASPTM loci. Importantly, we also report that SNPs solcap_snp_c1_13698 and PotVar0053460 are significantly associated with self-compatibility in this germplasm. These SNPs could serve as an efficient selection aid in breeding programs where clones are already routinely SNP genotyped with the Illumina Array. The Sli KASPTM markers described here also provide a valuable tool to select sources of self-compatibility other than the Sli-mediated self-compatibility found in M6. Sli Contribution to Self-compatibility in Dihaploids Extracted from Cultivated Potato Because nearly all S. tuberosum-derived dihaploids are male sterile, it is not possible to identify SC clones by self-pollinating them. Instead, by identifying dihaploids that carry SC alleles, it will be possible to select those genotypes and make more rapid progress toward the development of SC breeding populations. We identified SC Sli alleles in dihaploids of ‘Atlantic’, ‘Superior’ and breeding clone NY148. The homozygous SC Sli haplotype in ‘Atlantic’ dihaploid ATL.M.170 most likely indicates that ‘Atlantic’ is duplex for Sli. Interrogating the Sli haplotype of dihaploids allows efficient selection of individuals that will contribute to self-compatibility while donating favorable combinations of S. tuberosum alleles. Likewise, examining the Sli haplotype of cultivated tetraploid varieties could inform the self-compatibility potential of extracted dihaploids. 259 Challenges of Increasing Sli-based Self-compatibility in a Backcross Population The pool of S. tuberosum dihaploids used as recurrent parents in the backcross population was dominated by clones with heterozygous Sli and homozygous SI Sli haplotypes (Figure 3, S2). As a result, the frequency of the homozygous SC Sli haplotype at the six Sli KASPTM loci was low in the F1 hybrids. Use of a diverse set of S. tuberosum dihaploids as recurrent parents slightly increased the frequency of the homozygous SC Sli haplotype in the BC1 generation. To simultaneously improve agronomic traits and Sli-mediated self-compatibility through backcrossing it will be crucial to prioritize the use of dihaploids with the homozygous SC Sli haplotype. Prospects of Improving Self-fertility in Diploid Potato Self-compatibility is only one of many components that contribute to self-fertility. Improvement of self-fertility in diploid potato also depends upon concurrent improvement of male and female fertility and fecundity. The occurrence of male sterile, female sterile and non-flowering plants in diploid germplasm obscures the identification of genetic factors involved in reliable self- compatibility transmission and excludes potentially valuable genotypes from use in diploid variety development. This phenomenon is exemplified when using the clone US-W4 as a parent. Although it is readily SC, selfed offspring exhibit poor vigor and fertility (data not shown). In addition, there are a number of parameters in addition to the self-compatibility reaction itself that constitute good self-fertility for practical breeding purposes, such as berry set, seed set, and interactions with the environment, especially temperature. Conclusion Our characterization of the Sli haplotype in diploid self-compatibility donors used in North American breeding programs informs their use as parents and underscores the genetic complexity 260 of SC. We explored the feasibility of using six Sli KASPTM markers to select SC individuals in two genetically diverse populations. The Sli KASPTM markers used in this study were developed and validated in 45 clones from two S. tuberosum populations (Clot et al., 2020). We found that only the homozygous Sli SC genotype at five Sli KASPTM markers reliably predicted SC phenotype. Thus, when deploying Sli-based self-compatibility in breeding populations, selection based on the homozygous Sli SC KASPTM marker genotype will be efficient. However, we observed discrepancies between the heterozygous and homozygous SI Sli genotypes and SC phenotype. This observation illustrates both the necessity of robust self-compatibility phenotyping procedures and the diversity of genetic features contributing to self-compatibility in the germplasm used in this study. 261 APPENDICES 262 APPENDIX A: Chapter 4 Tables Table 4.1. KASPTM marker genotypes of eight self-compatible (SC) diploid clones at six tested marker loci (homozygous SC genotype (AA), heterozygous genotype (AB) and homozygous self-incompatible genotype (BB)) (NC = No Call). Markers are shown in order of their physical position on chromosome 12 of the potato doubled monoploid S. phureja clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03). Marker Genotype Clone Sli_090 Sli_561 Sli_304 Sli_626 Sli_898 Sli_424 S. chacoense M6 S. chacoense ‘524-8’ US-W4 XD3 RH DMRH-89 1S1 S. chacoense PI 133664-40 AA AB AB AB AB BB BB BB AA AB AB AB AB BB BB BB AA AB AB AB AB BB BB BB AA AB AB AB AB BB BB BB AA AB AB AB AB BB BB BB AA AB AA BB AB NC BB BB 263 Table 4.2. Chi-squared likelihood ratio testing of association between the genotype at six marker loci and a self-compatible phenotype in a diploid recurrent selection population. Markers are shown in order of their physical position on chromosome 12 of the potato doubled monoploid S. phureja clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03). Asterisks denote significance at a significance level of ∝ = 0.001. Marker Sli_090 Sli_561 Sli_304 Sli_626 Sli_898 Sli_424 N 159 138 164 153 147 163 Chi-square Prob>ChiSq 27.735 31.737 33.548 31.654 32.677 12.009 <.0001* <.0001* <.0001* <.0001* <.0001* 0.0025 R2 (U) 0.27 0.35 0.32 0.32 0.32 0.12 264 APPENDIX B: Chapter 4 Figures Figure 4.1. Physical position of nine KASPTM markers and two SNP markers reported in this study on chromosome 12 of the potato doubled monoploid S. phureja clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03). The six informative KASPTM markers that produced non- ambiguous genotype calls in ≥ 85% of the diploid germplasm tested (triangle) and three uninformative KASPTM markers (+) are shown. The two SNP markers (solcap_snp_c1_13698 and PotVar0053460) associated with the self-compatible (SC) phenotype are also given by a triangle. Significance of marker association with the SC phenotype, reported as the p-value of the Chi- squared test, is plotted in a blue-red gradient. The 333 kb Sli region reported by Clot et al. (2020) is shaded beige and delimited by black lines. Figure created using JMP® software (Version Pro 13. SAS Institute Inc., Cary, NC). 265 Figure 4.2. The frequency of the homozygous self-compatible (SC) genotype (AA) at six KASPTM marker loci spanning the Sli region on chromosome 12 (58,960,090-59,040,898 bp) increased over the course of five generations in a recurrent selection population. Mean genotype frequency of the homozygous SC genotype (AA), the heterozygous genotype (AB) and the homozygous self- incompatible genotype (BB) of six KASPTM markers within the Sli candidate region on chromosome 12 were plotted over the five generations (N=228; 39 cycle 0 clones, 25 cycle 1 clones, 50 cycle 2 clones, 85 cycle 3 clones and 29 cycle 4 clones). Figure created using JMP® software (Version Pro 13. SAS Institute Inc., Cary, NC). 266 Figure 4.3. Genotype frequencies of individuals from the diploid backcross population at KASPTM marker Sli_561, which delivered the best prediction accuracy of a self-compatible (SC) phenotype in the recurrent selection population. Mean genotype frequency of the homozygous SC genotype (AA), the heterozygous genotype (AB) and the homozygous self-incompatible genotype (BB) are shown for four SC donors, 17 recurrent dihaploid parents, 35 F1 hybrids, 24 BC1 individuals from a diploid backcross population. Figure created using JMP® software (Version Pro 13. SAS Institute Inc., Cary, NC). 267 APPENDIX C: Chapter 4 Supplementary Data Figure S4.1. Proportion of flowers that set fruit upon selfing under greenhouse conditions plotted against the marker genotype [homozygous self-compatible genotype (AA), heterozygous genotype (AB) and homozygous SI genotype (BB)] of six KASPTM markers in 178 individuals of a diploid recurrent selection population (18 cycle 0 clones, 15 cycle 1 clones, 42 cycle 2 clones, 74 cycle 3 clones, and 29 cycle 4 clones). Markers are shown in order of their physical position on chromosome 12 of the potato doubled monoploid S. phureja clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03). Figure created using JMP® software (Version Pro 13. SAS Institute Inc., Cary, NC). 268 y c n e u q e r F e p y t o n e G 1.0 0.5 0.0 1.0 0.5 0.0 1.0 0.5 0.0 1.0 0.5 0.0 Sli_090 Sli_304 Sli_561 AA AB BB No Call S C D o n o r P a r e n t s P a r e n t s i D h a p o d R e c u r r e n t l i C y c e l F 1 B C 1 Sli_626 Sli_898 Sli_424 Marker Figure S4.2. Stacked bar graphs show the frequency of the homozygous self-compatible (SC) (AA; black), heterozygous (AB; blue), homozygous self-incompatible (BB; grey), and No Call (magenta) genotype at six KASPTM marker loci in the backcross population Horizontal panels contain self-compatible Donor Parents (n=5), Dihaploid Recurrent Parents (n=17), F1 clones (n=36), and BC1 clones (n=24). Markers are shown in order of their physical position on chromosome 12 of the potato doubled monoploid S. phureja clone DM1-3 (DM) pseudomolecules (PGSC Version 4.03). Figure created using JMP® software (Version Pro 13. SAS Institute Inc., Cary, NC) 269 Table S4.1. Pedigree and self-compatibility data of 241 lines from a diploid recurrent selection population used in this study. Bulk pollen was created by mixing equal portions of self-compatible selections. 0 9 0 _ i l S 1 6 5 _ i l S 4 0 3 _ i l S 6 2 6 _ i l S 8 9 8 _ i l S 4 2 4 _ i l S B B A B A A A B A A A B B B A B B B B B B B B B B B A B N C B B A B A A A B A A A B N C A B N C B B B B B B B B A B B B B B A B A A A B A A A B B B A B B B B B B B B B B B A B B B B B A B A A A B A A A B B B B B B B B B B B N C B B N C N C B B A B A A A B A A A B B B B B B B B B B B B B B B A B B B N C A B A A A B A A B B B B B B A B B B B B B B B B A B A A Line DMRH -89 RH Scab4- 48 Solanu m chacoe nse '524-8' Solanu m chacoe nse M6 XD3 2xLB- 75 84SD2 2 Ber83 HS66 M269- 1Y MRC20 5 S703-5 BB900- BB901- A A Flowe rs Frui ts Fracti on of flower s setting fruit Fertili ty SC SC SC SC SC SC SI SI SI SI SI SI SI SC NP Cycle Parent al Parent al Parent al Parent al Parent al Parent al Parent al Parent al Parent al Parent al Parent al Parent al Parent al cycle cycle 0 0 Fema le Mal e Fema le 2 Mal e 2 Fema le 3 Mal e 3 Fema le 4 Mal e 4 Fema le 5 Mal e 5 2xLB -75 Scab4 -48 M6 M6 - - - - - - - - - - - - - - - - 11 10 0.91 270 Table S4.1 (cont’d) A B A A A B A B A B A B A B A B A B A B A B A B A A A B A B A B A B A B A B A B A B A B A B A A A B A B A B A B A B A B A B A B A B A B A A A B A B A B A B A B A B A B A B A B A B A A A B A B A B A B A B A B A B A B A B N C BB BB BB BB A A B B A A B B A B A B A B A B A B A B BB921-B BB BB BB BB BB BB922- N C BB BB BB A A B B A A B B A B N C A B A B BB A B A B BB BB BB BB BB A B N C A B A B BB927-A BB BB902-A BB902-B BB902-C BB902-D BB903-A BB907-A BB908-A BB909-A BB910-A BB912-A BB917-A BB918-A BB919-A BB921-A 01 BB924-A BB925-A BB927 N C A B A B A B A B A B A B A B B B A B A B A B A B A B A B B B B B N C B B N C 3 3 1.0 0 3 2 1 1 2 4 4 4 4 3 0 0 0 0 0 0 1.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 SC NP NP NP NF SC SI NP NP SI NP NP NP s s s s cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 271 Ambiguou cycle Ambiguou cycle Solanum chacoense '524- Ambiguou cycle Solanum chacoense '524- Ambiguou cycle Solanum chacoense '524- S703-5 S703-5 S703-5 S703-5 Ber83 A151-16 L308-A HS66 A133-134 MRC205 M267-B M269-1Y MRC205 S703-5 S703-5 US-W1712 A133-57 8' 8' 8' M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 8' 8' 8' 8' 8' 8' 8' Solanum chacoense '524- Solanum chacoense '524- Solanum chacoense '524- Solanum chacoense '524- Solanum chacoense '524- Solanum chacoense '524- Solanum chacoense '524- Ber83 2xLB-75 2xLB-75 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Table S4.1 (cont’d) 0.00 SC SI SC SC NP NF 0.00 Ambiguous 6 0 0 0 0 0 8 2 2 0 6 1.00 0.00 0.00 Ambiguous 0.00 Ambiguous BB929-A NC AB AB NC AB BB 6 BB930-B AB BB BB BB BB BB 10 BB934-A BB BB BB BB BB BB 8 3 BB935-A AB AB AB AB AB BB BB936-A AB NC AB AB AB BB BB938-A AB AB AB AB AB BB BB939-C BB BB BB BB BB BB 2 BB940-A BB BB BB BB BB BB BB941-A AB AB AB AB AB BB BB943-A AB AB AB AB AB BB 14 BB944-A AB AB AB AB AB BB BB945-A BB BB BB BB BB NC BB946-A AA AA AA AA AA BB 1.00 8 BB946-B BB BB BB BB BB AB 1.00 BB953-10 AB AB AB AB AB BB 2 1.00 2 BB953-A AB AB AB AB AB BB 0.00 Ambiguous BB953-B AB AB AB AB AB BB 2 0.32 CC804-01 AB NC AB AB AB AB 19 CC804-05 NC AA AA AA AA AA 27 19 0.70 9 CC805-03 NC BB BB BB BB AB 8 CC806-05 AB AB AB AB AB AB CC807-01 BB BB BB BB BB NC CC807-04 AA AA AA AA AA BB CC809-02 AB AB AB AB AB AB 18 CC809-04 AB AB AB AB NC AA 23 0.00 Ambiguous 1.00 SI NF SC SC SC SC NF NF SC SC 0 8 3 8 0.17 0.35 RH HS66 Scab4-48 XD3 XD3 84SD22 Ber83 XD3 XD3 XD3 XD3 XD3 XD3 cycle 0 cycle 0 cycle 0 cycle 0 cycle 0 M269-1Y cycle 0 cycle 0 A133-134 cycle 0 M267-B cycle 0 A133-57 DMRH-89 DMRH-89 XD3 cycle 0 DMRH-89 cycle 0 L308-A cycle 0 DMRH-89 84SD22 cycle 0 DMRH-89 MRC205 cycle 0 DMRH-89 MCR205 cycle 0 MRC205 cycle 0 MRC205 cycle 0 MRC205 cycle 1 BB901-A cycle 1 BB901-A cycle 1 BB902-A cycle 1 BB902-B cycle 1 BB902-C cycle 1 BB902-C cycle 1 BB909-A cycle 1 BB909-A XD3 XD3 XD3 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Scab4-48 M6 Scab4-48 M6 S703-5 M6 S703-5 M6 S703-5 M6 S703-5 M6 HS66 M6 HS66 M6 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 272 Table S4.1 (cont’d) N C B B A B B B A B A B B B A B N C A A A B B B B B A B A B B B B B A B A B A A B B B B N C B B A B A B B B A B A B A A N C B B B B A B A B B B B B A B N C A A B B N C A B A B A B A B B B A B A B N C A B B B B B A B A B B B B B A B A B A A B B B B A B B B A B A B B B A B A A A A A B B B B B A B A B B B B B A B A B A A B B B B A B B B A B A B B B A B A A A A A B B B B B A B A B B B B B A B A B A A B B B B A A A B N C A B A B A B N C B B B B B B B B B B N C B B B B A A A A B B 1 0 1 2 9 1 2 3 2 4 3 3 1 7 7 4 9 3 5 3 8 6 1 7 0 4 2 7 0 0 7 0 0 2 7 5 0.1 0 0.5 8 0.0 0 0.3 3 0.6 7 0.2 9 0.0 0 0.0 0 1.0 0 0.0 0 0.0 0 0.6 7 0.8 8 0.8 3 NF SC SC NF Ambigu ous NF NF NF SC SC SC SI SI SC SI SI NF SC SC SC CC810- CC811- CC811- CC812- CC815- CC815- CC819- CC820- CC822- CC822- CC823- CC823- CC825- CC825- CC827- CC831- CC832- 01 04 05 02 01 02 03 03 01 05 03 05 02 04 06 03 06 01 04 05 DD802- DD802- DD803- cycle BB925- Solanum chacoense cycle BB927- Solanum chacoense MRC205 MRC205 MRC205 M267-B RH RH '524-8' '524-8' XD3 XD3 XD3 XD3 Scab4-48 Scab4-48 HS66 MRC205 MRC205 BB901-A BB901-A BB901-A Solanum chacoense Solanum chacoense Solanum chacoense M6 M6 M6 '524-8' '524-8' '524-8' Ber83 2xLB-75 Ber83 Ber83 Ber83 Ber83 XD3 XD3 XD3 XD3 XD3 Bulk1 Bulk1 Bulk1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Scab4- 48 Scab4- 48 Scab4- 48 - M 6 M 6 M 6 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 A B B A A A A A A A B B A A A A B cycle BB912- cycle BB912- cycle BB912- cycle BB917- cycle BB920- cycle BB920- cycle BB930- cycle BB930- cycle BB930- cycle BB930- cycle BB934- cycle BB934- cycle BB938- cycle BB953- cycle BB953- cycle CC804- cycle CC804- cycle CC804- 01 01 05 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 273 A B A A A B A B A B B B A B A B A B N C A B A B A B A B N C A B A B A A A B B B A B A A A B A B N C B B N C A B A B A B N C A B A B N C A B N C A B A A A B B B A B A A A B A B A B B B A B A B A B A B A B A B A B A B A B A B A B A A A B B B Table S4.1 (cont’d) DD804 -06 DD804 -09 DD805 -05 DD805 -08 DD807 -03 DD807 -05 DD807 -06 DD808 -10 DD809 -09 DD812 -01 DD812 -02 DD812 -03 DD812 -05 DD814 -04 DD821 -09 DD821 -10 DD824 -01 DD825 -01 DD829 -01 DD829 -07 A B A A A B A B A B B B A B A B A B A B A B A B A B A B A B A B A B A A A B B B A B A A A B N C A B B B A B N C N C N C A B A A A B A B A B N C A B N C A B N C A A A B A B A B A B A A B B B B A A A A A A A A B B A A A B A A B B A A B B B B 1 3 9 6 6 6 1 1 1 6 1 0 6 5 5 8 1 2 5 6 5 1 7 1 4 0.0 0 0.6 7 1.0 0 0.8 3 0.8 3 0.2 7 0.3 1 0.3 0 1.0 0 0.4 0 0.4 0 0.0 0 0.5 0 1.0 0 1.0 0 1.0 0 0.3 5 0.3 6 0 6 6 5 5 3 5 3 6 2 2 0 6 5 6 5 6 5 SI SC SC SC SC SC SC SC SC NF SC SC Ambigu ous SC SC SC SC SC SC NF cycle CC806- cycle CC806- cycle CC806- cycle CC806- cycle CC807- cycle CC807- cycle CC807- cycle CC809- cycle CC809- cycle CC811- cycle CC811- cycle CC811- cycle CC811- cycle CC813- cycle CC817- cycle CC817- cycle CC819- cycle CC820- cycle CC825- cycle CC825- 02 02 05 05 04 04 04 02 04 05 05 05 05 04 02 02 03 03 02 02 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 BB902 -B BB902 -B BB902 -B BB902 -B BB902 -C BB902 -C BB902 -C BB909 -A BB909 -A BB912 -B BB912 -B BB912 -B BB912 -B BB918 -A BB921 -B BB921 -B BB925 -A BB927 -A BB934 -A BB934 -A Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 S703-5 S703-5 S703-5 S703-5 S703-5 S703-5 S703-5 HS66 HS66 MRC205 MRC205 MRC205 MRC205 M269-1Y MRC205 MRC205 Solanum chacoense Solanum chacoense '524-8' '524-8' Scab4-48 Scab4-48 274 M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 Solanum chacoense '524-8' M6 M6 Ber83 2xLB-75 XD3 XD3 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Table S4.1 (cont’d) B B A B B B A B A B A B B B A B A B A B B B A A A B A A A B A B A A A A A A A B B B A B N C A B A B A B B B N C N C A B B B A A N C A A A B N C A A A A A A A B B B A B N C A B A B A B B B A B A B A B B B A A A B A A A B A B A A A A A A A B N C A B B B A B N C A B B B A B A B N C N C A A A B A A A B A B A A A A A A N C B B A B B B B B B B N C B B B B B B B B B B A A B B A A B B N C A A A A A A N C B B A B B B B B B B A B B B A A A A A B B B A A B B A A B B A A A A B B A A A B 2 5 3 2 1 5 7 6 5 1 2 1 0 1 3 5 1 6 9 8 1 2 6 8 1 2 7 7 0.0 0 0.6 7 0.0 0 0.6 0 0.0 0 0.0 0 0.0 0 0.3 3 0.0 0 0.3 8 0.0 0 0.1 3 0.2 2 1.0 0 0.5 0 1.0 0 0.3 8 0.8 3 0.7 1 0.7 1 0 2 0 3 0 0 0 4 0 5 0 2 2 8 6 6 3 1 0 5 5 DD829- DD829- DD831- DD837- DD838- DD838- DD844- DD845- DD845- DD847- DD847- DD848- DD848- DD849- 09 10 01 08 01 02 03 02 03 05 06 01 02 06 07 03 06 06 08 04 DD849- DD850- DD850- DD851- DD851- DD852- Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 2 Bulk 1 Bulk 1 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 BB934- BB934- BB938- BB953- BB953- BB953- BB930- BB930- BB930- BB902- BB902- BB902- BB902- BB902- BB902- BB909- BB909- BB912- BB912- BB917- A A A A B B B B B A A B B B B A A A A A Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Scab4- 48 Scab4- 48 HS66 MRC20 MRC20 MRC20 5 5 5 XD3 XD3 XD3 S703-5 S703-5 S703-5 S703-5 S703-5 S703-5 HS66 HS66 MRC20 MRC20 5 5 B cycle CC825- cycle CC825- cycle CC827- cycle CC831- Ambiguo cycle CC832- Ambiguo cycle CC832- Ambiguo cycle CC823- SI SC SI SC us us us SC SI SC us Ambiguo SC SC SC SC SC SC SC SC SC 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 02 02 06 03 06 06 04 05 05 03 03 02 02 05 05 04 04 cycle CC823- cycle CC823- cycle CC805- cycle CC805- cycle CC806- cycle CC806- cycle CC806- cycle CC806- cycle CC809- cycle CC809- cycle cycle cycle CC810 CC810 CC812- 02 275 XD3 XD3 XD3 XD3 XD3 XD3 Ber83 Ber83 Ber83 M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M267- Solanum chacoense '524-8' Table S4.1 (cont’d) A B A B A B A B A B A A A B A A A A A B A B A B A A A B A B A A A B A B A B A A DD852 -08 DD853 -04 DD853 -05 DD855 -01 DD855 -03 DD857 -03 DD865 -03 EE700 -01 EE700 -03 EE701 -02 EE701 -06 EE702 -05 EE703 -07 EE704 -03 EE704 -06 EE704 -08 EE705 -04 EE705 -06 EE705 -08 EE706 -03 N C N C N C A B A B A A A B A A A A A B N C A B A A A B A B A A A B A B A B A A A B A B A B A B A B A A A B A A A A A B A B A B A A A B A B A A A B A B A B A A N C N C A B A B A B N C N C A A A A N C A B N C N C A B A B A A A B N C A B A A B B B B N C A B N C A A N C A A A A A B A B N C A A N C N C A A A B N C N C A A B B A A A A A A A B A A A B A A A A A B A B A B A A A B A B A A A B A A A A A A 1 7 5 2 1 0 3 9 1 8 1 5 5 4 3 1 4 3 3 1 7 2 2 7 1 6 5 4 6 0. 00 0. 20 0. 00 0. 00 0. 33 0. 56 0. 11 0. 20 0. 60 0. 25 1. 00 0. 36 0. 21 0. 18 0. 32 0. 29 0. 13 1. 00 1. 00 0. 33 0 1 0 0 1 5 2 3 3 1 3 5 7 3 7 2 2 5 4 2 SI SC Ambigu ous SI SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC BB91 7-A BB91 8-A BB91 8-A BB92 1-B BB92 1-B BB92 7-A BB92 0-A CC80 4-1 CC80 4-1 CC80 4-1 CC80 4-1 CC80 4-5 CC80 6-2 CC80 6-5 CC80 6-5 CC80 6-5 CC80 6-5 CC80 6-5 CC80 6-5 CC80 7-4 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 M267-B M269-1Y M269-1Y S703-5 S703-5 Solanum chacoense '524-8' RH BB901-A BB901-A BB901-A BB901-A BB901-A BB902-B BB902-B BB902-B BB902-B BB902-B BB902-B BB902-B BB902-C cycl e 2 cycl e 2 cycl e 2 cycl e 2 cycl e 2 cycl e 2 cycl e 2 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 CC812 -02 CC813 -04 CC813 -04 CC817 -02 CC817 -02 CC820 -03 CC815 -02 DD802 -01 DD802 -01 DD802 -04 DD802 -04 DD803 -05 DD804 -09 DD805 -05 DD805 -05 DD805 -05 DD805 -08 DD805 -08 DD805 -08 DD807 -03 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 276 Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' 2xLB-75 Solanum chacoense '524-8' Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 Bulk1 - - - - - - - Scab4 -48 Scab4 -48 Scab4 -48 Scab4 -48 Scab4 -48 S703- S703- S703- S703- S703- S703- S703- S703- 5 5 5 5 5 5 5 5 - - - - - - - M 6 M 6 M 6 M 6 M 6 M 6 M 6 M 6 M 6 M 6 M 6 M 6 M 6 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Table S4.1 (cont’d) A A A B A B A B A B A B A B A B A A A A A A A A A B A B A A A B A B A A A A A A A A A B A B N C A B A B A B A B A A A A A A N C A B N C A A A B A B A A A A A A A A A B N C A B A A A A A B A B A A A A A A A A B B A B A A A B A B A A A A A A A A B B A B A A A A A A A A B B A A A A A A A A B B B B A A B B A A B B A A A A 1 9 1 5 8 2 0 6 6 4 6 5 6 1 3 7 3 1 5 2 0 6 1 5 5 1 4 3 7 2 0 0 4 3 3 4 3 7 3 2 8 1 4 5 5 2 3 0. 16 0. 47 0. 25 0. 00 0. 00 0. 67 0. 75 0. 50 0. 80 0. 50 0. 54 0. 43 0. 67 0. 53 0. 70 0. 83 0. 33 0. 40 0. 21 SC SC SC SI NP Ambig uous SC SC SC SC SC SC SC SC SC SC SC SC SC SC cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 DD807 -05 DD808 -10 DD808 -10 DD809 -09 DD809 -09 DD809 -09 DD812 -02 DD812 -02 DD812 -02 DD812 -02 DD812 -03 DD814 -04 DD814 -04 DD821 -10 DD821 -10 DD821 -10 DD825 -01 DD829 -01 DD829 -01 DD829 -01 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k7 Bul k8 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 277 CC80 7-4 CC80 9-2 CC80 9-2 CC80 9-4 CC80 9-4 CC80 9-4 CC81 1-5 CC81 1-5 CC81 1-5 CC81 1-5 CC81 1-5 CC81 3-4 CC81 3-4 CC81 7-2 CC81 7-2 CC81 7-2 CC82 0-3 CC82 5-2 CC82 5-2 CC82 5-2 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 BB90 2-C BB90 9-A BB90 9-A BB90 9-A BB90 9-A BB90 9-A BB91 2-B BB91 2-B BB91 2-B BB91 2-B BB91 2-B BB91 8-A BB91 8-A BB92 1-B BB92 1-B BB92 1-B BB92 7-A BB93 4-A BB93 4-A BB93 4-A Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 S703-5 HS66 HS66 HS66 HS66 HS66 MRC205 MRC205 MRC205 MRC205 MRC205 M269-1Y M269-1Y S703-5 S703-5 S703-5 Solanum chacoense '524-8' Scab4-48 Scab4-48 Scab4-48 M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' 2xLB-75 XD3 XD3 XD3 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A A A B A B A B A B A B A B A B A A A A A A A A A B N C A A A B A B A A N C A A A A A B A B A B A B A B N C N C A A A A A A A A A B A B N C N C N C A A A A A A EE707 -05 EE708 -02 EE708 -06 EE709 -01 EE709 -03 EE709 -04 EE710 -03 EE710 -06 EE710 -09 EE710 -10 EE711 -01 EE712 -02 EE712 -07 EE713 -07 EE713 -08 EE714 -04 EE716 -06 EE717 -01 EE717 -03 EE717 -05 Table S4.1 (cont’d) A B A B A A A A N C A A A B A B A B A A A A A A A A A A A A A A A B A B A A N C A B A B A A A A A A A A A B A B A B A A A A A A A A A A A A A A A B A B A A A B A B A B A A A A A A A A A B A B N C A A A A A A A A A A A A A A A B A B A A A B A B A B A A A A A A A A A B N C N C A A A A A A A A A A A A A A N C N C A A A B A B A A A A N C A A A A A B A B A B A A A A A A A A A A A A A A A B A B A A A B 0.4 3 0.5 0 0.2 0 0.1 1 1.0 0 0.3 3 0.2 5 0.0 0 0.0 0 0.0 0 0.0 8 0.5 0 1.0 0 1.0 0 0.5 0 0.4 0 0.7 5 0.1 7 0.6 0 3 2 3 2 8 3 2 0 0 0 1 4 7 5 7 2 3 3 3 7 4 1 5 1 9 8 9 8 9 6 5 1 2 8 7 5 1 4 5 4 1 8 5 SC SC SC SC SC SC SC us us NF us SC SC SC SC SC SC SC SC SC A B A B A A A A A A N C A B A B A B A A A A A A A A A A A A A A A B A B A A A B EE718- EE719- EE721- EE721- EE722- EE723- EE725- EE726- 01 02 03 06 07 06 02 02 09 02 04 02 03 04 03 08 09 03 06 04 EE726- EE727- EE727- EE728- EE729- EE729- EE730- EE730- EE730- EE732- EE732- EE733- Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 CC825- CC827- CC831- CC831- CC832- CC832- CC805- CC805- CC805- CC805- CC805- CC823- CC806- CC806- CC806- CC806- CC806- CC809- CC809- 2 6 3 3 6 6 3 3 3 3 3 5 5 5 5 5 5 4 4 CC810 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 1 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 Bulk 2 BB934- BB938- BB953- BB953- BB953- BB953- BB902- BB902- BB902- BB902- BB902- BB930- BB902- BB902- BB902- BB902- BB902- BB909- BB909- BB912- A A A A B B A A A A A B B B B B B A A A Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Scab4- 48 HS66 MRC20 MRC20 MRC20 MRC20 5 5 5 5 XD3 XD3 XD3 XD3 XD3 XD3 S703-5 M6 S703-5 M6 S703-5 M6 S703-5 M6 S703-5 M6 Ber8 3 XD3 S703-5 M6 S703-5 M6 S703-5 M6 S703-5 M6 S703-5 M6 HS66 HS66 MRC20 5 M6 M6 M6 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - DD829-10 DD831-01 SI DD837-08 DD837-08 DD838-01 SI SI SI SI SI SI SI cycle DD838-02 Ambiguo cycle DD847-05 DD847-06 Ambiguo cycle DD847-06 cycle DD847-06 Ambiguo cycle DD847-06 cycle DD845-03 cycle cycle cycle cycle cycle cycle 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 cycle cycle cycle cycle cycle cycle cycle cycle DD849-06 DD849-06 DD849-07 DD849-07 DD849-07 DD850-06 DD850-06 DD851-06 278 Table S4.1 (cont’d) A A A B A A A B A B A B A A A A A A A B A A A B A B N C A B N C A A A A A B A A A A N C A A A B A B A B A A A A A A A B A A A B A B A A A B B B A A A A A B A A A A B B A A A B A B A B A A A A A A A B A A N C A B A A A B B B A A A A A B A A N C B B A A B B A A A B A A A A A A A A A A A A A B A A A A B B A A A A A B A A 1 0 5 5 3 2 7 5 6 1 2 4 3 2 4 5 6 1 0 9 9 3 1 2 1 4 2 6 3 3 2 0 3 4 1 7 0. 90 0. 60 0. 20 0. 67 0. 04 0. 80 0. 33 0. 50 0. 75 1. 00 1. 00 0. 00 0. 60 0. 67 0. 10 0. 78 NF SC SC SC SC SC SC SC SC SC SC SC Ambig uous SC SC SC SC cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 cycl e 3 DD851- DD851- 08 08 04 04 DD852- DD852- DD852- 08 SI DD853- DD853- 04 04 DD853- 05 SI DD853- 05 SI DD855- 01 SI DD855- 01 SI DD855- 01 SI DD855- 01 SI DD855- DD855- DD857- DD865- DD865- DD865- DD802- 03 03 03 02 02 03 02 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 CC81 0 CC81 0 CC81 2-2 CC81 2-2 CC81 2-2 CC81 3-4 CC81 3-4 CC81 3-4 CC81 3-4 CC81 7-2 CC81 7-2 CC81 7-2 CC81 7-2 CC81 7-2 CC81 7-2 CC82 0-3 CC81 5-2 CC81 5-2 CC81 5-2 CC80 4-1 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k1 BB91 2-A BB91 2-A BB91 7-A BB91 7-A BB91 7-A BB91 8-A BB91 8-A BB91 8-A BB91 8-A BB92 1-B BB92 1-B BB92 1-B BB92 1-B BB92 1-B BB92 1-B BB92 7-A BB92 0-A BB92 0-A BB92 0-A BB90 1-A Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 279 MRC205 MRC205 M267-B M267-B M267-B M269-1Y M269-1Y M269-1Y M269-1Y S703-5 S703-5 S703-5 S703-5 S703-5 S703-5 Solanum chacoense '524-8' RH RH RH M6 M6 Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' 2xLB-75 Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Scab4-48 M6 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N C A B A A A B A B A B A A A A A A A B A A A B A B A A A B B B A A A A A B A A A B A B A A A B A B A B A A A A A A A B A A A B A B A A N C B B A A A A A B A A EE734 -01 EE734 -04 EE735 -01 EE735 -03 EE736 -01 EE737 -05 EE737 -09 EE738 -01 EE738 -04 EE739 -01 EE739 -02 EE739 -04 EE739 -05 EE740 -01 EE740 -04 EE741 -08 EE742 -06 EE742 -07 EE743 -04 EE744 -06 Table S4.1 (cont’d) A A A B A B A B A B A B B B B B A A A B A A A A A A A A A A A A A A A A A A A A A A A B N C A B A A A B B B B B A A A B A A A A A A A A A A A A A A A A A A A A EE745- EE747- EE747- EE747- EE748- EE749- EE749- EE749- EE749- EE749- EE827- EE827- 10 04 09 13 06 02 03 04 05 06 05 08 01 FF600- FF603 FF604- FF609- FF611- FF611- FF612- FF613- 01 02 02 03 03 03 A A A B A B A B A A A B B B B B A A A B A A A A A A A A A A A A A A A A N C A A A A A B A B A B A A N C B B B B A A A B A A A A A A A A A A A A A A A A A A A A A A N C A B A B A A B B B B B B A A N C A A A A A A A A A A A A A A A A A A A A A A A B A B N C A A A B B B B B A A A B A A A A A A A A A A A B A A A A A A A A 1 0 8 2 7 1 7 9 6 3 1 3 1 1 7 5 6 1 1 1 6 3 1 6 2 4 8 5 0.5 0 0.5 0 0.0 4 0.0 6 0.3 3 0.0 0 0.0 0 0.1 5 0.0 0 0.4 3 1.0 0 0.6 7 0.4 5 1.0 0 1.0 0 0.1 3 0.2 9 1.0 0 1.0 0 5 4 1 1 3 0 0 2 0 3 5 4 5 1 6 3 2 7 8 5 SC SC SC SC SC Ambigu ous Ambigu ous NF SC SI SC SC SC SC SC SC SC SC SC SC 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle cycle DD804-06 cycle DD812-05 cycle DD812-05 cycle DD812-05 SI SI SI SI DD807-06 DD812-01 DD812-01 DD812-01 DD812-01 DD812-01 DD847-03 DD847-03 EE700-01 EE702-06 EE703-07 EE705-06 EE707-05 EE707-05 EE708-02 EE708-04 280 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 CC806- CC811- CC811- CC811- CC807- CC811- CC811- CC811- CC811- CC811- CC805- CC805- 2 5 5 5 4 5 5 5 5 5 3 3 DD802- DD803- 01 05 09 08 05 05 10 10 DD804- DD805- DD807- DD807- DD808- DD808- Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 2 Bulk 2 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 BB902 -B BB912 -B BB912 -B BB912 -B BB902 -C BB912 -B BB912 -B BB912 -B BB912 -B BB912 -B BB902 -A BB902 -A CC804 CC804 CC806 CC806 CC807 CC807 CC809 CC809 -1 -5 -2 -5 -4 -4 -2 -2 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 Bulk 1 M6 S703-5 M6 MRC2 05 MRC2 05 MRC2 05 M6 M6 M6 M6 S703-5 M6 MRC2 05 MRC2 05 MRC2 05 MRC2 05 MRC2 05 M6 M6 M6 S703-5 M6 S703-5 M6 Bulk BB901 1 -A Bulk BB901 1 -A BB902 Bulk 1 -B Bulk BB902 1 -B Bulk BB902 -C 1 Bulk BB902 1 -C Bulk BB909 -A 1 Bulk BB909 -A 1 - - - - - - - - - - - - Scab4- 48 Scab4- 48 S703-5 S703-5 S703-5 S703-5 HS66 HS66 - - - - - - - - - - - - M 6 M 6 M 6 M 6 M 6 M 6 M 6 M 6 Table S4.1 (cont’d) FF62 0-03 FF62 7-01 FF62 7-02 FF63 1-01 FF63 1-02 FF63 1-08 FF63 2-01 FF63 4-01 FF63 4-02 FF63 6-01 FF64 1-01 FF64 8-02 FF65 1-01 FF65 3-01 FF65 5-03 FF65 6-01 FF65 8-02 FF65 8-04 FF68 3-02 FF68 4-01 FF68 9-02 A B A A A A A A A A N C N C A A A A A A N C N C A A A A A A A A A B A B A A A A A A A B A A A A A A A A A B A B A A A A A A A A A B A A A A A A A A A B A B A A A A A A A B N C A A A A A A A B A B A A A A A A A A A B A A A A A A A A A B A B A A A A A A A B A A A A A A A A A B A B A A A A A A A A A B A A A A A A A A A B A B A A A A A A A B A A A A A A A A A B A B A A A A A A A A A B A A A A A A A A A B A B A A A A A A B B B B A A A A A A A A A A A A A A A A A A B B A A A A A A A A A B A B A A A A A A 2 5 5 5 8 1 3 5 5 8 1 6 5 1 0 8 8 1 8 8 3 4 7 5 2 4 5 5 0 3 5 8 1 2 5 4 1 7 3 6 8 6 1 8 7 2 7 5 1 7 4 4 0. 00 0. 60 1. 00 1. 00 0. 92 1. 00 0. 80 0. 13 0. 44 0. 60 0. 60 1. 00 0. 75 1. 00 0. 88 0. 06 1. 00 1. 00 0. 71 0. 80 0. 