GENETIC DISSECTION OF APHID RESISTANCE IN WILD SOYBEAN GLYCINE SOJA ACCESSION 85-32 By Shichen Zhang 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 2017 ABSTRACT GENETIC DISSECTION OF APHID RESISTANCE IN WILD SOYBEAN GLYCINE SOJA ACCESSION 85-32 By Shichen Zhang The soybean aphid, an invasive species, has been posing a substantial threat on soybean production in North America since its first discovery in 2000. Two novel aphid-resistance quantitative loci (QTLs) were previously revealed as controlling aphid resistance in the wild soybean, Glycine soja 85-32. Therefore, the first objective was to validate these QTLs under different genetic backgrounds. Using single nucleotide polymorphism (SNP) markers, discovered from whole genome resequencing data or mined from the SoySNP50K iSelect BeadChip, two aphid-resistance QTLs were successfully validated and designated Rag6 and Rag3c, respectively. The second objective was to fine map these two loci and identify structural variants within the candidate genes. Rag6 was refined to a 49-kb interval with four candidate genes, including three clustered nucleotide-binding site leucine-rich repeat (NBS-LRR) genes and an amine oxidase encoding gene. Rag3c was refined to a 150-kb interval with eleven candidate genes, two of which are a LRR gene and a lipase gene. By aligning the sequencing reads from the whole genome exome-capture of the resistant source to the soybean reference genome (aphid-susceptible), structural variants (including frame shifts, deletions and nonsynonymous coding changes) were identified within the candidate genes of Rag6 and Rag3c, and new SNPs and insertion/deletions were discovered in the exon regions. The variability and dynamics of aphid population limits the effectiveness of host-resistance gene(s). Therefore, the third objective was to develop and evaluate soybean advanced breeding lines integrated with different aphid-resistance genes. Based on the responses from the indicator lines, Biotype 3 was determined as a major component of aphid populations collected in Michigan during 2015 2016. The different performance of Rag-‘Jackson’ and Rag1-‘Dowling’ along with the breakdown of resistance in plant introductions (PIs) 567301B and 567324 may be explained by Biotype 3 or an unknown virulent biotype establishing in Michigan. Lines with rag1c, Rag3d, Rag6, Rag3c+Rag6, rag1b+rag3, rag1c+rag4, rag1c+rag3+rag4, rag1c+Rag2+rag3+rag4 and rag1b+rag1c+rag3+rag4 demonstrated strong and consistent resistance in five trials across 2015 - 2017. Due to the variability of virulent aphid populations, different combinations of Rag genes may perform differently across geographies. However, advanced breeding lines pyramided with three or four Rag genes will likely provide broader and more durable resistance to diverse and dynamic aphid populations across many geographic regions. This dissertation is dedicated to my father (Qinggen Zhang), mother (Meirong Cheng), sister (Shihou Zhang) and my fiancé (Sean F. Biehn) iv ACKNOWLEDGEMENTS Throughout my journey, up to this point, I have considered myself blessed with many wonderful teachers, mentors and friends. I am grateful for the open-minded atmosphere I had during my undergraduate years, where I had opportunities to join different laboratories and explore the agricultural sciences. My undergraduate research apprenticeship with Dr. Zeng and Dr. Zhou was invaluable. They gave me lots of training in agronomy and plant pathology. A special thanks to Dr. Chen for leading me to the fantastic realm of plant breeding and genetics. I am thankful to be a part of the PBGB program at MSU, where I met a lot of great professors and fellow students. It is a strong community where I gained knowledge and skills from different areas and witnessed collaborations among scientists. Thank you Drs Russell Freed, Dave Douches, Ning Jiang, Eric Olson, Cholani Weebadde, Tylor Johnston, Robin Buell, Robert Last and Sheng-Yang He for your guidance and training. I greatly appreciated all the discussions, advice and support from my committee members, Drs. Amy Iezzoni, Chris DiFonzo and James Kelly. My sincere gratitude to my advisor, Dr. Dechun Wang, for being the best mentor I could hope for. Thank you for training me to be independent and a critical thinker in research, and giving me opportunities to explore and implement my research ideas. I am also grateful for having my wonderful lab mates, Kate Zhang, Carmille Bales, Desmi Chandrasena, Zixiang Wen, Ruijuan Tan, Paul Collins, Feng Lin, John Boyse, Randy Laurenz and Cherry Gu, who all helped and supported my research projects. My appreciation also goes to PBGB, Graduate School, PSM and CANR for their financial support of my Ph.D. fellowship; and to United Soybean Board and Michigan Soybean Promotion v Committee for my research funding and assistantship. I would also like to extend my appreciation to the PSM staff and to my colleagues from other labs. Thank you for providing your generous help when needed. I would also like to acknowledge my friends, who have made my Ph.D. study a great adventure. A special thanks to Tiffany Liu for being studious with me, discussing scientific and philosophical questions about life, and pushing each other through tough times. The most important thing I learned from Tiffany was that the meaning of life is not only about enriching your own life but others as well. I would also like to thank Qianwei Jiang for always being there, supporting me, and giving me great advice. Last, but not least, I am grateful for all the love, support and tremendous advice from my family and the Biehn family. I am fortunate to have parents and a sister, who continue to support and remind me to pursue my dreams. I am also thankful for my grandparents, uncles, aunts and cousins for their love and encouragement. A special thanks to Sean Biehn, for your unconditional love and support, helping me to get through all the challenging times. Thank you again, to everyone, for all your support along my journey. I cannot wait to explore new adventures in science and do my best to enrich people’s lives. vi TABLE OF CONTENTS LIST OF TABLES ix LIST OF FIGURES x CHAPTER 1: LITERATURE REVIEW Soybean and its economic importance The soybean aphid and its impacts Soybean aphid management Host-plant resistance Biochemical mechanisms of host-plant resistance against aphids Identification of aphid-resistance genes in soybean germplasm Genetic approaches to sustain host-plant resistance against soybean aphids Candidate genes for aphid resistance in soybean Next-gen sequencing technologies applied to soybean genomics study Aims of dissertation research REFERENCES 1 2 2 4 5 6 7 15 16 17 18 19 CHAPTER 2