80 S I S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C S C cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 EE71 2-07 EE71 7-05 EE71 7-05 EE71 9-02 EE71 9-02 EE71 9-02 EE72 0-04 EE72 2-06 EE72 2-06 EE72 4-06 EE72 9-04 EE73 3-05 EE73 6-01 EE73 7-09 EE74 0-01 EE74 0-04 EE74 1-08 EE74 1-08 EE73 8-02 EE73 5-01 EE82 7-05 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 DD814- DD829- DD829- 04 01 01 DD831- 01 SI DD831- 01 SI DD831- 01 SI DD837- 02 DD838- 01 SI DD838- 01 SI DD845- DD849- DD851- DD852- 08 SI DD853- 02 06 06 DD855- DD855- DD857- DD857- DD853- 05 SI DD852- DD847- 04 03 04 03 03 03 03 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 CC81 3-4 CC82 5-2 CC82 5-2 CC82 7-6 CC82 7-6 CC82 7-6 CC83 1-3 CC83 2-6 CC83 2-6 CC82 3-5 CC80 6-5 CC81 0 CC81 2-2 CC81 3-4 CC81 7-2 CC81 7-2 CC82 0-3 CC82 0-3 CC81 3-4 CC81 2-2 CC80 5-3 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 Bul k2 BB91 8-A BB93 4-A BB93 4-A BB93 8-A BB93 8-A BB93 8-A BB95 3-A BB95 3-B BB95 3-B BB93 0-B BB90 2-B BB91 2-A BB91 7-A BB91 8-A BB92 1-B BB92 1-B BB92 7-A BB92 7-A BB91 8-A BB91 7-A BB90 2-A Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 Bul k1 281 M269-1Y Scab4-48 Scab4-48 HS66 HS66 HS66 MRC205 MRC205 MRC205 XD3 S703-5 MRC205 M267-B M269-1Y S703-5 S703-5 Solanum chacoense '524-8' Solanum chacoense '524-8' M269-1Y M267-B S703-5 Solanum chacoense '524-8' XD3 XD3 XD3 XD3 XD3 XD3 XD3 XD3 Ber83 M6 M6 Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' Solanum chacoense '524-8' 2xLB-75 2xLB-75 Solanum chacoense '524-8' Solanum chacoense '524-8' M6 Table S4.2. Pedigree and self-compatibility data of 86 lines from a diploid backcross population used in this study. Line BB912-B BB920-A BB930-A BB932-A Solanum chacoense M6 ATL.M.120 ATL.M.170 ATL.M.188 ATL.M.198 ATL.M.403 ATL.M.404 ATL.M.405 ATL.M.424 ATL.M.429 ATL.V.023 ATL.V.033 NY148DH-01 R127DH-02 VT-SUP-08 0 9 0 _ i l S 1 6 5 _ i l S 4 0 3 _ i l S 6 2 6 _ i l S 8 9 8 _ i l S 4 2 4 _ i l S Fertilit y Cycle Female Male Female 2 Male 2 Female 3 Male 3 A B B B A B A A A A B B A A B B A B A B B B B B A B B B B B B B A B B B B B A B B B N C A A A A B B A A N C A B N C B B B B A B B B B B B B A B B B B B A B B B A B A A A A B B N C N C A B A B B B B B A B B B B B B B A B B B B B A B B B A B N C A A B B A A B B A B A B B B B B A B B B B B B B A B B B B B A B B B A B A A A A B B A A B B A B B B B B B B A B B B B B B B A B B B B B A B B B B B N C A A B B A A A B B B N C B B A B N C B B A B B B N C B B B B SC SC SC SC SC Parental/SC donor/Bulk 3 Parental/SC donor/Bulk 3 Parental/SC donor/Bulk 3 Parental/SC donor/Bulk 3 Parental/SC donor Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid Parental/dihaploid 282 MRC20 5 RH XD3 XD3 M6 Solanum chacoense '524-8' Ber83 S703-5 Table S4.2 (cont’d) SI SI NP NP NP VT-SUP-19 BB BB BB BB BB BB VT-SUP-70 AB AB AB AB AB NC VT-SUP-96 BB BB BB BB BB BB BB BB BB BB BB AB CC841-04 BB BB BB BB BB AA Ambiguous CC842-01 CC842-03 AB AB AB AB AB AB NC AB AB AB AB NC CC843-01 BB BB BB BB BB AA CC844-01 CC845-01 BB BB BB BB BB AB NC AB AB AB AB AB CC846-05 AB AB AB AB AB AB Ambiguous CC846-07 CC848-05 AB AB AB AB AB AA CC849-01 BB BB BB BB BB AB CC849-04 AA AA AA AA AA BB Ambiguous BB BB BB BB BB AB Ambiguous CC851-02 CC852-02 AB AB AB AB AB AB Ambiguous BB BB BB BB BB AB Ambiguous CC852-08 BB BB BB BB BB AB CC853-02 CC856-03 BB BB BB BB BB BB AB AB AB AB AB BB CC859-04 BB NC NC AB AB AB CC863-01 CC863-12 BB BB BB BB BB BB BB BB BB BB BB BB CC863-13 AB AB AB AB AB BB Ambiguous CC863-17 CC863-77 AB AB AB AB AB BB Ambiguous BB BB BB BB BB BB Ambiguous CC864-01 NF NF SI SI Parental/dihaploid Parental/dihaploid Parental/dihaploid F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 ATL.M.113 ATL.M.114 ATL.M.114 ATL.M.128 ATL.M.159 ATL.M.170 ATL.M.179 ATL.M.179 ATL.M.192 ATL.M.198 ATL.M.198 ATL.M.403 ATL.M.422 ATL.M.422 ATL.M.423 ATL.V.023 ATL.V.033 ATL.M.170 ATL.M.170 ATL.M.170 ATL.M.170 ATL.M.170 ATL.M.403 BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B BB912-B Bulk3 Bulk3 Bulk3 Bulk3 Bulk3 Bulk3 283 Table S4.2 (cont’d) SI NP BB AB AB AB AB AB Ambiguous BB BB BB BB BB BB AB AB AB AB AB AB AB NC AB AB AB AB AB AB AB AB AB BB BB BB BB BB BB BB Ambiguous CC864-06 CC864-17 CC864-18 CC864-26 CC864-28 CC864-29 DD881-01 AB AB AB AB NC AB DD881-05 AB AB AB AB NC AB DD881-07 AB AB AB NC NC AB DD881-09 AB AB AB NC NC AB DD881-14 AB AB AB NC NC AB DD881-17 AB AB AB NC NC AB DD883-05 NC AB AB NC AB AA AB AB AB AB AB AA FF696-01 AB AB AB AB AB AA FF716-03 FF744-01 NC AB AB AB AB BB AB AB AB AB AB BB FF752-04 AB AB AB AB AB NC EE757-01 EE769-03 AB AB AB AB NC AB AB AB AB AB AB AB EE790-04 AB AB AB AB AB AB EE790-05 EE791-03 BB BB BB BB BB BB AB AB AB NC AB NC Ambiguous EE798-02 AA AA AA AA AA AA Ambiguous EE803-02 EE809-01 NC AB AB AB AB AB BB BB BB BB BB BB Ambiguous EE812-05 SC SC SC SC SC SC SC SC SC SC SC SC NP SC SC SI SC F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 BC2 BC2 BC2 BC2 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 Bulk3 Bulk3 Bulk3 Bulk3 Bulk3 Bulk3 M6 M6 M6 M6 M6 M6 M6 ATL.M.403 ATL.M.403 ATL.M.403 ATL.M.403 ATL.M.403 ATL.M.403 ATL.M.170 ATL.M.170 ATL.M.170 ATL.M.170 ATL.M.170 ATL.M.170 ATL.M.403 VT-SUP-19 R127DH-02 ATL.M.404 VT-SUP-70 ATL.M.404 ATL.M.120 ATL.V.023 ATL.V.023 NY148DH-01 CC863-25 ATL.M.170 NY148DH-01 CC864-20 ATL.M.403 CC864-20 ATL.M.403 ATL.M.405 ATL.M.403 CC864-20 ATL.M.403 CC864-20 ATL.M.403 VT-SUP-08 EE758-03 ATL.M.404 CC858-03 ATL.V.030 BB912-B EE815-07 VT-SUP-19 CC858-03 ATL.V.030 BB912-B EE757-01 ATL.M.404 DD883-05 ATL.M.403 EE757-01 ATL.M.404 DD883-05 ATL.M.403 DD883-05 ATL.M.403 CC845-06 ATL.M.170 BB912-B CC858-03 ATL.V.030 BB912-B CC858-03 ATL.V.030 BB912-B M6 M6 M6 Bulk3 Bulk3 Bulk3 Bulk3 Bulk3 284 Table S4.2 (cont’d) NF NF SC SI SC SC SI NF BB BB BB BB BB NC AB AB AB AB NC AB BB BB BB BB BB BB AB AB AB AB AB BB AB AB AB AB AB NC AB AB AB AB AB AB NC AA AA AA AA AA AB AB AB AB NC AB AB AB AB AB AB AB Ambiguous AB AB AB AB AB AA NC AB AB AB AB AA AB AB AB AB AB AB AA AA AA AA AA AA BB BB BB NC BB BB Ambiguous BB BB BB BB BB BB SC SI SI SC SI EE815-06 EE823-04 EE824-04 EE825-08 EE834-02 EE835-01 EE847-02 EE847-03 EE848-01 EE851-06 EE853-11 EE853-27 EE862-03 EE870-06 EE872-03 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 BC1 CC858-03 ATL.V.030 BB912-B VT-SUP-19 CC857-05 ATL.V.024 BB912-B VT-SUP-96 CC863-25 ATL.M.170 VT-SUP-96 CC864-20 ATL.M.403 VT-SUP-96 DD881-01 ATL.M.170 ATL.M.405 DD881-01 ATL.M.170 ATL.M.120 NY148DH-01 DD881-17 ATL.M.170 NY148DH-01 DD881-17 ATL.M.170 ATL.M.170 DD881-17 ATL.M.170 ATL.M.403 DD881-17 ATL.M.170 NY148DH-01 DD883-05 ATL.M.403 NY148DH-01 DD883-05 ATL.M.403 DD883-05 ATL.M.403 ATL.M.198 DD883-05 ATL.M.403 VT-SUP-08 VT-SUP-96 DD883-05 ATL.M.403 Bulk3 Bulk3 M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 M6 285 Table S4.3. Kompetitive Allele Specific PCR Primers used in this study. DM v 4.03 Chrom osome 12 Positio n (bp) Self-Incompatible Allele Primer Self-Compatible Allele Primer Common Primer 589600 GAAGGTGACCAAGTTCATGCTGTTGACTTGTT GAAGGTCGGAGTCAACGGATTATAGTTGACTT 589625 GAAGGTGACCAAGTTCATGCTGTTGAGATTGA GAAGGTCGGAGTCAACGGATTGTTGAGATTG AATCTAGCATATTGGC GACACTCTAGTAGG GTTAATCTAGCATATTGGT AGACACTCTAGTAGA 590073 GAAGGTGACCAAGTTCATGCTAAAATTGATAA GAAGGTCGGAGTCAACGGATTAAAAAAATTG ACTCATTGCAAAGTCTAG CTCATTTTCAACTTCCCTG ATAACTCATTTTCAACTTCCCTA AGACATTACAAA 590257 GAAGGTGACCAAGTTCATGCTAGTCGAGTTTC GAAGGTCGGAGTCAACGGATTGTAGTCGAGTT AATCTTCGGTTCGTTTGT AATCTTCCCTCG TCAATCTTCCCTCT 590396 GAAGGTGACCAAGTTCATGCTCAAATATGTTG GAAGGTCGGAGTCAACGGATTCAAATATGTTG ATGCATTAGCAATTTCTA TTTATTTGGTGTTCAAATTGG TTTATTTGGTGTTCAAATTGT 590408 GAAGGTGACCAAGTTCATGCTGTAAAGYTTTT GAAGGTCGGAGTCAACGGATTAAAGYTTTTAC GGATAAATCCGYGGGGA 590564 GAAGGTGACCAAGTTCATGCTGAAKATGGGA GAAGGTCGGAGTCAACGGATTGAAKATGGGA ACCAGATGATTATGAAGATATT CAGATGATTATGAAGATATC TGGAAATTGGGATCG TGGAAATTGGGATCC 591552 GAAGGTGACCAAGTTCATGCTTCCCACTGGTT GAAGGTCGGAGTCAACGGATTCCCACTGGTTG 591844 GAAGGTGACCAAGTTCATGCTCCTCCGGGATA GAAGGTCGGAGTCAACGGATTCCTCCGGGAT CATGGTAATC AAATTCAGGACC GCATGGTAATA AATTCAGGACT 90 61 04 37 26 98 10 91 24 ID Sli_ 090 Sli_ 561 Sli_ 304 Sli_ 737 Sli_ 626 Sli_ 898 Sli_ 410 Sli_ 291 Sli_ 424 Exclud ed from analysi s (Ambi guous calls in >15% of lines) x x x CTCAGAATTGCCAAGAA ACTCCATCAAAT GTTCAATACCACCTAAGC TTGGAAATCTT TGGCTTGATAT GTCCATCATGAT GACATAT AGTAACAATGGCGGAAT CTAAARCTTCAAA AGGAAAGACCTCAATCA ACATTGCAGAAT CCACAGGTATGCATGTTA CCCCATA 286 Table S4.4. Self-compatibility phenotype and SNP marker genotype at four SNP loci from the Illumina Infinium V1 8.3K Array in 164 recurrent selection clones SNP Chromosome 12 Position (DM v4.03) 58047558 58983259 59129520 58047342 Fertility SI SI SI SC SI SI SI SC SI SI SC SC SC SC SC SC SI SI SC SI SI SC SC SC SC Cycle Parental Parental Parental Parental Parental Parental Parental Parental Parental Parental Parental Parental Parental cycle 0 cycle 0 cycle 0 cycle 0 cycle 0 cycle 0 cycle 0 cycle 0 cycle 0 cycle 0 cycle 0 cycle 1 solcap_snp_c1_2689 solcap_snp_c1_2690 solcap_snp_c1_13698 solcap_snp_c2_46213 AA AA AA AB AA AA AA AB AA AA AA AA AA AA AA AA AA AA AA AA AB AB AA AA AA AB BB BB BB AB AB AB AB AA BB AB AB BB BB AB BB AB BB AB AB BB BB AB AA AB BB AB AA AB AA AA AA AB AB AB AB AA AA AB BB AA AA AA AB AB AB AB BB BB AB BB AB AB AA AB AA BB AB AA AB AB BB AB AB BB AB BB Line 2xLB-75 84SD22 Ber83 DMRH-89 HS66 M269-1Y MRC205 RH S703-5 Scab4-48 Solanum chacoense 524-8 Solanum chacoense M6 XD3 BB900-A BB902-A BB908-A BB909-A BB918-A BB929-A BB930-B BB943-A BB946-B BB953-10 BB953-A CC804-01 287 Table S4.4 (cont’d) CC804-05 CC806-05 CC809-02 CC809-04 CC811-04 CC811-05 CC822-01 CC822-05 CC823-03 CC823-05 CC825-02 CC825-04 CC827-06 CC831-03 DD802-01 DD802-04 DD803-05 DD804-06 DD804-09 DD805-05 DD805-08 DD807-03 DD807-05 DD807-06 DD808-10 DD809-09 DD812-02 DD812-03 SC SC SC SC SC SC SC SC SC SI SI SC SI SI SC SC SC SI SC SC SC SC SC SC SC SC SC SC cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 1 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 AA AA AB AA AA AA AA AA AA AA AB AB AA AA AA AA AA AA AA AA AA AA AA AA AB AA AA AA 288 AB BB BB BB BB AB BB BB AB AB AB AA AB AA AB AB AB AA AA AB BB AA AA AB BB AA AB AA AB AA AB AB AA AB AA AA AB AB AA AB AB AB AB BB AA BB BB AB AA BB BB AB AB Table S4.4 (cont’d) DD814-04 DD821-09 DD821-10 DD824-01 DD825-01 DD829-01 DD829-09 DD829-10 DD831-01 DD837-08 DD845-02 DD845-03 DD847-05 DD848-01 DD848-02 DD849-06 DD849-07 DD850-03 DD850-06 DD851-06 DD851-08 DD852-04 DD852-08 DD853-04 DD855-01 DD855-03 DD857-03 DD865-03 SC SC SC SC SC SC SI SC SI SC SC SI SC SC SC SC SC SC SC SC SC SC SI SC SI SC SC SC cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 cycle 2 AA AA AA AB AA AB AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA 289 AA AB AB AA AA AA AA AA AB AB AB AB AB AA AB AA AB AB AA AA AA AA AB AB AB AB AA AA AB Table S4.4 (cont’d) EE700-01 EE701-02 EE701-06 EE702-05 EE703-07 EE704-03 EE704-08 EE705-04 EE705-06 EE706-03 EE707-05 EE708-02 EE710-03 EE710-06 EE710-09 EE710-10 EE712-02 EE712-07 EE713-07 EE713-08 EE714-04 EE716-06 EE717-01 EE717-03 EE717-05 EE718-01 EE719-02 EE721-03 SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AB AA AA AA AA AA AA AA 290 BB AB BB AB AB AB AB AA AB AB AB AB BB AB AA BB AB AB BB AB AB AB AA AA AA AB AA AA AB AA AB AB AA AA AB AB AB AA AA AA AB AA AA AB AB AA AA AA AA AB AA AB AB AA AB AA AA AB AA AB AB AB BB AA AB AB AA AA AB BB AA AB AA BB AB AB AB AA AB Table S4.4 (cont’d) EE721-06 EE722-07 EE723-06 EE725-02 EE729-03 EE729-04 EE730-03 EE730-09 EE732-03 EE732-06 EE736-01 EE737-05 EE737-09 EE738-01 EE738-04 EE739-02 EE739-04 EE739-05 EE740-01 EE740-04 EE742-06 EE743-04 EE744-06 EE745-10 EE747-09 EE747-13 EE748-06 EE749-05 SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 cycle 3 AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA 291 AB BB AB BB AB AB AB BB AB AB BB AB BB BB AB AB BB AB BB AA AA AA AB AA AA AA AA AB AA AB AB AA AA AA AA AB AB AA AB AA AA AA AA AB AB AA AA BB AB AA AA AA AB AB AA AA AB AA AB AB AA AA AA AB AB AB AA AB BB AB AA AB AB AA AA Table S4.4 (cont’d) FF600-01 FF609-02 FF611-02 FF611-03 FF612-03 FF613-03 FF620-03 FF627-01 FF627-02 FF631-01 FF631-02 FF631-08 FF632-01 FF634-01 FF634-02 FF636-01 FF641-01 FF648-02 FF651-01 FF653-01 FF655-03 FF656-01 FF658-02 FF658-04 FF683-02 FF684-01 FF689-02 SC SC SC SC SC SC SI SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC SC cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AB AB AA AA AA BB AA AA AB AB AB AA AA AB AB BB BB AB BB BB AB BB AB BB AB AA AB BB BB BB BB AB 292 AA AA AA AA AA AA AB AA AA AA AA AB AB AA AA AA AA AB AA AA AA AA AA AA AA AA AB AB AB AB AB AB AB BB AB AA AA AA AA AB AA AA AA AB AA AB AB AA AB AB AA AA AA Table S4.5. Self-compatibility phenotype and SNP marker genotype at eleven SNP loci from the Illumina Infinium V3 22K Array in 31 recurrent selection clones SNP Chromosome 12 Position (DM v4.03) 5898368 5898357 5898311 58047342 solcap_snp _c1_2689 58047558 solcap_snp _c1_2690 58154032 solcap_snp _c2_7839 9 PotVar0 053483 58983259 solcap_snp _c1_13698 Line 84SD22 Ber83 RH Solanum chacoense M6 FF600-01 FF609-02 FF611-02 FF611-03 FF612-03 FF613-03 FF620-03 FF627-01 FF627-02 FF631-01 FF631-02 FF631-08 FF632-01 Fert ility SI SI SC SC SC SC SC SC SC SC SI SC SC SC SC SC SC Cyc le Pare ntal Pare ntal Pare ntal Pare ntal cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 cycl e 4 293 5898370 5898636 5898648 5 5 4 5 9 PotVar0 053460 PotVar0 053456 PotVar0 053453 PotVar0 053309 PotVar0 053291 AA AA AB AA AA AA AA AA AA AA AA AA AA AA AA AA AA AB BB AB BB BB AA AA AB AB AB AA AA AB AB BB BB AB BB BB AB BB BB BB BB BB BB BB BB BB BB BB BB BB BB AA AB AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA BB AB AA AA AA AA AA AA AA AA AB AA AA AA AB AB AB AA AA AB BB BB BB BB BB BB BB AB BB BB BB AB AB AB BB AB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB AB AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AB BB AB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB AB BB BB BB BB BB BB BB BB BB BB BB BB BB BB 59129520 solcap_snp _c2_46213 AB AB AB AA AB AB AB AB AB AB AB BB AB AA AA AA AA Table S4.5 (cont’d) FF634-01 FF634-02 FF636-01 FF641-01 FF648-02 FF651-01 FF653-01 FF655-03 FF656-01 FF658-02 FF658-04 FF683-02 FF684-01 FF689-02 SC SC SC SC SC SC SC SC SC SC SC SC SC SC cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 cycle 4 AA AA AA AA AA AA AA AA AA AB AB AA AA AA BB BB AB BB AB BB AB AA AB BB BB BB BB AB BB BB BB BB BB BB BB BB BB AB AB BB BB BB AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AB AA AA AA AA AA AA AA AA BB BB BB BB AB BB BB BB BB AB AB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB AA AA AA AA AA AA AA AA AA AA AA AA AA AA BB BB BB BB BB BB BB BB BB AB AB BB BB BB BB BB BB BB BB BB BB BB BB AB AB BB BB BB AB AA AA AA AB AA AB AB AA AB AB AA AA AA 294 APPENDIX D: Chapter 4 Copyright Permissions RightsLink Printable License https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=0ffa8793-273c... SPRINGER NATURE LICENSE TERMS AND CONDITIONS Feb 22, 2021 This Agreement between Dr. Natalie Kaiser ("You") and Springer Nature ("Springer Nature") consists of your license details and the terms and conditions provided by Springer Nature and Copyright Clearance Center. License Number 5014120959424 License date Feb 22, 2021 Licensed Content Publisher Springer Nature Licensed Content Publication American Journal of Potato Research Licensed Content Title Assessing the Contribution of Sli to Self-Compatibility in North American Diploid Potato Germplasm Using KASP™ Markers Licensed Content Author N. R. Kaiser et al Licensed Content Date Feb 19, 2021 Type of Use Thesis/Dissertation Requestor type academic/university or research institute Format Portion print and electronic full article/chapter 1 of 6 Figure S4.6 Springer Copyright Permission for inclusion of Chapter 4 in this dissertation. 2/22/21, 5:22 AM 295 REFERENCES 296 REFERENCES Ai, Y., Kron, E., & Kao, T. H. (1991). 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R., Shang, Y., Li, C., & Huang, S. (2019). The genetic basis of inbreeding depression in potato. Nature Genetics, 51, 374- 378. 300 CHAPTER 5 SELF-FERTILITY AND RESISTANCE TO THE COLORADO POTATO BEETLE (LEPTINOTARSA DECEMLINEATA) IN A DIPLOID SOLANUM CHACOENSE RECOMBINANT INBRED LINE POPULATION This chapter was submitted as a manuscript for review to Crop Science on February 7, 2021 (Kaiser, et al., 2021). 301 Abstract A diploid potato recombinant inbred line population was derived from a cross between Solanum chacoense inbred line M6 and S. chacoense accession USDA8380-1 (80-1) to identify loci associated with self-compatibility and Colorado potato beetle resistance. Individuals from the F4 and F5 generations were genotyped on the Illumina Infinium V3 22K Single Nucleotide Polymorphism (SNP) Array and a genetic map constructed. All F5 individuals contain at least one copy of the dominant S-locus inhibitor (Sli) haplotype; however, not all F5 individuals set fruit. Pollen tubes reached the ovaries of both self-fruitful and self-unfruitful plants, indicating that the presence of the dominant Sli allele is not sufficient for selfed fruit and seed production. Loci on chromosomes 3, 5, 6 and 12 were identified as novel targets for self-fertility improvement. Evaluation of fruit and seed set upon selfing in the F4 generation over two greenhouse seasons revealed environmental influence on self-fertility. Loci exhibiting residual heterozygosity were found on all chromosomes except chromosomes 3 and 11 in F5 inbreds, but none of the measured self-fertility traits were correlated with the level of heterozygosity based on SNP genotyping. Four SNPs on chromosome 2 between 22,151,711-22,381,719 bp were associated with foliar leptine glycoalkaloid synthesis and Colorado potato beetle resistance in the recombinant inbred line population. Robust inbred lines carrying Colorado potato beetle resistance were developed without field selection during the inbreeding process and beetle resistance was introgressed into diploid breeding lines. Introduction Conducting cultivated potato (Solanum tuberosum L.) improvement at the diploid level (2n = 2x = 24) allows the use of tools, technologies and breeding approaches that are not possible or efficient in cultivated tetraploid (2n = 4x = 48) potato breeding. For example, the creation of inbred 302 diploid lines would permit breeders to purge deleterious alleles and stack economically important traits. Unfortunately, inbred line development in potato is limited by the self-infertility prevalent in diploid germplasm (Cipar, 1964), excessive heterozygosity in selfed progeny (Hosaka & Sanetomo, 2020; Leisner et al., 2018; Marand et al., 2019; Peterson et al., 2016), and inbreeding depression due to the presence of deleterious recessive alleles (De Jong & Rowe, 1971; Zhang et al., 2019). Self-fertility describes an array of traits necessary to reliably produce offspring upon self-pollination including self-compatibility, male and female fertility, fruit set, and seed set. Genetic investigation of self-compatibility in diploid potato has largely focused on inheritance and transmission of the dominant self-incompatibility inhibitor gene Sli located on chromosome 12, originally identified in Solanum chacoense (Clot et al., 2020; Hosaka & Hanneman, 1998a, 1998b; Phumichai et al., 2006; Phumichai et al., 2005). In an effort to improve self-compatibility in diploid breeding programs, the highly fertile inbred line S. chacoense M6 (Jansky et al., 2014) has been used as an Sli donor. Introgression of the self-compatibility present in select diploid genotypes, such as M6, also expands the possible types of genetic mapping populations available in potato (Endelman & Jansky, 2016; Jansky et al., 2016; Kaiser et al., 2020). In the absence of widespread self-compatible (SC) potato germplasm, genetic mapping studies have been largely limited to F1 and pseudo-F2 populations derived from crosses between heterozygous parents (Lindhout et al., 2018). Development of diploid F2 and recombinant inbred line (RIL) mapping populations from homozygous, SC parental lines would allow examination of the inheritance of complex traits (Jansky, 2020). In both F2 and RIL populations selfing restores the homozygous condition and can expose the contribution of recessive genes. The additional number of recombination events in each genotype during recurrent selfing can increase the genetic map resolution for detecting quantitative 303 trait loci (QTLs). Furthermore, seed immortalized RIL populations permit replicated phenotypic evaluation in multiple environments to probe genotype-by-environment interactions. The purpose of this study was to exploit M6-mediated self-compatibility to establish vigorous, inbred diploid potato lines for use in potato varietal improvement. The fixation of desirable epistatic complexes of alleles in inbred lines offers unique potential for transmitting quantitative traits, such as disease and pest resistance. Glycoalkaloid-mediated host plant insect resistance is an excellent example of a target trait for this breeding approach. Glycoalkaloids are produced in all potato tissues except the tuber pith (Friedman, 2006). In large doses, glycoalkaloids induce nausea and vomiting in mammals with an industry standard for glycoalkaloid levels in tubers intended for human consumption of 20 mg/100 g fresh weight (commonly expressed as 20 mg% fresh weight). Host plant resistance efficacy is dependent on both total leaf glycoalkaloid accumulation and the production of specific glycoalkaloids (Lachman et al., 2001; Tingey, 1984). For example, the common glycoalkaloids a-chaconine and a-solanine are present in leaves of insect susceptible potato varieties in insufficient amounts to inhibit Colorado potato beetle feeding. Wild species relatives of potato produce the potent glycoalkaloids leptines and leptinines which effectively reduce Colorado potato beetle feeding and reproduction through a cholinesterase inhibiting and cell membrane disruption mechanism (Sanford et al., 1994; Sanford et al., 1996; Sinden et al., 1980). Unlike the common glycoalkaloids a-chaconine and a-solanine, leptines and leptinines are present only in aerial tissue and as such do not pose a hazard to human health (Mweetwa et al., 2012). The high leptine-producing diploid S. chacoense accession USDA8380-1 (80-1) has demonstrated strong antibiosis properties against the Colorado potato beetle (Sinden et al., 1986). Unfortunately, the presence of multiple loci contributing to leptine production and the recessive inheritance of key functional and/or regulatory genes in the leptine biosynthesis pathway 304 (Boluarte-Medina et al., 2002; Hutvágner et al., 2001; Kaiser et al., 2020; Manrique-Carpintero et al., 2014; Ronning et al., 1998; Ronning et al., 1999; Sagredo et al., 2009; Sagredo et al., 2006) has prevented the successful introgression of Colorado potato beetle resistance into cultivated potato. For this study, a diploid intraspecific S. chacoense RIL population was created from a cross between the SC M6 inbred S. chacoense clone and the high leptine producing self-incompatible (SI) 80-1 S. chacoense clone to i) evaluate the practicality of inbred line development and the progression toward homozygosity through selfed generations, ii) examine genetic features contributing to self-fertility, glycoalkaloid content and Colorado potato beetle resistance and iii) develop SC Colorado potato beetle resistant germplasm to be deployed in breeding. Materials and Methods Plant material Creating Inbred Lines. The diploid RIL population was generated from a cross between the S. chacoense SC inbred line M6 (Jansky et al. 2014) (female) and the SI S. chacoense clone USDA8380-1 (PI 458310, 80-1) (male). Twenty F1 plants were grown under greenhouse conditions (16-hr photoperiod at 20 ˚C) and a single SC, Colorado potato beetle resistant F1 individual was selected for self-pollination to produce 700 diploid F2 seedlings. Of these, 325 individuals grew and developed and 305 were determined to be SC. Self-compatibility was determined by fruit set and evaluated by a maximum of 50 self-pollinations of each individual under greenhouse conditions in the winter/spring of 2016 (16-hr photoperiod at 20 ˚C). Progeny were advanced to the F5 generation by selfing. At each generation, five plants of each family were grown to ensure identification of a SC individual to perpetuate the lineage. Parental lines, the F1 hybrid and individuals from the F2, F4 and F5 generations were maintained in tissue culture on 305 Murashige and Skoog (MS) (Murashige & Skoog, 1962) medium (MS salts at 8.8g/L, 3% sucrose, pH 5.8 and 0.6% plant agar) at 22 ˚C and 16-hr photoperiod. Transmitting Leptine-based Colorado Potato Beetle Resistance. A hybrid family (referred to hereafter as MSHH786B) was created by crossing a Colorado potato beetle resistant, leptine- producing F4 individual 472_04_06, from the recombinant inbred line population described above, to a diploid breeding line MSDD880-03S2-263-01-04. MSDD880-03S2-263-01-04 is a SC, beetle susceptible S2 selection from a cross between inbred line M6 and the Solanum berthaultii PI 473334 clone SB1. A total of 15 SC F1 hybrids from the MSHH786B family were evaluated for Colorado potato beetle resistance under field conditions. Self-fertility phenotyping Evaluation of F4 Fruit and Seed Set. In the fall of 2018, 48 F4 individuals (representing 45 unique families) were planted in the greenhouse and grown at 20˚C under high-pressure sodium lights set to a 16-hr photoperiod. Self-pollinations were made by extruding pollen from the anthers of 2-3 flowers per plant onto a glass slide that was gently applied to the stigmas of non-emasculated flowers. As flowering time in this population is not synchronized, pollinations were made between 17 December 2018 and 10 April 2019. However, because age-dependent plasticity in the SI response has been observed in Solanum (Travers et al., 2004), self-pollinations were always initiated using the first cohort of flowers for each individual genotype. Fruits were harvested five weeks post-pollination. The number of developed fruits and the total number of seeds were recorded for each genotype and used to calculate the fraction of flowers setting fruit and the average number of seeds per fruit. Evaluation of F4 and F5 Self-Fertility. Tissue culture plantlets of 62 F4 individuals (representing 57 unique families), 77 F5 individuals (representing 55 unique families) and the SI 306 line 80-1 (N = 137) were transplanted into 3.8 L pots in the greenhouse (16-hr photoperiod at 20 ˚C) on 21 August 2020. Unlike in 2018-2019, in 2020 the greenhouse was equipped with Philips GreenPower light-emitting diode (LED) DR/W-MB lights (Philips Lighting Holding B.V., Netherlands). As each plant flowered, a sample was collected for analysis of pollen viability and pollen tube growth in the style. Pollen harvested from 1-5 anthers was extruded onto a glass slide and used to self-pollinate the genotype. Remaining pollen was then stained with acetocarmine, covered with a cover slip, sealed with clear nail polish and stored at room temperature in the dark prior to visualization. Slides were imaged at 10x using Leica imaging software coupled to a Leica DM750 binocular microscope. The number of total pollen grains and the number of viable pollen grains were quantified using a custom macro in Fiji (Schindelin et al., 2012) to discriminate between unstained, shriveled pollen and stained, turgid pollen. Percent viable pollen was calculated using a minimum of 100 total pollen grains. Styles receiving the pollen used for pollen viability assessment were collected 48 hours post-pollination. At least two stylar samples were collected per genotype. Petals, sepals and anthers were removed. The remaining style and ovary were stored in 1.5 mL Eppendorf tubes containing a fixation solution of 3:1 ethanol/acetic acid in the dark at room temperature for at least 24 hours. Styles and ovaries were then softened in an 8N NaOH solution at 60 °C for 1 hour. Samples were subsequently triple rinsed with water and stained with 0.1% aniline blue in 0.1N K3PO4 for 1 hour, shaking, in dark conditions. Styles and ovaries were placed on a microscope slide, gently squashed with a cover slip and visualized at 4x using a Nikon Eclipse SMZ1270i Stereo Upright microscope with a SOLA light engine. Photographs were taken with an ANDOR Zyla sCMOS camera and NIS-Elements BR software. Images for each stylar sample were stitched together using Microsoft Image Composite Editor software. Pollen tube growth was given a score between 0-3: 0 = no 307 pollen visible on stigma, 1= ungerminated pollen visible on stigma, 2 = all pollen tube growth aborted in style, 3 = majority of pollen tubes reach the ovary. The average pollen tube growth score was calculated for each genotype, excluding samples with a score of 0. Self-pollinations were made between 14 October 2020 and 2 December 2020, using the first cohort of flowers for each genotype. Plants that did not flower in this period were designated as non-flowering (NF). Fruits were harvested five weeks post-pollination. The number of developed fruits, total fruit weight, and total seeds were recorded for each genotype. The fraction of flowers setting fruit, average fruit weight and average number of seeds per fruit were then calculated. Selfed fruit and seed set data for F4 individuals evaluated in both 2019 and 2020 can be found in Table S1 and self-fertility trait data for F4 and F5 individuals evaluated in 2020 can be found in Table S2. Glycoalkaloid analysis Sample Preparation of Foliar Glycoalkaloids from Greenhouse Grown Plants. Foliar glycoalkaloid content was measured in the 62 F4 individuals, 74 F5 individuals, and the leptine- producing parental line 80-1 grown under greenhouse conditions for self-fertility evaluation in the fall/winter of 2020. The tetraploid cultivated variety ‘Atlantic’ was included as a check (N = 138) and was transplanted from tissue culture at the same time as the other lines used in this study. Samples were taken from one plant of each genotype at anthesis. Five leaflets from the fourth fully-expanded leaf of each genotype were placed in a 15 mL plastic conical centrifuge tube (Corning, Inc., Corning, NY), flash frozen and stored at -80°C prior to lyophilization for 72 hrs in a SP VirTis Genesis Pilot Lyophilizer (SP Scientific Products, Stone Ridge, NY). Sample Preparation of Tuber and Foliar Glycoalkaloids from Field Grown Plants. Foliar tissue samples were collected from each of two replicates for the parental line 80-1, nine 308 beetle resistant F5 lines, and two beetle resistant MSHH786B hybrid lines (MSHH786B_01 and MSHH786B_09) grown in the Michigan State University Montcalm Research Center (Lakeview, MI) Colorado potato beetle nursery. For each replicate, one leaflet from the fourth fully-expanded leaf was taken from each plant in a five-plant plot and placed in a 15 mL plastic conical centrifuge tube (Corning, Inc., Corning, NY) seven weeks after transplanting. Tissue was immediately flash frozen and stored at -80°C prior to lyophilization for 72 hrs. Tubers were harvested from field- grown plants 20 weeks after transplanting. A minimum of five randomly selected tubers were used for each replicate of each genotype. Because the tubers produced by these genotypes are uniformly small (~3.8 cm diameter), a size criterium was not imposed. Diced tuber pieces were placed in a 50mL Corning tube, flash frozen and stored at -80°C prior to lyophilization for 72 hrs. Glycoalkaloid Extraction. The same glycoalkaloid extraction procedure was used for all samples. The freeze-dried tissue was ground, and 30 mg of powder was extracted in 600 uL of solution (49% HPLC grade methanol, 49% sterile water, 1% glacial acetic acid, 0.1% formic acid). The samples were briefly vortexed and incubated at 60°C for 30 minutes before centrifugation for one minute at 14,000 rpm. The supernatant was filtered through a 0.22 um Corning® Costar® Spin-X® centrifuge tube and diluted 1:100 in extraction solution containing internal standard Telmisartan at a final concentration of 0.5 uM. Glycoalkaloid Quantification. Glycoalkaloids were analyzed using Waters Acquity (Waters Corporation, MA, USA) high performance liquid chromatography coupled with Xevo TQ-S Micro Tandem Quadrupole (Waters Corporation, MA, USA) mass spectrometry (HPLC– MS/MS). Compounds were separated on a Waters Acquity BEH-C18 UPLC column (2.1 x 50mm). Glycoalkaloids were eluted in a binary gradient system composed of Solvent A (LC-MS grade water, 0.1% formic acid) and Solvent B (LC-MS grade acetonitrile) at a flow rate of 0.4 309 mL/min at 25 °C. The following stepwise gradient was implemented: 90% A, 10% B; 2:00 min, 40% A, 60% B; 2:01, 0% A, 100%B; 3:01, 90% A, 10% B. Each sample was injected at a volume of 5 uL in duplicate. The mass spectrometer was operated in positive ion mode. Mass spectrometry data were acquired by the Waters MassLynx software and processed using Waters Quanlynx MS Software. Molar concentrations were determined using standard curves of purified α-solanine and α-chaconine (Sigma-Aldrich) in a range from 0.01-10.0 uM. The response factors for α-chaconine and α-solanine were used for leptine I and leptine II (referred hereafter as leptine I/II), respectively. Colorado potato beetle resistance phenotyping Field trials were conducted in 2019 and 2020 in the Montcalm Research Center Colorado potato beetle nursery. The RIL progenitor F2 population was previously evaluated for Colorado potato beetle field defoliation resistance at this location in 2017 (Kaiser et al., 2020). The beetle nursery has been naturally infested with an overwintering Colorado potato beetle population for at least four decades (Coombs et al., 2003) and is planted annually with susceptible potatoes. Tuber seed pieces of the commercial cultivar ‘Atlantic’ were planted in border rows around the trial and in alternate rows within the trial to provide sufficient fodder for emerging beetles and to encourage uniform densities throughout the field on 30 April 2019 and on 28 April 2020. Adult beetles emerged from the soil the weeks of 20 May 2019 and 25 May 2020. In each year, plants of each line were transplanted to the field in a randomized complete block design when transplants were approximately the same age and maturity of the ‘Atlantic’ spreader rows that had been planted from tuber pieces. Trial plots of ‘Atlantic’ transplants were also included as a susceptible check within trial rows. In 2019, nine stem cuttings were taken from each of two parental lines, 98 F4 individuals, and the susceptible check ‘Atlantic’, rooted and grown in trays for 6 weeks in the greenhouse and 310 transplanted in the field on 21 June 2019 (N = 909) in a randomized complete block design. In 2020, 10 in vitro plantlets of the resistant parent 80-1, 74 F5 individuals, and the susceptible check ‘Atlantic’ were first grown under greenhouse conditions (16-hr photoperiod, 20 °C) and then transplanted to the field on 3 June 2020 (N = 740). The randomized complete block design consisted of three replications of three plants in 2019 and two replications of five plants in 2020. Percent defoliation of each plot was assessed visually each week beginning on 9 July in 2019 and on 24 June 2020. Defoliation evaluation continued for a total of 5 weeks in 2019 and 4 weeks in 2020 at which point the ‘Atlantic’ check was completely defoliated. In each year, defoliation was caused by overwintered adults, first-generation larvae, and second-generation adults and larvae. Defoliation data were used to calculate the area under the defoliation curve (AUDC), comparable to the area under the disease progression curve (Coombs et al., 2003; Shaner & Finney, 1977) (e.g. 4900 if 100% of the plot was defoliated by the 49th day of the trial). To determine the relative AUDC (RAUDC) for each plot over the observational period, the AUDC for each plot was divided by the maximum defoliation observation for that plot. Data were analyzed in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). Non-parametric Kruskal-Wallis tests were used for pairwise comparison (α = 0.05) due to unequal variances (Levene’s test p < 0.0001). Single Nucleotide Polymorphism (SNP) genotyping Genomic DNA was extracted from freeze-dried leaf tissue of 113 F4 individuals (representing 97 unique families) and 80 F5 individuals (representing 54 unique families) following the Mag-Bind® Plant DNA Plus 96 Kit protocol (Omega Bio-tek, Norcross, GA). SNP genotyping was performed using the Illumina Infinium Potato 22K V3 Array (Felcher et al., 2012; Hamilton et al., 2011; Vos et al., 2015) at Michigan State University and allele calls were made 311 and manually curated in GenomeStudio software (Illumina, Inc). SNP genotypes of the two parental lines, their F1 hybrid and 236 F2 individuals had been previously generated (Kaiser et al., 2020). To augment genotype data for the parental lines, 80-1 and M6 were also genotyped in duplicate using the Illumina Infinium V4 SNP Array (Illumina, Inc.) and a consensus call was created for each parental line from the total unfiltered 30,991 V4 SNPs. The V4 contains approximately 9,000 more SNPs than the V3 array which were selected to represent genetic diversity of landraces and wild diploid species from the secondary and tertiary potato genepools. A total of 31 potato genotypes from 18 species were re-sequenced and SNP identification was based on the alignment of the Illumina reads to the potato doubled monoploid S. tuberosum clone DM1-3 516 R44 (DM) pseudomolecules (PGSC Version 4.03) (PGSC, 2011; Sharma et al., 2013) and de novo assemblies of reads that failed to map to the reference genome. After applying filtering parameters on mapping quality, base quality, SNP quality, and coverage, V4 SNPs were selected for uniform distribution across the genome and in genomic regions/genes of interest. Manual filtering of the V3 and V4 SNP data in this study removed uninformative and poor-quality SNPs. The physical position of SNPs from both the V3 and V4 Arrays was previously determined by alignment of the contextual sequence to the DM pseudomolecules (PGSC Version 4.03) (Hamilton et al., 2011; Hirsch et al., 2013, Vos et al., 2015). Linkage Analysis of F4 individuals Linkage analysis of the F4 population was conducted in JoinMap® 4.1 (Van Ooijen, 2006) using 97 individuals with non-identical SNP genotype profiles using the RIL population type (x = 4). Non-identical SNPs were grouped using independence logarithm of odds (LOD) function. A LOD threshold of 6 was used to produce a stable configuration of linkage groups. The map order 312 was calculated using the maximum likelihood map algorithm. The physical position of mapped SNPs from the Illumina V3 Array on the DM pseudomolecules (PGSC Version 4.03) was used to compare genetic and physical maps. The physical length of each chromosome was calculated by subtracting the first megabase (Mb) position of mapped loci on each chromosome from the last position. Total physical map length was the sum of the physical map lengths for each of the 12 chromosomes. Map coverage for each chromosome was reported as the total distance in Mb covered by SNP positions divided by the total length of each DM Version 4.03 assembled chromosome. Total map coverage was reported as the total distance (Mb) covered by all 12 chromosomes divided by the total distance of all 12 DM Version 4.03 assembled chromosomes. Average distance between loci mapped in each chromosome was calculated by summing all the individual interlocus intervals in cM and dividing by the total number of intervals and by the average from chromosome average intervals for the overall genome. Linkage groups were visualized in MapChart (Voorrips, 2002). Statistical analysis and SNP marker trait association Glycoalkaloid Analysis and Field Colorado Potato Beetle Defoliation Trait Data. The mean of technical replicates was used to calculate correlations coefficients for compound concentrations (mg/g dry weight (DW)) of individually measured glycoalkaloids, total measured leptines and the ratio of acetylated glycoalkaloids to non-acetylated glycoalkaloids [mean total leptines (mg/g DW)]/ [mean α-chaconine (mg/g DW) + mean α-solanine (mg/g DW)]. For samples collected from field-grown plants, the total measured fresh weight (FW) and DW of each sample were used to calculate the mg% FW for each compound. The total glycoalkaloid mg% FW for tubers was reported. In the 32 F4 and 65 F5 individuals evaluated for Colorado potato beetle resistance under field conditions and grown in the greenhouse for glycoalkaloid analysis, the 313 Spearman’s rank correlation coefficients were calculated in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC) to test the relationship between individual foliar glycoalkaloid content and RAUDC as well as the ratio of acetylated to non-acetylated compounds and RAUDC. Self-Fertility Trait Data. Non-flowering plants recorded in the 2020 greenhouse growing season were not included in further analysis for both generations. In all instances, the average number of seeds per fruit was only determined for individuals setting fruit. The relationship between the fraction of flowers setting fruit and the average number of seeds per fruit was compared with Spearman’s rank correlation coefficients using two measures of fruit set in the 62 F4 individuals and 74 F5 individuals evaluated in 2020: the proportion of total self-pollinated flowers setting fruit and the fraction of flowers setting fruit from the number of self-pollinated flowers only on days that resulted in fruit set in that genotype. For the 48 F4 individuals assessed in 2019 and 2020, Spearman’s rank correlation coefficients were used to test for correlation of traits between years. SNP Marker-Trait Analysis. One-way ANOVA was performed in R software (version 4.0.1) (R:A Language and Environment for Statistical Computing, 2010) to test for association between each individual segregating SNP coded as AA, AB or BB and the following phenotypes: 2020 greenhouse glycoalkaloid traits (compound concentrations of individually measured glycoalkaloids, total measured leptines, the ratio of acetylated glycoalkaloids to non-acetylated glycoalkaloids), 2020 and 2019 RAUDC under field conditions, and 2020 greenhouse self-fertility traits (the fraction of total flowers setting fruit, average fruit weight, the average number of seeds per fruit, the fraction of viable pollen, and the mean pollen tube growth rating). To identify loci associated with leptine synthesis, leptine accumulation data were converted to presence/absence, coded 1/0 and Fisher’s exact T test in R was used to test for SNP association. The SNPs with a p- 314 value < 0.001 for each trait were further analyzed in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC) to discard SNPs where a single genotype (AA, AB or BB) was responsible for significance and was represented by < 5 individuals. The contingency analysis chi-squared test p- value was reported for the presence of leptines. The number of individuals in each generation used for each SNP-trait association test can be found in Table S3. The -log(p-value) of loci significantly associated with the presence of leptines in F4 and F5 individuals was plotted against the physical position on the DM pseudomolecules PGSC v4.03 chromosome 2 using PhenoGram Synthesis View (Wolfe et al., 2013). For all other traits, the 80-1 parental genotype (a), recombinant genotype (h), and M6 parental genotype (b) mean trait value as well as the Kruskal-Wallis p-value was calculated and visualized with PhenoGram Synthesis View (Wolfe et al., 2013). Distorted segregation analysis Deviation from the expected 1:2:1 (homozygous:heterozygous:homozygous) genotypic class frequencies was calculated by chi-squared tests of each SNP in the F4 and F5 generations. The significance of distorted segregation was reported as the chi-squared test p-value on a scale from 0 to 7 where 0 = p > 0.01; 1 = 0.01 ≥ p > 0.001; 2 = 0.001 ≥ p > 1e-4; 3 = 1e-4 ≥ p >1e-5; 4 = 1e-5 ≥ p > 1e-6; 5 = 1e-6 ≥ p > 1e-7; 6 = 1e-7 ≥ p > 1e-8; 7 =1e-8 ≥ p (Velmurugan et al., 2018). The frequency of the parental genotype (80-1 = a; M6 = b) and recombinant genotype (h) at each locus in the F4 and F5 generations was examined to establish patterns of preferential inheritance. Heterozygosity retention analysis Heterozygosity was calculated based on the frequency of heterozygous SNP genotype calls at each locus in the 113 F4 and 80 F5 genotyped individuals. Loci with four or more times the expected level of heterozygosity (assuming a 50% reduction in heterozygosity with each generation of selfing) were considered excessively heterozygous. The physical location of 315 excessively heterozygous loci in each generation were plotted on the PGSC v4.03 pseudomolecules using PhenoGram (Wolfe et al., 2013). Sli KASP marker genotyping Parental lines 80-1 and M6, their F1 hybrid, as well as 22 F4 individuals and 49 F5 individuals were genotyped with two Kompetitive Allele Specific PCR (KASP)TM markers Sli_626 and Sli_898 designed by Clot et al. (2020) within a candidate Sli region on chromosome 12. The dominant homozygous genotype (Sli/Sli) at these two marker loci is significantly associated with self-compatibility in diverse diploid potato backgrounds (Kaiser et al., 2021). Each KASPTM assay is composed of an allele-specific SC and SI forward primer and a common reverse primer (Table S4). KASPTM assays were conducted according to methods described previously by Kaiser et al., (2021). Results Producing the F4 and F5 generation Production of inbred lines strictly by single seed descent was prevented by self-infertility, poor seed germination, and plant mortality in each generation. Reduction in fecundity and plant seedling vigor traits was expected due to the homozygous condition of deleterious and lethal alleles (Hosaka & Sanetomo, 2020; Phumichai & Hosaka., 2006, Zhang et al., 2019). In addition to plant death related to low vigor, wide variation in susceptibility to Alternaria alternata (Fries.) Keissl under greenhouse conditions impeded self-pollination of some individuals before they succumbed to disease. A. alternata is a fungal pathogen that causes the destructive and rapidly advancing disease potato brown leaf spot (Rotem, 1994; Thomma, 2003). Consequently, only 92 and 55 unique F2 families are represented in the F4 and F5 generations, respectively. There exists wide variation in plant morphology between families in the F5 generation (Figure 1a-f). Dwarf 316 phenotypes arose in the F4 and F5 generations. Features such as leaf trichome production, plant growth habit, stolon production, leaf pigment, and leaf shape were generally maintained within each familial lineage (Figure 1g). Inbred F5 lines demonstrating robust self-fertility were identified (Figure 1h-i). Although glandular trichomes confer host plant resistance to the Colorado potato beetle (Groden & Casagrande, 1986; Pelletier & Dutheil, 2006; Yencho et al., 1994), dense leaf trichomes were only observed in a single family in this study and trichome density was not quantified in the RIL population. Characterizing self-fertility traits Overall, self-fertility was diminished in the F5 individuals compared to the F4 individuals that had been evaluated in the 2020 greenhouse season. A greater proportion of individuals in the F5 generation (40%) failed to flower within the pollination period compared to the F4 generation (15%). Although there was broad variation in both generations, the mean fraction of flowers setting fruit in the F5 individuals (0.11) was significantly lower than in F4 individuals (0.29) (t = -3.981, p = 0.0001) (Figure S1). The average fruit weight of each genotype ranged from 0.1-3.84 g in the F4 generation and from 0.22-2.49 g in the F5 generation (Figure S1). A total of 7,346 seeds resulted from self-pollination of F4 individuals and 1,865 seeds from self-pollination of F5 individuals. The total number of seeds per genotype ranged from 5 - 850 seeds and 5 - 430 seeds in the F4 and F5 generations, respectively, with a significantly lower average number of seeds per fruit in the F5 generation (t = -3.13, p = 0.0030) (Figure 2a). The number of seeds per fruit was not significantly correlated to the proportion of self-pollinated flowers setting fruit. Parthenocarpic fruits were produced by five lines each in the F4 and F5 generations (Table S2). Lines producing parthenocarpic fruit in each generation were not from the same families either within a generation or between 317 generations. There was not an individual genotype that produced both seeded and parthenocarpic fruit. Pollen viability was similarly high in both generations in 2020 (mean F4 fraction of viable pollen = 0.84, mean F5 fraction of viable pollen = 0.77) (Figure 2b). Pollen viability was positively correlated to the fraction of flowers setting fruit in F5 individuals (r = 0.5239, p = 0.0372) but not in the F4 generation (a = 0.05). The number of seeds per fruit was not significantly correlated to pollen viability in either generation (a = 0.05). Pollen tube growth in the style was assessed 48 hours after self-pollination in the SI parent 80-1, 50 F4 individuals and 38 F5 individuals where pollen was present. Pollen tube growth was aborted in the style of all replicates of 80-1 (Figure 3a). No progeny displaying pollen tube growth abortion in the style in all replicates were identified. Consequently, mean pollen tube growth ratings were similarly high in both generations. The F4 mean pollen tube growth rating (2.97) was slightly higher than the F5 mean pollen tube growth rating (2.92), although not statistically different. Pollen tubes reaching the ovary were observed not only in genotypes that set seed upon selfing, but also genotypes that produced parthenocarpic fruit or no fruit at all after self-pollination. (Figure 3b-d). The 48 F4 individuals that were evaluated in two greenhouse seasons were similarly self- fruitful in 2019 and 2020 (Figure 2c). However, significantly more seeds per fruit were produced in the 48 F4 lines during the selfing period in 2020 (t = 3.59, p = 0.0007) (Figure 2c), perhaps attributable to the use of LED lights in this year. Seed set was not significantly correlated to the fraction of flowers setting fruit in either year and there was no significant correlation between years for either trait. 318 Foliar glycoalkaloid content of greenhouse grown plants Leptine I/II was detected in 28 F4 lines, ranging from 0.13-9.07 mg/g DW, and in 26 F5 lines, ranging from 0.05-7.03 mg/g DW, grown under greenhouse conditions (Figure 4a, Figure S2, Table S5). The presence of leptines was not significantly different from a 1:1 ratio in the F4 generation (c2 = 0.4460) or in the F5 generation, when considering only a single F5 individual from a family (c2 = 0.1161) (a = 0.05). Families represented by multiple F5 individuals did not contain both leptine producing and non-leptine producing individuals. Ten F4 lines and five F5 lines had greater total leptines than parent 80-1 (4.27 mg/g DW). The foliar concentration of the non- acetylated glycoalkaloids solanine and chaconine varied widely in both generations (Figure 4a, Figure S2). The mean ratio of acetylated to non-acetylated compounds was significantly higher in F2 individuals (0.39) than those observed in the F4 (0.07) or F5 (0.04) generations (p < 0.0001) (Table S5). Colorado potato beetle field defoliation resistance In field trials under natural beetle pressure, the susceptible cv. ‘Atlantic’ was completely defoliated during the summer of 2019 and 2020, resulting in RAUDC values of 28.7 and 30.9, respectively (Table S6). The parental line 80-1 remained robustly resistant with a RAUDC of 0.2 in 2020 and 0.5 in 2019 (Table S6). Field defoliation of F4 and F5 lines was continuously distributed with RAUDC values ranging from 0.2-86.7 in the F4 generation evaluated during 2019 and from 0.0-48.1 in the F5 generation assessed during 2020 (Figure 4c, Table S6). Although the mean RAUDC was reduced in each generation, from 20.01 in the F2 generation to 15.79 in the F5 generation, this decrease was not statistically significant. In the F4 and F5 individuals containing leptines, leptines I/II, total leptines and the ratio of acetylated to non-acetylated compounds was significantly negatively correlated to field defoliation (Table 1). 319 Generally, the F1 hybrid individuals exhibited strong defoliation resistance throughout the first larval generation in 2020. However, once the susceptible foliage in the field was completely consumed, the hybrids were defoliated by second generation larval feeding resulting in RAUDC values ranging from 5.7-55.7 (Figure S3a, Table S6). Two hybrids (MSHH786B_01 and MSHH786B_09) retained robust foliage until the end of the evaluation period and were used for field glycoalkaloid sampling. Glycoalkaloid content of foliar and tuber tissue of field-grown plants Leptines I/II were present in the foliar tissue sampled from each of the nine Colorado potato beetle resistant F5 inbred lines as well as the two F1 hybrid individuals grown in the Montcalm Research Center beetle nursery in 2020 (Figure S3b, Table S7). Tuber mean total glycoalkaloids ranged from 57.2 mg% fresh weight to 609.0 mg% fresh weight in the five F5 lines that tuberized in the field (Figure S4, Table S7). No genotypes evaluated contained tuber glycoalkaloid levels below the commercial industry threshold of 20.0 mg% fresh weight (Figure S4, Table S7). V3 22K SNP Array genotyping of the RIL population The V3 SNPs with a missing call in either parent were removed (n = 662). A total of 17,879 V3 SNPs that were monomorphic between parental lines (either AA or BB in both parents) and 134 V3 SNPs heterozygous in both parents were removed. A total of 694 V3 SNPs that were heterozygous in one parent (AAxAB, BBxAB, ABxAA or ABxBB) but non-segregating in the F1 hybrid (AA or BB) were removed. The V3 SNPs previously identified as uninformative for three- cluster calling in GenomeStudio (Hirsch et al., 2013) and poor-quality V3 SNPs determined by manual curation in GenomeStudio were removed (n = 638). This filtering process resulted in 1,020 informative V3 SNPs distributed across the 12 chromosomes for further analysis (Table S8). 320 SNP marker loci associated with self-fertility Of the 1,020 informative segregating SNP markers, nine total SNPs were associated with self-fertility traits in the F4 and F5 generations (Kruskal-Wallis p-value < 0.05). The distal portion of chromosome 12 harboring Sli was not associated with fruit or seed set. Instead, SNP PotVar0079948 on chromosome 5 was associated with increased fruit set in F4 individuals (p = 0.0005) while a SNP (solcap_snp_c2_39463) on chromosome 4 was associated with fruit set in F5 individuals (p = 0.0013) (Table 2). SNP PotVar0079948 on chromosome 5 resides within PGSC0003DMG400018434 (DM v6.1 annotation = Soltu.DM.05G005290) annotated as a photosynthetic gene. Gene Soltu.DM.05G005300 7.5 kb downstream on chromosome 5 is a NAC transcription factor activated by APETALA 3 and PISTALLATA and described to function in flower, embryo and fruit development. The closest gene (PGSC0003DMG400006402; Soltu.DM.04G029620) to SNP solcap_snp_c2_39463 lies 2 kb upstream on chromosome 4. Seed set in the F5 generation was associated with SNP solcap_snp_c1_6157 on chromosome 4 (p = 0.0102) and SNP solcap_snp_c2_23308 on chromosome 12 (p = 0.0102), where the recombinant genotype resulted in significantly more seeds per fruit than either parental genotype (Table 2). SNP solcap_snp_c1_6157 on chromosome 4 is positioned within a gene (PGSC0003DMG400005353; Soltu.DM.04G018190) encoding a glycosyl hydrolase family 31 protein. SNP solcap_snp_c2_23308 on chromosome 12 falls within gene PGSC003DMG400028845 (Soltu.DM.12G008730.1) annotated as an ovule receptor-like kinase. A single SNP on chromosome 3 (solcap_snp_c2_50372), in a ~30 kb region without annotated genes, was associated with both seed set and fruit weight in F4 individuals (Table 2). The M6 parental genotype contributed to increased pollen tube growth in the style in F5 individuals at four 321 SNPs in a 582 kb region on chromosome 3 (58,295,011-58,877,163 bp) containing 69 annotated genes (Table 2). Sli KASPTM marker genotyping The self-compatible parent M6 is homozygous for the dominant Sli genotype (Sli/Sli) while SI parent 80-1 has the homozygous recessive genotype (sli/sli) at the two KASPTM marker loci tested (Table S9). The F1 hybrid is heterozygous (Sli/sli) for both markers (Table S9). The homozygous recessive genotype was not detected in any of the inbred progeny. The frequency of the homozygous dominant Sli genotype at each marker locus (≥ 0.88) was much greater than the frequency of the heterozygous Sli/sli genotype in F4 and F5 individuals (Table S9). SNP marker loci associated with glycoalkaloid content A total of 23 loci associated with the presence of leptines in the F4 and F5 generations were located on chromosomes 2, 6, 7 and 8 (Table S10). Six SNPs on chromosome 2 between 14041901-22381719 bp were detected in both generations (p < 0.0001) (Table S10, Figure S5). Of these, four SNPs (PotVar0039036, PotVar0039005, solcap_snp_c2_32460 and solcap_snp_c2_32462) were also significantly associated with leptine accumulation in both generations (p < 0.0001) (Table S10). Several known genes involved in primary metabolism upstream of the secondary glycoalkaloid pathway including sterol side chain reductase (SSR) 1 (PGSC0003DMG400011801; Soltu.DM.02G003240), 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG2) (PGSC0003DMG400003461; Soltu.DM.02G004910), and squalene epoxidase (SQE) (PGSC0003DMG400003324; Soltu.DM.02G007460) reside within this region. Contingency analysis revealed that two SNPs (solcap_snp_c2_32462 and PotVar0039036) 230 kb apart on chromosome 2 were the best predictors of leptine synthesis in the F5 (R = 0.6279, p < 0.0001) and F4 (R = 0.55, p < 0.0001) generations, respectively. The SNP on chromosome 6 322 (solcap_snp_c2_57292) was also associated with solanine accumulation in the F4 generation (Table S10). Loci associated with leptine I/II accumulation and the ratio of acetylated to non-acetylated compounds were only detected on chromosome 2 (Table S10). Between 14.0 – 22.4 Mb on chromosome 2 there are three recombination blocks in F5 individuals (Table S8) that correspond to different patterns of genotype trait mean values (Table S10). The first block contains three SNPs (solcap_snp_c2_32239, solcap_snp_c2_41874 and solcap_snp_c2_30945) at which the 80-1 parental genotype contributes the highest mean leptine I/II content (Table S10, Figure 5). In the next block, the recombinant genotype at two SNP loci (PotVar0039036 and PotVar0039005) results in lower mean leptine I/II content than either parental genotype (Figure 5). Finally, the recombinant genotype at SNPs solcap_snp_c2_32460 and solcap_snp_c2_32462 has the highest mean trait value (Table S10, Figure 5). Two loci on chromosome 6 were found to be associated with a-chaconine accumulation in the F5 generation while SNPs associated with a-solanine accumulation were detected on chromosome 1, 6, 10, 12 and within a 419 kb region on chromosome 7 (Table S10). None of these a-chaconine and a-solanine associated SNPs were found to be significantly associated with field defoliation in either generation. SNP marker loci associated with Colorado potato beetle resistance Four SNP markers (PotVar0039036, PotVar0039005, solcap_snp_c2_32460, and solcap_snp_c2_32462) were significantly associated with Colorado potato beetle resistance in F4 individuals. These four SNPs span 230 kb on chromosome 2 and were each also significantly associated with leptine synthesis and accumulation (Table S10). The seven significant SNP markers for Colorado potato beetle resistance in F5 individuals were located in an overlapping 323 region on chromosome 2. However, these SNPs were disqualified from further analysis because a single genotype (AA, AB or BB) was responsible for significance and was represented by < 5 individuals. Genetic map construction A collection of 286 SNPs and 97 F4 individuals was used to create a linkage map covering 77% of the 12 assembled DM pseudomolecules (PGSC Version 4.03) (Table 3, Table S11). The map spanned a genetic distance of 805.2 cM with an average of 24 SNP markers per chromosome, distributed at an average distance of 3.64 cM between SNPs (Table 3). All mapped SNPs were in the expected position based on the DM v4.03 physical map (Table S11). Chromosomes 1, 7, 9 and 11 were split into multiple genetically unlinked groups (Figure S6). The small F5 population size combined with the low number of non-redundant SNP markers prevented creation of an informative linkage map in the F5 generation. V4 32K SNP Array genotyping of parental lines Both parental lines 80-1 and M6 were genotyped on the V4 SNP Array to determine whether the platform captures a greater number of polymorphisms between the two S. chacoense lines useful for mapping. V4 SNPs with a missing call in either parental line (n = 1,159), V4 SNPs monomorphic between the two lines (n = 25,894), V4 SNPs heterozygous in both parents (n = 236), and V4 SNPs previously determined to be uninformative for three-cluster calling in GenomeStudio (n = 1,186) were removed. A further 1,593 V4 SNPs heterozygous in one parent were removed. After filtering, 923 V4 SNPs with a homozygous call in either parent were retained. A total of 385 were not present on the V3 Array and were distributed across the twelve chromosomes (Table S8). Of these, the greatest number of SNPs were located on chromosomes 12 (N = 67), 6 (N = 61), 11 (N = 44) and 1 (N = 43) (Table S8). 324 Heterozygosity retention Parent 80-1 was less heterozygous (3.8%) than parent M6 (4.8%) based on the mean percent heterozygosity at 20,357 V3 SNP markers. The mean percent heterozygosity based on the 29,832 V4 SNP markers was higher for both parental lines, but the relative relationship remained similar (80-1 = 4.3%; M6 = 5%). The percent heterozygosity of the 20,357 V3 SNP markers decreased from 1.4% in the F4 generation to 0.6% in the F5 generation. The frequency of the heterozygous SNP genotype (AB) at the 1,020 segregating loci analyzed in this study was also lower in parent 80-1 (0.19) compared to parent M6 (0.33). The frequency of the heterozygous SNP marker genotype (AB) at the 1,020 segregating loci analyzed in this study decreased significantly over the course of inbreeding from 0.58 in the F2 generation (N = 236) to 0.26 in the F4 generation (N =113) and finally to 0.17 in the F5 generation (N = 80) (Figure S7). However, there was considerable variation in the level of homozygosity within the F5 individuals (Table 4). The level of homozygosity was not significantly correlated with any one of the fertility trait values measured in the F5 generation. A total of 229 loci in the F4 generation (Figure 6a) and 307 loci in the F5 generation (Figure 6b) exhibited four or more times the expected level of heterozygosity (Table S12). In both generations, excessively heterozygous loci were found on chromosomes 1, 4, 6, 7, 8, 9, 10 and 12, although the majority of loci were located on chromosome 8 and 12 (Figure 6a, b). At 15 of these loci on chromosome 12 the parental 80-1 genotype was not present in any of the F5 progeny (Figure 6b). There were no loci heterozygous in every family. Distorted segregation Loci exhibiting distorted segregation at a significance level of p ≤ 0.001 were detected on chromosomes 1, 2, 3, 7, 8, and 12 in the F2, F4 and F5 generations (Figure 7). Generally, 325 the significance of segregation distortion increased over the course of inbreeding and the most distorted loci were localized to chromosomes 1, 8, and 12 (Table 5, Figure 7). Although the percentage of loci displaying severe segregation distortion (p < 1e-8) on chromosome 1 remained very similar throughout the inbreeding process, there were 32.0% and 80.7% more loci departing from Mendelian segregation ratios on chromosomes 7 and 8, respectively, between the F2 and F5 generation (Table 5). The M6 homozygous genotype was preferentially inherited on large pericentromeric regions of chromosomes 1 and 3 (Figure S8). Over the course of inbreeding, the majority of chromosome 8 became disproportionally enriched for the recombinant genotype (Figure S8). On chromosome 12, two distinct patterns of preferential inheritance occur. Between 11,041,151-48,851,340 bp the frequency of the homozygous 80-1 genotype was suppressed, while the recombinant and M6 homozygous genotype were preferentially inherited in each generation (Figure S8). In this region, the frequency of the 80-1 homozygous genotype did not exceed 0.03, the mean frequency of the recombinant genotype was 0.36 and the mean M6 homozygous genotype frequency was 0.61 in the F5 inbreds (Figure S8). The homozygous 80-1 genotype appeared only in F4 and F5 individuals of three families (93, 268 and 641) in this region (Table S8). On the most distal portion of the long arm of chromosome 12, in the vicinity of the candidate Sli region (Clot et al., 2020), a different signature of distorted segregation was present. The frequency of M6 homozygous genotype at SNP PotVar0053309 (58,986,365 bp) was increased to 0.86 and 0.90 in the F4 and F5 generations, respectively (Figure S8). The 80-1 homozygous genotype at this SNP locus was not present in any individuals of the F2, F4 or F5 generations (Figure S8). 326 Discussion Creating diploid inbred lines for use in potato breeding Establishing a diverse set of diploid potato inbred lines by conventional breeding first requires the introduction of self-compatibility from a limited number of known SC donors into germplasm with the desired agronomic traits. Subsequent selfing imposes selection for self- fertility, seed germination and vigor traits that have not been refined through inbreeding in the SI background. This study applied the process to construct SC, Colorado potato beetle resistant inbred lines and to explore the cost of inbreeding depression in diploid potato. Unlike in SC diploid crops, where two completely homozygous parental lines are used as founders, the host plant resistance donor parent 80-1 of our RIL population is SI. Parent 80-1 most likely harbors deleterious alleles contributing to plant mortality, low vigor, and a non-flowering phenotype that manifested over the course of inbreeding. As a result, individuals carrying potentially valuable host plant resistance alleles were lost. Contending with the appearance of low- vigor traits in each generation of inbreeding also introduced increased labor and greenhouse space requirements. The inefficiencies of producing even this relatively low number (55) of F5 inbreds cannot be overstated. However, F5 inbreds with strong plant growth habit and functional self- fertility were produced (Figure 1c,f,h-i). A year effect for selfed seed was observed by evaluating the same 48 F4 genotypes under greenhouse conditions in two years (2019 and 2020). Self- pollination resulted in 1.7x more selfed seeds per fruit in 2020 than in 2019. Complex genotype- by-environment interactions mediate SC/SI responses in tomatoes (Webb & Williams, 1988) and have been observed in SC S. phureja – S. stenotomum populations (Haynes & Guedes, 2018). Optimizing and tightly regulating environmental greenhouse conditions will be crucial to economical inbred line development. 327 Self-fertility Multiple factors appear to mediate the SC response in this RIL population. Sli is proposed to inhibit the cytotoxic effect of S-RNase, encoded by the S-locus on chromosome 1, permitting self-pollen tube growth in the style (Eggers, 2020; McClure et al., 2011; Sanwen et al., 2019). Preferential inheritance of the homozygous M6 genotype over a lengthy stretch of chromosome 1 that coincides with the location of the S-locus (Gebhardt et al., 1991) and S-RNase in potato (Enciso-Rodriguez et al., 2019) could suggest that Sli is incapable of interacting with 80-1 S-RNase alleles. Although segregation distortion signatures on chromosome 12 and Sli KASP marker genotyping show that all of the individuals in the F5 generation carried at least one copy of the dominant Sli allele, not all F5 individuals set fruit. Pollen tubes reached the ovaries of both self- fruitful and self-unfruitful plants, demonstrating that the presence of the dominant Sli allele permits self pollen tube growth in the style but does not ensure fruit/seed set upon selfing. The existence of post-stylar self-compatibility barriers was previously observed in cultivated diploid potato germplasm (Peterson et al., 2016). Distinct loci on multiple chromosomes were significantly associated with selfed seed and fruit set in the same growing season, indicating multigenic control of these traits. Kaiser et al. (2021) recently described several SC clones from diverse genetic backgrounds lacking dominant Sli alleles. Candidate genes encoding high-top protein (HT) (Goldraij et al., 2006) (chromosome 12), arabinogalactan (120K) (Lee et al., 2009) (chromosome 8), and Kunitz-type proteinase inhibitors (NaStEP) (Jiménez-Durán et al., 2013) (chromosome 3) have been proposed to modulate the strength of the SC response in Solanum. Molecular characterization of these loci has primarily been conducted in tomato and their role in potato remains to be determined. The SNP markers significantly associated with self-fertility traits in this study did not colocalize with these particular 328 modifier loci but could instead serve as novel targets for self-fertility improvement. Chromosome 12 in particular is a rich source of self-fertility genes in potato, containing genes encoding HT proteins (54 Mb), Sli (58 Mb) and the ovule receptor like kinase (53 Mb) associated with selfed seed set in this study. It is important to note that the recombinant genotype at five of the nine SNPs significantly associated with fertility traits in this study resulted in the best mean trait values for selfed seed set and fruit set. This finding illustrates that SI clones can contribute to self-fertility and the potential to exploit heterosis for these traits. Taken together, improvement of self-fertility and inbred germplasm may not be easily accomplished through marker assisted selection for Sli alone. Utility of RILs in potato genetics RIL populations are an immortal genetic resource that allow phenotypic evaluation distributed over geographical location and time. Seed immortalization of a potato mapping population is especially novel in this vegetatively propagated crop where tuber seed pieces have a limited storage life and require considerable storage space. As one practical example, the genetic basis of the wide variation in A. alternata resistance observed in this RIL population could be further investigated in replicated disease trials. No major resistance genes against A. alternata have been identified to date and control of potato brown leaf spot relies on the use of protective fungicides (Soleimani & Kirk, 2012; Stevenson et al., 2001). Progress toward homozygosity in the RIL population developed in this study varied between families and was slowed by retention of large heterozygous regions on chromosomes 8 and 12. Unique combinations of beneficial self-fertility alleles in each family could explain the differences in homozygosity levels observed between families. Two of the most homozygous (97% 329 and 95%) F5 inbreds in this study belong to the same family. Selection for these superior haplotypes could improve the efficiency of inbred development. A slower than expected approach to homozygosity has been reported in selfed S. chacoense (Hosaka & Sanetomo, 2020; Phumichai et al., 2005) and S. tuberosum Group Phureja (Peterson et al., 2016) populations, with persistent heterozygosity on chromosome 8. The large heterozygous blocks identified here on chromosome 8 in F5 inbreds do not coincide with gene-dense heterozygous areas of elevated recombination previously reported in M6 (Marand et al., 2019). Residual heterozygosity on chromosome 8 in this population may be a function of reduced recombination due to structural genomic differences in parental lines rather than a prerequisite for functional gamete production. The necessity of heterozygosity for plant vigor and self-fertility in potato has been questioned. That no heterozygous loci were conserved in all families is promising for the development of completely homozygous potato lines. Phumichai & Hosaka (2006) describe a positive correlation between the heterozygosity level at 62 restriction length fragment polymorphism markers and fertility traits in a S3 family created by use of Sli. However, heterozygosity was not linked to self-fertility performance in this S. chacoense population. Further rounds of selfing are needed to develop completely homozygous lines from this population. The exact number of selfed generations required to deliver true breeding inbred lines in all families remains to be determined. Hosaka et al. (2020) recently developed S10 diploid potato lines, posited to be 100% homozygous based on a lack of segregation for SNP marker genotype of V3 SNPs in the selfed S11 offspring. Availability of genetically uniform lines from the RIL population described here would permit maintenance of inbred lines by open-pollination and would provide a valuable resource to further study host plant resistance to the Colorado potato beetle. 330 Colorado potato beetle host plant resistance in F5 inbred lines This study demonstrates that robust inbred lines carrying Colorado potato beetle resistance equivalent to the resistant donor parent can be developed without field selection during the inbreeding process. Selection for self-fertility may have inadvertently caused the reduction in foliar leptine content observed over the course of inbreeding yet field beetle defoliation resistance remained stable across generations. Although the most field resistant F5 individuals contained leptines, leptine content was not proportional to the level of field resistance. The sufficiency of leptine accumulation for a strong Colorado potato beetle host plant resistance response has been posed previously (Kaiser et al., 2020; Lorenzen et al., 2001; Sagredo et al., 2009). A higher ratio of acetylated (leptines I/II) to non-acetylated (α-solanine and α-chaconine) glycoalkaloids measured under greenhouse conditions was significantly correlated to lower field defoliation in this study and confirms the same observation in the F2 generation (Kaiser et al., 2020). The ratio of acetylated to non-acetylated glycoalkaloids can be measured in a single spectrometry analysis and represents a powerful metabolite marker to predict field performance without incurring the costs of conducting a Colorado potato beetle field trial. Loci significantly associated with leptine synthesis and accumulation in three generations (F2 (Kaiser et al., 2020), F4 and F5) were localized to chromosome 2. SNPs associated with accumulation of the common glycoalkaloids a-solanine and a-chaconine in the RIL population were only found on chromosomes 1, 6, 7, 10 and 12, suggesting that the region on chromosome 2 is specific to leptine production rather than general glycoalkaloid metabolism. Despite increased opportunities for recombination in this population structure, the identified region is quite large. The size is likely a function of both the limited population size and dearth of SNP marker density. 331 Introducing Colorado potato beetle resistance to diploid breeding lines Leptine production was successfully introduced into diploid breeding germplasm using a RIL individual from the F4 generation. Continuous variation in field defoliation resistance among the F1 MSHH786B hybrids supports the hypothesis that multiple recessive genes contribute to the Colorado potato beetle resistance phenotype (Boluarte-Medina et al., 2002; Hutvágner et al., 2001; Manrique-Carpintero et al., 2014; Ronning et al., 1999). Since Colorado potato beetle resistance is more likely fixed in the strongly resistant, highly homozygous F5 inbreds, they are a more efficient vehicle to introgress host plant resistance into cultivated diploid backgrounds. However, introgression hybrids will likely require selfing to recover the homozygous condition of recessive loci contributing to Colorado potato beetle resistance. Tuber glycoalkaloid content varied in the limited number of beetle resistant inbreds created in this study. Further work is needed to fully characterize the relationship between host plant resistance and glycoalkaloid tissue partitioning. Prioritization of the beetle resistant inbreds with low tuber glycoalkaloids for further breeding will be crucial to ensure a safe food supply. Limitations of utilizing the SNP Array platform to genotype small populations The conclusions drawn from this study are limited by the small RIL population size and the number and origin of SNP markers used. The increased number of recombination events in a RIL population can provide improved map resolution when coupled with sufficient marker density to capture these events. Genetic investigation in this population was constrained by the large number of SNPs (17,879; 85% of total SNPs) that were monomorphic between parental lines, despite inclusion of SNPs with low minor allele frequency in tetraploid cultivars on the V3 array (Vos et al., 2015). Significant trait variation within S. chacoense (Bamberg et al., 1996; Christensen et al., 2017; Hosaka & Hanneman, 1991) and a simple sequence repeat survey of 10 332 natural S. chacoense populations (Haynes et al., 2017) suggest that there is genetic heterogeneity between S. chacoense accessions (Bamberg & del Rio, 2020). The intrinsic ascertainment bias of the SNP array platform could prevent detection of these polymorphisms and hamper detection of loci integral to self-fertility in the heterozygous condition. The partial heterozygosity of SI parent 80-1 and inbred line parent M6 (Leisner et al., 2018; Marand et al., 2019) also constricted the number of informative SNP markers to those that were homozygous in both parents. The additional 385 SNP markers with contrasting homozygous states in 80-1 and M6 on the V4 Array suggest that marker saturation could be increased incrementally by genotyping the RIL population with the V4 array. Alternatively, genotyping-by-sequencing is a viable cost-effective alternative to SNP array genotyping in diploid potato (Endelman & Jansky, 2015) could combat the overestimation of recombination frequency caused by multiple cycles of meiotic events inherent to RIL development. In addition, to providing more suitable levels of marker density, a next generation resequencing approach would more accurately characterize the approach to homozygosity through inbreeding. Conclusion This study is the first report of a RIL population in potato. The work highlights the challenges of establishing inbred germplasm, reinforces the complexity of selecting for self- fertility in diploid potato, and lays the foundation for optimization of potato RIL development. The inbred lines described here also have utility in diploid breeding as self-fertility donors. Equally importantly, crossing these self-fertile F5 inbred lines to inbred material derived from other SC sources will begin to inform combining ability for self-fertility at the diploid level. The availability 333 of highly homozygous Colorado potato beetle resistant lines will enable genomic inquiry of the loci contributing to this trait. 334 APPENDICES 335 APPENDIX A: Chapter 5 Tables Table 5.1. Spearman’s rank correlation coefficients among measured traits in the F4 (left) and F5 (right) generations of a diploid recombinant inbred line population derived from Solanum chacoense lines USDA8380-1 and M6. F4 Leptine Ia Leptine IIa Total Leptinea α-Solaninea α-Chaconinea Acetylated/Non- Acetylatedb Field Defoliationc -0.67*** -0.63*** -0.68*** ns ns -0.61** α- Chaconinea α- Solaninea -0.44* -0.50** -0.44* 0.88*** ns ns ns Leptine II 0.96*** F5 Leptine Ia Leptine IIa Total Leptinea α-Solaninea α-Chaconinea Acetylated/Non- Acetylatedb Field Defoliationd -0.68*** -0.60*** -0.68*** ns ns -0.69*** α- Chaconinea α- Solaninea ns ns ns 0.85*** ns ns ns *** p < 0.0001, **p < 0.01, *p < 0.05, ns not significant, n = 32 *** p < 0.0001, **p < 0.01, *p < 0.05, ns not significant, n = 65 Leptine II 0.92*** a Data represent the mean of two technical replicates mg/g DW b Data represent [mean total leptines (mg/g DW)]/ [mean α-chaconine (mg/g DW) + mean α-solanine (mg/g DW)] c Data represent the mean of the relative area under the defoliation progression curve (RAUDC) measured in three biological replicate field plots in 2019 d Data represent the mean of the relative area under the defoliation progression curve (RAUDC) measured in two biological replicate field plots in 2020 336 Table 5.2. Significant single nucleotide polymorphisms (SNPs) associated with fertility traits by Kruskal-Wallis testing in the F4 and F5 generations of a diploid recombinant inbred line population derived from Solanum chacoense lines USDA8380-1 and M6. The physical position of each significant SNP on the S. tuberosum clone DM1-3 516 R44 PGSC v4.03 pseudomolecules (Chromosome) is given (Position). The number of individuals (N) used in each SNP marker-trait association is given. Trait Generation Kruskal-Wallis p value SNP Chromosome solcap_snp_c2_50372 solcap_snp_c2_50372 solcap_snp_c2_148 CH03 CH03 CH03 Position (bp) 2524231 2524231 58295011 PotVar0014064 CH03 58519761 solcap_snp_c2_99 CH03 58520073 Average Fruit Weight Seeds per fruit Pollen tube growth rating Pollen tube growth rating Pollen tube growth rating Pollen tube growth rating 58877163 30716444 Seeds per fruit 63406121 4701617 53190167 Fraction of Flowers Setting Fruit Fraction of Flowers Setting Fruit Seeds per fruit solcap_snp_c2_616 solcap_snp_c1_6157 solcap_snp_c2_39463 PotVar0079948 solcap_snp_c2_23308 CH03 CH04 CH04 CH05 CH12 80-1 Parental Genotype Trait Mean 1.20 26.20 2.50 2.50 2.50 2.53 10.64 0.22 0.11 14.48 Recombinant Genotype Trait Mean 2.48 39.21 2.97 2.97 2.97 3.00 9.38 0.03 0.42 25.92 M6 Parental Genotype Trait Mean 1.14 18.24 2.98 2.98 2.98 2.98 24.82 0.03 0.36 9.46 N 41 36 34 34 34 34 17 51 50 17 0.0055 0.0028 0.0045 0.0045 0.0045 0.0003 0.0102 0.0013 0.0005 0.0102 F4 F4 F5 F5 F5 F5 F5 F5 F4 F5 337 Table 5.3. Summary of the Solanum chacoense USDA8380-1 x M6 F4 population linkage map based on 97 individuals and 288 single nucleotide polymorphism (SNP) markers Chromosomea No. mapped Map Length Map Length SNPs (cM)b (Mb)c Map coveragec Average interlocus distance (cM)b chr01.1 chr01.2 chr02 chr03 chr04 chr05 chr06 chr07.1 chr07.2 chr08 chr09.1 chr09.2 chr09.3 chr10 chr11.1 chr11.2 chr12 Total 12 14 31 7 14 35 22 7 11 17 4 6 4 12 35 15 40 286 45.01 78.06 112.25 20.61 105.48 85.17 10.03 32.75 25.36 77.81 23.47 3.35 2.45 75.26 28.94 41.97 37.26 805.22 58.89 18.60 38.52 34.22 67.00 49.96 39.71 40.01 3.55 48.95 2.63 16.94 0.61 55.29 5.23 25.62 51.12 556.85 0.66 0.66 0.79 0.55 0.93 0.96 0.67 0.70 0.06 0.86 0.04 0.28 0.01 0.93 0.12 0.56 0.84 0.77 3.78 6.00 3.74 3.43 8.11 2.50 0.48 5.46 2.54 4.86 7.82 0.67 0.82 6.84 0.85 3.00 0.96 3.64 aChromosomes 1, 7, 9 and 11 were split into multiple genetically unlinked groups bGenetic distance in given in centimorgan (cM) cMap length (Mb) and map coverage (Mb) are based on the assembled Solanum tuberosum group Phureja DM1-3 pseudomolecules (PGSC Version 4.03) 338 Table 5.4. Individual mean, minimum (Min) and maximum (Max) genotype frequencies at 1,020 SNP markers segregating in the F2 (N = 236), F4 (N = 113) and F5 (N=80) generations of a Solanum chacoense recombinant inbred line population. The homozygous genotype represents both AA and BB SNP marker genotypes. Homozygous Genotype Heterozygous Genotype F2 F4 F5 Parental Generation Mean Min Max Mean Min Max 0.84 0.69 0.48 0.33 0.79 0.95 0.98 0.81 0.58 0.26 0.17 0.26 0.42 0.74 0.83 0.74 0.16 0.31 0.52 0.67 0.21 0.05 0.02 0.19 339 Table 5.5. The number of loci exhibiting distorted segregation at the p<1e-8 level of significance in the F2, F4 and F5 generations of a Solanum chacoense recombinant inbred line population. Total SNP loci evaluated = 1,020. Generation F2 Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 Total F4 F5 No. SNPs % Total No. SNPs % Total No. SNPs % Total 27 0 4 0 0 0 0 1 2 2 1 147 184 38.6 0.0 7.3 0.0 0.0 0.0 0.0 0.6 2.1 5.4 1.1 95.5 18.0 26 0 7 4 3 2 33 137 2 2 1 145 362 37.1 0.0 12.7 8.2 6.4 1.8 44.0 82.5 2.1 5.4 1.1 94.2 35.5 26 0 3 2 0 4 24 135 2 2 0 132 330 37.1 0.0 5.5 4.1 0.0 3.7 32.0 81.3 2.1 5.4 0.0 85.7 32.4 340 APPENDIX B: Chapter 5 Figures Figure 5.1. Characterizing the F4 and F5 generation of a Solanum chacoense recombinant inbred line population. Morphological traits, including (a) trichome production, (b) stolon production, (c) plant height, (d,f) leaf size and leaf shape (b-g), segregating in the F5 generation. Similar morphological features in a F4 (right) and F5 (left) individual from the same family (g). Fruit (h) and seed (i) of a self-compatible F5 individual. Photos taken by Luca M. Kaiser. 341 b) c) ns ** a) / t i u r F s d e e S Generation Generation *** ns Year Figure 5.2. Self-fertility in the F4 and F5 generation of a Solanum chacoense recombinant inbred line (RIL) population. (a) Box plots of the average number of seeds per fruit for the 37 F4 and 18 F5 individuals setting fruit upon self-pollination under greenhouse conditions in 2020. (b) Box plots of the mean fraction of viable pollen determined by image analysis of acetocarmine stained pollen samples (F4 N = 53; F5 N = 55). Paternal parent of the RIL population S. chacoense USDA8380-1 is self-incompatible, setting no fruit upon self-pollination, and maternal parent S. chacoense M6 is highly self-compatible, setting fruit and seed upon self-pollination. (c) Box plots of the fraction of fruit setting fruit upon self-pollination (bottom) and the average number of seeds per fruit in the 48 F4 individuals evaluated under greenhouse conditions in both 2019 and 2020. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = no significance. Charts created using JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 342 Figure 5.3. Pollen tube growth in the style 48 hrs post pollination of self-incompatible parent Solanum chacoense 80-1 (a), an F5 recombinant inbred line (095_04_01_01) that failed to set fruit upon selfing (b), an F5 recombinant inbred line (199_02_01_01) that produced only parthenocarpic fruit (c), and an F5 recombinant inbred line (495_01_05_04) that set fruit and seed (d). Pollen tubes visualized by staining with aniline blue. 343 a) ) b) W D g g m / ( t n e t n o c r a i l o f n a e M - n o n o t d e t a y t e c a s d n u o p m o c d e t a y t e c a l l f o o i t a R c) C D U A R n a e M Generation Generation Generation Figure 5.4. Foliar glycoalkaloid content of greenhouse grown plants and Colorado potato beetle resistance under field conditions. (a) Box plots of mean foliar content of individual compounds in leptine-producing parent Solanum chacoense USDA8380-1 (80-1), S. tuberosum tetraploid cultivar ‘Atlantic’, and 62 F4 and 74 F5 recombinant inbred individuals. (b) Box plots of the ratio of acetylated to non-acetylated compounds in 80-1, ‘Atlantic’ and the 62 F4 and 74 F5 individuals grown in 2020. Data represent the mean of two technical replicates. (c) Colorado potato beetle defoliation resistance of 80-1, ‘Atlantic’, F4 (n = 54), and F5 (n = 72) individuals under field conditions represented by mean relative area under the defoliation progression curve (RAUDC) in 2019 and 2020. The mean RAUDC was not significantly significant between generations. Data represent the means of replicated field plots in choice trials with high beetle pressure at the Michigan State University Montcalm Research Center CPB Nursery (Lakeview, MI). Charts created using JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 344 1 1 7 1 5 1 2 2 4 4 1 2 5 1 2 2 3 6 5 1 8 3 2 2 9 1 7 1 8 3 2 2 1 0 9 1 4 0 4 1 5 4 7 8 1 5 5 1 0 2 2 9 2 9 5 1 a) b) l ) e u a v - p ( 0 1 g o l - I e n i t p e L ) / W D g g m n a e m ( c) I I e n i t p e L ) W D g / g m n a e m ( 8 7 1 2 6 9 3 3 3 5 2 3 6 9 3 3 Leptine I Leptine II 80-1 parental genotype Recombinant genotype M6 parental genotype 80-1 parental genotype Recombinant genotype M6 parental genotype Figure 5.5. Loci associated with leptine I/II accumulation in the F5 recombinant inbred line population (N =26). The physical position (bp) of each SNP on the Solanum tuberosum clone DM1-3 516 R44 PGSC v4.03 chromosome 2 is shown at the top of the figure. The significance of each SNP is plotted for leptine I (green) and leptine II (yellow) content expressed as the -log10 of the ANOVA p-value (a). The red line denotes a p-value of 0.0001. The mean trait value of the S. chacoense 80-1 parental genotype (purple circle), the recombinant genotype (grey square) and the 345 S. chacoense M6 parental genotype (blue triangle) are plotted for leptine I (b) and leptine II (c). Charts created using PhenoGram Synthesis Viewer (Wolfe et al., 2013). 346 Figure 5.6. Excessive heterozygosity (red) at 229 loci in the F4 generation (a) and 307 loci in the F5 generation (b) of a Solanum chacoense recombinant inbred line population are plotted according to their absolute position on the 12 S. tuberosum clone DM1-3 516 R44 PGSC v4.03 pseudomolecules. The 15 loci on chromosome 12 where the S. chacoense USDA8380-1 homozygous parental genotype was not present in any F5 inbred lines is shown in blue (b). Charts created with PhenoGram (Wolfe et al., 2013). 347 Figure 5.7. Physical location of 1020 SNPs segregating in the F2, F4 and F5 generations of a Solanum chacoense recombinant inbred line population. The color scale indicates the significance of segregation distortion based on p-values associated with the chi-squared test of segregation in each generation. The significance of segregation distortion increases from blue (no distortion) to red (significant distortion at p<1e-8) according to the following scale: 0 = p > 0.01; 1 = 0.01 ≥ p > 0.001; 2 = 0.001 ≥ p > 1e-4; 3 = 1e-4 ≥ p > 1e-5; 4 = 1e-5 ≥ p > 1e-6; 5 = 1e-6 ≥ p > 1e-7; 6 = 1e- 7 ≥ p > 1e-8; 7 =1e-8 ≥ p. Charts created in JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 348 APPENDIX C: Chapter 5 Supplementary Data 2020 F4 N=41 F5 N=22 2019 F4 N=48 2020 F4 N=53 F5 N=55 t i h g e W t i u r F e g a r e v A s r e w o F l f o n o i t c a r F t i u r F g n i t t e S Figure S5.1. Distribution of average fruit weight in grams (grey) and the fraction of flowers setting fruit (black) in the F4 and F5 generation under greenhouse conditions in 2020. The fraction of flowers setting fruit (black) in F4 individuals under greenhouse conditions in 2019 is also shown. A quartile outlier boxplot displaying the sample median (horizontal line within box plot), a confidence diamond of the sample mean, and the shortest half or most dense 50% of the observations (red bracket) is shown to the right of each distribution. Charts created using JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 349 a) Leptine I Leptine II α-Solanine α-Chaconine W D g g m / r a i l o F n a e M 80-1 Atlantic 80-1 Atlantic Atlantic 80-1 Atlantic 80-1 b) Leptine I Leptine II α-Solanine α-Chaconine W D g g m / r a i l o F n a e M 80-1 Atlantic 80-1 Atlantic Atlantic 80-1 Atlantic 80-1 Figure S5.2. Foliar glycoalkaloid content in mg/g dry weight (DW) of greenhouse grown F4 (N = 62) (a) and F5 (N = 74) (b) individuals. Values for the leptine-producing parent Solanum chacoense USDA8380-1 (80-1) and S. tuberosum tetraploid cultivar ‘Atlantic’ (Atlantic) grown in the same conditions are shown. Data represent the mean of two technical replicates. A quartile outlier boxplot displaying the sample median (horizontal line within box plot), a confidence diamond of the sample mean, and the shortest half or most dense 50% of the observations (red bracket) is shown to the right of each distribution. Charts created using JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 350 a) C D U A R n a e M Atlantic 80-1 b) / g g m i ) t h g e W y r D ( t n e t n o C r a i l o F n a e M Figure S5.3. a) Distribution of Colorado potato beetle resistance under field conditions, represented by the mean relative area under the defoliation progression curve (RAUDC), in the 15 MSHH786B F1 individuals. Values for the leptine-producing parent Solanum chacoense USDA8380-1 (80-1) and S. tuberosum tetraploid cultivar ‘Atlantic’ (Atlantic) in the same field trial are shown. b) Mean foliar glycoalkaloid content of individual compounds in mg/g dry weight of foliar tissue collected from plants (resistant parent Solanum chacoense 80-1, F5 inbred lines, and two F1 hybrids [MSHH786B_09; MSHH786B_01] resulting from a cross between the beetle resistant F4 line 472_04_06 and a self-compatible diploid S2 selection DD880-03S2-263-01-04 with agronomic traits) grown under field conditions in the Montcalm Research Center Colorado potato beetle nursery during the summer of 2020. Data represent the mean of two biological replicates, each with two technical replicates. A quartile outlier boxplot displaying the sample median (horizontal line within box plot), a confidence diamond of the sample mean, and the shortest half or most dense 50% of the observations (red bracket) is shown to the right of the distribution. Charts created using JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 351 Figure S5.4. Mean tuber glycoalkaloid content expressed as total mg% fresh weight of Colorado potato beetle resistant F5 recombinant inbred lines, resistant parent Solanum chacoense 80-1, and an F1 hybrid (MSHH786_01) resulting from a cross between a beetle resistant F4 line and a self- compatible diploid breeding line with desirable agronomic traits. Each bar is the mean of three replicates ± SEM. The industry standard for safe tuber glycoalkaloid levels (20 mg% fresh weight) is given by a dashed black line. Charts created using JMP® (Version Pro 13. SAS Institute Inc., Cary,NC). 352 F4 F5 -log10(p-value) SNP Position (bp) 0 bp b M 9 2 2 . Chromosome 2 48,614,681bp Figure S5.5. Loci significantly associated with the presence of leptines I/II in F5 (blue) and F4 (red) recombinant inbred individuals are plotted by their physical position (bp) on PGSC v4.03 chromosome 2. The position on the y-axis represents the significance of the association for each single nucleotide polymorphism (SNP) expressed as the -log10 of the Fisher’s exact test p-value. The red line denotes a p-value of 0.001. SNP marker loci also significantly associated with Colorado potato beetle resistance under field conditions in the F4 generation of the recombinant inbred line population are shown in blue text. Charts created using PhenoGram Synthesis Viewer (Wolfe et al., 2013). 353 1.1 1.2 1.1 1.2 2 2 3 3 2 4 4 3 1.1 0.6 1.5 4.5 11.1 11.7 18.2 18.8 34.9 37.2 38.3 38.7 45.6 1.1 PotVar0119966 solcap_snp_c1_2425 PotVar0071966 PotVar0045000 solcap_snp_c2_21233 solcap_snp_c1_6114 PotVar0045593 solcap_snp_c2_43973 solcap_snp_c1_13814 PotVar0122493 solcap_snp_c1_6787 solcap_snp_c2_45301 0.6 1.5 4.5 11.1 11.7 18.2 18.8 34.9 37.2 38.3 38.7 45.6 PotVar0119966 solcap_snp_c1_2425 PotVar0071966 PotVar0045000 solcap_snp_c2_21233 solcap_snp_c1_6114 PotVar0045593 solcap_snp_c2_43973 solcap_snp_c1_13814 PotVar0122493 solcap_snp_c1_6787 solcap_snp_c2_45301 6 7.1 0.0 0.6 1.5 4.5 11.1 11.7 18.2 18.8 21.8 22.4 PotVar0043831 PotVar0119966 solcap_snp_c1_2425 PotVar0071966 PotVar0045000 solcap_snp_c2_21233 solcap_snp_c1_6114 PotVar0045593 PotVar0041329 solcap_snp_c2_14350 36.5 34.9 37.2 38.3 38.7 45.6 49.9 50.5 51.8 70.0 70.6 72.4 74.0 74.6 0.0 77.4 78.1 1.2 1.2 PotVar0028786 solcap_snp_c2_43973 solcap_snp_c1_13814 PotVar0122493 solcap_snp_c1_6787 solcap_snp_c2_7062 solcap_snp_c2_45301 solcap_snp_c2_7068 solcap_snp_c2_5076 PotVar0126949 PotVar0126587 solcap_snp_c2_53077 PotVar0110932 solcap_snp_c2_14741 PotVar0099779 PotVar0100004 PotVar0043831 21.8 22.4 36.5 49.9 50.5 51.8 70.0 70.6 72.4 74.0 74.6 77.4 78.1 PotVar0041329 solcap_snp_c2_14350 PotVar0028786 solcap_snp_c2_7062 solcap_snp_c2_7068 solcap_snp_c2_5076 PotVar0126949 PotVar0126587 solcap_snp_c2_53077 PotVar0110932 solcap_snp_c2_14741 PotVar0099779 PotVar0100004 7.1 7.1 7.2 2 0.0 21.8 22.4 36.5 49.9 50.5 51.8 70.0 70.6 72.4 74.0 74.6 77.4 78.1 0.0 8.3 15.1 22.7 23.6 24.2 29.9 31.4 33.3 33.6 37.8 38.5 46.0 62.7 63.0 66.4 66.7 68.3 68.6 75.4 77.3 78.3 0.0 79.4 8.3 79.5 15.1 79.8 22.7 85.4 23.6 86.0 24.2 86.7 29.9 88.1 31.4 33.3 33.6 37.8 111.7 38.5 112.2 46.0 62.7 63.0 66.4 66.7 68.3 68.6 75.4 77.3 78.3 79.4 79.5 79.8 85.4 86.0 86.7 88.1 PotVar0028786 PotVar0043831 PotVar0041329 solcap_snp_c2_14350 solcap_snp_c2_7062 solcap_snp_c2_7068 solcap_snp_c2_5076 solcap_snp_c2_4521 PotVar0038974 PotVar0117640 solcap_snp_c1_13459 solcap_snp_c1_12329 PotVar0123847 PotVar0062500 PotVar0062424 solcap_snp_c1_13920 solcap_snp_c2_46915 PotVar0094234 solcap_snp_c1_13240 PotVar0038051 solcap_snp_c1_16727 PotVar0046549 solcap_snp_c2_42169 solcap_snp_c1_16171 solcap_snp_c2_55632 solcap_snp_c2_42172 solcap_snp_c2_53034 solcap_snp_c2_42166 solcap_snp_c2_40635 solcap_snp_c2_40638 PotVar0010429 PotVar0010382 PotVar0009651 solcap_snp_c2_25143 PotVar0009673 PotVar0009997 PotVar0126949 PotVar0126587 solcap_snp_c2_53077 solcap_snp_c2_4521 PotVar0110932 PotVar0038974 solcap_snp_c2_14741 PotVar0117640 PotVar0099779 solcap_snp_c1_13459 PotVar0100004 solcap_snp_c1_12329 PotVar0123847 PotVar0062500 PotVar0062424 solcap_snp_c1_13920 solcap_snp_c2_46915 PotVar0094234 solcap_snp_c2_15068 solcap_snp_c1_13240 solcap_snp_c1_7873 PotVar0038051 solcap_snp_c1_16727 PotVar0046549 solcap_snp_c2_42169 solcap_snp_c1_16171 solcap_snp_c2_55632 solcap_snp_c2_42172 solcap_snp_c2_53034 solcap_snp_c2_42166 solcap_snp_c2_40635 solcap_snp_c2_40638 PotVar0010429 PotVar0010382 PotVar0009651 solcap_snp_c2_25143 PotVar0009673 PotVar0009997 8 8 9.2 9.1 7.2 111.7 112.2 solcap_snp_c2_15068 solcap_snp_c1_7873 0.0 0.3 1.5 4.8 0.0 6.0 18.2 19.4 20.6 23.7 28.9 31.8 32.1 36.5 solcap_snp_c2_36232 PotVar0084678 solcap_snp_c2_51389 solcap_snp_c1_15783 solcap_snp_c1_6898 PotVar0109399 solcap_snp_c1_2051 solcap_snp_c1_12745 solcap_snp_c1_12749 solcap_snp_c2_53779 solcap_snp_c2_54077 solcap_snp_c2_56256 solcap_snp_c2_7770 solcap_snp_c1_14440 4 solcap_snp_c2_39463 solcap_snp_c2_43735 solcap_snp_c2_26757 PotVar0075681 solcap_snp_c1_6898 PotVar0109399 solcap_snp_c1_10167 0.0 0.3 1.5 4.8 18.2 19.4 20.6 0.0 8.3 15.1 22.7 23.6 24.2 29.9 31.4 33.3 33.6 37.8 38.5 46.0 62.7 63.0 66.4 66.7 68.3 68.6 75.4 77.3 78.3 79.4 0.0 79.5 0.3 79.8 1.5 85.4 4.8 86.0 86.7 18.2 88.1 19.4 20.6 111.7 112.2 3 solcap_snp_c2_36232 PotVar0084678 solcap_snp_c2_51389 solcap_snp_c1_15783 solcap_snp_c2_4521 PotVar0038974 PotVar0117640 solcap_snp_c1_13459 solcap_snp_c1_12329 PotVar0123847 PotVar0062500 solcap_snp_c1_2051 PotVar0062424 solcap_snp_c1_12745 solcap_snp_c1_13920 solcap_snp_c1_12749 solcap_snp_c2_46915 PotVar0094234 solcap_snp_c1_13240 PotVar0038051 solcap_snp_c1_16727 PotVar0046549 solcap_snp_c2_42169 solcap_snp_c1_16171 solcap_snp_c2_55632 solcap_snp_c2_42172 solcap_snp_c2_53034 solcap_snp_c2_42166 solcap_snp_c2_40635 solcap_snp_c2_40638 solcap_snp_c2_36232 PotVar0010429 PotVar0084678 PotVar0010382 solcap_snp_c2_51389 PotVar0009651 solcap_snp_c1_15783 solcap_snp_c2_25143 PotVar0009673 solcap_snp_c1_2051 PotVar0009997 solcap_snp_c1_12745 solcap_snp_c1_12749 solcap_snp_c2_15068 solcap_snp_c1_7873 5 5 solcap_snp_c2_57149 PotVar0114684 solcap_snp_c2_11696 PotVar0024787 PotVar0025592 solcap_snp_c1_6898 solcap_snp_c1_3786 PotVar0026113 PotVar0109399 PotVar0079374 PotVar0079702 PotVar0079935 PotVar0080669 solcap_snp_c2_53779 PotVar0116931 solcap_snp_c2_54077 PotVar0117259 solcap_snp_c2_56256 PotVar0089663 solcap_snp_c2_7770 PotVar0083800 solcap_snp_c1_14440 PotVar0084164 PotVar0085522 PotVar0091177 PotVar0091041 PotVar0014376 solcap_snp_c1_15690 solcap_snp_c2_47393 PotVar0106493 solcap_snp_c1_12008 solcap_snp_c2_40774 solcap_snp_c1_12414 solcap_snp_c2_10358 PotVar0082112 PotVar0123206 solcap_snp_c2_55240 PotVar0128236 solcap_snp_c2_8521 solcap_snp_c2_8513 PotVar0034819 solcap_snp_c2_3451 solcap_snp_c2_39463 solcap_snp_c2_43735 PotVar0016517 PotVar0017188 solcap_snp_c2_26757 solcap_snp_c1_10167 PotVar0075681 4 0.0 0.6 3.8 9.4 11.7 14.5 16.4 16.8 17.2 17.6 17.9 19.4 20.6 23.1 30.7 31.6 33.8 35.4 38.8 44.1 44.4 45.0 45.9 49.4 52.8 59.5 67.2 68.4 70.0 70.3 71.2 75.7 76.0 84.1 85.2 0.0 6.0 23.7 28.9 31.8 32.1 36.5 57.3 59.8 67.5 82.1 89.3 57.3 59.8 67.5 0.0 82.1 6.0 89.3 23.7 105.2 28.9 105.5 31.8 32.1 36.5 57.3 59.8 67.5 82.1 89.3 9.2 105.2 105.5 PotVar0016517 PotVar0017188 solcap_snp_c2_53779 solcap_snp_c2_54077 solcap_snp_c2_56256 solcap_snp_c2_7770 solcap_snp_c1_14440 105.2 105.5 solcap_snp_c2_39463 solcap_snp_c2_43735 solcap_snp_c2_26757 PotVar0075681 6 6 0.0 3.5 3.8 4.1 4.4 4.7 5.0 5.3 6.0 6.1 6.2 6.5 6.8 7.4 8.0 8.5 8.8 9.1 9.4 10.0 solcap_snp_c2_27565 solcap_snp_c2_24266 solcap_snp_c2_33932 PotVar0069473 solcap_snp_c2_33933 PotVar0004038 solcap_snp_c2_11303 solcap_snp_c2_50186 solcap_snp_c1_3689 solcap_snp_c2_32918 solcap_snp_c1_8594 solcap_snp_c2_24322 solcap_snp_c2_27865 PotVar0104740 solcap_snp_c2_51761 solcap_snp_c2_56059 PotVar0134018 solcap_snp_c2_40242 solcap_snp_c2_33297 PotVar0127173 solcap_snp_c2_57412 solcap_snp_c1_11276 7.1 7.2 8 0.0 0.9 1.8 11.4 13.3 29.5 32.7 PotVar0022751 PotVar0022524 PotVar0022336 solcap_snp_c2_26167 PotVar0102276 solcap_snp_c2_47004 PotVar0069646 0.0 4.2 11.1 12.6 17.2 21.4 22.0 22.9 23.2 23.8 25.4 PotVar0044278 solcap_snp_c1_4029 solcap_snp_c2_12603 PotVar0043855 solcap_snp_c2_30428 PotVar0037150 PotVar0037035 solcap_snp_c2_28846 PotVar0037011 PotVar0036990 solcap_snp_c2_28849 0.0 0.6 0.9 16.3 17.2 26.7 38.6 42.5 43.8 46.0 46.9 50.3 56.8 59.0 76.3 76.9 77.8 solcap_snp_c1_14166 solcap_snp_c2_17305 solcap_snp_c2_57003 PotVar0077179 PotVar0125352 solcap_snp_c1_13094 PotVar0100216 PotVar0081239 solcap_snp_c2_19079 solcap_snp_c2_19211 solcap_snp_c2_34604 PotVar0119089 solcap_snp_c1_8300 PotVar0023704 solcap_snp_c2_28433 PotVar0023284 solcap_snp_c2_28480 12 12 10 11.1 solcap_snp_c2_47004 PotVar0069646 solcap_snp_c2_27565 solcap_snp_c2_24266 solcap_snp_c2_33932 PotVar0069473 PotVar0022751 solcap_snp_c2_33933 PotVar0022524 PotVar0004038 PotVar0022336 solcap_snp_c2_11303 solcap_snp_c2_50186 solcap_snp_c2_26167 solcap_snp_c1_3689 PotVar0102276 solcap_snp_c2_32918 solcap_snp_c1_8594 solcap_snp_c2_24322 solcap_snp_c2_27865 PotVar0104740 solcap_snp_c2_51761 solcap_snp_c2_56059 PotVar0134018 solcap_snp_c2_40242 solcap_snp_c2_33297 PotVar0127173 solcap_snp_c2_57412 solcap_snp_c1_11276 PotVar0022751 PotVar0022524 PotVar0022336 solcap_snp_c2_26167 PotVar0102276 solcap_snp_c2_47004 PotVar0069646 0.0 0.9 1.8 11.4 13.3 29.5 32.7 7.1 0.0 3.5 3.8 4.1 4.4 4.7 5.0 5.3 6.0 6.1 6.2 6.5 6.8 7.4 8.0 8.5 8.8 9.1 9.4 10.0 0.0 0.9 1.8 11.4 13.3 solcap_snp_c2_27565 solcap_snp_c2_57149 solcap_snp_c2_24266 PotVar0114684 solcap_snp_c2_33932 solcap_snp_c2_11696 PotVar0069473 PotVar0024787 solcap_snp_c2_33933 PotVar0025592 PotVar0004038 solcap_snp_c1_3786 solcap_snp_c2_11303 PotVar0026113 solcap_snp_c2_50186 PotVar0079374 solcap_snp_c1_3689 PotVar0079702 solcap_snp_c2_32918 PotVar0079935 solcap_snp_c1_8594 PotVar0080669 solcap_snp_c2_24322 PotVar0116931 solcap_snp_c2_27865 PotVar0117259 PotVar0104740 PotVar0089663 solcap_snp_c2_51761 PotVar0083800 solcap_snp_c2_56059 PotVar0084164 PotVar0134018 PotVar0085522 solcap_snp_c2_40242 PotVar0091177 solcap_snp_c2_33297 PotVar0091041 PotVar0127173 PotVar0014376 solcap_snp_c2_27565 solcap_snp_c2_57412 solcap_snp_c1_15690 solcap_snp_c2_24266 solcap_snp_c1_11276 solcap_snp_c2_47393 solcap_snp_c2_33932 PotVar0106493 PotVar0069473 solcap_snp_c1_12008 solcap_snp_c2_33933 solcap_snp_c2_40774 PotVar0004038 solcap_snp_c1_12414 solcap_snp_c2_11303 solcap_snp_c2_10358 solcap_snp_c2_50186 PotVar0082112 solcap_snp_c1_3689 PotVar0123206 solcap_snp_c2_32918 solcap_snp_c2_55240 solcap_snp_c1_8594 PotVar0128236 solcap_snp_c2_24322 solcap_snp_c2_8521 solcap_snp_c2_27865 solcap_snp_c2_8513 PotVar0104740 PotVar0034819 solcap_snp_c2_51761 solcap_snp_c2_3451 solcap_snp_c2_56059 PotVar0134018 solcap_snp_c2_40242 solcap_snp_c2_33297 PotVar0127173 solcap_snp_c2_57412 solcap_snp_c1_11276 29.5 32.7 0.0 4.2 11.1 12.6 17.2 21.4 22.0 22.9 23.2 23.8 25.4 0.0 0.9 1.8 11.4 13.3 29.5 32.7 0.0 2.8 0.0 PotVar0114494 0.6 PotVar0130582 0.9 PotVar0022751 PotVar0022524 PotVar0022336 solcap_snp_c2_26167 PotVar0102276 PotVar0044278 solcap_snp_c1_4029 solcap_snp_c2_12603 PotVar0043855 solcap_snp_c2_30428 PotVar0037150 PotVar0037035 solcap_snp_c2_28846 PotVar0037011 PotVar0036990 solcap_snp_c2_28849 solcap_snp_c2_47004 PotVar0069646 0.0 4.2 11.1 12.6 17.2 21.4 22.0 22.9 23.2 23.8 25.4 16.3 17.2 PotVar0011742 PotVar0012073 26.7 20.4 23.5 7.2 7.2 0.0 4.2 11.1 12.6 17.2 21.4 22.0 22.9 23.2 23.8 25.4 PotVar0044278 solcap_snp_c1_4029 solcap_snp_c2_12603 PotVar0043855 solcap_snp_c2_30428 9.1 PotVar0037150 PotVar0037035 solcap_snp_c2_28846 PotVar0037011 PotVar0036990 solcap_snp_c2_28849 0.0 2.8 20.4 23.5 PotVar0114494 PotVar0130582 PotVar0011742 PotVar0012073 0.0 0.6 0.9 PotVar0007613 PotVar0007606 solcap_snp_c1_14166 solcap_snp_c2_12760 solcap_snp_c2_17305 solcap_snp_c2_44814 solcap_snp_c2_57003 solcap_snp_c1_6192 solcap_snp_c1_6196 0.0 PotVar0044278 0.6 solcap_snp_c1_4029 0.9 solcap_snp_c2_12603 1.8 PotVar0043855 2.7 solcap_snp_c2_30428 3.4 PotVar0037150 PotVar0037035 solcap_snp_c2_28846 PotVar0037011 PotVar0036990 solcap_snp_c2_28849 PotVar0077179 PotVar0125352 solcap_snp_c1_13094 16.3 17.2 26.7 9.1 PotVar0100216 PotVar0081239 solcap_snp_c2_19079 solcap_snp_c2_19211 solcap_snp_c2_34604 PotVar0119089 solcap_snp_c1_8300 PotVar0023704 0.0 2.8 38.6 42.5 43.8 46.0 46.9 50.3 56.8 59.0 PotVar0114494 PotVar0130582 solcap_snp_c1_14166 solcap_snp_c2_17305 solcap_snp_c2_57003 solcap_snp_c2_28433 PotVar0023284 solcap_snp_c2_28480 76.3 76.9 77.8 PotVar0011742 PotVar0012073 20.4 9.2 PotVar0077179 23.5 PotVar0125352 0.0 0.6 0.9 1.8 2.7 3.4 solcap_snp_c1_13094 PotVar0007613 PotVar0007606 solcap_snp_c2_12760 solcap_snp_c2_44814 solcap_snp_c1_6192 solcap_snp_c1_6196 PotVar0100216 PotVar0081239 solcap_snp_c2_19079 solcap_snp_c2_19211 solcap_snp_c2_34604 PotVar0119089 solcap_snp_c1_8300 PotVar0023704 38.6 42.5 43.8 8 46.0 46.9 50.3 56.8 59.0 76.3 76.9 77.8 0.0 0.6 0.9 16.3 17.2 26.7 38.6 42.5 43.8 46.0 46.9 50.3 56.8 59.0 76.3 76.9 77.8 solcap_snp_c2_28433 PotVar0023284 solcap_snp_c2_28480 9.1 9.1 9.3 8 solcap_snp_c1_10167 10 PotVar0016517 10 PotVar0017188 11.1 11.2 11.1 9.3 0.0 2.8 0.0 0.3 0.6 2.4 0.0 solcap_snp_c2_55483 0.6 solcap_snp_c1_14166 PotVar0114494 PotVar0108622 0.9 solcap_snp_c2_17305 PotVar0108619 PotVar0130582 1.8 solcap_snp_c2_57003 solcap_snp_c2_20879 2.7 3.4 PotVar0077179 PotVar0125352 PotVar0011742 PotVar0012073 solcap_snp_c1_13094 20.4 23.5 0.0 PotVar0007613 PotVar0007606 solcap_snp_c2_12760 solcap_snp_c2_44814 solcap_snp_c1_6192 solcap_snp_c1_6196 0.0 0.3 solcap_snp_c2_1113 0.6 2.4 PotVar0108198 13.7 9.2 PotVar0100216 9.2 PotVar0081239 solcap_snp_c2_19079 solcap_snp_c2_19211 solcap_snp_c2_34604 PotVar0119089 PotVar0007613 solcap_snp_c1_8300 PotVar0007606 PotVar0023704 solcap_snp_c2_12760 solcap_snp_c2_44814 solcap_snp_c1_6192 solcap_snp_c1_6196 solcap_snp_c2_28433 PotVar0023284 solcap_snp_c2_28480 9.3 0.0 0.6 0.9 1.8 2.7 3.4 9.3 33.8 9.3 45.2 55.7 56.3 56.9 57.5 62.8 63.1 73.7 75.3 0.0 0.3 0.6 2.4 10 solcap_snp_c2_55819 solcap_snp_c2_15483 PotVar0005097 PotVar0005344 solcap_snp_c2_48091 solcap_snp_c2_48145 solcap_snp_c2_29749 solcap_snp_c1_9066 solcap_snp_c1_7187 PotVar0058165 solcap_snp_c2_55483 PotVar0108622 PotVar0108619 solcap_snp_c2_20879 0.0 0.3 0.6 2.4 solcap_snp_c2_55483 PotVar0108622 PotVar0108619 solcap_snp_c2_20879 0.0 solcap_snp_c2_1113 13.7 PotVar0108198 33.8 45.2 55.7 56.3 56.9 57.5 62.8 63.1 73.7 75.3 solcap_snp_c2_55819 solcap_snp_c2_15483 PotVar0005097 PotVar0005344 solcap_snp_c2_48091 solcap_snp_c2_48145 solcap_snp_c2_29749 solcap_snp_c1_9066 solcap_snp_c1_7187 PotVar0058165 PotVar0064694 PotVar0064415 solcap_snp_c2_13473 solcap_snp_c1_4347 solcap_snp_c2_37189 PotVar0066236 solcap_snp_c2_33657 PotVar0066299 PotVar0066338 solcap_snp_c2_33653 solcap_snp_c1_10062 solcap_snp_c1_2187 solcap_snp_c1_2153 solcap_snp_c1_2150 PotVar0066824 solcap_snp_c2_5960 PotVar0067342 PotVar0067424 solcap_snp_c2_6249 solcap_snp_c2_55972 PotVar0067504 PotVar0067827 PotVar0067501 PotVar0110427 solcap_snp_c1_2210 solcap_snp_c2_6302 solcap_snp_c2_47382 solcap_snp_c2_47386 PotVar0106087 PotVar0105621 solcap_snp_c2_20947 solcap_snp_c2_21015 solcap_snp_c2_21050 solcap_snp_c2_21066 solcap_snp_c2_23923 0.0 13.7 33.8 45.2 55.7 56.3 56.9 57.5 62.8 63.1 73.7 75.3 11.1 PotVar0108198 solcap_snp_c2_1113 solcap_snp_c2_55819 0.0 0.3 0.7 3.1 0.0 5.9 4.5 7.4 5.4 12.5 9.0 15.8 9.6 19.0 10.2 20.3 11.4 20.6 12.0 23.5 13.2 34.9 13.5 35.2 14.1 10 37.7 14.4 40.2 14.7 40.8 15.6 42.0 16.5 16.8 18.9 20.1 20.4 22.9 23.2 23.5 25.3 25.6 25.8 25.9 0.0 26.2 0.3 27.4 0.7 27.7 3.1 28.3 28.9 5.9 7.4 9.0 9.6 10.2 11.4 12.0 13.2 0.0 13.5 4.5 14.1 5.4 14.4 12.5 14.7 15.8 15.6 19.0 16.5 20.3 16.8 20.6 18.9 23.5 34.9 20.1 35.2 20.4 37.7 22.9 40.2 23.2 40.8 23.5 42.0 25.3 25.6 25.8 25.9 26.2 27.4 27.7 28.3 28.9 PotVar0064694 PotVar0064415 solcap_snp_c2_13473 solcap_snp_c1_4347 PotVar0021975 solcap_snp_c2_55483 solcap_snp_c2_37189 solcap_snp_c2_13636 PotVar0108622 0.0 PotVar0066236 PotVar0134713 PotVar0108619 solcap_snp_c2_33657 PotVar0047209 solcap_snp_c2_20879 PotVar0066299 PotVar0047371 PotVar0066338 solcap_snp_c2_3737 13.7 solcap_snp_c2_33653 PotVar0112779 solcap_snp_c1_10062 PotVar0112395 solcap_snp_c1_2187 solcap_snp_c2_15364 solcap_snp_c1_2153 PotVar0008725 solcap_snp_c1_2150 PotVar0008637 33.8 PotVar0066824 solcap_snp_c2_43865 solcap_snp_c2_5960 solcap_snp_c2_51284 PotVar0067342 PotVar0124390 PotVar0067424 45.2 solcap_snp_c2_34198 solcap_snp_c2_6249 55.7 solcap_snp_c2_55972 56.3 PotVar0067504 56.9 PotVar0067827 57.5 PotVar0067501 62.8 PotVar0110427 63.1 solcap_snp_c1_2210 73.7 solcap_snp_c2_6302 75.3 solcap_snp_c2_47382 solcap_snp_c2_47386 PotVar0106087 PotVar0105621 PotVar0064694 solcap_snp_c2_20947 PotVar0064415 solcap_snp_c2_21015 solcap_snp_c2_13473 solcap_snp_c2_21050 solcap_snp_c1_4347 solcap_snp_c2_21066 solcap_snp_c2_15483 solcap_snp_c2_23923 solcap_snp_c2_37189 PotVar0066236 PotVar0005097 solcap_snp_c2_33657 PotVar0005344 PotVar0066299 solcap_snp_c2_48091 PotVar0066338 solcap_snp_c2_48145 solcap_snp_c2_33653 solcap_snp_c2_29749 solcap_snp_c1_9066 solcap_snp_c1_10062 solcap_snp_c1_2187 solcap_snp_c1_7187 solcap_snp_c1_2153 PotVar0058165 PotVar0021975 solcap_snp_c1_2150 solcap_snp_c2_13636 PotVar0066824 PotVar0134713 solcap_snp_c2_5960 PotVar0047209 PotVar0067342 PotVar0047371 PotVar0067424 solcap_snp_c2_3737 solcap_snp_c2_6249 PotVar0112779 solcap_snp_c2_55972 PotVar0112395 PotVar0067504 solcap_snp_c2_15364 PotVar0008725 PotVar0067827 PotVar0008637 PotVar0067501 solcap_snp_c2_43865 PotVar0110427 solcap_snp_c2_51284 solcap_snp_c1_2210 PotVar0124390 solcap_snp_c2_6302 solcap_snp_c2_34198 solcap_snp_c2_47382 solcap_snp_c2_47386 PotVar0106087 PotVar0105621 solcap_snp_c2_20947 solcap_snp_c2_21015 solcap_snp_c2_21050 solcap_snp_c2_21066 solcap_snp_c2_23923 11.2 11.2 0.0 0.3 0.7 3.1 5.9 7.4 9.0 9.6 10.2 11.4 12.0 13.2 13.5 14.1 14.4 14.7 15.6 16.5 16.8 18.9 20.1 20.4 22.9 23.2 23.5 25.3 25.6 25.8 25.9 26.2 27.4 27.7 28.3 28.9 0.0 0.3 2.8 3.7 4.3 4.6 5.2 5.7 11.1 5.8 6.7 7.0 7.3 7.6 7.9 8.2 8.7 9.0 9.9 11.5 12.4 13.6 25.6 27.1 28.6 32.5 32.8 34.3 34.9 35.5 35.8 0.0 36.4 0.3 2.8 37.3 3.7 4.3 12 PotVar0108198 solcap_snp_c2_1113 solcap_snp_c2_55819 PotVar0027707 0.0 solcap_snp_c2_53244 0.3 solcap_snp_c2_27379 0.7 PotVar0027811 3.1 solcap_snp_c2_44924 5.9 solcap_snp_c2_45743 7.4 solcap_snp_c2_51098 9.0 solcap_snp_c2_5952 9.6 solcap_snp_c2_14413 10.2 solcap_snp_c2_16908 11.4 solcap_snp_c2_16294 12.0 solcap_snp_c2_16919 13.2 solcap_snp_c2_21336 13.5 solcap_snp_c2_48392 14.1 solcap_snp_c2_13933 14.4 solcap_snp_c2_5953 14.7 PotVar0036410 solcap_snp_c2_15483 PotVar0064694 15.6 solcap_snp_c2_17623 PotVar0064415 PotVar0005097 16.5 solcap_snp_c1_14870 solcap_snp_c2_13473 PotVar0005344 16.8 solcap_snp_c2_18791 solcap_snp_c1_4347 solcap_snp_c2_48091 18.9 solcap_snp_c2_18825 solcap_snp_c2_37189 solcap_snp_c2_48145 20.1 solcap_snp_c2_18836 PotVar0066236 solcap_snp_c2_29749 20.4 PotVar0109256 solcap_snp_c2_33657 solcap_snp_c1_9066 22.9 solcap_snp_c1_7495 PotVar0066299 solcap_snp_c1_7187 23.2 PotVar0066338 PotVar0110851 PotVar0058165 23.5 solcap_snp_c2_33653 solcap_snp_c2_57627 25.3 solcap_snp_c1_10062 solcap_snp_c2_23254 25.6 solcap_snp_c1_2187 solcap_snp_c2_50821 25.8 solcap_snp_c1_2153 solcap_snp_c2_48482 25.9 solcap_snp_c1_2150 PotVar0053460 26.2 PotVar0066824 solcap_snp_c2_46213 27.4 solcap_snp_c2_5960 PotVar0053168 PotVar0067342 27.7 PotVar0052695 PotVar0067424 28.3 PotVar0052507 solcap_snp_c2_6249 28.9 solcap_snp_c1_1923 solcap_snp_c2_55972 PotVar0052374 PotVar0067504 PotVar0052284 PotVar0067827 solcap_snp_c2_5704 PotVar0067501 solcap_snp_c2_5684 PotVar0110427 solcap_snp_c2_5507 solcap_snp_c1_2210 solcap_snp_c2_6302 solcap_snp_c2_47382 solcap_snp_c2_47386 PotVar0106087 PotVar0105621 solcap_snp_c2_20947 solcap_snp_c2_21015 solcap_snp_c2_21050 solcap_snp_c2_21066 solcap_snp_c2_23923 PotVar0027707 solcap_snp_c2_53244 solcap_snp_c2_27379 PotVar0027811 solcap_snp_c2_44924 solcap_snp_c2_45743 solcap_snp_c2_51098 solcap_snp_c2_5952 solcap_snp_c2_14413 solcap_snp_c2_16908 solcap_snp_c2_16294 solcap_snp_c2_16919 solcap_snp_c2_21336 solcap_snp_c2_48392 solcap_snp_c2_13933 solcap_snp_c2_5953 PotVar0036410 solcap_snp_c2_17623 solcap_snp_c1_14870 solcap_snp_c2_18791 solcap_snp_c2_18825 solcap_snp_c2_18836 PotVar0109256 solcap_snp_c1_7495 PotVar0110851 solcap_snp_c2_57627 solcap_snp_c2_23254 solcap_snp_c2_50821 solcap_snp_c2_48482 PotVar0053460 solcap_snp_c2_46213 PotVar0053168 PotVar0052695 PotVar0052507 solcap_snp_c1_1923 PotVar0052374 PotVar0052284 solcap_snp_c2_5704 solcap_snp_c2_5684 solcap_snp_c2_5507 4.6 5.2 5.7 5.8 6.7 7.0 7.3 7.6 7.9 8.2 8.7 9.0 9.9 11.5 12.4 13.6 25.6 27.1 28.6 32.5 32.8 34.3 34.9 35.5 35.8 36.4 37.3 Figure S5.6. Genetic map of the F4 recombinant inbred line population based on 97 individuals and 288 SNP markers. The unlinked groups of chromosomes 1, 7, 9 and 11 are shown separately. Figure created in MapCharts (Voorrips, 2002). 354 a) b) Figure S5.7. Locus genotype frequency (a) and individual genotype frequency (b) of the two homozygous genotypes (AA:grey, BB:black) and the heterozygous genotype (AB:red) at 1,020 SNPs segregating in 236 F2 individuals, 113 F4 individuals, and 80 F5 individuals. 01 03 Chromosome 07 08 12 M6 (Distorted Segregation) Recombinant (Distorted Segregation) 80-1 (Distorted Segregation) M6 Recombinant 80-1 F 5 F 4 G e n e r a t i o n F 2 y c n e u q e r F e p y t o n e G 0.8 0.6 0.4 0.2 0.0 0.8 0.6 0.4 0.2 0.0 0.8 0.6 0.4 0.2 0.0 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 PGSC v4.03 Physical Position (Mb) Figure S5.8. Genotype frequency of the M6 parental (grey), recombinant (black) and 80-1 parental (blue) genotype of 519 SNPs plotted against their physical position on the PGSC v4.03 pseudomolecules. Loci exhibiting distorted segregation at the p<0.0001 significance threshold are shown with an asterisk (*) for each genotype. 355 Table S5.1. Selfed Fruit per flower and seed per fruit data for the 48 F4 recombinant inbred lines evaluated under greenhouse conditions in 2019 and 2020. Line 2019 2019 Fruit/Flower Seed/Fruit Fruit/Flower 2020 2020 Seed/Fruit EE501F4_002_04_03 EE501F4_019_04_04 EE501F4_028_04_05 EE501F4_036_04_04 EE501F4_062_02_01 EE501F4_076_03_01 EE501F4_082_01_02 EE501F4_093_02_02 EE501F4_095_04_06 EE501F4_113_03_01 EE501F4_161_02_05 EE501F4_173_03_08 EE501F4_182_04_01 EE501F4_183_02_05 EE501F4_196_01_01 EE501F4_230_03_05 EE501F4_233_01_01 EE501F4_234_01_01 EE501F4_268_02_01 EE501F4_296_02_04 EE501F4_319_02_01 EE501F4_321_04_02 EE501F4_334_03_08 EE501F4_348_01_05 EE501F4_365_02_03 EE501F4_378_02_05 EE501F4_382_01_07 EE501F4_397_01_01 EE501F4_411_02_02 EE501F4_470_02_09 EE501F4_471_01_04 EE501F4_478_04_08 EE501F4_492_04_04 EE501F4_492_04_07 EE501F4_495_01_03 EE501F4_533_05_03 EE501F4_537_01_01 0.38 0.60 0.50 0.00 0.21 0.13 0.00 1.00 0.20 0.21 0.00 0.86 0.25 0.00 0.17 0.21 0.15 0.50 0.83 0.00 0.20 0.29 1.00 0.27 0.06 0.08 0.90 0.44 0.64 0.50 0.31 0.24 0.33 1.00 0.00 0.46 0.60 4.00 20.00 12.50 8.33 0.00 15.71 25.00 27.78 12.50 6.25 0.00 17.50 8.75 6.67 13.33 10.00 25.00 16.67 6.67 10.00 1.00 11.11 18.13 5.71 6.25 19.50 15.00 20.00 5.83 11.54 33.33 0.17 0.30 0.00 0.00 0.09 0.09 0.60 0.55 0.31 0.62 0.07 0.46 0.55 0.33 0.17 0.65 0.00 0.54 0.25 0.18 0.57 0.00 0.47 0.17 0.06 0.84 0.00 0.19 0.07 0.23 0.17 0.16 0.67 0.67 0.75 0.37 0.00 5.00 9.23 3.00 18.67 18.18 11.25 45.31 18.75 43.75 15.00 50.00 36.36 7.57 20.00 20.00 31.25 30.00 3.13 20.00 2.50 25.00 50.00 45.00 32.50 16.21 12.50 30.00 356 Table S5.1 (cont’d) EE501F4_540_04_02 EE501F4_540_04_04 EE501F4_570_04_03 EE501F4_611_04_03 EE501F4_615_03_01 EE501F4_636_02_04 EE501F4_641_01_05 EE501F4_653_02_01 EE501F4_653_02_03 EE501F4_672_04_03 EE501F4_706_02_05 0.25 0.00 0.85 0.73 0.00 0.43 1.00 0.60 0.04 0.17 0.25 20.00 30.91 3.64 16.67 5.00 16.67 25.00 8.33 10.00 0.42 0.33 1.00 0.38 0.64 0.00 0.00 0.09 0.50 0.08 0.00 26.00 21.25 37.50 2.00 35.42 20.00 21.00 20.00 357 Table S5.2. Self-fertility trait data for the F4 and F5 recombinant inbred line individuals measured during the 2020 greenhouse season. Fruit Gener ation Classificati Ye ar line Tota l Flo wers Flowe ring (F) / Non- flowe ring (NF) on # To tal Fr uit Aver age Frui t Wei ght Fru it per flo wer See ded Fru it Wei ght # Fr uit wi th see d # To tal Se ed # Se eds pe r Fr uit Mea n Frac tion of Viab le Polle n Mea n Poll en Tub e Gro wth Rati ng chacoense USDA8380- S. 1 EE501F4_0 28_04_05 EE501F4_0 36_04_04 EE501F4_0 62_02_01 EE501F4_0 63_04_04 EE501F4_0 76_03_01 EE501F4_0 82_01_02 EE501F4_0 89_02_04 EE501F4_0 93_02_02 EE501F4_0 95_04_06 EE501F4_1 06_02_01 EE501F4_1 13_03_01 EE501F4_1 56_05_02 EE501F4_1 61_02_05 EE501F4_1 73_03_08 EE501F4_1 82_04_01 EE501F4_1 82_04_04 EE501F4_1 83_02_05 EE501F4_1 96_01_01 EE501F4_2 30_03_05 EE501F4_2 33_01_01 EE501F4_2 34_01_01 EE501F4_2 68_02_01 Parent F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 50 3 4 11 12 22 25 3 40 26 0 26 0 14 26 29 29 3 6 34 14 13 20 Fruitless Fruitless Fruitless Parthenocar pic Fruit Fruitless Seeded Fruit Seeded Fruit Fruitless Seeded Fruit Seeded Fruit Seeded Fruit Parthenocar pic Fruit Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit Fruitless Seeded Fruit Seeded Fruit 0 0 0 1 0 2 0.54 0.30 15 1.99 0 22 2.45 8 2.07 16 2.42 1 12 16 10 1 1 0.73 1.33 2.42 1.49 0.45 3.84 22 1.89 0 7 5 0.86 0.86 0.0 0 0.0 0 0.0 0 0.0 9 0.0 0 0.0 9 0.6 0 0.0 0 0.5 5 0.3 1 0.6 2 0.0 7 0.4 6 0.5 5 0.3 4 0.3 3 0.1 7 0.6 5 0.0 0 0.5 4 0.2 5 F F F F F F F F F F NF F NF F F F F F F F F F F 2.00 1.00 1.00 3.00 0.61 3.00 0.85 3.00 1.00 2.67 0.89 2 3.00 0.94 15 3.00 0.90 3.00 0.82 22 3.00 0.53 8 3.00 0.93 16 3.00 1.00 3.00 1.00 3.00 0.72 3.00 0.66 0.98 3.00 0.96 12 16 10 1 1 3.00 0.95 22 3.00 0.92 3.00 0.69 3.00 0.77 7 5 0.60 29.9 0 53.8 9 16.5 7 38.6 4 15.9 0 38.7 1 14.9 2 0.45 3.84 41.6 2 6.02 4.28 6 28 0 40 0 90 72 5 22 5 70 0 40 0 15 50 80 0 53 10 0 3.0 0 18. 67 18. 18 11. 25 45. 31 18. 75 43. 75 40. 00 15. 00 50. 00 36. 36 7.5 7 20. 00 358 Table S5.2 (cont’d) EE501F4_296_02_ EE501F4_297_01_ EE501F4_319_02_ EE501F4_321_04_ EE501F4_334_03_ EE501F4_348_01_ EE501F4_365_02_ EE501F4_378_02_ EE501F4_382_01_ EE501F4_397_01_ EE501F4_411_02_ EE501F4_454_01_ EE501F4_470_02_ EE501F4_471_01_ EE501F4_478_04_ EE501F4_478_04_ EE501F4_492_04_ EE501F4_492_04_ EE501F4_495_01_ EE501F4_499_01_ EE501F4_533_05_ EE501F4_537_01_ EE501F4_540_04_ EE501F4_540_04_ EE501F4_543_03_ EE501F4_565_02_ EE501F4_570_04_ EE501F4_611_04_ EE501F4_615_03_ F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 04 01 01 02 08 05 03 05 07 01 02 02 09 04 05 08 04 07 03 04 03 01 02 04 02 04 03 03 01 3 4 1 3 7 6 1 7 6 1 7 1 9 2 0 1 6 2 9 0 1 3 6 0 2 5 1 5 2 1 2 0 6 4 1 8 1 2 1 2 0 0 8 2 9 4 5 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 F F F F F F F F F F F N F F F N F F F F F F F F F F N F N F F F F Seeded Fruit Fruitless Parthenocarpic Fruit Fruitless Seeded Fruit Parthenocarpic Fruit Seeded Fruit Seeded Fruit Fruitless Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit Parthenocarpic Fruit Seeded Fruit Fruitless Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit Seeded Fruit 1.6 8 0.2 8 1.9 3 0.2 0 2.8 7 0.8 7 1.3 8 0.8 8 1.6 0 1.3 7 2.2 5 2.8 7 2.5 7 0.6 0 0.1 0 2.6 5 1.4 2 0.7 8 1.1 5 0.4 1 1.6 8 6 0 4 0 8 1 1 1 6 0 3 2 3 1 4 1 0 1 4 1 5 2 1 5 0 5 4 1 1 1 1 2 9 0.1 8 0.0 0 0.5 7 0.0 0 0.4 7 0.1 7 0.0 6 0.8 4 0.0 0 0.1 9 0.0 7 0.2 3 0.1 7 0.1 6 0.6 7 0.6 7 0.7 5 0.3 3 0.3 7 0.0 0 0.4 2 0.3 3 1.3 8 0.3 8 0.6 4 3.0 0 3.0 0 3.0 0 3.0 0 2.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 0.8 4 0.9 7 0.5 7 0.9 8 0.7 8 1.0 0 0.9 1 0.5 7 0.7 2 0.8 5 0.7 4 0.9 9 0.8 8 0.0 5 0.9 2 0.9 6 0.8 7 0.6 5 0.9 7 0.8 8 0.9 4 0.6 4 0.9 5 0.8 1 10.0 8 15.4 4 2.87 13.8 7 12 0 25 0 30 50 4.15 60 1.76 4.81 1.37 9.01 28.7 4 36.0 1 5 75 50 18 0 32 5 22 7 4.63 75 39.7 3 7.10 45 0 13 0 3.13 85 9.76 3.32 41.6 8 15 0 10 85 0 20.0 0 31.2 5 30.0 0 3.13 20.0 0 2.50 25.0 0 50.0 0 45.0 0 32.5 0 16.2 1 12.5 0 30.0 0 26.0 0 21.2 5 37.5 0 2.00 35.4 2 6 8 1 1 6 3 2 3 1 4 1 0 1 4 6 1 5 5 4 4 5 2 4 359 Table S5.2 (cont’d) F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 EE501F4_636_02_04 EE501F4_641_01_05 EE501F4_642_03_01 EE501F4_653_02_01 EE501F4_653_02_03 EE501F4_672_04_03 EE501F4_701_01_03 EE501F4_706_02_05 EE501F5_002_04_03 EE501F5_019_04_04 EE501F5_062_02_01 EE501F5_064_03_08 EE501F5_081_02_06 EE501F5_089_02_08 EE501F5_093_02_05 EE501F5_095_04_01 EE501F5_095_04_01 EE501F5_113_03_01 EE501F5_154_02_05 EE501F5_156_01_03 EE501F5_156_01_03 EE501F5_160_03_03 EE501F5_161_02_06 EE501F5_173_03_08 EE501F5_173_03_08 EE501F5_196_01_05 EE501F5_199_02_01 EE501F5_220_03_01 EE501F5_221_05_08 _01 _01 _05 _01 _01 _03 _01 _01 _03 _02 _02 _01 _03 _03 _01 _01 _05 _01 _01 _02 _01 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 5 3 0 1 1 2 0 4 9 0 5 1 0 0 0 6 4 5 3 0 2 3 1 2 2 9 0 2 0 1 6 1 4 2 6 8 1 7 1 3 1 0 3 F F N F F F F N F F F N F N F F F F F F F F N F F N F F F F F F F F F Fruitless Fruitless Seeded Fruit Seeded Fruit Seeded Fruit Fruitless Parthenocarpic Fruit Fruitless Fruitless Parthenocarpic Fruit Seeded Fruit Fruitless Fruitless Fruitless Fruitless Seeded Fruit Fruitless Seeded Fruit Seeded Fruit Seeded Fruit Parthenocarpic Fruit Fruitless Fruitless 360 0 0 1 1 0 4 0 2 0 0 1 1 0 0 0 0 0 7 0 4 3 5 1 0 0 0.7 2 0.6 9 0.4 6 0.2 2 0.4 4 2.4 9 1.4 8 1.2 0 0.5 7 2.0 7 1.2 3 0.0 0 0.0 0 0.0 9 0.5 0 0.0 8 0.0 0 0.2 0 0.0 0 0.0 0 0.2 0 0.3 3 0.0 0 0.0 0 0.0 0 0.0 0 0.4 4 0.0 0 0.1 5 0.3 8 0.2 9 0.0 8 0.0 0 0.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 2.0 0 2.6 7 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 0.9 8 0.9 5 0.8 0 0.7 9 0.7 7 0.8 8 0.7 8 0.6 1 0.0 0 0.8 7 0.6 2 0.5 9 0.7 0 0.4 3 0.9 3 0.9 5 0.7 7 0.8 8 0.9 0 0.9 6 0.9 2 0.8 3 0.9 3 1 1 0 1 9 2 4 3 4 0.72 6.92 0.46 20 21 0 20 20.0 0 21.0 0 20.0 0 24.4 3 19 5 21.6 7 2.61 25 4.80 1.71 9.89 75 15 50 12.5 0 18.7 5 5.00 12.5 0 Table S5.2 (cont’d) EE501F5_230_03_05_ EE501F5_233_01_01_ EE501F5_234_01_01_ EE501F5_234_01_01_ EE501F5_237_01_09_ EE501F5_278_02_01_ EE501F5_278_02_01_ EE501F5_297_01_06_ EE501F5_297_01_06_ EE501F5_311_01_08_ EE501F5_321_04_02_ EE501F5_321_04_02_ EE501F5_337_01_01_ EE501F5_361_03_04_ F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 03 01 01 02 01 01 03 01 02 01 01 03 01 01 01 01 04 01 06 01 02 01 03 10 02 01 01 02 01 EE501F5_365_02_03_ EE501F5_370_02_02_ EE501F5_370_02_02_ EE501F5_378_02_04_ EE501F5_380_01_04_ EE501F5_382_01_06_ EE501F5_382_01_06_ EE501F5_397_01_04_ EE501F5_397_01_04_ EE501F5_402_04_06_ EE501F5_431_02_04_ EE501F5_455_01_02_ EE501F5_470_02_06_ EE501F5_470_02_06_ EE501F5_471_01_04_ 2 3 6 9 6 0 3 0 0 0 0 0 0 0 6 7 0 0 2 0 1 0 7 7 3 2 8 3 2 0 6 0 1 8 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 F F F F N F F N F N F N F N F N F N F N F F F N F N F F F N F F F F F F N F F N F F Seeded Fruit Fruitless Fruitless Fruitless Fruitless Seeded Fruit Seeded Fruit Fruitless Fruitless Fruitless Fruitless Seeded Fruit Fruitless Seeded Fruit Fruitless Fruitless 1 5 0 0 0 0 1 1 0 0 0 0 4 0 5 0 0 1.4 9 0.7 8 1.7 0 0.5 3 1.8 7 0.6 5 0.0 0 0.0 0 0.0 0 0.0 0 0.1 7 0.1 4 0.0 0 0.0 0 0.0 0 0.0 0 0.1 3 0.0 0 0.1 6 0.0 0 0.0 0 3.0 0 3.0 0 3.0 0 3.0 0 2.0 0 3.0 0 3.0 0 3.0 0 2.5 0 3.0 0 1.0 0 0.8 8 0.5 3 0.6 4 0.8 9 0.8 8 0.5 5 0.9 7 0.8 4 0.8 2 0.8 8 0.7 4 0.9 6 1 5 22.3 6 43 0 28.6 7 1 1 2 5 0.78 1.70 10 10 1.06 25 9.34 10 0 10.0 0 10.0 0 12.5 0 20.0 0 361 _01 _04 _01 _01 _04 _01 _02 _01 _01 _03 _01 _01 _05 _01 _01 _03 _01 _01 _05 _01 _01 _03 _01 _01 _04 _01 _02 EE501F5_492_04_04 EE501F5_495_01_02 EE501F5_495_01_05 EE501F5_499_01_07 EE501F5_499_01_07 EE501F5_533_05_03 EE501F5_540_04_02 EE501F5_540_04_02 EE501F5_543_03_01 EE501F5_570_04_03 EE501F5_570_04_03 EE501F5_611_04_03 EE501F5_636_02_03 EE501F5_636_02_03 EE501F5_641_01_07 EE501F5_641_01_09 EE501F5_641_01_09 EE501F5_642_03_01 EE501F5_672_04_03 EE501F5_672_04_03 EE501F5_673_01_04 EE501F5_701_01_03 EE501F5_701_01_03 EE501F5_706_02_05 EE501F5_706_02_05 Table S5.2 (cont’d) EE501F5_478_03_08 EE501F5_478_03_08 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 0 4 0 2 4 0 0 4 0 1 2 0 1 4 8 3 0 0 1 0 0 0 0 1 0 1.0 3 0.5 8 1.7 7 1.6 0 2.1 1 1.6 5 0.2 9 0.3 2 0.2 2 0.0 0 0.2 2 0.0 0 0.7 7 0.0 0 0.0 0 0.2 5 0.0 0 0.5 7 0.0 0 0.4 7 0.2 9 0.1 7 0.0 0 0.0 0 0.0 5 0.0 0 0.0 0 0.0 0 0.0 0 0.1 3 0.0 0 3.0 0 3.0 0 3.0 0 2.6 7 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 3.0 0 0.9 4 0.6 9 0.9 3 0.9 3 0.1 4 0.7 8 0.6 6 0.8 1 0.7 5 0.8 8 0.9 8 0.8 9 0.3 6 0.7 7 0.7 8 0.6 9 0.8 0 0.9 3 0.8 3 0.9 7 0.8 7 0.8 7 4 1 8 4 1 2 1 4 8 4.11 10.6 2 5 10 0 7.09 75 19.1 7 29.4 7 13.1 6 11 5 42 0 20 0 1.25 5.56 18.7 5 9.58 30.0 0 25.0 0 1 0.32 5 5.00 3 2 0 1 8 7 3 1 0 0 8 5 1 6 0 1 2 2 1 1 1 3 0 2 8 1 8 1 3 7 2 1 7 0 2 4 1 3 8 8 1 3 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 F N F F F F N F N F F F F N F F F F F F F F F F F N F F F F F F Fruitless Seeded Fruit Fruitless Seeded Fruit Fruitless Fruitless Seeded Fruit Fruitless Seeded Fruit Fruitless Seeded Fruit Seeded Fruit Parthenocarpic Fruit Fruitless Fruitless Seeded Fruit Fruitless Fruitless Fruitless Fruitless Parthenocarpic Fruit Fruitless 362 Table S5.3. Summary of the number of individuals (N) used for single nucleotide polymorphism (SNP) marker-trait association in the F4 and F5 generations of a Solanum chacoense recombinant inbred line population in 2020. Trait alpha-Solanine foliar content alpha-Chconine foliar content Leptine I foliar content Leptine II foliar content Ratio of acetylated to non-acetylated compounds Colorado potato beetle field defoliation (RAUDC) Pollen viability Fraction of flowers setting fruit Average fruit weight Seeds per fruit Pollen tube growth in the style alpha-Solanine foliar content alpha-Chconine foliar content Leptine I foliar content Leptine II foliar content Ratio of acetylated to non-acetylated compounds Colorado potato beetle field defoliation (RAUDC) Pollen viability Fraction of flowers setting fruit Average fruit weight Seeds per fruit Pollen tube growth in the style Generation N 69 69 69 69 69 61 48 51 21 17 34 62 62 62 62 62 44 51 52 41 36 47 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 363 Table S5.4. Primer information for the two KASPTM markers used to genotype the Sli candidate region in this study. Self-Incompatible Allele Primer Self-Compatible Allele Primer Common Primer GAAGGTGACCAAGTTCATGCTCAAATATGTTGTTT GAAGGTCGGAGTCAACGGATTCAAATATGTTGTT ATGCATTAGCAATTTCTAG 5904089 GAAGGTGACCAAGTTCATGCTGTAAAGYTTTTACC GAAGGTCGGAGTCAACGGATTAAAGYTTTTACC GGATAAATCCGYGGGGAG DM v 4.03 Chromo some 12 Position (bp) 5903962 6 8 ID Sli_ 626 Sli_ 898 ATTTGGTGTTCAAATTGG AGATGATTATGAAGATATT TATTTGGTGTTCAAATTGT AGATGATTATGAAGATATC TCCATCATGAT ACATAT 364 Mean Leptin e I (mg/g Dry weight ) Mean Leptin e II (mg/g Dry weight ) Total Leptin es (mg/g Dry weight) Mean α- Solani ne (mg/g Dry weight) Ratio of Acetylate d to non- acetylate compoun d ds Mean α- Chaconi ne (mg/g Dry weight) Table S5.5. Foliar glycoalkaloid data for greenhouse grown individuals from the F4 and F5 generations of the recombinant inbred line population. Generati on Parent Check F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 Growth Conditio n GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH Yea r 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 202 0 Line S. chacoense USDA8380-1 S. tuberosum 'Atlantic' (4x) EE501F4_002_04_03 EE501F4_019_04_04 EE501F4_028_04_05 EE501F4_036_04_04 EE501F4_062_02_01 EE501F4_063_04_04 EE501F4_076_03_01 EE501F4_082_01_02 EE501F4_089_02_04 EE501F4_093_02_02 EE501F4_095_04_06 EE501F4_106_02_01 EE501F4_113_03_01 EE501F4_156_05_02 EE501F4_161_02_05 EE501F4_173_03_08 EE501F4_182_04_01 EE501F4_182_04_04 EE501F4_183_02_05 EE501F4_196_01_01 EE501F4_230_03_05 EE501F4_233_01_01 EE501F4_234_01_01 4.27 1.74 0.66 0.00 12.03 11.94 7.89 16.07 0.00 0.18 2.43 12.57 0.00 33.07 0.00 27.58 0.00 15.59 0.00 32.38 6.57 21.20 4.47 22.96 0.00 18.25 3.87 27.72 0.00 12.90 13.72 13.93 0.00 23.17 0.00 28.71 2.75 19.49 2.50 20.79 3.18 16.78 0.32 0.39 6.39 9.18 0.00 28.62 4.91 22.73 0.00 31.58 9.92 0.06 9.05 23.91 23.54 14.95 23.47 15.48 17.29 19.78 17.37 17.17 6.34 16.39 18.53 14.46 17.83 13.54 4.19 9.15 22.89 13.31 22.98 1.78 0.00 0.30 0.00 0.11 0.00 0.00 0.00 0.00 0.18 0.11 0.00 0.09 0.00 0.68 0.00 0.00 0.08 0.06 0.10 0.03 0.02 0.00 0.14 0.00 1.30 0.00 2.18 0.00 0.64 0.00 0.00 0.00 0.00 1.55 1.06 0.00 1.15 0.00 4.66 0.00 0.00 0.69 0.53 0.67 0.02 0.00 0.00 1.39 0.00 2.97 0.00 5.71 0.00 1.79 0.00 0.00 0.00 0.00 5.02 3.41 0.00 2.73 0.00 9.07 0.00 0.00 2.07 1.98 2.51 0.30 0.39 0.00 3.52 0.00 365 Table S5.5 (cont’d) EE501F4_268_02_01 EE501F4_271_03_03 EE501F4_296_02_04 EE501F4_297_01_01 EE501F4_311_01_06 EE501F4_319_02_01 EE501F4_321_04_02 EE501F4_334_03_08 EE501F4_348_01_05 EE501F4_363_05_04 EE501F4_365_02_03 EE501F4_378_02_05 EE501F4_382_01_07 EE501F4_397_01_01 EE501F4_411_02_02 EE501F4_454_01_02 EE501F4_470_02_09 EE501F4_471_01_04 EE501F4_478_04_05 EE501F4_478_04_08 EE501F4_492_04_04 EE501F4_492_04_07 EE501F4_495_01_03 EE501F4_499_01_04 EE501F4_533_05_03 EE501F4_537_01_01 EE501F4_540_04_02 EE501F4_540_04_04 EE501F4_543_03_02 EE501F4_570_04_03 EE501F4_611_04_03 EE501F4_615_03_01 EE501F4_636_02_04 EE501F4_641_01_02 EE501F4_653_02_01 EE501F4_653_02_03 EE501F4_672_04_03 EE501F4_701_01_03 EE501F4_706_02_05 EE501F5_002_04_03_01 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F5 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 0.00 0.00 5.85 0.00 0.00 2.20 0.00 0.00 1.31 0.00 1.11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.63 6.55 2.99 0.00 0.00 3.70 0.00 0.56 0.00 0.00 3.83 0.00 0.92 0.13 5.93 2.16 0.00 0.00 0.00 2.05 0.93 7.03 0.00 0.00 2.31 0.00 0.00 0.71 0.00 0.00 0.37 0.00 0.15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.03 2.83 1.19 0.00 0.00 1.34 0.00 0.04 0.00 0.00 1.62 0.00 0.26 0.00 3.62 0.64 0.00 0.00 0.00 0.48 0.09 3.06 0.00 0.00 8.16 0.00 0.00 2.91 0.00 0.00 1.68 0.00 1.26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.66 9.38 4.17 0.00 0.00 5.04 0.00 0.60 0.00 0.00 5.45 0.00 1.18 0.13 9.55 2.80 0.00 0.00 0.00 2.52 1.02 10.08 0.00 12.66 11.61 33.44 23.25 15.34 15.32 20.67 21.97 9.61 14.33 11.82 31.35 23.08 20.99 16.22 12.79 1.10 15.54 9.52 25.60 21.36 22.24 17.40 42.78 2.29 24.28 26.33 19.43 11.21 28.01 16.63 23.38 6.23 27.93 33.98 9.56 11.28 15.28 16.59 0.00 8.83 6.15 22.61 16.58 10.12 13.83 17.46 16.12 7.19 15.99 8.56 27.28 16.83 16.39 9.67 10.25 0.00 14.98 6.02 15.43 17.15 20.71 13.38 30.05 5.03 16.87 18.47 12.15 7.26 16.83 20.24 8.73 5.28 20.21 23.39 13.46 8.35 15.05 9.26 -- 0.00 0.46 0.00 0.00 0.11 0.00 0.00 0.04 0.00 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.60 0.10 0.00 0.00 0.16 0.00 0.08 0.00 0.00 0.17 0.00 0.03 0.00 0.30 0.24 0.00 0.00 0.00 0.13 0.03 0.39 366 Table S5.5 (cont’d) EE501F5_019_04_04_01 EE501F5_064_03_08_01 EE501F5_081_02_06_01 EE501F5_089_02_08_03 EE501F5_093_02_05_01 EE501F5_095_04_01_01 EE501F5_095_04_01_03 EE501F5_113_03_01_02 EE501F5_154_02_05_02 EE501F5_156_01_03_01 EE501F5_156_01_03_03 EE501F5_160_03_03_03 EE501F5_161_02_06_01 EE501F5_173_03_08_01 EE501F5_173_03_08_05 EE501F5_196_01_05_01 EE501F5_199_02_01_01 EE501F5_220_03_01_02 EE501F5_221_05_08_01 EE501F5_230_03_05_03 EE501F5_233_01_01_01 EE501F5_234_01_01_01 EE501F5_234_01_01_02 EE501F5_268_02_01_01 EE501F5_278_02_01_01 EE501F5_278_02_01_03 EE501F5_297_01_06_01 EE501F5_297_01_06_02 EE501F5_311_01_08_01 EE501F5_321_04_02_01 EE501F5_321_04_02_03 EE501F5_361_03_04_01 EE501F5_365_02_03_01 EE501F5_370_02_02_01 EE501F5_370_02_02_04 EE501F5_378_02_04_01 EE501F5_380_01_04_06 EE501F5_382_01_06_01 EE501F5_382_01_06_02 EE501F5_397_01_04_01 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 0.00 0.00 0.00 0.78 0.00 4.69 0.30 2.99 0.00 0.00 0.00 0.05 5.29 1.77 1.88 3.51 3.61 0.00 0.00 0.00 0.89 0.00 0.00 0.00 0.69 1.60 0.00 0.00 0.00 0.00 0.00 0.00 0.70 0.00 0.00 0.00 0.93 0.00 0.00 0.00 0.00 0.00 0.00 0.16 0.00 1.81 0.00 1.08 0.00 0.00 0.00 0.00 2.31 0.42 0.71 1.18 1.14 0.00 0.00 0.00 0.34 0.00 0.00 0.00 0.04 0.22 0.00 0.00 0.00 0.00 0.00 0.00 0.04 0.00 0.00 0.00 0.17 0.00 0.00 0.00 0.00 0.00 0.00 0.94 0.00 6.50 0.30 4.07 0.00 0.00 0.00 0.05 7.60 2.19 2.59 4.69 4.75 0.00 0.00 0.00 1.23 0.00 0.00 0.00 0.73 1.82 0.00 0.00 0.00 0.00 0.00 0.00 0.74 0.00 0.00 0.00 1.10 0.00 0.00 0.00 0.07 6.88 10.87 16.35 0.81 11.80 9.06 14.23 18.20 18.18 9.84 8.18 13.95 22.73 15.30 20.93 14.58 10.17 13.49 30.34 26.09 29.94 26.38 0.00 14.30 19.48 17.86 19.42 14.97 8.59 24.09 11.03 15.27 11.93 18.54 21.95 9.53 9.62 29.97 13.96 0.00 6.54 9.96 12.10 0.27 7.43 9.62 10.65 15.15 12.52 7.59 12.15 6.33 20.01 11.07 14.11 9.14 10.57 10.14 25.04 17.31 21.88 19.53 0.00 14.32 19.18 15.13 16.61 11.19 7.89 19.91 8.95 18.03 16.34 20.32 17.48 7.62 9.06 22.33 9.74 0.00 0.00 0.00 0.03 0.00 0.34 0.02 0.16 0.00 0.00 0.00 0.00 0.37 0.05 0.10 0.13 0.20 0.00 0.00 0.00 0.03 0.00 0.00 -- 0.03 0.05 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.06 0.00 0.00 0.00 367 Table S5.5 (cont’d) EE501F5_397_01_04_03 EE501F5_402_04_06_10 EE501F5_431_02_04_02 EE501F5_455_01_02_01 EE501F5_470_02_06_01 EE501F5_470_02_06_02 EE501F5_471_01_04_01 EE501F5_478_03_08_01 EE501F5_478_03_08_04 EE501F5_492_04_04_01 EE501F5_495_01_02_01 EE501F5_495_01_05_04 EE501F5_533_05_03_01 EE501F5_537_01_01_01 EE501F5_540_04_02_01 EE501F5_540_04_02_03 EE501F5_543_03_01_01 EE501F5_570_04_03_01 EE501F5_570_04_03_05 EE501F5_611_04_03_01 EE501F5_636_02_03_01 EE501F5_636_02_03_03 EE501F5_641_01_07_01 EE501F5_641_01_09_01 EE501F5_641_01_09_05 EE501F5_642_03_01_01 EE501F5_672_04_03_01 EE501F5_672_04_03_03 EE501F5_673_01_04_01 EE501F5_701_01_03_01 EE501F5_701_01_03_04 EE501F5_706_02_05_01 EE501F5_706_02_05_02 EE501F2_002 EE501F2_003 EE501F2_007 EE501F2_010 EE501F2_011 EE501F2_015 EE501F2_019 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F2 F2 F2 F2 F2 F2 F2 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2017 2017 2017 2017 2017 2017 2017 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 0.00 2.96 1.17 0.00 0.00 0.00 0.00 1.22 2.84 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.34 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.12 1.28 1.57 0.39 0.30 23.61 0.02 5.00 2.18 11.22 22.27 6.44 0.00 0.94 0.27 0.00 0.00 0.00 0.00 0.25 0.94 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.33 0.17 0.31 0.00 0.00 25.29 0.03 6.70 2.17 12.55 28.78 5.03 0.00 3.90 1.44 0.00 0.00 0.00 0.00 1.47 3.78 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.34 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.44 1.45 1.88 0.39 0.30 48.90 0.06 11.71 4.35 23.77 51.06 11.48 27.87 13.15 17.74 22.73 3.74 13.63 0.43 12.36 14.46 32.54 17.27 14.43 29.31 19.22 11.53 13.24 27.12 13.97 13.58 15.73 31.75 31.66 19.92 17.43 0.00 18.55 1.68 0.25 20.26 12.07 21.70 10.97 9.18 20.24 64.07 14.13 31.53 11.05 23.92 22.62 17.58 9.08 11.77 12.36 3.16 10.54 0.00 10.07 11.15 22.14 15.96 14.57 24.71 23.82 11.38 8.06 19.79 9.45 8.60 9.68 21.59 22.92 16.24 15.47 0.00 16.17 1.89 0.26 12.53 9.76 15.37 11.86 13.14 12.68 50.13 7.76 27.38 4.90 12.50 17.48 0.00 0.18 0.05 0.00 0.00 0.00 0.00 0.07 0.15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 -- 0.00 0.00 0.00 0.04 0.07 0.05 0.02 0.01 1.49 0.00 0.53 0.07 1.49 1.40 0.29 368 Table S5.5 (cont’d) EE501F2_021 EE501F2_026 EE501F2_028 EE501F2_036 EE501F2_037 EE501F2_038 EE501F2_041 EE501F2_044 EE501F2_045 EE501F2_047 EE501F2_050 EE501F2_051 EE501F2_054 EE501F2_056 EE501F2_059 EE501F2_061 EE501F2_062 EE501F2_063 EE501F2_064 EE501F2_066 EE501F2_069 EE501F2_071 EE501F2_075 EE501F2_076 EE501F2_080 EE501F2_081 EE501F2_082 EE501F2_089 EE501F2_092 EE501F2_093 EE501F2_099 EE501F2_105 EE501F2_106 EE501F2_113 EE501F2_116 EE501F2_121 EE501F2_123 EE501F2_124 EE501F2_126 EE501F2_128 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 11.25 0.01 18.63 5.37 0.02 0.80 11.16 9.07 12.39 10.40 22.75 15.45 11.45 0.03 9.04 5.30 0.03 0.02 9.00 4.94 0.04 21.37 15.96 2.22 7.00 0.02 4.11 6.46 6.70 0.02 0.03 11.87 0.04 5.37 11.46 7.99 11.21 7.19 0.02 27.35 17.64 0.02 18.92 7.00 0.01 1.27 10.43 10.28 10.99 12.38 16.29 19.84 11.26 0.03 11.02 8.27 0.04 0.03 8.43 6.20 0.04 25.80 24.05 3.29 9.19 0.02 4.39 7.37 7.75 0.03 0.03 11.18 0.03 5.79 12.06 8.89 11.63 10.67 0.03 25.31 28.89 0.03 37.55 12.38 0.03 2.06 21.59 19.35 23.38 22.79 39.04 35.29 22.71 0.06 20.06 13.57 0.07 0.06 17.43 11.14 0.08 47.17 40.02 5.51 16.19 0.04 8.50 13.84 14.44 0.06 0.06 23.05 0.07 11.16 23.53 16.89 22.84 17.86 0.04 52.66 17.45 33.21 22.47 22.84 34.45 22.03 27.70 28.07 30.99 44.71 41.19 31.16 12.71 36.60 9.87 15.05 54.40 38.68 20.19 42.24 53.84 60.61 41.33 20.59 12.86 46.17 19.14 13.79 17.58 47.30 55.79 28.62 52.22 11.22 15.36 31.08 23.78 17.69 36.23 38.61 8.47 32.61 14.39 18.48 39.40 17.47 17.02 17.92 23.34 27.02 37.75 17.33 7.75 36.89 5.29 8.16 48.05 40.89 13.98 26.05 48.61 34.53 24.32 14.48 7.86 41.36 14.94 11.05 11.89 52.17 52.03 18.45 55.81 5.59 7.90 17.08 14.78 10.79 33.31 23.29 1.11 0.00 1.02 0.30 0.00 0.05 0.48 0.42 0.43 0.32 0.49 0.73 1.11 0.00 1.32 0.58 0.00 0.00 0.51 0.16 0.00 0.50 0.61 0.16 0.78 0.00 0.25 0.56 0.49 0.00 0.00 0.49 0.00 0.66 1.01 0.35 0.59 0.63 0.00 0.85 369 Table S5.5 (cont’d) EE501F2_131 EE501F2_137 EE501F2_139 EE501F2_140 EE501F2_142 EE501F2_143 EE501F2_145 EE501F2_154 EE501F2_155 EE501F2_160 EE501F2_173 EE501F2_179 EE501F2_180 EE501F2_182 EE501F2_183 EE501F2_195 EE501F2_196 EE501F2_199 EE501F2_202 EE501F2_214 EE501F2_215 EE501F2_217 EE501F2_220 EE501F2_221 EE501F2_226 EE501F2_230 EE501F2_231 EE501F2_233 EE501F2_242 EE501F2_259 EE501F2_260 EE501F2_262 EE501F2_264 EE501F2_268 EE501F2_270 EE501F2_271 EE501F2_275 EE501F2_278 EE501F2_291 EE501F2_294 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 8.43 0.03 7.55 12.92 4.33 9.63 0.03 14.33 28.98 10.34 5.74 20.37 5.85 9.18 7.39 25.66 11.26 0.02 0.02 15.07 0.03 0.02 10.87 0.01 5.33 10.71 0.01 0.02 5.67 3.95 5.23 3.12 5.79 17.10 3.38 9.43 15.98 9.38 0.02 3.65 8.31 0.03 8.64 15.42 4.65 9.00 0.03 14.44 23.76 6.79 4.21 19.13 8.15 8.56 8.99 25.83 10.57 0.03 0.04 17.67 0.04 0.03 10.30 0.01 6.20 11.33 0.01 0.03 6.44 5.18 6.25 4.44 3.97 17.21 3.86 9.12 19.56 7.37 0.03 3.80 16.74 0.06 16.18 28.34 8.97 18.63 0.06 28.77 52.74 17.13 9.95 39.50 14.00 17.74 16.38 51.49 21.83 0.04 0.06 32.74 0.07 0.06 21.17 0.02 11.53 22.04 0.02 0.04 12.10 9.13 11.48 7.57 9.76 34.31 7.24 18.54 35.54 16.75 0.04 7.44 21.80 28.22 12.88 47.14 9.06 18.44 42.63 28.18 32.85 16.45 26.90 28.70 30.82 17.88 20.82 20.93 35.51 34.14 59.00 18.28 42.67 48.15 16.99 33.37 33.03 23.08 18.67 45.01 18.62 20.35 6.17 28.29 9.95 22.01 27.97 32.66 22.17 31.98 37.47 16.46 17.71 28.28 7.80 25.93 5.98 12.12 42.32 16.61 20.90 15.37 24.39 17.08 18.70 12.44 13.88 12.41 23.52 32.70 45.95 8.99 39.79 31.32 11.43 26.89 20.02 15.57 20.30 31.44 12.67 13.95 4.05 18.98 8.18 13.20 21.07 22.10 10.87 26.84 34.13 12.16 0.42 0.00 0.78 0.39 0.60 0.61 0.00 0.64 0.98 0.54 0.19 0.86 0.28 0.59 0.47 1.54 0.37 0.00 0.00 1.20 0.00 0.00 0.74 0.00 0.22 0.57 0.00 0.00 0.39 0.27 1.12 0.16 0.54 0.97 0.15 0.34 1.08 0.28 0.00 0.26 370 Table S5.5 (cont’d) EE501F2_296 EE501F2_305 EE501F2_311 EE501F2_312 EE501F2_313 EE501F2_316 EE501F2_319 EE501F2_320 EE501F2_321 EE501F2_322 EE501F2_326 EE501F2_334 EE501F2_335 EE501F2_338 EE501F2_341 EE501F2_343 EE501F2_348 EE501F2_349 EE501F2_361 EE501F2_363 EE501F2_365 EE501F2_370 EE501F2_371 EE501F2_375 EE501F2_377 EE501F2_378 EE501F2_380 EE501F2_382 EE501F2_386 EE501F2_387 EE501F2_391 EE501F2_397 EE501F2_401 EE501F2_402 EE501F2_405 EE501F2_407 EE501F2_408 EE501F2_410 EE501F2_411 EE501F2_417 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 8.56 0.02 6.94 1.09 0.01 4.92 4.39 1.01 1.18 0.02 0.70 8.59 6.05 3.21 0.02 0.02 11.01 0.01 0.01 0.01 1.80 0.02 4.18 8.12 3.57 2.96 6.68 0.01 4.53 4.45 0.02 2.40 0.02 6.68 6.08 0.06 10.06 12.99 4.56 5.52 9.67 0.01 7.76 1.09 0.01 8.80 3.40 1.82 1.20 0.02 1.17 12.58 8.63 4.98 0.01 0.01 17.97 0.02 0.01 0.01 2.01 0.02 5.64 12.69 4.61 3.98 9.61 0.01 7.42 7.00 0.02 2.77 0.02 6.68 8.66 0.10 18.24 11.74 8.02 5.50 18.23 0.02 14.70 2.17 0.02 13.72 7.79 2.82 2.38 0.04 1.87 21.17 14.68 8.19 0.02 0.03 28.98 0.03 0.02 0.02 3.80 0.04 9.82 20.81 8.18 6.94 16.29 0.02 11.94 11.46 0.04 5.18 0.04 13.36 14.74 0.16 28.31 24.73 12.57 11.02 10.12 22.81 26.79 17.05 19.42 31.98 15.26 17.18 16.86 29.82 23.44 24.88 31.68 12.60 38.16 38.26 23.71 21.70 18.40 12.42 18.83 30.83 17.57 26.53 15.55 23.80 14.14 16.23 24.24 18.56 41.55 6.73 29.55 13.15 15.08 34.69 10.88 30.21 29.41 14.91 5.00 28.35 15.99 17.46 16.62 18.98 12.09 13.75 12.97 25.53 24.09 15.97 23.48 8.67 31.84 27.14 13.82 16.43 22.06 14.88 20.48 31.21 12.16 14.70 9.98 15.90 8.67 16.98 15.82 11.12 31.57 4.96 29.36 8.33 10.04 33.18 4.46 22.96 15.86 12.17 1.21 0.00 0.34 0.06 0.00 0.27 0.28 0.09 0.08 0.00 0.04 0.52 0.27 0.38 0.00 0.00 0.77 0.00 0.00 0.00 0.10 0.00 0.33 0.50 0.32 0.17 0.71 0.00 0.30 0.39 0.00 0.44 0.00 0.62 0.59 0.00 1.85 0.47 0.28 0.41 371 Table S5.5 (cont’d) EE501F2_419 EE501F2_421 EE501F2_423 EE501F2_431 EE501F2_438 EE501F2_446 EE501F2_447 EE501F2_449 EE501F2_451 EE501F2_454 EE501F2_455 EE501F2_456 EE501F2_457 EE501F2_462 EE501F2_465 EE501F2_470 EE501F2_471 EE501F2_474 EE501F2_475 EE501F2_478 EE501F2_483 EE501F2_488 EE501F2_491 EE501F2_492 EE501F2_494 EE501F2_498 EE501F2_499 EE501F2_500 EE501F2_501 EE501F2_503 EE501F2_504 EE501F2_505 EE501F2_507 EE501F2_508 EE501F2_511 EE501F2_514 EE501F2_516 EE501F2_519 EE501F2_520 EE501F2_521 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 2.94 10.33 2.52 3.79 16.20 3.44 26.37 17.22 7.42 0.01 6.99 0.02 1.89 0.02 4.91 0.92 0.03 10.83 6.97 9.73 1.99 13.84 27.28 10.09 12.17 8.25 7.63 0.02 0.02 7.65 0.02 11.06 15.55 3.43 0.02 6.77 7.09 8.39 3.80 1.52 2.41 13.17 2.50 3.53 16.92 4.61 22.95 20.92 10.18 0.04 6.50 0.03 2.61 0.02 7.66 1.25 0.03 15.63 10.29 14.31 2.16 11.12 24.69 12.07 29.13 11.75 12.01 0.03 0.05 11.21 0.03 14.69 27.94 5.25 0.04 16.32 14.73 21.60 13.20 4.65 5.34 23.50 5.02 7.31 33.13 8.05 49.32 38.14 17.60 0.05 13.49 0.05 4.50 0.05 12.57 2.16 0.06 26.46 17.27 24.04 4.15 24.97 51.97 22.16 41.30 20.00 19.64 0.06 0.07 18.86 0.06 25.75 43.50 8.68 0.05 23.08 21.82 29.99 17.01 6.17 17.42 9.44 30.71 26.95 31.11 24.01 20.58 18.07 24.05 36.07 53.94 54.76 42.74 30.94 41.48 19.61 40.51 29.43 20.88 23.93 32.80 30.09 34.51 25.73 44.80 18.20 19.72 26.75 46.29 21.34 30.48 15.06 29.66 8.48 26.93 31.08 13.49 36.75 31.74 19.05 18.08 5.93 28.32 32.39 16.84 17.05 12.52 9.32 14.88 26.29 50.78 41.63 32.64 36.49 31.58 17.91 45.34 17.85 14.63 15.96 32.50 24.14 23.35 16.18 20.54 12.33 13.84 25.32 45.76 10.71 27.01 8.93 17.32 4.76 30.07 19.03 7.47 19.27 17.05 11.13 0.15 1.53 0.09 0.12 0.69 0.20 1.49 1.39 0.45 0.00 0.13 0.00 0.06 0.00 0.17 0.06 0.00 0.56 0.49 0.60 0.06 0.46 0.90 0.53 0.63 0.66 0.59 0.00 0.00 0.59 0.00 1.07 0.93 0.66 0.00 0.46 1.04 0.54 0.35 0.20 372 Table S5.5 (cont’d) EE501F2_523 EE501F2_524 EE501F2_526 EE501F2_527 EE501F2_528 EE501F2_532 EE501F2_533 EE501F2_534 EE501F2_537 EE501F2_538 EE501F2_540 EE501F2_543 EE501F2_544 EE501F2_545 EE501F2_545 EE501F2_549 EE501F2_551 EE501F2_557 EE501F2_558 EE501F2_565 EE501F2_570 EE501F2_572 EE501F2_584 EE501F2_587 EE501F2_589 EE501F2_597 EE501F2_599 EE501F2_603 EE501F2_604 EE501F2_608 EE501F2_610 EE501F2_611 EE501F2_615 EE501F2_621 EE501F2_622 EE501F2_623 EE501F2_625 EE501F2_636 EE501F2_636 EE501F2_641 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 0.01 0.01 0.01 0.85 2.90 1.45 0.01 0.02 2.01 0.02 0.01 4.30 1.31 6.04 7.96 4.04 3.83 3.54 6.19 3.20 0.01 0.01 4.22 0.04 4.96 4.53 2.29 23.36 0.03 20.93 7.55 0.75 0.02 22.34 6.59 6.22 1.75 0.02 0.02 17.34 0.02 0.02 0.03 2.51 7.62 6.49 0.02 0.05 6.23 0.03 0.02 18.12 4.14 24.50 30.38 14.07 16.49 15.12 25.53 14.51 0.02 0.04 12.33 0.21 19.53 20.98 11.64 26.86 0.03 18.28 44.45 0.93 0.03 29.04 4.59 8.38 1.93 0.02 0.03 22.61 0.03 0.03 0.05 3.36 10.52 7.94 0.03 0.07 8.24 0.05 0.03 22.42 5.45 30.54 38.34 18.11 20.32 18.66 31.72 17.70 0.03 0.05 16.55 0.25 24.49 25.51 13.92 50.22 0.06 39.21 52.00 1.69 0.05 51.38 11.18 14.61 3.69 0.04 0.05 39.95 17.78 49.56 25.16 21.16 12.15 12.50 41.58 38.59 32.51 36.84 37.60 23.66 13.25 9.65 11.71 50.45 16.01 18.24 23.90 8.87 20.71 34.60 32.45 21.97 19.47 9.57 15.18 34.68 44.30 38.84 21.38 30.71 46.00 30.89 55.44 20.97 61.42 45.92 35.55 54.57 17.73 40.58 22.20 16.02 7.29 9.18 36.23 32.44 24.30 34.59 31.88 12.47 10.86 4.44 5.29 37.84 10.89 10.11 15.92 5.98 18.45 25.12 25.87 18.66 11.90 5.37 9.35 16.45 49.66 25.46 10.30 24.52 49.51 14.64 50.89 11.78 55.03 35.37 26.42 37.75 0.00 0.00 0.00 0.09 0.54 0.37 0.00 0.00 0.15 0.00 0.00 0.62 0.23 2.17 2.26 0.21 0.76 0.66 0.80 1.19 0.00 0.00 0.28 0.01 0.78 1.71 0.57 0.98 0.00 0.61 1.64 0.03 0.00 1.13 0.11 0.45 0.03 0.00 0.00 0.43 373 Table S5.5 (cont’d) EE501F2_642 EE501F2_646 EE501F2_647 EE501F2_652 EE501F2_653 EE501F2_653 EE501F2_654 EE501F2_656 EE501F2_670 EE501F2_670 EE501F2_672 EE501F2_672 EE501F2_673 EE501F2_701 EE501F2_703 EE501F2_706 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 S. chacoense USDA8380-1 S. tuberosum 'Atlantic' (4x) S. chacoense M6 Parent Check Parent 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH GH 0.01 16.77 5.94 17.60 18.25 29.33 4.38 10.03 0.03 0.09 0.05 3.95 9.03 16.14 1.32 18.66 15.01 0.00 0.01 0.02 19.12 5.64 19.85 17.49 28.32 6.71 15.41 0.03 0.12 0.04 19.25 12.34 17.55 1.22 25.05 13.07 0.01 0.01 0.03 35.89 11.58 37.45 35.74 57.65 11.09 25.44 0.06 0.22 0.09 23.21 21.37 33.69 2.53 43.71 28.08 0.02 0.02 15.91 36.93 38.84 40.31 24.73 45.75 57.91 43.70 27.45 86.97 120.05 17.36 31.24 33.55 10.64 45.23 6.54 15.82 22.37 18.77 20.09 32.50 26.00 18.59 33.50 35.24 24.78 22.25 70.87 115.31 8.99 17.43 21.30 8.19 28.17 3.17 13.07 27.57 0.00 0.63 0.16 0.56 0.82 0.73 0.12 0.37 0.00 0.00 0.00 0.88 0.44 0.61 0.13 0.60 2.89 0.00 0.00 374 Table S5.6. Field Colorado potato beetle defoliation data for genotypes in the F4 and F5 generations of the recombinant inbred line population evaluated at the Montcalm Research Center in 2019 and 2020. Line Generation Growth conditions S. tuberosum 'Atlantic' (4x) S. chacoense M6 S. chacoense USDA8380-1 Check Parent Parent EE501F4_019_04_04 EE501F4_028_04_05 EE501F4_036_04_04 EE501F4_082_01_02 EE501F4_093_02_02 EE501F4_106_02_01 EE501F4_113_03_01 EE501F4_161_02_05 EE501F4_202_03_04 EE501F4_203_03_04 EE501F4_214_02_01 EE501F4_215_01_01 EE501F4_230_03_05 EE501F4_233_01_01 EE501F4_234_01_01 EE501F4_279_01_01 EE501F4_296_02_04 EE501F4_297_01_01 EE501F4_311_01_06 EE501F4_319_04_02 EE501F4_321_04_02 EE501F4_334_03_08 EE501F4_378_02_01 EE501F4_378_02_05 EE501F4_397_01_01 EE501F4_397_01_02 EE501F4_454_01_02 EE501F4_454_02_02 EE501F4_471_01_04 EE501F4_474_04_02 EE501F4_492_94_04 EE501F4_533_05_03 EE501F4_537_01_01 EE501F4_538_05_02 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field 375 Year Mean RAUDC 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 28.67 11.46 0.49 40.21 2.43 86.71 11.92 8.57 58.43 0.93 11.40 50.07 31.93 0.19 0.41 17.66 0.98 8.85 9.00 1.71 62.11 21.50 13.43 18.25 57.14 27.20 9.86 23.49 25.77 15.99 31.36 31.14 9.86 16.43 27.68 24.06 11.39 Table S5.6 (cont’d) EE501F4_540_04_02 EE501F4_540_04_03 EE501F4_540_04_04 EE501F4_543_03_02 EE501F4_570_04_03 EE501F4_574_04_03 EE501F4_615_03_01 EE501F4_615_93_02 EE501F4_624_03_01 EE501F4_635_02_01 EE501F4_635_02_03 EE501F4_636_03_04 EE501F4_642_03_01 EE501F4_653_02_01 EE501F4_653_02_03 EE501F4_672_04_03 EE501F4_673_01_04 EE501F4_673_02_04 EE501F4_701_01_03 EE501F4_706_02_05 S. tuberosum 'Atlantic' (4x) S. chacoense USDA8380-1 EE501F5_002_04_03_01 EE501F5_019_04_04_01 EE501F5_062_02_01_05 EE501F5_064_03_08_01 EE501F5_081_02_06_01 EE501F5_089_02_08_03 EE501F5_093_02_05_01 EE501F5_095_04_01_01 EE501F5_095_04_01_03 EE501F5_113_03_01_02 EE501F5_154_02_05_02 EE501F5_156_01_03_01 EE501F5_156_01_03_03 EE501F5_160_03_03_03 EE501F5_161_02_06_01 EE501F5_173_03_08_01 EE501F5_173_03_08_05 EE501F5_196_01_05_01 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 Check Parent F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2019 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 12.70 20.07 22.96 0.69 21.86 24.36 2.37 1.63 13.06 2.93 6.06 17.50 24.21 5.29 33.84 29.50 2.98 2.84 5.92 0.39 30.93 0.23 7.00 29.75 33.25 14.58 18.08 2.22 35.58 2.10 1.11 4.43 22.75 21.29 27.13 21.88 0.06 0.64 1.05 7.88 MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field 376 Table S5.6 (cont’d) EE501F5_199_02_01_01 EE501F5_220_03_01_02 EE501F5_221_05_08_01 EE501F5_230_01_01_01 EE501F5_230_03_05_03 EE501F5_234_01_01_01 EE501F5_234_01_01_02 EE501F5_278_02_01_01 EE501F5_278_02_01_03 EE501F5_297_01_06_01 EE501F5_297_01_06_02 EE501F5_311_01_08_01 EE501F5_321_04_02_01 EE501F5_321_04_02_03 EE501F5_365_02_03_01 EE501F5_370_02_02_01 EE501F5_370_02_02_04 EE501F5_378_01_04_01 EE501F5_378_02_04_01 EE501F5_380_01_04_06 EE501F5_382_01_06_01 EE501F5_382_01_06_02 EE501F5_397_01_04_01 EE501F5_397_01_04_03 EE501F5_402_04_06_01 EE501F5_431_02_04_02 EE501F5_455_01_02_01 EE501F5_470_02_06_01 EE501F5_470_02_06_02 EE501F5_471_01_04_01 EE501F5_478_03_08_01 EE501F5_478_03_08_04 EE501F5_492_04_04_01 EE501F5_495_01_02_01 EE501F5_495_01_05_04 EE501F5_499_01_07_01 EE501F5_533_05_03_01 EE501F5_537_01_01_01 EE501F5_540_04_02_01 EE501F5_540_04_02_03 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 1.11 11.96 19.43 24.79 25.08 18.96 20.42 16.33 8.93 11.67 22.75 23.92 25.38 21.00 7.35 20.13 24.79 5.60 7.00 14.58 32.03 3.50 37.92 29.17 7.00 0.70 14.88 19.02 11.55 26.25 4.08 0.47 22.17 48.13 21.00 4.67 18.38 1.75 12.13 31.50 MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field 377 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 2020 26.83 28.00 26.54 5.25 9.22 6.18 10.21 23.33 10.50 19.43 26.83 11.67 0.00 4.96 23.86 37.08 33.48 5.71 55.73 14.83 27.40 38.54 46.46 29.42 46.35 26.92 33.44 45.31 53.28 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 Table S5.6 (cont’d) EE501F5_540_04_03_05 EE501F5_543_03_01_01 EE501F5_570_04_03_01 EE501F5_570_04_03_05 EE501F5_611_04_03_01 EE501F5_636_02_03_01 EE501F5_636_02_03_03 EE501F5_641_01_09_04 EE501F5_642_03_01_01 EE501F5_672_04_03_01 EE501F5_672_04_03_03 EE501F5_673_01_04_01 EE501F5_706_02_05_01 EE501F5_706_02_05_02 HH786B_01 HH786B_03 HH786B_08 HH786B_09 HH786B_10 HH786B_11 HH786B_12 HH786B_14 HH786B_15 HH786B_16 HH786B_19 HH786B_20 HH786B_21 HH786B_22 HH786B_23 MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field MRC Field 378 Table S5.7. Leaf and tuber glycoalkaloid data of field-grown F5 inbreds and MSHH786B hybrids evaluated for Colorado potato beetle field resistance in 2020. Mean Lepti ne I (mg % Mean Lepti ne II (mg % Mean Total Solani ne Glycoalkal Tiss ue Mean Mean Line α- Mean Lepti ne I (mg/g Dry Weig ht) Mean Lepti ne II (mg/g Dry Weig ht) Mean α- Chacon ine (mg/g Dry Weight Mean α- Chacon ine (mg% Fresh Weight ) oids (mg% Fresh Weight) 46.77 137.21 71.04 86.84 78.33 42.33 47.36 74.65 93.25 46.98 16.19 18.73 126.21 25.84 67.75 92.66 85.31 243.28 122.21 393.37 399.84 284.65 345.57 300.60 161.14 197.64 284.39 284.80 260.27 89.75 52.51 335.63 57.23 144.48 231.29 169.11 608.99 371.77 α- Solani ne (mg% Fresh Weig ht) 112.5 6 170.9 7 102.1 6 122.4 6 120.2 1 60.95 72.37 114.0 4 114.1 8 84.03 45.50 22.60 209.4 1 31.39 76.73 138.6 3 83.80 365.7 1 249.5 7 Fresh Weig ht) 165.7 2 Fresh Weig ht) 68.32 70.43 21.23 83.89 104.1 6 75.35 42.81 57.14 27.55 32.11 26.71 15.06 20.77 73.06 22.64 62.43 14.95 91.72 19.32 11.18 0.00 37.54 8.75 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 ) 3.02 8.62 5.05 4.89 5.99 3.81 3.62 5.24 6.90 3.65 1.08 1.93 3.69 0.79 1.82 2.64 2.59 6.61 3.45 Solanum chacoense USDA8380-1 Leaf 10.76 F5_095_04_01_01 Leaf 4.38 F5_095_04_01_03 Leaf 5.95 F5_113_03_01_02 Leaf 5.87 F5_161_02_06_01 F5_173_03_08_01 F5_173_03_08_05 F5_199_02_01_01 F5_431_02_04_02 F5_478_03_08_04 MSHH786B_01 MSHH786B_09 Solanum chacoense USDA8380-1 F5_095_04_01_03 F5_113_03_01_02 F5_161_02_06_01 F5_431_02_04_02 F5_478_03_08_01 MSHH786B_01 Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Tub er Tub er Tub er Tub er Tub er Tub er Tub er 5.70 3.83 4.34 5.15 4.65 6.94 1.29 1.15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.44 1.32 1.96 1.81 2.02 1.35 1.58 1.59 1.11 2.83 0.58 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (mg/g Dry Weig ht) 7.26 10.74 7.25 6.90 9.16 5.48 5.50 8.01 8.45 6.48 3.03 2.33 6.13 0.96 2.08 3.95 2.55 9.93 7.04 379 Table S5.8. Graphical genotype data for F2, F4, and F5 recombinant inbred line individuals at the 1020 V3 SNP loci used for analysis in this study. [This data set can be accessed with the online version of this publication.] 380 Table S5.9. KASPTM marker genotyping data of parental lines, their F1 hybrid, F4 recombinant inbred lines and F5 recombinant inbred lines at two loci within the Sli candidate region associated with self-compatibility. Marker Name Generation Parental Parental Line Solanum chacoense 80-1 Solanum chacoense M6 EE501F1_02 EE501F4_028_04_05 EE501F4_076_03_01 EE501F4_082_01_02 EE501F4_093_02_02 EE501F4_113_03_01 EE501F4_161_02_05 EE501F4_196_01_01 EE501F4_233_01_01 EE501F4_234_01_01 EE501F4_321_04_02 EE501F4_378_02_01 EE501F4_454_01_02 EE501F4_471_01_04 EE501F4_492_04_04 EE501F4_533_05_03 EE501F4_537_01_01 EE501F4_540_04_04 EE501F4_543_03_02 EE501F4_611_04_03 EE501F4_615_03_01 EE501F4_636_03_04 EE501F4_672_04_03 EE501F5_028_04_05_01 EE501F5_064_03_08_01 EE501F5_076_03_06_01 EE501F5_081_02_06_01 EE501F5_089_02_08_01 EE501F5_093_02_05_01 EE501F5_095_04_01_01 EE501F5_154_02_05_01 EE501F5_156_01_03_01 EE501F5_160_03_03_01 EE501F5_161_02_06_01 EE501F5_173_03_08_01 EE501F5_182_04_01_01 F1 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F4 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 Sli_898 sli/sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/Sli -- Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/sli Sli/sli Sli/Sli Sli/Sli Sli/Sli Sli_626 sli/sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/Sli Sli/sli -- Sli/Sli Sli/Sli -- Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli -- Sli/Sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli -- Sli/sli Sli/Sli Sli/Sli Sli/Sli 381 Table S5.8 (cont’d) F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 EE501F5_196_01_05_01 EE501F5_199_02_01_01 EE501F5_220_03_01_01 EE501F5_233_01_01_01 EE501F5_268_02_01_01 EE501F5_278_02_01_01 EE501F5_297_01_06_01 EE501F5_311_01_08_01 EE501F5_321_04_04_01 EE501F5_348_01_05_01 EE501F5_361_03_04_01 EE501F5_365_02_03_01 EE501F5_370_02_02_01 EE501F5_378_02_04_01 EE501F5_380_01_04_01 EE501F5_382_01_06_01 EE501F5_397_01_04_01 EE501F5_454_01_02_01 EE501F5_470_02_06_01 EE501F5_478_03_08_01 EE501F5_492_04_04_01 EE501F5_495_01_02_01 EE501F5_495_01_05_01 EE501F5_499_01_07_01 EE501F5_533_05_03_01 EE501F5_537_01_01_01 EE501F5_540_04_02_01 EE501F5_543_03_01_01 EE501F5_570_04_03_01 EE501F5_636_02_03_01 EE501F5_641_01_09_01 EE501F5_642_03_01_01 EE501F5_672_04_03_01 EE501F5_673_01_04_01 EE501F5_701_01_03_01 EE501F5_706_02_05_01 Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/Sli Sli/sli Sli/Sli -- Sli/sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli -- Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli -- Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli Sli/Sli 382 Table S5.10. SNPs significantly associated with leptine I/II, alpha-solanine, alpha-chaconine, the ratio of acetylated to non-acetylated compounds, and the presence of leptines in the F4 and F5 generation of the recombinant inbred line population. Chromosomes are separated by a thick, black line. Cell shading (grey or white) is used to group identical SNPs that are associated with different traits/indifferrent generations. RAUDC = Relative area under the defoliation curve evaluated under field conditions. 80-1 Parent al Genoty pe Trait Mean M6 Parent al Genoty pe Trait Mean Recombin ant Genotype Trait Mean 16.12 12.06 23.34 1.46 0.48 0.08 1.53 0.50 0.08 1.46 0.48 0.08 2.62 1.56 0.50 0.09 6.94 2.62 1.64 0.52 0.09 6.94 2.52 1.17 0.22 0.15 0.05 7.79 1.27 0.31 0.06 1.27 0.31 0.06 1.27 0.31 0.06 0.41 0.09 0.03 0.01 0.10 0.02 0.00 0.09 0.02 0.00 0.10 0.02 0.00 1.82 0.97 0.19 0.04 26.91 11.57 0.41 0.09 0.03 0.01 1.82 0.97 0.19 0.04 26.91 11.57 2.51 1.56 0.50 0.15 0.09 7.74 0.39 0.09 0.03 0.02 0.01 26.51 Position (bp) Generat ion 80003968 14041901 14041901 14041901 15518745 15518745 15518745 15929220 15929220 15929220 22151711 22151711 22151711 22151711 22151711 22152144 22152144 22152144 22152144 22152144 22381563 22381563 22381563 22381563 22381563 22381563 F5 F5 F5 F5 F5 F5 F5 F5 F5 F5 F4 F5 F5 F5 F4 F4 F5 F5 F5 F4 F4 F5 F5 F4 F5 F4 Chr CH 01 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 Trait Solani ne Leptin e I Leptin e II Ratio Leptin e I Leptin e II Ratio Leptin e I Leptin e II Ratio Leptin e I Leptin e I Leptin e II Ratio RAUD C Leptin e I Leptin e I Leptin e II Ratio RAUD C Leptin e I Leptin e I Leptin e II Ratio Ratio RAUD C solcap_snp_c2_ SNP 7344 32239 32239 32239 41874 41874 41874 30945 30945 30945 solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ PotVar0039036 PotVar0039036 PotVar0039036 PotVar0039036 PotVar0039036 PotVar0039005 PotVar0039005 PotVar0039005 PotVar0039005 PotVar0039005 solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ solcap_snp_c2_ 32460 32460 32460 32460 32460 32460 solcap_snp_c2_ Kind of Test Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- Wilcoxon/Kruskal- wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis wallis 383 P- value 0.002 5 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 0.006 6 <0.00 01 <0.00 01 <0.00 01 <0.00 01 0.005 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 0.004 9 R 2 (U ) N 6 9 6 9 6 9 6 7 6 9 6 9 6 7 6 9 6 9 6 7 6 2 6 9 6 9 6 7 4 4 6 2 6 9 6 9 6 7 4 4 6 2 6 9 6 9 6 1 6 7 4 4 Table S5.9 (cont’d) solcap_snp_c2_3 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 02 CH 06 CH 06 CH 06 CH 07 CH 07 CH 07 CH 07 CH 07 CH 07 CH 07 CH 07 CH 07 CH 07 CH 07 CH 10 CH 10 CH 12 CH 12 CH 02 CH 02 CH 02 CH 02 223817 223817 223817 223817 223817 223817 339621 339621 339632 339632 19 19 19 19 19 19 78 78 53 53 8 58 83 227060 506206 512703 586600 637671 637812 783017 795288 795856 100206 100224 100270 100577 748091 457177 462274 7 4 4 3 9 9 5 544263 547279 91 08 01 01 45 45 140419 140419 155187 155187 F 4 F 5 F 5 F 4 F 5 F 4 F 5 F 5 F 5 F 5 F 4 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 5 F 4 F 5 F 5 F 5 F 5 F 4 F 5 F 4 F 5 Leptine I Leptine I Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Leptine II Wilcoxon/Kruskal-wallis Ratio Ratio RAUDC Leptine I Ratio Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Leptine I Wilcoxon/Kruskal-wallis Ratio Wilcoxon/Kruskal-wallis Solanine Wilcoxon/Kruskal-wallis Chaconine Chaconine Solanine Solanine Solanine Solanine Solanine Solanine Solanine Solanine Solanine Solanine Solanine Solanine Solanine Solanine Solanine Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Wilcoxon/Kruskal-wallis Contingency Analysis Likelihood Ratio Contingency Analysis Likelihood Ratio Contingency Analysis Likelihood Ratio Contingency Analysis Likelihood Ratio 384 <0.00 01 <0.00 01 <0.00 01 <0.00 01 <0.00 01 0.004 9 <0.00 01 <0.00 01 <0.00 01 <0.00 01 0.000 5 0.001 5 0.000 4 0.004 2 0.004 2 0.004 2 0.004 2 0.004 2 0.004 2 0.004 2 0.004 2 0.004 2 0.004 2 0.000 2 0.001 3 0.001 5 0.007 7 0.007 7 <0.00 01 <0.00 01 <0.00 01 <0.00 01 2.52 2.51 0.39 1.17 1.64 0.09 0.22 0.52 0.03 0.15 0.15 0.02 0.05 0.09 7.79 7.74 0.01 26.5 1 1.53 0.52 0.16 0.08 0.03 0.01 1.40 0.16 0.52 0.08 18.1 1 10.7 5 10.5 0 11.6 1 11.6 1 11.6 1 11.6 1 11.6 1 11.6 1 11.6 1 11.6 1 11.6 1 11.6 1 13.1 2 19.6 0 19.6 0 11.8 2 11.8 2 0.01 24.1 3 14.4 5 14.7 7 16.2 2 16.2 2 16.2 2 16.2 2 16.2 2 16.2 2 16.2 2 16.2 2 16.2 2 16.2 2 22.5 4 11.0 4 11.7 0 25.2 7 25.2 7 0.03 12.1 5 20.0 3 19.0 5 20.6 0 20.6 0 20.6 0 20.6 0 20.6 0 20.6 0 20.6 0 20.6 0 20.6 0 20.6 0 17.4 9 12.7 4 12.3 7 15.7 8 15.7 8 6 2 6 9 6 9 6 2 6 7 4 4 6 9 6 7 6 9 6 7 6 2 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 2 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 0.39 88 0.43 7 0.40 85 0.47 04 solcap_snp_c2_3 solcap_snp_c2_3 solcap_snp_c2_3 solcap_snp_c2_3 solcap_snp_c2_3 2462 2462 2462 2462 2462 2462 PotVar0038051 PotVar0038051 PotVar0038136 PotVar0038136 solcap_snp_c2_5 solcap_snp_c2_5 7292 828 PotVar0073914 PotVar0022997 PotVar0022719 PotVar0022711 solcap_snp_c2_3 solcap_snp_c2_3 8867 8871 PotVar0022575 solcap_snp_c1_1 solcap_snp_c2_5 solcap_snp_c1_1 5906 4652 5911 PotVar0022478 solcap_snp_c2_4 5799 PotVar0108198 solcap_snp_c2_2 solcap_snp_c2_5 solcap_snp_c2_4 solcap_snp_c2_3 solcap_snp_c2_3 solcap_snp_c2_4 solcap_snp_c2_4 4747 0821 8482 2239 2239 1874 1874 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 2 CH0 6 CH0 6 CH0 6 CH0 7 CH0 8 159292 159292 221517 221517 221521 223815 223815 223817 223817 250037 253328 270974 270976 270997 276020 276033 20 20 11 11 44 63 63 19 19 03 05 48 16 43 74 01 78 78 53 84 276186 339621 339632 369565 764530 764621 227060 8 526938 68 665936 4 F 4 F 5 F 4 F 5 F 5 F 4 F 5 F 4 F 5 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 4 F 5 F 5 F 5 F 4 F 4 F 4 F 5 F 5 Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Presence of Leptines Table S5.9 (cont’d) solcap_snp_c2_30 solcap_snp_c2_30 945 945 PotVar0039036 PotVar0039036 PotVar0039005 solcap_snp_c2_32 solcap_snp_c2_32 solcap_snp_c2_32 solcap_snp_c2_32 solcap_snp_c2_21 PotVar0117640 solcap_snp_c2_45 solcap_snp_c2_45 solcap_snp_c2_45 460 460 462 462 759 316 319 323 PotVar0123847 PotVar0123826 solcap_snp_c1_12 329 PotVar0038051 PotVar0038136 PotVar0046764 solcap_snp_c2_30 solcap_snp_c2_30 solcap_snp_c2_57 solcap_snp_c1_40 solcap_snp_c1_13 594 595 292 29 230 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 Contingency Analysis Likelihood <0.000 1 1 1 1 1 1 1 1 1 1 1 Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood Contingency Analysis Likelihood 0.0002 0.0002 0.0002 0.0004 0.0004 0.0001 <0.000 1 1 0.0018 0.0005 0.0005 <0.000 1 0.0004 0.0003 Contingency Analysis Likelihood <0.000 Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 6 9 0.441 0.437 0.55 0.529 6 0.568 3 0.477 7 0.589 2 0.477 7 0.627 9 0.328 7 0.326 2 0.205 6 0.205 6 0.205 6 0.184 9 0.184 9 0.214 1 0.331 7 0.271 1 0.142 3 0.178 8 0.178 8 0.268 2 0.173 6 0.184 385 Table S5.11. Genetic map based on 97 F4 individuals from the recombinant inbred line population and 288 SNPs. SNP Locus Name SNP Locus PGSC v4.03 Physical Position (bp) 354295 472297 472549 1028869 1158715 2857296 2956117 3693421 4041479 13546944 35834252 29682798 37684708 59364003 68952404 74248443 74628218 77047246 80230736 80262082 80598198 85586812 85465494 85897859 86449422 86602967 87245902 87548665 7676939 22152699 25332805 27143975 27618678 27602074 29473175 29707398 30142847 29955410 31352403 31839875 33962178 36879127 36941026 37438491 37474969 37474756 37439083 38688454 39194958 39073504 PotVar0120099 PotVar0120085 PotVar0119966 solcap_snp_c1_2425 PotVar0071966 PotVar0045000 solcap_snp_c2_21233 solcap_snp_c1_6114 PotVar0045593 solcap_snp_c2_43973 solcap_snp_c1_13814 PotVar0122493 solcap_snp_c1_6787 solcap_snp_c2_45301 PotVar0043831 PotVar0041329 solcap_snp_c2_14350 PotVar0028786 solcap_snp_c2_7062 solcap_snp_c2_7068 solcap_snp_c2_5076 PotVar0126949 PotVar0126587 solcap_snp_c2_53077 PotVar0110932 solcap_snp_c2_14741 PotVar0099779 PotVar0100004 solcap_snp_c2_4521 PotVar0038974 PotVar0117640 solcap_snp_c1_13459 solcap_snp_c1_12329 PotVar0123847 PotVar0062500 PotVar0062424 solcap_snp_c1_13920 solcap_snp_c2_46915 PotVar0094234 solcap_snp_c1_13240 PotVar0038051 solcap_snp_c1_16727 PotVar0046549 solcap_snp_c2_42169 solcap_snp_c1_16171 solcap_snp_c2_55632 solcap_snp_c2_42172 solcap_snp_c2_53034 solcap_snp_c2_42166 solcap_snp_c2_40635 Map Distance (cM) Linkage Group 0 0.299 0.596 1.5 4.546 11.147 11.745 18.188 18.797 34.939 37.188 38.342 38.7 45.607 0 21.84 22.441 36.507 49.881 50.5 51.801 69.991 70.588 72.435 73.972 74.568 77.438 78.06 0 8.287 15.065 22.675 23.625 24.221 29.881 31.408 33.254 33.55 37.831 38.476 46.025 62.665 62.964 66.365 66.722 68.315 68.615 75.404 77.344 78.257 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.1 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 1.2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 386 Table S5.10 (cont’d) solcap_snp_c2_40638 PotVar0010429 PotVar0010382 PotVar0009651 solcap_snp_c2_25143 PotVar0009673 PotVar0009997 solcap_snp_c2_15068 solcap_snp_c1_7873 solcap_snp_c2_36232 PotVar0084678 solcap_snp_c2_51389 solcap_snp_c1_15783 solcap_snp_c1_2051 solcap_snp_c1_12745 solcap_snp_c1_12749 solcap_snp_c1_6898 PotVar0109399 solcap_snp_c2_53779 solcap_snp_c2_54077 solcap_snp_c2_56256 solcap_snp_c2_7770 solcap_snp_c1_14440 solcap_snp_c2_39463 solcap_snp_c2_43735 solcap_snp_c2_26757 PotVar0075681 solcap_snp_c1_10167 PotVar0016517 PotVar0017188 solcap_snp_c2_57149 PotVar0114684 solcap_snp_c2_11696 PotVar0024787 PotVar0025592 solcap_snp_c1_3786 PotVar0026113 PotVar0079374 PotVar0079702 PotVar0079935 PotVar0080669 PotVar0116931 PotVar0117259 PotVar0089663 PotVar0083800 PotVar0084164 PotVar0085522 PotVar0091177 PotVar0091041 PotVar0014376 39073798 39079305 39079979 40253455 40435644 40252701 39963506 45695644 46195190 419098 833348 983008 1276863 10654105 34492320 34638581 4586164 6340399 9941686 10968547 12425864 47904770 55384468 63406121 64055406 65600243 68141339 70185081 71336433 71590759 1738100 1956664 2261080 3358775 3813315 3960507 4250232 4495794 4549568 4701481 4764411 5364099 5690795 5942512 7541185 7670627 8807655 10109724 10113512 12237074 21920999 42190428 43219233 45360490 45545898 47122961 48541183 79.443 79.471 79.782 85.406 86.002 86.699 88.148 111.652 112.249 0 0.308 1.542 4.763 18.178 19.387 20.605 0 6.013 23.705 28.937 31.789 32.086 36.541 57.303 59.778 67.516 82.07 89.253 105.166 105.477 0 0.606 3.788 9.386 11.682 14.525 16.369 16.759 17.151 17.563 17.859 19.387 20.596 23.086 30.699 31.6 33.766 35.388 38.774 44.105 44.401 44.998 45.942 49.394 52.841 59.451 67.221 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 2 chr 3 chr 3 chr 3 chr 3 chr 3 chr 3 chr 3 chr 4 chr 4 chr 4 chr 4 chr 4 chr 4 chr 4 chr 4 chr 4 chr 4 chr 4 chr 4 chr 4 chr 4 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 solcap_snp_c1_15690 solcap_snp_c2_47393 PotVar0106493 solcap_snp_c1_12008 solcap_snp_c2_40774 solcap_snp_c1_12414 solcap_snp_c2_10358 387 Table S5.10 (cont’d) PotVar0082112 PotVar0123206 solcap_snp_c2_55240 PotVar0128236 solcap_snp_c2_8521 solcap_snp_c2_8513 PotVar0034819 solcap_snp_c2_3451 solcap_snp_c2_27565 solcap_snp_c2_24266 solcap_snp_c2_33932 PotVar0069473 solcap_snp_c2_33933 PotVar0004038 solcap_snp_c2_11303 solcap_snp_c2_50186 solcap_snp_c1_3689 solcap_snp_c2_32918 solcap_snp_c1_8594 solcap_snp_c2_24322 solcap_snp_c2_27865 PotVar0104740 solcap_snp_c2_51761 solcap_snp_c2_56059 PotVar0134018 solcap_snp_c2_40242 solcap_snp_c2_33297 PotVar0127173 solcap_snp_c2_57412 solcap_snp_c1_11276 PotVar0022751 PotVar0022524 PotVar0022336 solcap_snp_c2_26167 PotVar0102276 solcap_snp_c2_47004 PotVar0069646 PotVar0044278 solcap_snp_c1_4029 solcap_snp_c2_12603 PotVar0043855 solcap_snp_c2_30428 PotVar0037150 PotVar0037035 solcap_snp_c2_28846 PotVar0037011 PotVar0036990 solcap_snp_c2_28849 solcap_snp_c1_14166 solcap_snp_c2_17305 solcap_snp_c2_57003 PotVar0077179 PotVar0125352 solcap_snp_c1_13094 PotVar0100216 PotVar0081239 solcap_snp_c2_19079 48662020 49045164 49467229 49728003 50584500 50584053 51319479 51697156 3470372 8312329 6903226 29622205 6903541 26013204 30365962 7262487 30466255 31830854 35194342 36214491 34893352 37869257 38211801 38291420 38707476 40303649 38901318 42867595 43086245 43183046 637293 1002452 1350553 2610382 3172006 7139104 40644479 52337275 52693868 53106014 53252078 54329836 55382464 55469145 55887265 55736464 55736778 55889177 7836331 9320406 9975029 45008776 45869730 49240884 51138331 52158230 52490868 68.433 69.958 70.254 71.168 75.734 76.035 84.141 85.169 0 3.486 3.505 3.811 4.079 4.377 4.669 4.675 4.971 5.267 6.043 6.102 6.164 6.46 6.757 7.353 7.95 8.547 8.843 9.14 9.436 10.033 0 0.916 1.829 11.351 13.275 29.496 32.749 0 4.244 11.095 12.622 17.23 21.448 22.045 22.945 23.242 23.801 25.364 0 0.597 0.893 16.266 17.228 26.659 38.582 42.474 43.845 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 5 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 6 chr 7.1 chr 7.1 chr 7.1 chr 7.1 chr 7.1 chr 7.1 chr 7.1 chr 7.2 chr 7.2 chr 7.2 chr 7.2 chr 7.2 chr 7.2 chr 7.2 chr 7.2 chr 7.2 chr 7.2 chr 7.2 chr 8 chr 8 chr 8 chr 8 chr 8 chr 8 chr 8 chr 8 chr 8 388 solcap_snp_c2_19211 solcap_snp_c2_34604 PotVar0119089 solcap_snp_c1_8300 PotVar0023704 solcap_snp_c2_28433 PotVar0023284 solcap_snp_c2_28480 PotVar0114494 PotVar0130582 PotVar0011742 PotVar0012073 PotVar0007613 PotVar0007606 solcap_snp_c2_12760 solcap_snp_c2_44814 solcap_snp_c1_6192 solcap_snp_c1_6196 solcap_snp_c2_55483 PotVar0108622 PotVar0108619 solcap_snp_c2_20879 solcap_snp_c2_1113 PotVar0108198 solcap_snp_c2_55819 solcap_snp_c2_15483 PotVar0005097 PotVar0005344 solcap_snp_c2_48091 solcap_snp_c2_48145 solcap_snp_c2_29749 solcap_snp_c1_9066 solcap_snp_c1_7187 Table S5.10 (cont’d) PotVar0058165 PotVar0064694 PotVar0064415 solcap_snp_c2_13473 solcap_snp_c1_4347 solcap_snp_c2_37189 PotVar0066236 solcap_snp_c2_33657 PotVar0066299 PotVar0066338 solcap_snp_c2_33653 solcap_snp_c1_10062 solcap_snp_c1_2187 solcap_snp_c1_2153 solcap_snp_c1_2150 PotVar0066824 solcap_snp_c2_5960 PotVar0067342 PotVar0067424 solcap_snp_c2_6249 solcap_snp_c2_55972 PotVar0067504 PotVar0067827 PotVar0067501 52800147 53138349 53457367 54295146 54777820 56382148 56628142 56781388 531917 50724 2469771 2679615 30812635 30812503 45924103 46740222 47753124 47754984 59483310 59586577 59586631 60097041 2382807 4571779 53235977 54996383 55842116 55856581 55898404 55919521 56366011 56395440 57468777 57670512 811667 786787 922248 1163793 1748103 2060890 2274063 2261267 2261684 2289760 2349165 2562328 2702156 2736790 2771287 2854841 2973385 3072948 3252697 3182994 3262580 3268257 3262453 46.01 46.917 50.314 56.793 58.993 76.279 76.908 77.808 0 2.813 20.373 23.47 0 0.603 0.899 1.8 2.716 3.35 0 0.307 0.603 2.447 0 13.674 33.829 45.249 55.703 56.336 56.925 57.521 62.79 63.097 73.693 75.257 0 0.297 0.66 3.08 5.913 7.437 8.958 8.985 9.559 10.157 11.366 11.963 13.171 13.468 14.064 14.361 14.657 15.558 16.459 16.755 18.914 20.122 20.419 chr 8 chr 8 chr 8 chr 8 chr 8 chr 8 chr 8 chr 8 chr 9.1 chr 9.1 chr 9.1 chr 9.1 chr 9.2 chr 9.2 chr 9.2 chr 9.2 chr 9.2 chr 9.2 chr 9.3 chr 9.3 chr 9.3 chr 9.3 chr 10 chr 10 chr 10 chr 10 chr 10 chr 10 chr 10 chr 10 chr 10 chr 10 chr 10 chr 10 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 389 PotVar0110427 solcap_snp_c1_2210 solcap_snp_c2_6302 solcap_snp_c2_47382 solcap_snp_c2_47386 PotVar0106087 PotVar0105621 solcap_snp_c2_20947 solcap_snp_c2_21015 solcap_snp_c2_21050 solcap_snp_c2_21066 solcap_snp_c2_23923 PotVar0021975 solcap_snp_c2_13636 PotVar0134713 PotVar0047209 PotVar0047371 Table S5.10 (cont’d) solcap_snp_c2_3737 PotVar0112779 PotVar0112395 solcap_snp_c2_15364 PotVar0008725 PotVar0008637 solcap_snp_c2_43865 solcap_snp_c2_51284 PotVar0124390 solcap_snp_c2_34198 PotVar0027707 solcap_snp_c2_53244 solcap_snp_c2_27379 PotVar0027811 solcap_snp_c2_44924 solcap_snp_c2_45743 solcap_snp_c2_51098 solcap_snp_c2_5952 solcap_snp_c2_14413 solcap_snp_c2_16908 solcap_snp_c2_16294 solcap_snp_c2_16919 solcap_snp_c2_21336 solcap_snp_c2_48392 solcap_snp_c2_13933 solcap_snp_c2_5953 PotVar0036410 solcap_snp_c2_17623 solcap_snp_c1_14870 solcap_snp_c2_18791 solcap_snp_c2_18825 solcap_snp_c2_18836 PotVar0109256 solcap_snp_c1_7495 PotVar0110851 solcap_snp_c2_57627 solcap_snp_c2_23254 solcap_snp_c2_50821 solcap_snp_c2_48482 PotVar0053460 3657926 3732947 3943269 4250278 4365454 4346145 4666571 5051445 5295465 5464471 5735381 6017871 19468350 36652714 37384590 38554619 39786075 40397444 40484801 40627337 41501023 43867510 43790644 44295451 44620835 44648784 45092064 9357042 9357754 10256960 11041151 11627023 15695850 13183157 37123358 14690892 21202485 13267302 21328539 14931696 20487184 43326456 37123412 45972509 50361328 50896643 51258447 51732789 51862256 52758176 53048469 53313788 53583976 53793483 54426391 54727908 58983574 22.902 23.199 23.495 25.313 25.628 25.758 25.923 26.22 27.429 27.725 28.322 28.938 0 4.533 5.446 12.466 15.77 19.039 20.266 20.563 23.532 34.884 35.181 37.674 40.157 40.754 41.969 0 0.296 2.781 3.684 4.28 4.577 4.596 4.618 4.624 4.631 4.637 5.172 5.743 5.759 5.764 5.768 5.77 6.669 6.965 7.262 7.558 7.854 8.151 8.748 9.044 9.948 11.469 12.373 13.648 25.584 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.1 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 11.2 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 390 solcap_snp_c1_1923 PotVar0052374 PotVar0052284 solcap_snp_c2_5704 solcap_snp_c2_5684 solcap_snp_c2_5507 Table S5.10 (cont’d) solcap_snp_c2_46213 PotVar0053168 PotVar0052695 PotVar0052507 59129520 59208079 59680999 59793920 59870038 59957211 59979506 59986990 60036258 60477931 27.105 28.626 32.456 32.752 34.274 34.87 35.467 35.763 36.36 37.261 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 chr 12 391 Table S5.12. Excessively heterozygous loci in F4 and F5 individuals of the recombinant inbred line population. SNP Locus F4 Ratio of Actual to F5 Ratio of Actual to DM PGSC v4.03 Chromosome Position (bp) Expected Heterozygosity Expected Heterozygosity solcap_snp_c2_54581 PotVar0081045 solcap_snp_c2_46521 solcap_snp_c1_3866 PotVar0060988 PotVar0060978 solcap_snp_c2_35699 PotVar0101389 solcap_snp_c1_370 solcap_snp_c2_34890 PotVar0114684 PotVar0114686 PotVar0024652 solcap_snp_c1_13641 solcap_snp_c2_50207 solcap_snp_c1_8132 solcap_snp_c2_31893 PotVar0086012 solcap_snp_c2_16817 PotVar0090785 solcap_snp_c2_46103 solcap_snp_c1_11534 PotVar0022997 PotVar0022978 solcap_snp_c1_11520 PotVar0022977 PotVar0022817 solcap_snp_c2_38893 PotVar0022719 PotVar0022711 PotVar0022691 solcap_snp_c2_38900 solcap_snp_c2_38867 solcap_snp_c2_38871 PotVar0022575 solcap_snp_c1_15906 solcap_snp_c2_54652 solcap_snp_c1_15911 PotVar0022478 solcap_snp_c1_15914 solcap_snp_c1_10783 PotVar0022336 PotVar0022114 solcap_snp_c2_26197 PotVar0130044 solcap_snp_c2_26167 PotVar0102547 solcap_snp_c2_26248 PotVar0102276 solcap_snp_c1_7989 PotVar0134027 solcap_snp_c2_34179 01 01 01 01 01 01 02 04 04 04 05 05 05 06 06 06 06 06 06 06 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 07 08 24708276 25032740 59974382 71223852 81815329 81815464 47326597 4920111 52558547 67953469 1956664 1956763 2959035 1832292 7407390 46026590 46408984 47308813 48131679 48429967 564582 583570 586600 587066 587066 587123 588855 637024 637671 637812 638125 638125 783017 795288 795856 1002067 1002244 1002704 1005773 1005913 1318666 1350553 1503268 2498400 2546763 2610382 3039313 3118896 3172006 47655133 49455578 4174544 392 3.26 3.17 4.74 4.50 5.95 4.29 4.68 3.19 3.04 3.04 3.21 3.12 4.39 5.20 5.13 13.77 3.40 7.90 7.60 3.20 8.80 5.00 6.80 3.40 3.40 3.40 3.80 4.00 3.60 3.80 3.20 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.80 3.60 3.40 3.40 3.24 3.04 9.60 4.54 3.40 Table S5.11 (cont’d) solcap_snp_c1_8380 solcap_snp_c1_13230 solcap_snp_c2_12748 solcap_snp_c2_52857 solcap_snp_c2_47906 solcap_snp_c1_14166 solcap_snp_c1_14165 solcap_snp_c2_19646 solcap_snp_c2_19645 solcap_snp_c2_19639 solcap_snp_c2_19638 solcap_snp_c2_19631 solcap_snp_c2_17283 solcap_snp_c2_17284 solcap_snp_c1_5707 solcap_snp_c2_17289 solcap_snp_c2_17290 solcap_snp_c2_17294 solcap_snp_c1_5713 solcap_snp_c2_17305 solcap_snp_c2_17318 solcap_snp_c2_17321 solcap_snp_c2_17322 solcap_snp_c2_57003 solcap_snp_c2_57002 solcap_snp_c2_48358 solcap_snp_c2_33771 solcap_snp_c1_9474 solcap_snp_c2_30904 solcap_snp_c2_30905 solcap_snp_c2_30907 solcap_snp_c2_7782 solcap_snp_c1_2684 solcap_snp_c1_2686 solcap_snp_c2_7785 solcap_snp_c2_28516 solcap_snp_c2_57849 PotVar0088658 solcap_snp_c2_50849 solcap_snp_c2_34090 solcap_snp_c2_53878 solcap_snp_c2_53881 solcap_snp_c1_15689 solcap_snp_c2_34085 solcap_snp_c2_34078 solcap_snp_c2_34075 solcap_snp_c2_34069 solcap_snp_c2_34564 solcap_snp_c2_42298 solcap_snp_c2_42297 solcap_snp_c2_42293 solcap_snp_c2_2178 solcap_snp_c2_2183 solcap_snp_c2_19949 solcap_snp_c2_19946 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 3.47 3.43 3.42 3.43 3.60 3.54 3.47 3.50 3.40 3.32 3.23 3.24 3.36 3.32 3.33 3.33 3.47 3.33 3.33 3.40 3.33 3.33 3.33 3.33 3.21 3.26 3.24 3.26 3.21 3.26 3.21 3.26 3.57 3.26 3.26 3.26 3.26 3.17 3.26 3.26 3.26 3.26 3.26 3.26 3.26 3.26 3.13 3.26 3.29 3.33 3.26 3.26 3.26 3.03 3.26 5810262 6659364 7277409 7445492 7788444 7836331 7837521 8092928 8134055 8195963 8196002 8354399 8980876 8980940 9087306 9087656 9088040 9091210 9226857 9320406 9554691 9558581 9559536 9975029 9975056 10277914 10833062 11597854 12698558 12698757 12699036 13195806 13208418 13212735 13213183 13920231 14440357 15138225 15139435 15880448 15892521 15911353 15912416 15953548 16000177 16000723 16001908 16652660 17060414 17060690 17061437 18395020 19348560 19827882 19942842 393 5.60 5.60 5.60 5.60 5.60 5.60 5.60 5.00 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.00 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.27 5.20 5.20 5.20 5.27 5.20 5.20 5.20 5.20 5.20 5.20 5.27 5.20 5.06 5.00 5.00 5.06 5.00 5.00 5.00 5.00 5.20 5.27 5.20 5.20 5.20 5.20 5.20 5.20 Table S5.11 (cont’d) solcap_snp_c2_19942 PotVar0076451 solcap_snp_c2_19934 solcap_snp_c1_6252 solcap_snp_c2_2852 solcap_snp_c2_2843 solcap_snp_c2_2842 solcap_snp_c2_2840 solcap_snp_c2_2839 solcap_snp_c2_2837 solcap_snp_c1_846 solcap_snp_c1_845 solcap_snp_c2_2832 solcap_snp_c2_2830 solcap_snp_c2_8167 solcap_snp_c2_8172 solcap_snp_c2_5914 solcap_snp_c2_5913 solcap_snp_c2_5909 solcap_snp_c2_5907 solcap_snp_c2_20307 solcap_snp_c2_29489 solcap_snp_c2_29490 solcap_snp_c2_29491 solcap_snp_c2_29492 solcap_snp_c2_29494 solcap_snp_c2_29495 solcap_snp_c1_8987 solcap_snp_c2_29477 solcap_snp_c2_29478 solcap_snp_c2_29479 solcap_snp_c2_29284 solcap_snp_c2_29283 solcap_snp_c2_29282 solcap_snp_c2_19426 solcap_snp_c1_6130 solcap_snp_c1_6131 solcap_snp_c2_19431 solcap_snp_c2_19433 solcap_snp_c1_6136 solcap_snp_c2_19437 solcap_snp_c2_19439 solcap_snp_c2_47923 solcap_snp_c1_14174 solcap_snp_c2_47922 solcap_snp_c2_47921 solcap_snp_c2_47920 solcap_snp_c1_14959 solcap_snp_c2_30254 solcap_snp_c1_9169 solcap_snp_c2_30288 solcap_snp_c2_30293 solcap_snp_c2_33766 PotVar0060614 solcap_snp_c1_11719 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 3.53 3.26 3.03 3.26 3.26 3.21 3.16 3.13 3.21 3.29 3.26 3.20 3.17 3.17 3.26 3.26 3.30 3.29 3.21 3.21 3.26 3.29 3.26 3.26 3.26 3.17 3.19 3.21 3.26 3.26 3.26 3.26 3.29 3.26 3.29 3.26 3.21 3.38 3.26 3.26 3.17 3.21 3.26 3.29 3.26 3.26 3.27 3.21 3.29 3.26 3.26 3.26 3.24 3.26 3.54 19950036 19950697 20246896 20465382 20914451 21132862 21132878 21133008 21388321 21434617 21435247 21435300 21444550 21568049 21906383 22239641 23895246 23895303 23895925 23950203 25466161 26137205 26139555 26141458 26158698 26158842 26158884 26161643 26163253 26163279 26170060 28480153 28480198 28480293 29755831 29756364 29756431 29758299 29758730 29812858 30148056 30148252 30353879 30362485 30365828 30367119 30367162 31977011 32229217 32311520 32544771 32685897 33597174 34288273 35379060 394 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.27 5.20 8.40 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.27 5.20 5.20 5.20 5.20 5.20 5.20 5.20 5.00 5.06 5.00 5.00 5.00 5.06 4.60 4.60 Table S5.11 (cont’d) solcap_snp_c2_49246 solcap_snp_c2_49245 solcap_snp_c2_41467 solcap_snp_c2_41463 solcap_snp_c1_12161 solcap_snp_c2_2816 solcap_snp_c1_838 solcap_snp_c2_2757 solcap_snp_c2_2748 solcap_snp_c2_32300 solcap_snp_c2_32310 solcap_snp_c2_51328 solcap_snp_c2_51329 solcap_snp_c2_45770 solcap_snp_c2_44319 solcap_snp_c2_44305 solcap_snp_c2_44304 solcap_snp_c1_13043 solcap_snp_c2_44334 solcap_snp_c2_18894 solcap_snp_c2_18895 solcap_snp_c2_53733 solcap_snp_c2_57300 PotVar0123288 PotVar0125449 solcap_snp_c1_14393 solcap_snp_c1_3613 solcap_snp_c1_219 solcap_snp_c2_689 PotVar0101834 solcap_snp_c1_9060 solcap_snp_c2_29741 solcap_snp_c2_6104 solcap_snp_c2_24612 solcap_snp_c2_34774 solcap_snp_c2_34762 solcap_snp_c2_34806 solcap_snp_c2_27379 PotVar0027811 solcap_snp_c2_44924 solcap_snp_c2_51098 solcap_snp_c2_16298 solcap_snp_c1_403 solcap_snp_c1_404 solcap_snp_c1_15034 solcap_snp_c2_14411 solcap_snp_c2_14413 solcap_snp_c2_21336 solcap_snp_c2_45743 solcap_snp_c1_8084 solcap_snp_c1_14767 solcap_snp_c1_14768 solcap_snp_c1_8913 solcap_snp_c2_29296 solcap_snp_c2_29293 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 09 09 09 09 09 10 10 11 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 35651364 35651378 35921407 35922019 36109444 36714006 37259543 37467447 37492252 38685106 38687057 38876238 38877340 39115651 39579286 39591467 39591478 39670914 39698106 43102244 43106686 43760647 43848943 43897045 45873794 739884 1556721 38263840 38266787 52568992 56310959 56311171 3886997 6936540 7863490 8063612 8519787 10256960 11041151 11627023 13183157 13270097 13617956 13617978 14219727 14689798 14690892 14931696 15695850 16345513 18919522 18919535 19181173 19187649 19188907 395 3.04 3.45 3.42 3.04 3.46 3.54 3.24 3.19 3.29 3.19 3.60 3.14 3.05 3.26 3.26 4.18 3.57 3.54 4.18 4.14 4.00 3.04 3.21 3.07 3.19 3.07 3.07 3.21 3.23 3.13 3.12 3.07 3.09 3.07 3.12 3.21 4.20 4.20 4.20 4.25 4.40 4.40 4.20 4.20 4.40 4.40 4.40 4.60 4.60 4.40 4.60 4.40 4.40 4.80 4.86 4.00 4.00 4.00 4.00 3.80 6.00 3.40 3.20 5.00 5.00 3.20 7.00 7.00 3.20 3.60 3.80 3.20 3.60 5.47 5.40 6.20 5.80 5.80 5.80 5.80 6.00 6.00 5.80 5.80 6.00 5.80 5.87 5.87 5.80 6.00 Table S5.11 (cont’d) solcap_snp_c2_53383 solcap_snp_c2_48392 solcap_snp_c2_48391 solcap_snp_c2_56163 solcap_snp_c2_16908 solcap_snp_c1_5458 solcap_snp_c2_16911 solcap_snp_c2_16915 solcap_snp_c1_5468 PotVar0044793 PotVar0044772 solcap_snp_c2_10060 solcap_snp_c2_10059 solcap_snp_c1_3326 solcap_snp_c2_58209 solcap_snp_c1_16695 solcap_snp_c2_40524 solcap_snp_c2_50139 solcap_snp_c2_50137 solcap_snp_c2_9491 solcap_snp_c2_27921 solcap_snp_c2_27922 solcap_snp_c2_27923 solcap_snp_c1_3172 solcap_snp_c2_27674 solcap_snp_c2_27673 solcap_snp_c2_27670 solcap_snp_c2_44369 solcap_snp_c1_13066 solcap_snp_c2_57161 solcap_snp_c2_45825 solcap_snp_c2_4214 solcap_snp_c2_27779 solcap_snp_c2_27780 solcap_snp_c2_27782 solcap_snp_c2_27788 solcap_snp_c2_27774 solcap_snp_c2_57686 solcap_snp_c2_57687 solcap_snp_c2_57692 PotVar0012912 PotVar0013114 solcap_snp_c2_45808 solcap_snp_c2_18996 solcap_snp_c2_18994 solcap_snp_c2_38375 solcap_snp_c2_19722 solcap_snp_c2_33619 PotVar0031972 solcap_snp_c2_13933 solcap_snp_c2_13958 solcap_snp_c2_53701 PotVar0036493 solcap_snp_c2_33630 PotVar0036483 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 20336319 20487184 20492079 20520046 21202485 21202779 21204474 21327580 21526751 21736671 21737124 22135725 22137171 22905890 24386302 24386805 26157851 27348155 27416356 27626001 28906171 28906568 28908226 29224615 29830607 29830883 29832277 30573417 30582910 32522905 33208343 35283808 35531719 35531765 35532613 35538152 35675157 36059700 36059760 36060641 38538120 38543349 40126394 40365310 40655220 41076744 41670282 42295551 43307930 43326456 43552012 45788710 45970798 45970798 45971166 396 3.16 3.03 3.07 3.07 3.14 3.21 3.07 3.07 3.21 3.21 3.12 3.07 3.40 4.42 3.07 3.07 3.07 3.07 3.21 3.07 3.07 3.07 3.07 3.07 3.07 3.21 3.07 3.07 3.14 3.12 3.12 3.07 3.13 3.07 3.03 3.21 3.13 3.07 3.07 3.21 3.07 3.07 3.07 3.07 3.10 3.07 3.07 3.07 3.17 3.12 3.07 3.07 3.07 3.17 5.80 5.80 5.80 5.80 6.00 6.00 5.80 5.80 6.00 6.00 5.80 5.87 5.80 12.15 5.80 5.80 5.80 5.80 6.00 5.87 5.80 5.80 5.80 5.80 5.80 6.00 5.80 5.80 5.80 5.80 5.80 5.80 5.80 6.00 5.80 5.80 6.00 5.87 5.80 5.80 6.00 5.87 5.80 5.80 5.80 5.80 5.80 5.80 5.80 5.80 5.80 5.80 5.80 5.80 6.00 Table S5.11 (cont’d) PotVar0036410 solcap_snp_c2_33628 solcap_snp_c2_21077 solcap_snp_c2_53205 solcap_snp_c2_51046 solcap_snp_c2_51047 solcap_snp_c2_49683 solcap_snp_c2_48686 solcap_snp_c2_48687 solcap_snp_c2_48008 solcap_snp_c2_43152 solcap_snp_c2_43141 solcap_snp_c2_43147 solcap_snp_c2_17623 solcap_snp_c2_17613 PotVar0037431 solcap_snp_c2_17741 solcap_snp_c2_43909 solcap_snp_c2_43910 solcap_snp_c2_43913 solcap_snp_c2_43916 PotVar0101568 solcap_snp_c2_43899 solcap_snp_c2_50527 solcap_snp_c1_14870 solcap_snp_c1_14869 solcap_snp_c2_50522 solcap_snp_c1_6004 solcap_snp_c2_18791 solcap_snp_c2_18803 solcap_snp_c1_6006 solcap_snp_c2_18825 solcap_snp_c2_18836 solcap_snp_c2_18848 solcap_snp_c2_18855 solcap_snp_c2_42342 PotVar0109256 PotVar0109153 PotVar0109073 solcap_snp_c1_7495 solcap_snp_c2_23308 3.07 3.07 3.21 3.07 3.07 3.07 3.07 3.12 3.12 3.14 3.02 3.03 3.64 3.24 3.07 3.49 5.80 5.80 6.00 5.80 5.80 5.80 5.80 5.80 5.80 5.60 4.80 4.80 5.00 4.80 4.86 5.00 5.00 4.80 4.80 4.80 4.80 4.66 4.40 4.40 4.40 4.40 4.40 4.60 4.40 4.40 4.60 4.40 4.20 4.20 4.20 4.20 4.20 4.20 4.20 3.60 3.04 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 45972509 45977120 46657785 47233586 48177592 48177610 48605748 48734091 48851340 49624425 49994628 50145601 50248982 50361328 50389681 50391416 50573932 50632815 50633303 50639385 50643474 50739899 50796916 50896387 50896643 50896655 50902386 51251314 51258447 51271405 51728044 51732789 51862256 51908158 51931824 52580682 52758176 52760874 52761637 53048469 53190167 397 APPENDIX D: HT-B Sequence Analysis >Solanum_chacoense_80-1_1 ACAAACTCATATAAAATGGCATTCAAGGCAAATATTTTGCTTATATTTTCTTTGGTTC TTAATTATATCATCAGAAGTTATTGCAAGGGAGATAGTTGAGCCTTCACTTCCATTG CTTGAGGGTAAGTTGGTTTTTTAATTGTAGTTTTGCTATATTTAATTTTTGAACATAT AATTTCGTTATCGATCTAAAAGAGTTGTATATATTGGTGTAACAAATGAAATTCAGA ATACAGAAATGAACAATCCAACGCTCCAAAAATAATAATAATAATAATAACAACAA CAATAATGATGACGATGATTTCGTTAGTAATGTTTGTAAAGCCGCTTGTTGTTAG >Solanum_chacoense_80-1_2 AAACTCATATAAAATGGCATTCAAGGCAAATATTTTGCTTATATTTTCTTTGGTTCTT AATTATATCATCAGAAGTTATTGGGGCAAGGGAGATAGTTGAGCCTTCACTTCCATT GCTTGAGGGTAAGTTGGTTCCTTTTTTTTAATTGTAGTTTTGCTATATTTAATTTTTTA CACGAACATATAATTTCGTTATCGATCTAAAAGAGTTGTATATATTGGTGTAACAAA TGAAATTCAGAAATACAGAAATGAACAATCCACTTACGCTCCAAAAATAATAATAA TAAAATAATAACAACAACAATAATGATGACGATGATTTCGTTAGTAATGTTTGTAAA GCCGCTTGTTGTTAG >Solanum_chacoense_M6 ACAAACTCATATAAAATGGCAAATATTTTGCTTATATTTTCTTTGGTTCTTATGATTA TATATATCATCAGAAGTTATTGCAAGGGAGATAGTTGAGCCTTCACTTCACTTCACT TCATTGCTTGAGGGTAAGTTGTTTTTTAATTGTAGTTTTGCTATATTTAATTTTTGAAC ATATAATTTCGTTATCGATCTAAAAGAGTTGTATATATTGGTGTAACAAATGAAATT CAGAATACAGAAATGAAAAAAAAGGTGGGAAATGGCCGGGATGGATTTTTGATGCA GCGTGTTCACGTTGCCCTTGCCCAAGCAAAGATAATAATAATAATAATAACAACAAC AATAATGATGACGATGATTTCGTTAGTAATGTTTGTAAAGCCGCTTGTTGTTAG 398 CLUSTAL O(1.2.4) multiple sequence alignment M6_1 ACAAACTCATATAAAAT---------GGCAAATATTTTGCTTATATTTTCTTTGGTTCTT 80-1_1 ACAAACTCATATAAAATGGCATTCAAGGCAAATATTTTGCTTATATTTTCTTTGGTTCTT 80-1_2 --AAACTCATATAAAATGGCATTCAAGGCAAATATTTTGCTTATATTTTCTTTGGTTCTT *************** ********************************** M6_1 ATGATTATATATATCATCAGAAGTTA---TTGCAAGGGAGATAGTTGAGCCTTCACTTCA 80-1_1 AA------TTATATCATCAGAAGTTAT---TGCAAGGGAGATAGTTGAGCCTTCACTTC- 80-1_2 AA------TTATATCATCAGAAGTTATTGGGGCAAGGGAGATAGTTGAGCCTTCACTTC- *: :***************** **************************** M6_1 CTTCACTTCATTGCTTGAGGGTAAGTTGTTT-------TTTAATTGTAGTTTTGCTATAT 80-1_1 --------CATTGCTTGAGGGTAAGTTGGTTT------TTTAATTGTAGTTTTGCTATAT 80-1_2 --------CATTGCTTGAGGGTAAGTTGGTTCCTTTTTTTTAATTGTAGTTTTGCTATAT ******************** ** ********************** M6_1 TTAATTT-----TTGAACATATAATTTCGTTATCGATCTAAAAGAGTTGTATATATTGGT 80-1_1 TTAATTT-----TTGAACATATAATTTCGTTATCGATCTAAAAGAGTTGTATATATTGGT 80-1_2 TTAATTTTTTACACGAACATATAATTTCGTTATCGATCTAAAAGAGTTGTATATATTGGT ******* : ********************************************** M6_1 GTAACAAATGAAATTCAGAAT-ACAGAAATGAAAAAAAAGGTGGGAAATGGCCGGGATGG 80-1_1 GTAACAAATGAAATTCAGA-ATACAGAAATGAAC-------------------------- 80-1_2 GTAACAAATGAAATTCAGAAATACAGAAATGAAC-------------------------- ******************* : ***********. M6_1 ATTTTTGATGCAGCGTGTTCACGTTGCCCTTGCCCAAGCAAAGATAATA--ATAATAATA 80-1_1 ----------------------AATCCA---ACGCT-CCAAAAATAATAATAAT--AATA 80-1_2 ----------------------AATCCACTTACGCT-CCAAAAATAATAATAATAAAATA .:* *. .* *: ****.****** *:: **** M6_1 ATAACAACAACAATAATGATGACGATGATTTCGTTAGTAATGTTTGTAAAGCCGCTTGTT 80-1_1 ATAACAACAACAATAATGATGACGATGATTTCGTTAGTAATGTTTGTAAAGCCGCTTGTT 80-1_2 ATAACAACAACAATAATGATGACGATGATTTCGTTAGTAATGTTTGTAAAGCCGCTTGTT ************************************************************ M6_1 GTTAG 80-1_1 GTTAG 80-1_2 GTTAG ***** 398 341 359 51 60 58 108 110 111 161 156 163 216 211 223 275 244 257 333 276 294 393 336 354 Figure. S5.9. Sequence analysis of the high-top B (HT-B) open reading frame (ORF) in parental lines Solanum chacoense USDA8380-1 (80-1) and Solanum chacoense M6. The HT-B ORF was amplified in genomic DNA from M6 and 80-1 using the forward primer 5’- CAACAAACTCATATAAAATGGC-3’ and reverse primer 5’- CTAACAACAAGCGGCTTTACA-3’ with the Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, United States). The purified PCR products were cloned into the pGEM®-T Easy cloning vector, and transformed into DH5α competent cells (Thermo Fisher, Carlsbad, CA, United States) and 12 colonies Sanger sequenced. Sequences were aligned using Clustal Omega (v.1.2.4) (Sievers & Higgins, 2018). The sequences in FASTA format and the alignments are provided. The predicted start (green) and stop (red) codons are shown for each allele in each genotype. 399 >S.chacoense 80-1 HTB_1 Predicted Protein Sequence MAFKANILLIFSLVLNYIIRSYCKGDS-AFTSIA-G-VGFLIVVLLYLIFEHIISLSI- KSCIYWCNK-NSEYRNEQSNAPKIIIIIITTTIMMTMISLVMFVKPLVV >S.chacoense 80-1 HTB_2 Predicted Protein Sequence MAFKANILLIFSLVLNYIIRSYWGKGDS-AFTSIA-G-VGSFFLIVVLLYLIFYTNI- FRYRSKRVVYIGVTNEIQKYRNEQSTYAPKIIIIK--QQQ---R-FR--CL-SRLLL >S.chacoense M6 HTB Predicted Protein Sequence MANILLIFSLVLMIIYIIRSYCKGDS-AFTSLHFIA-G-VVF-L-FCYI-FLNI- FRYRSKRVVYIGVTNEIQNTEMKKKVGNGRDGFLMQRVHVALAQAKIIIIIITTTIMMTM ISLVMFVKPLVV Figure S5.10. Predicted protein sequences generated from genomic high-top B (HT-B) sequence in parental lines Solanum chacoense USDA8380-1 (80-1) and S. chacoense M6. Open reading frames are highlighted in grey. 400 APPENDIX E: S-RNase Sequence Analysis >Solanum_chacoense_S-RNase_80-1 GGGGAAACTGGAAAATGGTTAAACCACAACTCACATCAGCTCTCTTCATTGTGCTTT TTGCTCTTTCTCCCGCTTATGGGGATTTCGATTCCCTCCAACTGGTATTAACATGGCC AGCATCATTTTGCCATGTTAATGATTGTGTGCGAATAGCTCCAAAAAACTTCACGAT TCACGGGCTTTGGCCGAATAAAGAGGGAACGGTGCTGCAGAACTGCAAGCCAAAAC CTAAGTATGTTAATTTCAAGGTAAGCAATAGCATTTTTTTAGAGCCCGCTTTTCCGCT CAGTTCAATTTACTTGAAAGATTCTTTTCGAAATGCTTACAGGATAAGATGTTCAAC GATCTTGACAAACACTGGATTCAGTTGAAGTTTGATGAAGATTATGGTGAAAAGGA ACAACCTTTATGGCTCTATCAATATTTTAAGCATGGATCTTGTTGTCAGAAAATGTAC AACCAAAACACGTATTTTAGTCTAGCCTTGCGCTTAAAAGACAGGTTTGATCTTCTG AGAACTCTCCAAATACATCATATTTTTCCTGGATCAAGTTATACATTTAAGAAAATC TTTGATGCCGTCAAGACAGCTACTCAAATGGATCCTGACCTTAAGTGTACTAAAGGA GTACCGGAACTATATGAAATAGGCATATGTTTCACCCCAAATGCAGATGCTCTGATT CCATGTCGTCAAAGTAATACATGCGATAGGACAGGAAAAATCTTTTTTCGCTGAACA ACTTCACAT >Solanum_chacoense_S-RNase_M6 AAACTGGAAAATGGTTAAACCACAACTCACATCAGCTCTCTTCATTGTGCTTTTTGCT CTTTCTCCCGCTTATGGGGATTTCGATTCCCTCCAACTGGTATTAACATGGCCAGCAT CATTTTGCCATGTTAATGATTGTGTGCGAATAGCTCCAAAAAACTTCACGATTCACG GGCTTTGGCCGGATAAAGAGGGAACGGTGCTGCAGAACTGCAAGCCAAAACCTAAG TATGTTAATTTCAAGGTAAGCAATAGCATTTTTTTAGAGCCCGCTTTTCCGCTCAGTT CAATTTACTTGAAAGATTCTTTTCGAAATGCTTACAGGATAAGATGTTCAACGATCT TGACAAACACTGGATTCAGTTGAAGTTTGATGAAGATTATGGTGAAAAGGAACAAC CTTTATGGCTCTATCAATATTTTAAGCATGGATCTTGTTGTCAGAAAATGTACAACCA AAACACGTATTTTAGTCTAGCCTTGCGCTTAAAAGACAGGTTTGATCTTCTGAGAAC TCTCCAAATACATCATATTTTTCCTGGATCAAGTTATACATTTAAGAAAATCTTTGAT GCCGTCAAGACAGCTACTCAAATGGATCCTGACCTTAAGTGTACTAAAGGAGTACC GGAACTATATGAAATAGGCATATGTTTCACCCCAAATGCAGATGCTCTGATTCCATG TCGTCAAAGTAATACATGCGATAGGACAGGAAAAATCTTTTTT 401 60 56 720 716 360 356 420 416 240 236 300 296 120 116 180 176 CLUSTAL O(1.2.4) multiple sequence alignment Solanum_chacoense_S-RNase_80-1 GGGGAAACTGGAAAATGGTTAAACCACAACTCACATCAGCTCTCTTCATTGTGCTTTTTG Solanum_chacoense_S-RNase_M6 ----AAACTGGAAAATGGTTAAACCACAACTCACATCAGCTCTCTTCATTGTGCTTTTTG ******************************************************** Solanum_chacoense_S-RNase_80-1 CTCTTTCTCCCGCTTATGGGGATTTCGATTCCCTCCAACTGGTATTAACATGGCCAGCAT Solanum_chacoense_S-RNase_M6 CTCTTTCTCCCGCTTATGGGGATTTCGATTCCCTCCAACTGGTATTAACATGGCCAGCAT ************************************************************ Solanum_chacoense_S-RNase_80-1 CATTTTGCCATGTTAATGATTGTGTGCGAATAGCTCCAAAAAACTTCACGATTCACGGGC Solanum_chacoense_S-RNase_M6 CATTTTGCCATGTTAATGATTGTGTGCGAATAGCTCCAAAAAACTTCACGATTCACGGGC ************************************************************ Solanum_chacoense_S-RNase_80-1 TTTGGCCGAATAAAGAGGGAACGGTGCTGCAGAACTGCAAGCCAAAACCTAAGTATGTTA Solanum_chacoense_S-RNase_M6 TTTGGCCGGATAAAGAGGGAACGGTGCTGCAGAACTGCAAGCCAAAACCTAAGTATGTTA ******** *************************************************** Solanum_chacoense_S-RNase_80-1 ATTTCAAGGTAAGCAATAGCATTTTTTTAGAGCCCGCTTTTCCGCTCAGTTCAATTTACT Solanum_chacoense_S-RNase_M6 ATTTCAAGGTAAGCAATAGCATTTTTTTAGAGCCCGCTTTTCCGCTCAGTTCAATTTACT ************************************************************ Solanum_chacoense_S-RNase_80-1 TGAAAGATTCTTTTCGAAATGCTTACAGGATAAGATGTTCAACGATCTTGACAAACACTG Solanum_chacoense_S-RNase_M6 TGAAAGATTCTTTTCGAAATGCTTACAGGATAAGATGTTCAACGATCTTGACAAACACTG ************************************************************ Solanum_chacoense_S-RNase_80-1 GATTCAGTTGAAGTTTGATGAAGATTATGGTGAAAAGGAACAACCTTTATGGCTCTATCA Solanum_chacoense_S-RNase_M6 GATTCAGTTGAAGTTTGATGAAGATTATGGTGAAAAGGAACAACCTTTATGGCTCTATCA ************************************************************ Solanum_chacoense_S-RNase_80-1 ATATTTTAAGCATGGATCTTGTTGTCAGAAAATGTACAACCAAAACACGTATTTTAGTCT Solanum_chacoense_S-RNase_M6 ATATTTTAAGCATGGATCTTGTTGTCAGAAAATGTACAACCAAAACACGTATTTTAGTCT ************************************************************ Solanum_chacoense_S-RNase_80-1 AGCCTTGCGCTTAAAAGACAGGTTTGATCTTCTGAGAACTCTCCAAATACATCATATTTT Solanum_chacoense_S-RNase_M6 AGCCTTGCGCTTAAAAGACAGGTTTGATCTTCTGAGAACTCTCCAAATACATCATATTTT ************************************************************ Solanum_chacoense_S-RNase_80-1 TCCTGGATCAAGTTATACATTTAAGAAAATCTTTGATGCCGTCAAGACAGCTACTCAAAT Solanum_chacoense_S-RNase_M6 TCCTGGATCAAGTTATACATTTAAGAAAATCTTTGATGCCGTCAAGACAGCTACTCAAAT ************************************************************ Solanum_chacoense_S-RNase_80-1 GGATCCTGACCTTAAGTGTACTAAAGGAGTACCGGAACTATATGAAATAGGCATATGTTT Solanum_chacoense_S-RNase_M6 GGATCCTGACCTTAAGTGTACTAAAGGAGTACCGGAACTATATGAAATAGGCATATGTTT ************************************************************ Solanum_chacoense_S-RNase_80-1 CACCCCAAATGCAGATGCTCTGATTCCATGTCGTCAAAGTAATACATGCGATAGGACAGG Solanum_chacoense_S-RNase_M6 CACCCCAAATGCAGATGCTCTGATTCCATGTCGTCAAAGTAATACATGCGATAGGACAGG ************************************************************ Solanum_chacoense_S-RNase_80-1 AAAAATCTTTTTT Solanum_chacoense_S-RNase_M6 AAAAATCTTTTTT ************* Figure S5.11. Sequence analysis of the S-RNase open reading frame (ORF) in parental lines Solanum chacoense USDA8380-1 (80-1) and Solanum chacoense M6. The S-RNase ORF was amplified in genomic DNA from M6 and 80-1 using the forward primer 5’- 5’- ATGTGAAGTTGTTCAGCGAAA -3’ with the Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, United States). The purified PCR products were cloned into the pGEM®-T Easy cloning vector, and transformed into DH5α competent cells (Thermo Fisher, Carlsbad, CA, United States) and six colonies Sanger sequenced. Sequences were aligned using Clustal Omega (v.1.2.4) (Sievers & Higgins, 2018). The sequences in FASTA format and the alignments are provided. The predicted start (green) and stop (red) codons are shown for each genotype. GGGGAAACTGGAAAATGGTT reverse 600 596 660 656 480 476 540 536 733 729 -3’ and primer 402 >S.chacoense 80-1 S-RNase Predicted Protein Sequence MVKPQLTSALFIVLFALSPAYGDFDSLQLVLTWPASFCHVNDCVRIAPKNFTIHGLWPN KEGTVLQNCKPKPKYVNFKVSNSIFLEPAFPLSSIYLKDSFRNAYRIRCSTILTNTGFS- SLMKIMVKRNNLYGSINILSMDLVVRKCTTKTRILV-PCA-KTGLIF- ELSKYIIFFLDQVIHLRKSLMPSRQLLKWILTLSVLKEYRNYMK-AYVSPQMQML- FHVVKVIHAIGQEKSFFAEQLH >S.chacoense M6 S-RNase Predicted Protein Sequence MVKPQLTSALFIVLFALSPAYGDFDSLQLVLTWPASFCHVNDCVRIAPKNFTIHGLWPD KEGTVLQNCKPKPKYVNFKVSNSIFLEPAFPLSSIYLKDSFRNAYRIRCSTILTNTGFS- SLMKIMVKRNNLYGSINILSMDLVVRKCTTKTRILV-PCA-KTGLIF- ELSKYIIFFLDQVIHLRKSLMPSRQLLKWILTLSVLKEYRNYMK-AYVSPQMQML- FHVVKVIHAIGQEKSF Figure S5.12. Predicted protein sequences generated from genomic S-RNase sequence in parental lines Solanum chacoense USDA8380-1 (80-1) and S. chacoense M6. Open reading frames are highlighted in grey. 403 REFERENCES 404 REFERENCES Bamberg, J., & del Rio, A. (2020). 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BioData Mining, 6(18). doi:10.1186/1756‐0381‐6‐ 18 Zhang, C., Wang, P., Tang, D., Yang, Z., Lu, F., Qi, J., Tawari, N. R., Shang, Y., Li, C., & Huang, S. (2019). The genetic basis of inbreeding depression in potato. Nature Genetics, 51(3), 374-378. 410 CHAPTER 6 CHARACTERIZING THE TRANSCRIPTIONAL AND GLYCOALKALOID RESPONSE TO COLORADO POTATO BEETLE INFESTATION IN SOLANUM CHACOENSE 411 Abstract Accumulation of leptine glycoalkaloids in foliar tissue was previously associated with the strong beetle resistance observed in the S. chacoense line USDA8380-1 (80-1). However, leptine content alone did not completely explain resistance to the Colorado potato beetle under field conditions in an F2 mapping population derived from 80-1 segregating for leptine content and the role of an induced defense response to herbivory in S. chacoense remains unexplored. In this study, the transcriptional and glycoalkaloid response to adult Colorado potato beetle infestation was assessed in 80-1 and the susceptible line S. chacoense EE501F2_093 over a 48-hr period following beetle infestation. Foliar levels of a-solanine and a-chaconine were similar between genotypes and there was no treatment effect observed for foliar leptine concentration in the host plant resistant line 80-1. Gene expression profiling in response to Colorado potato beetle feeding revealed differentially upregulated genes with cell signaling and cellular stress response functions in 80-1. Genes differentially expressed between genotypes following beetle treatment did not contain key known genes in the glycoalkaloid biosynthesis pathway. The allelic sequences of a previously reported candidate acetyltransferase (Soltu.DM.02G006530) chromosome 2 associated with leptine biosynthesis and beetle resistance were characterized in 80-1 and M6, which does not produce leptines. A deletion identified in the M6 allele provides a target for modification of leptine glycoalkaloid production in 80-1 by genome editing. Introduction Plants have evolved chemical defense strategies to mitigate herbivory damage. Members of the Solanaceae family, such as cultivated potato (Solanum tuberosum L.) and tomato (Solanum lycopersicum), produce specialized metabolites with insecticidal properties known as steroidal 412 glycoalkaloids (Lachman et al., 2001; Tingey, 1984). These compounds protect against insect pests through cell membrane disruption and cholinesterase inhibition (Chowański et al., 2016). Glycoalkaloids are composed of a cholesterol-derived skeleton, or aglycone, and a glycosidic group. Biosynthesis of the two most common glycoalkaloids in potato, a-chaconine and a- solanine, is a multi-step process that involves many glycoalkaloid metabolism (GAME) genes (Cárdenas et al., 2015; Itkin et al., 2013; Umemoto et al., 2016). Glycoalkaloid accumulation in potato tissues is influenced by wounding, light exposure and temperature (Friedman & McDonald, 1997; Nie et al., 2019). Wounding in one part of the potato plant can prompt changes in glycoalkaloid content of other tissues and prime the plant for further defense. For example, tuber herbivory increases expression glycoalkaloid biosynthetic genes in potato leaves and delivers increased resistance to aphids (Kumar et al., 2016). In other instances, this herbivore-induced glycoalkaloid response can have undesirable consequences for human health. Tubers of plants defoliated by the Colorado potato beetle, one of the most economically important pests of potato, were found to have increased glycoalkaloid concentrations (Hlywka et al., 1994). Increased glycoalkaloid levels in response to external stimuli is mediated by the coordinated expression of glycoalkaloid biosynthetic genes (Nahar et al., 2017). This regulatory process is achieved by a set of jasmonic acid (JA)-responsive Ethylene Responsive Factor transcription factors (JREs) (Cárdenas et al., 2016; Thagun et al., 2016), including the master regulator JRE4/GAME9 (Nakayasu et al., 2018). Downregulation of the steroidal glycoalkaloid biosynthesis pathway, and altered Colorado potato beetle development, can also be accomplished in potato by RNA interference silencing of biosynthetic genes such as GAME4 (Paudel et al., 2017). 413 Transcriptional profiling of herbivore-attacked plants has revealed expression patterns elicited both by the physical damage and biochemical signature of the insect (Erb & Reymond, 2019). In a previous study, Kaiser et al. (2020) identified differences in constitutive glycoalkaloid and transcriptome profiles between Colorado potato beetle resistant and susceptible individuals from a diploid Solanum chacoense F2 population (Kaiser, Manrique-Carpintero, et al., 2020). Colorado potato beetle resistance under field conditions in this population was correlated to accumulation of the specialized glycoalkaloids leptines I/II (Kaiser, Manrique-Carpintero, et al., 2020). Leptines I and II are potent insecticidal compounds found only in the foliar tissue of select S. chacoense genotypes (Mweetwa et al., 2012; Sinden et al., 1986) and are acetylated forms of the ubiquitous glycoalkaloids a-chaconine and a-solanine, respectively (Ronning et al., 1998). However, the leptine I/II content alone does not explain host plant resistance in S. chacoense (Kaiser et al., 2021; Kaiser, Douches, et al., 2020; Lorenzen et al., 2001). The role of an induced response could explain disparities between glycoalkaloid measurements taken at a single point in time and observed insect resistance. Kaiser et al. (2020) also reported a candidate acetyltransferase (Soltu.DM.02G006530) within a quantitative trait locus (QTL) on chromosome 2 associated with leptine biosynthesis and beetle resistance (Kaiser, Manrique-Carpintero, et al., 2020; Manrique-Carpintero et al., 2014; Sagredo et al., 2006). The aim of this study was to i) determine if a distinct pattern of transcriptional and foliar glycoalkaloid response to Colorado potato beetle defoliation discriminates resistant from susceptible S. chacoense genotypes and ii) clarify the allelic sequences of Soltu.DM.02G006530 in the high-leptine producing line S. chacoense USDA8380-1 and S. chacoense M6, which does not produce leptines. 414 Materials and Methods Plant Material An F2 population segregating for foliar glycoalkaloid content and Colorado potato beetle resistance was previously created from a cross between the S. chacoense line USDA8380-1 (PI 458310, 80-1) and the S. chacoense self-compatible inbred line M6 (Kaiser, Manrique-Carpintero, et al., 2020). Line 80-1 produces high levels of leptines (Sanford et al., 1996) and is resistant to Colorado potato beetle defoliation (Sanford et al., 1997) while M6 does not produce leptines and is susceptible to Colorado potato beetle defoliation (Crossley et al., 2018). The F2 individual EE501F2_093 does not produce leptines and is highly susceptible to the Colorado potato beetle under field conditions and in detached leaf bioassays (Kaiser, Manrique-Carpintero, et al., 2020). All plant material was maintained in tissue culture on Murashige and Skoog (MS) (Murashige & Skoog, 1962) medium (MS salts at 8.8g/L, 3% sucrose, pH 5.8 and 0.6% plant agar) at 22 ˚C and 16-hr photoperiod. Whole Plant Colorado potato beetle time course bioassay Six in vitro plantlets of each genotype (80-1 and EE501F2_093) (N = 12 plantlets) with good roots were transplanted to 6” pots in SUREMIXTM media (Michigan Grower Products Inc., Galesburg, MI). Two plants of each genotype were placed in individual RESTCLOUDTM mesh insect cages (15.3” wide x 15.3” deep x 23.6” long) (N = 6 cages) in a growth chamber maintained under 60% relative humidity, a 16-hr photoperiod, 16°C night and 20°C day temperature and 250 415 mE of light intensity. After two weeks, plants received 20-20-20 Peters Professional General Purpose Fertilizer (ICL-SF USA, Summersville, SC) at a rate of 500mg/l twice weekly. For each genotype, two cages received adult Colorado potato beetles (treatment) and one cage did not receive beetles (control). Adult Colorado potato beetles were collected from untreated foliage of the commercial cultivar S. tuberosum ‘Atlantic’ at the Michigan State University Montcalm Research Center (Lakeview, MI) on July 16th, 2019. Adult beetles were starved for 6 hrs prior to being put on the experimental plants. When plants were 15-weeks old, 50 beetles were applied to treatment cages. To avoid bias in defoliation associated with initial beetle placement, an equal number of beetles was were placed uniformly on the leaves of each of the two plants inside each cage. Leaf tissue was harvested for RNA isolation in the following time course: T0 = immediately preceding beetle placement; T1 = 24 hrs after beetle placement; T2 = 48 hrs after beetle placement. At each of the three timepoints, two biological replicates were prepared by pooling equal quantities of tissue from the third fully expanded leaf of each genotype and condition (treatment or control) (N = 24). Total percent defoliation was visually determined for each treatment plant at T2. RNA extraction, quantification and sequencing RNA was isolated using the Qiagen RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Samples were Turbo DNase (Thermo Fisher Scientific, Waltham, MA, USA) treated, and RNA quantity and quality was assessed using an Agilent 4200 TapeStation (Agilent Technologies Inc., Santa Clara, CA). Libraries were prepared using the Illumina TruSeq Stranded mRNA Library Preparation Kit and approximately 30,000,000 50nt single-end Illumina reads were generated for each sample on the Illumina HiSeq 4000 platform. Raw reads were processed with Trimmomatic 416 (v0.35) (MINLEN = 36, LEADING = 20, TRAILING = 20) to remove low-quality bases, adapters, and primers (Bolger et al., 2014). Cleaned reads were aligned to the potato doubled monoploid S. tuberosum line DM1-3 516 R44 (DM) pseudomolecules (PGSC Version 6.1) using STAR (v2.7.3a) (Dobin et al., 2013). Alignments for each sample generated from separate Illuminia lanes were merged using the Picard tool MergeSamFiles (v2.18.27-Java-1.8) (broadinstitute.github.io/picard). Reads aligning to annotated DM reference genes were counted using HTSeq in union mode (v0.11.2) (Anders et al., 2015). Counts were then analyzed using R package DESeq2 to determine normalized expression values (Love et al., 2014). Count data was normalized by log transformation using the rlog function in the DESeq2 package. The first two principal components of log transformed counts for each sample were visualized using the R package ggplot2 (Wickham, 2016). Treatment was coded to contain the four levels ‘Control’, ‘Treatment 0hrs’, ‘Treatment 24hrs’, and ‘Treatment 48hrs’ and the design ~genotype + time + genotype:treatment was used. Contrasts between the four conditions (‘80-1 Treatment 24hrs’, ‘80-1 Treatment 48hrs’, ‘EE501F2_093 Treatment 24hrs’, and ‘EE501F2_093 Treatment 48hrs’) were performed. Differentially expressed genes were called significant using an adjusted P-value (Benjamini– Hochberg adjustment) and a false discovery rate of <5%. Lists of up- and downregulated genes from the contrasts of the conditions were compared. Heatmaps of the log transformed counts for differentially expressed genes were generated using the R package pheatmap (https://cran.r- project.org/web/packages/pheatmap/pheatmap.pdf). Differentially expressed genes were queried for transcripts of known genes in the glycoalkaloid pathway (Table S6.2). Gene ontology terms for differentially expressed genes were obtained from Spud DB (Hirsch et al., 2014). 417 Data availability. Raw expression and count data is available at NCBI GEO (accession # GSE169331). Identification of allelic variation in candidate leptine biosynthesis gene Primers were designed to amplify the open reading frame (ORF) of Soltu.DM.02G006530 in S. chacoense 80-1 and S. chacoense M6 (Forward: 5’ AGGTGCTGGAAGGAGTCTGA 3’; Reverse 5’ TCCAATGCTTGTATTGCTTTTGGA 3’) with the Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, United States). The purified PCR products were lined into the pGEM®-T Easy cloning vector, and transformed into DH5α competent cells (Thermo Fisher, Carlsbad, CA, United States) and eight colonies Sanger sequenced. To determine if Soltu.DM.02G006530 co-segregates with the presence of leptines, PCR was conducted using GoTaq DNA polymerase (Promega, Fitchburg, WI, United States), the Soltu.DM.02G006530 ORF primers and DNA previously isolated from 80-1, M6, their F1 hybrid and 51 individuals of the F2 population segregating for beetle resistance and leptine production (Kaiser, Manrique- Carpintero, et al., 2020). Of the 62 F2 individuals, 25 produced leptines and 26 did not produce leptines (Table S6.1). The following conditions were used: 94°C for 4 minutes, followed by 35 cycles of 94°C for 15 s, 55°C for 1 min and 72°C for 1 min before a final extension of 5 min at 72°C. PCR products were visualized on 1% agarose gels and scored for the presence or absence of the 80-1 allele 1,285 bp band. Results Whole plant Colorado potato beetle time course bioassay The transcriptional and glycoalkaloid response to Colorado potato beetle infestation was assessed in the host plant resistant line S. chacoense 80-1 and the susceptible line EE501F2_093 over a 48-hr period following beetle infestation. Defoliation damage inflicted by adult Colorado 418 potato beetle feeding during the observation period ranged from 50-100% on EE501F2_093 treatment plants with a mean of 84% and from 0-10% on 80-1 treatment plants with a mean of 3.8% (Table S6.3). Defoliation of 80-1 manifested as ‘leaf clipping’ where leaflets were sheared from the plant but not consumed. After 48 hrs, the majority of beetles in 80-1 treatment cages were located on the floor, walls and ceiling of the cages. Glycoalkaloid response to beetle feeding Leptines I/II were detected in both control and treatment samples of 80-1 but not in EE501F2_093 samples. Leptine content was highest in 80-1 at T1 (mean total leptines = 7.6 mg/g dry weight) but there was not significant difference in leptine content between time points or condition (Figure 6.1). Neither was there a significant difference in a-solanine or a-chaconine content in response to beetle feeding in either genotype at any of the three time points (Figure 6.1). Foliar a-solanine or a-chaconine content was higher in all EE501F2_093 samples than in the 80- 1 samples at each time point, although the difference was not statistically significant (Figure 6.1). Transcriptome response to beetle feeding RNA-seq yielded an average of 14 million clean, trimmed reads per sample of which ~83% mapped uniquely to the DM v6.1 potato genome. An average of 90% of the uniquely mapped reads in each sample were assigned to DM v6.1 annotated transcripts. Principal component analysis (PCA) of the RNA-seq data revealed that genotype accounted for 64% of the observed variance in gene expression (Figure 6.2). A time effect was also observed explaining 11% of variance in gene expression. Greater PCA separation was observed between time points for genotype EE501F2_093 than for 80-1. However, comparing EE501F2_093 gene expression in response to treatment between timepoints identified only a single differentially expressed gene (Soltu.DM.02G010430.1) annotated as a carbonic anhydrase and upregulated at 48 hrs. Pearson’s 419 correlations of biological replicates (log2 transformed counts) were all above 94%, suggesting good reproducibility (Figure S6.1). Correlation between all EE501F2_093 samples (>97%) was slightly higher than that between all 80-1 samples (>93%) (Figure S6.1). Pairwise contrasts between genotypes identified 26 and 12 differentially expressed genes at 24 hrs and 48 hrs after beetle infestation, respectively (Table 6.1, Figure 6.3). Three genes (Soltu.DM.03G000360.1, Soltu.DM.06G003170.1, and Soltu.DM.06G027610.1) differentially expressed genes were shared between the two timepoints and were each down regulated in 80-1 treatment samples. Of the genes displaying a treatment effect in 80-1 compared to EE501F2_093 at 24 hrs after beetle infestation, 11 were upregulated in 80-1 and 15 were downregulated in 80-1 (Table 6.1). A similar proportion of up and down regulated genes in response to treatment between genotypes was observed at 48 hrs after beetle infestation (Table 6.1). Examination of annotation and gene ontology for those genes upregulated in 80-1 treatment samples at 24 hrs revealed that most function in cellular stress response or as receptors (Table 6.1). The set of differentially expressed genes at both time points did not contain key known genes in the glycoalkaloid biosynthesis pathway or Soltu.DM.02G006530, hypothesized to be involved in leptine biosynthesis, (Table 6.1, Table S6.2). Identification of allelic variation in a candidate leptine biosynthesis gene The 80-1 allelic sequence of Soltu.DM.02G006530 is composed of a single 914 bp exon that encodes a predicted 302 amino acid protein (Figure 6.4a). The M6 Soltu.DM.02G006530 allele harbors a deletion spanning the 5’ untranslated region (UTR) and genic region (Figure 6.4a,b). The two M6 alleles share 97.3% identity to each other and 56% identity to the 80-1 exonic sequence (Figure 6.4b). Both M6 alleles are predicted to encode a 50 amino acid protein covering 420 59% of the 80-1 predicted protein with 98% identity. The 80-1 allele co-segregated with the presence of leptines in the 54 F2 progeny tested (Table S6.1). Discussion Beetle infestation does not alter S. chacoense foliar glycoalkaloid profile The difference in Colorado potato beetle feeding behavior and defoliation damage between the two S. chacoense genotypes over the 48-hr observation period was not explained by foliar a- solanine or a-chaconine content. This observation confirms previous reports that as a specialized Solanaceous herbivore, the Colorado potato beetle is well-adapted to these defense compounds in their host plant (Kowalski et al., 1999; Lyytinen et al., 2007). However, the results of this study are noteworthy because they demonstrate that unlike other external stressors, Colorado potato beetle herbivory of Solanum chacoense does not elicit elevated glycoalkaloid levels. Furthermore, Colorado potato beetle infestation did not prompt an increase in foliar leptine content of the resistant genotype 80-1, suggesting that leptines contribute to constitutive, rather than induced, host plant resistance. The fact that leptine content measured under greenhouse conditions could predict defoliation resistance to the Colorado potato beetle under field conditions supports this theory (Kaiser et al., 2021) and recommends leptine content as a metabolite marker for host plant resistance selection. Metabolite markers offer an appealing alternative to genetic markers when selecting for polygenic traits and in understudied crops such as potato (Haas et al., 2020; Melandri et al., 2020; Price et al., 2020; Sprenger et al., 2018). Transcriptional response to beetle infestation differs between S. chacoense genotypes A limited number of genes demonstrated a unique transcriptional response between the beetle resistant and susceptible genotypes examined in this study. The transcriptional response in resistant genotype 80-1 was characterized by an upregulation of genes with cell signaling and 421 cellular stress response functions and a downregulation of genes involved in ion exchange. The defense elicitors secreted by insect saliva or regurgitant are known to induce JA and salicylic acid (SA) biosynthesis and signaling as well as to activate protein kinases (Hogenhout & Bos, 2011; Wu & Baldwin, 2010). Colorado potato beetle elicitors in particular are suggested to both induce and repress gene expression in potato (Lawrence et al., 2008). Early detection and transcriptional response to Colorado potato beetle elicitors may contribute to the defoliation resistance of 80-1 leaves. Although significant changes in gene expression for each genotype between timepoints were not detected, early and transient transcriptional defense reprogramming may not have been captured in the 24-hr period between the first two timepoints used in this study. Allelic variation in a candidate leptine biosynthesis gene Characterization in this study of Soltu.DM.02G006530 allelic variation between S. chacoense lines 80-1 and M6 revealed a deletion in the M6 coding sequence. Spanning the deletion promoter region and start codon, this deletion could explain the previously reported lack of Soltu.DM.02G006530 expression reported by Kaiser et al. (2020) in M6 and beetle susceptible lines from the S. chacoense F2 population. Furthermore, co-segregation of the 80-1 allele with leptine production in individuals from the same F2 population suggests that Soltu.DM.02G006530 may be integral to leptine biosynthesis in this germplasm. Regulatory genes that permit leptine synthesis and/or biosynthetic genes responsible for the production of leptine precursors may be fixed in this population. Screening a diverse array of cultivated germplasm with the primers developed in this study will establish whether the presence of the 80-1 Soltu.DM.02G006530 allele alone is sufficient for leptine production. The sequence information generated in this study will facilitate efforts to modify the leptine biosynthesis pathway through genome editing. 422 APPENDICES 423 APPENDIX A: Chapter 6 Tables Table 6.1. Differentially expressed genes between Solanum chacoense USDA8380-1 (80-1) and the F2 line EE501F2_093 in response to Colorado potato beetle feeding at 24 hrs and 48 hrs post beetle infestation. Genes with an upregulated treatment effect in 80-1 compared to EE501F2_093 have negative log2foldChange while genes with a downregulated treatment effect in 80-1 compared to EE501F2_093 have a positive log2FoldChange. Gene Soltu.DM.08G00 5440.2 Soltu.DM.08G00 5480.1 Soltu.DM.10G02 5590.1 Soltu.DM.07G01 8560.1 Soltu.DM.03G03 3620.1 Soltu.DM.06G03 4550.2 Soltu.DM.11G00 8840.1 Soltu.DM.01G00 1260.1 Soltu.DM.10G02 3000.1 Soltu.DM.01G00 1280.1 Soltu.DM.01G00 Soltu.DM.06G02 1250.1 4680.1 6380.1 4220.1 2180.1 Soltu.DM.01G02 Soltu.DM.08G02 Soltu.DM.01G01 baseM ean log2FoldCh ange lfcS E 92.36 1493.2 4 -7.37 -6.16 323.07 -4.21 28.02 1401.2 6 1586.9 6 -4.04 -4.02 -3.61 167.79 -3.32 124.97 -2.61 344.99 -2.55 95.21 -2.06 162.85 162.94 34.24 66.25 271.83 -1.79 1.93 2.68 3.00 4.25 1.4 3 1.4 0 0.9 3 0.8 3 0.9 8 0.6 2 0.7 5 0.6 5 0.5 5 0.4 7 0.4 1 0.4 4 0.6 6 0.7 0 0.9 8 sta t - 5.1 6 - 4.3 9 - 4.5 4 - 4.8 6 - 4.1 1 - 5.7 8 - 4.4 2 - 4.0 2 - 4.6 2 - 4.3 5 - 4.3 3 4.4 1 4.0 6 4.3 1 4.3 5 padj DMv6.1 Functional Annotation Tim e lipoxygenase lipoxygenase conserved hypothetical protein receptor kinase cytochrome P450 family 77 subfamily A polypeptide 5 pseudogene sulfate transporter 3;1 nodulin MtN21 /EamA-like transporter family protein receptor like protein Major facilitator superfamily protein receptor like protein receptor like protein Sec14p-like phosphatidylinositol transfer family protein RmlC-like cupins superfamily protein RING/U-box superfamily protein Family of unknown function (DUF716) 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 6.5E- 04 1.4E- 02 7.9E- 03 2.5E- 03 3.7E- 02 5.1E- 05 1.3E- 02 4.9E- 02 6.7E- 03 1.5E- 02 1.5E- 02 1.3E- 02 4.4E- 02 1.6E- 02 1.5E- 02 424 Table 6.1 (cont’d) Soltu.DM.12G0 22170.1 Soltu.DM.03G0 31440.1 Soltu.DM.08G0 25490.1 Soltu.DM.03G0 27110.1 Soltu.DM.02G0 24260.1 Soltu.DM.09G0 25140.1 Soltu.DM.06G0 27610.1 Soltu.DM.06G0 03170.1 Soltu.DM.02G0 25270.1 Soltu.DM.03G0 00360.1 Soltu.DM.03G0 30480.1 Soltu.DM.05G0 21720.1 Soltu.DM.05G0 21710.1 Soltu.DM.08G0 07260.1 Soltu.DM.12G0 24650.1 Soltu.DM.09G0 29360.1 Soltu.DM.10G0 30300.1 Soltu.DM.01G0 49230.1 Soltu.DM.02G0 26930.1 Soltu.DM.03G0 23190.1 Soltu.DM.06G0 27610.1 Soltu.DM.06G0 03170.1 Soltu.DM.03G0 00360.1 127. 19 322. 79 64.3 8 97.3 4 360. 85 506. 63 792. 57 278. 36 160. 28 78.7 7 12.0 4 46.6 9 43.8 5 49.9 2 10.5 8 60.7 8 48.3 3 307. 51 267. 79 281. 67 792. 57 278. 36 78.7 7 1. 10 0. 93 1. 07 1. 31 1. 08 1. 01 0. 91 1. 39 1. 26 1. 55 2. 07 2. 07 1. 85 2. 01 1. 85 1. 03 1. 46 0. 52 0. 40 0. 47 0. 92 1. 39 1. 54 4.5 6 5.6 4 5.2 0 4.2 2 5.3 1 5.8 8 7.3 0 4.7 9 5.7 6 5.6 5 4.9 4 - 5.9 7 - 5.4 3 - 4.3 0 - 4.5 0 - 6.6 3 - 4.2 6 - 4.7 1 4.5 6 4.3 3 4.6 2 4.3 3 4.5 3 7.9E- 03 7.0E- 05 6.0E- 04 2.4E- 02 3.9E- 04 5.0E- 05 6.9E- 09 3.4E- 03 5.1E- 05 7.0E- 05 1.8E- 03 2.9E- 05 4.7E- 04 3.9E- 02 2.1E- 02 8.2E- 07 4.2E- 02 1.5E- 02 2.1E- 02 3.6E- 02 1.9E- 02 3.6E- 02 2.1E- 02 5.02 5.21 5.54 5.54 5.74 5.94 6.63 6.64 7.28 8.73 10.2 3 - 12.3 6 - 10.0 3 - 8.62 - 8.33 - 6.82 - 6.20 - 2.43 1.80 2.06 4.26 6.01 6.95 SPFH/Band 7/PHB domain-containing membrane-associated chlorophyllase protein family Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily protein voltage dependent anion channel Lactoylglutathione lyase / glyoxalase I family protein aspartic proteinase A1 conserved hypothetical protein cation exchanger Sulfite exporter TauE/SafE family protein aspartic proteinase A1 Late embryogenesis abundant protein (LEA) family protein Thioredoxin superfamily protein Thioredoxin superfamily protein hypothetical protein hypothetical protein Protein of unknown function (DUF803) Sec14p-like phosphatidylinositol transfer family protein conserved hypothetical protein global transcription factor group E7 P-loop containing nucleoside triphosphate hydrolases superfamily protein conserved hypothetical protein cation exchanger aspartic proteinase A1 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs 425 APPENDIX B: Chapter 6 Figures i t n e t n o c d o l a k l a o c y g r a l i l o f n a e M ) t h g i e w y r d g g m / ( I I / I s e n i t p e L i e n n o c a h C - α i e n n a o S - α l Figure 6.1. Foliar glycoalkaloid profiling of resistant and susceptible Solanum chacoense genotypes in response to Colorado potato beetle infestation under growth chamber conditions. The mean content (mg/g dry weight) is shown for a-solanine (bottom), a-chaconine (middle) and leptines I/II (top) in leaf tissue sampled from USDA8380-1 (80-1) and EE501F2_093 (F2_093) at three time points: T0 = before beetles were placed on treatment plants; T1 = 24 hrs after beetles were placed on treatment plants; T2 = 48 hrs after beetles were placed on treatment plants. Treatment plants were in cages that received beetles while control plants were in cages that did not receive beetles. Bars represent the mean of two biological replicates ± standard error of the mean. Chart created using JMP® (Version Pro 13. SAS Institute Inc., Cary, NC). 426 e c n a i r a v % 1 1 : 2 C P Condition and Time Point Genotype PC1: 64% variance Figure 6.2. Principal component analysis of log2 transformed counts of transcriptome data of Colorado potato beetle resistant diploid Solanum chacoense USDA8380-1 (80-1; green) and a susceptible F2 line (EE501F2_093; orange) from the S. chacoense recombinant inbred line population (Kaiser et al., 2021) in response to adult Colorado potato beetle feeding. Control samples at each time point (0 hrs; square, 24 hrs; circle, 48 hrs; triangle) after beetle infestation are given by solid symbols and treatment samples are shown in outline symbols. 427 80-1 F2 093 80-1 F2 093 Figure 6.3. Differential expression observed in leaf tissue of Colorado potato beetle resistant diploid Solanum chacoense USDA8380-1 (80-1) and a susceptible F2 line (EE501F2_093) from the S. chacoense recombinant inbred line population (Kaiser et al., 2021) in response to adult Colorado potato beetle feeding 24hrs and 48 hrs after infestation. The log2 transformed counts of the 36 differentially expressed genes in Colorado potato beetle Treatment (purple bar) and Control (blue bar) leaf samples of each variety (80-1; EE501F2_093 [F2093]) at sampled timepoints (24 and 48 hrs) post beetle placement are shown in the heatmap. Columns are organized by timepoint, genotype and treatment. The doubled monoploid (DM) version 6.1 transcript ID is shown to the right of each row. The color key to the right of the heatmap indicates log2 values. 428 a) 80-1 M6 b) 100 bp Figure 6.4 Gene structure and sequence of Soltu.DM.02G006530. (a) Gene structure of the 5’ untranslated region (UTR) and single exon of Soltu.DM.02G006530 in Solanum chacoense USDA8380-1 (80-1; top) and M6 (bottom). (b) Sequence alignment of 80-1 and M6 Soltu.DM.02G006530 5’ UTR and exon allelic sequences. A black arrow indicates the start codon in the 80-1 allele. Complete nucleotide conservation is shade light grey while blue shading indicates divergent nucleotide sequences. Nucleotide positions are listed in bp to the left and right of each sequence. 429 APPENDIX C: Chapter 6 Supplementary Data Figure S6.1 Pearson correlation of log2 transformed gene counts between samples in the RNAseq experiment. Samples were taken from leaves of Solanum chacoense USDA8380-1 (80- 1) and F2 line EE501F2_093 treatment (T) or control (C) plants at three time points: T0 = before beetle placement, T1 = 24 hrs after beetle placement, T2 = 48 hrs after beetle placement. Two biological replicates were used for each sample. 430 Table S6.1 The presence of leptines (1 = leptines present; 0 = no foliar leptine content) and the Soltu.DM.02G006530 ORF marker(‘+’ = band present; ‘-‘ = no band present) designed in this study. Sample Name Presence of Leptines Marker Generation Parent Parent Solanum chacoense USDA8380-1 EE501F1_02 Solanum chacoense M6 F1 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 F2 EE501F2_37 EE501F2_56 EE501F2_62 EE501F2_69 EE501F2_93 EE501F2_99 EE501F2_145 EE501F2_202 EE501F2_217 EE501F2_233 EE501F2_291 EE501F2_313 EE501F2_341 EE501F2_349 EE501F2_361 EE501F2_363 EE501F2_391 EE501F2_407 EE501F2_454 EE501F2_462 EE501F2_471 EE501F2_500 EE501F2_511 EE501F2_534 EE501F2_540 EE501F2_604 EE501F2_10 EE501F2_11 EE501F2_36 EE501F2_41 EE501F2_44 EE501F2_50 EE501F2_54 EE501F2_71 EE501F2_76 EE501F2_89 EE501F2_113 EE501F2_142 EE501F2_214 EE501F2_220 EE501F2_226 EE501F2_230 EE501F2_259 EE501F2_262 EE501F2_268 EE501F2_271 EE501F2_275 EE501F2_278 EE501F2_294 EE501F2_641 EE501F2_701 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 + + - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + + + + + + 431 Table S6.2 Gene names and representative transcript ID for the doubled monoploid (DM) v6.1 assembly of glycoalkaloid genes inspected in the RNAseq experiment. Gene Name DMv6.1 ID GAME9 SQS1 SSR1 HMGR2 SQE/QO2 SSR2 HMGR1 CD-S5 CAS SQE/QO1 SGT3/ GAME2 SGT1/GAME1 MYC2 SGT2 HMGS Soltu.DM.01G031000.1 Soltu.DM.01G050130.1 Soltu.DM.02G003240.1 Soltu.DM.02G004910.1 Soltu.DM.02G007460.1 Soltu.DM.02G012480.1 Soltu.DM.02G022190.1 Soltu.DM.02G026060.1 Soltu.DM.04G019820.1 Soltu.DM.04G032150.1 Soltu.DM.07G014160.1 Soltu.DM.07G014220.1 Soltu.DM.08G022770.1 Soltu.DM.08G022920.1 Soltu.DM.12G007770.1 Table S6.3 Percent defoliation caused by adult Colorado potato beetle feeding on Solanum chacoense USDA8380-1 (80-1) and EE501F2_093 whole plants at T2 (48 hrs after beetle placement). Treatment Cage Rep % Defoliation at T2 Genotype 80-1 80-1 80-1 80-1 EE501F2_093 EE501F2_093 EE501F2_093 EE501F2_093 1 1 2 2 1 1 2 2 1 2 1 2 1 2 1 2 0 5 0 10 50 95 90 100 432 REFERENCES 433 REFERENCES Anders, S., Pyl, P. T., & Huber, W. (2015). HTSeq—A Python framework to work with high‐ 166-169. 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Annual Reviews in Genetics, 44, 1-24. doi:doi:https://doi.org/10.1146/annurev- genet-102209-163500 437 CHAPTER 7 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS 438 Refining the genetic landscape of glycoalkaloid-mediated host plant resistance This study integrated parental linkage mapping and whole genome bulk segregant analysis in the F2 and F5 generations of a Solanum chacoense recombinant inbred line (RIL) population to identify a quantitative trait locus (QTL) on chromosome 2 with dominance effect associated with Colorado potato beetle resistance and leptine glycoalkaloid production. The ratio of acetylated (leptines) to non-acetylated (solanine and chaconine) glycoalkaloids, rather than leptine content alone, is a reliable predictor of field defoliation resistance. We detected previously uncharacterized single nucleotide polymorphism (SNP) loci associated with the ratio of acetylated to non- acetylated compounds. These SNPs can be used to support marker assisted selection for host-plant resistance using accessible genotyping platforms such as Kompetitive Allele-Specific PCR KASPTM. Furthermore, the deletion we identified in the S. chacoense M6 allele of a candidate acetyltransferase within the QTL on chromosome 2 (Soltu.DM.02G006530), provides a target for genome editing mediated modification of leptine glycoalkaloid production in our leptine- producing parental line S. chacoense USDA8380-1 (80-1). It is possible that in addition to sequence variants, SNPs and indels, accessions of S. chacoense exhibit structural variation including DNA copy number variation (CNV) and presence/absence variation (PAV) associated with Colorado potato beetle resistance. Examination of a diversity panel of wild species (including S. chacoense), landraces, and cultivars revealed that structural variation is widely distributed across tuber-bearing Solanum species (Hardigan et al., 2017). Structural variation has been directly implicated in glycoalkaloid production in potato (Aversano et al., 2017). Of particular interest to this study, a homolog of the gene encoding the functional enzyme responsible for leptinine, a leptine precursor, production in S. chacoense is not present in cultivated potato (Cárdenas et al., 2019). In addition, tandem triplication of the 439 solanidine galactosyltransferase (SGT1) gene in 80-1 was reported previously (Manrique- Carpintero et al., 2014) and segmental duplication gave rise to paralogs in the enzyme that catalyzes the first committed step of glycoalkaloid precursors in S. comersonii. (Villano et al., 2020). Availability of the genomic and transcriptomic profiling of the beetle resistant parental line S. chacoense 80-1 produced in this study enables high-throughput genotyping by whole genome resequencing of our RIL population (Huang et al., 2009). A next generation sequencing approach would allow suitable levels of marker density and recombination resolution to more narrowly define the region on chromosome 2 critical to Colorado potato beetle resistance and to comprehensively query sequence and structural variation in glycoalkaloid metabolism genes. Our transcriptomic and metabolite profiling in response to Colorado potato beetle herbivory suggests that in addition to constitutive leptine production, differential regulation of cell signaling and cellular stress response genes in 80-1 contribute to the strong host plant resistance in this genotype. In Arabidopsis, changes in plant defense signaling hormones, such as jasmonic acid (JA) and salicylic acid (SA), are detectable as early as 3 hours after insect attack and some hormone-responsive genes are activated within 12 hours after insect attack (De Vos et al., 2005). Furthermore, activation of JA signaling can prime plants against future biotic stresses and improve plant stress tolerance (Santino et al., 2013). Analysis of global gene expression and JA/SA kinetics in 80-1 at multiple intervals within the first 24 hours after beetle infestation could reveal if an initial transcriptional reprogramming event is integral to host plant resistance. Achieving sustainable Colorado potato beetle management We have created a diverse collection of diploid potato genetic resources with host plant resistance to the Colorado potato beetle (a mapping population, the first reported inbred lines with resistance, sequenced genomes, and transcriptome profiles of beetle infested plants) to study how 440 S. chacoense host plant resistance can be most effectively deployed in potato varieties. Namely, the inheritance of S. chacoense leptine-based host plant resistance should be further assessed in the diploid introgression hybrids created in this study to formulate the most appropriate introgression breeding strategy. Glycoalkaloid content and composition varies between potato species as a function of genetic variation in both functional biosynthesis enzymes and regulatory elements (Cárdenas et al., 2016; Itkin et al., 2013; Mariot et al., 2016; Shakya & Navarre, 2008). Modification of glycoalkaloid content in potato tissues has been a target of domestication, resulting in reduced levels of total tuber flesh glycoalkaloids (Hardigan et al., 2017; Johns & Alonso, 1990; Zhen et al., 2019) and the absence of certain novel foliar glycoalkaloids in S. tuberosum (Laurila et al., 1996; Paudel et al., 2019; Tai et al., 2015; Tai et al., 2014). Therefore, transmission of leptine-based S. chacoense host plant resistance should be evaluated in a panel of cultivated diploid clones to determine if diverse S. tuberosum backgrounds are permissive of leptine production. Before potato varieties with leptine-based Colorado potato beetle resistance are introduced into commercial production, it is imperative that we have a clearer picture of how leptine-based host plant resistance contributes to or detracts from efforts to intelligently manage the Colorado potato beetle and Colorado potato beetle control products. First, whole plant and detached leaf bioassays should be conducted to determine if the Colorado potato beetle resistant inbred lines developed in this study effectively protect plants from defoliation by a diverse panel of Colorado potato beetle populations. To date there is no evidence of how readily Colorado potato beetle develop resistance to S. chacoense host plant resistance. To provide a measure of the durability of this mechanism of plant protection, Colorado potato beetles could be reared for 10 successive generations on resistant inbred lines and changes in physiology and behavior of surviving beetles characterized at each generation (França et al., 1994). 441 Finally, it is hypothesized that co-evolution of the Colorado potato beetle and Solanaceous plants rich in glycoalkaloids is a contributing factor in the rapid and rampant insecticide resistance evolution observed in the Colorado potato beetle (Alyokhin et al., 2015). If this is true, then it is possible that exposure to glycoalkaloid-based host plant resistance could exacerbate insecticide resistance, making existing chemical control measures less effective. Little data directly supports this hypothesis, but a strain of Colorado potato beetle resistant to carbofuran, azinphosmethyl and permethrin exhibited increased sensitivity to the common glycoalkaloid a-chaconine, suggesting that insecticides and glycoalkaloids may interact at a common target site (Wierenga & Hollingworth, 1992; Wierenga & Hollingworth, 1993). This hypothesis could be tested by exposing Colorado potato beetle populations with and without resistance to commercially applied insecticides to the inbred lines created in this study. Understanding and deploying self-fertility in diploid potato breeding The results of this study illuminate the genetic complexity of self-fertility in diploid potato. We demonstrate in a S. chacoense RIL population that the presence of the dominant S-locus inhibitor (Sli) allele alone is not sufficient to yield fruit and seed set upon selfing. The presence of at least one dominant Sli allele appears to enable self-pollen tube growth to reach the ovary, overcoming the arrest of pollen tube growth in the style characteristic of S-locus RNase (S-RNase) action (Luu et al., 2000). A secondary reaction in the ovary, called late-acting self-incompatibility, is described in angiosperms and may be necessary in addition to Sli to allow pollen tubes to penetrate the ovary or to permit fertilization (Duarte et al., 2020; Gibbs, 2014; Seavey & Bawa, 1986). The mechanism of Sli remains unknown, but segregation distortion patterns in our RIL population support the hypothesis that Sli inhibits self-incompatibly by mediating the interaction 442 of S-RNase and S-locus F-box (SLF) or by interacting directly with S-RNase (McClure et al., 2011). S-RNase is expressed in pistils of the self-compatible (SC) S. chacoense parent M6 and encodes a protein with a predicted ribonuclease domain. Although an identical S-RNase sequence was found in the self-incompatible (SI) parent S. chacoense 8380-1 (80-1) (Chapter 5 Appendix E), we also observed preferential inheritance of the homozygous M6 parental genotype on chromosome 1 in the S-locus region. Post transcriptional modifications of 80-1 S-RNase may render it incapable of interacting with Sli. Crossing SC inbred lines homozygous for the dominant Sli allele to SC lines lacking functional S-RNase by targeted gene editing (Enciso-Rodriguez et al., 2019; Ye et al., 2018) and to their SI wild-type counterparts with functional S-RNase would further reveal the function of Sli. To efficiently assess S-RNase allelic diversity and inform the use of Sli in diploid breeding programs, pollen and pistil transcriptomes could be analyzed by a de novo RNA sequencing approach (Williams et al., 2014). Reduced expression of the self-compatibility modifier high top (HT-B) locus in S. chacoense has been previously shown to confer a SC phenotype (O’Brien et al., 2002). It is unlikely that HT-B contributes to the variation in self-fertility in our RIL lines. Although, we observed preferential inheritance of the homozygous M6 and recombinant genotype at the HT-B locus, both M6 and 80-1 express a predicted non-functional HT-B protein (Chapter 5 Appendix E). Further work should query the genetic composition at other known SC modifier loci in the F5 inbred lines developed in this study. By genotyping a diverse set of diploid SC clones, we identified novel sources of self- compatibility lacking the dominant Sli allele. Identification of these alternate sources of SC expands the genetic base available to breeders seeking to improve SC in breeding populations without introducing the unadapted traits present in M6. Although the F5 inbreds developed in this 443 study are derived from M6, they also contain beneficial self-fertility alleles contributed by parent 80-1 and represent a unique set of self-fertility donors. Crossing the inbred lines developed in this study to SC clones without Sli will assess the potential heterotic effect of combining different SC sources. To circumvent a cumbersome diallel crossing strategy, combining ability could be efficiently evaluated by pollinating individual females with bulk pollen, evaluating F1 hybrids for the desired traits and determining the paternal parent by paternity marker testing (Rudolf-Pilih et al., 2019). Ultimately self-compatibility must be enriched in cultivated diploid backgrounds with agronomic tuber traits. Dihaploids (2x = 2n = 24) produced from tetraploid varieties and breeding lines are a valuable resource to complement the use of wild diploid species. Unfortunately, dihaploids are often male sterile, even when extracted from male fertile tetraploids (Peloquin & Hougas, 1960; Ross et al., 1964). We propose that Sli genotyping is an efficient tool to prioritize dihaploids that harbor dominant Sli alleles. Efforts to disentangle the cytoplasmic male sterility from self-incompatibility are necessary to leverage SC in dihaploid backgrounds (Phumichai et al., 2006) and to facilitate diploid potato hybrid seed production (Anisimova & Gavrilenko, 2017). It is clear from the present work that functional use of SC requires concurrent improvement of male and female fertility traits. Further efforts to investigate the genetic bases of these environmentally influenced traits should consider substantially expanding the number of clonal replicates. Opportunities to accelerate genetic gain in diploid potato We demonstrate that vigorous lines carrying economic traits of interest can be produced by three successive rounds of inbreeding under greenhouse conditions. Because the S. chacoense genotypes used in this study do not tuberize under long-day conditions, we could not assess the 444 correlation between self-fertility and tuber traits. Future work is needed to determine the maximum number of selfed greenhouse generations possible without field evaluation for tuber characteristics. The inbred lines produced in this study represent an ideal set of germplasm in which to test several considerations relevant to launching an inbred/F1 hybrid breeding scheme. For example, the minimum level of homozygosity required for uniform expression of quantitative traits can be ascertained by screening the progeny of our F5 RILs, which exhibit varying degrees of homozygosity. 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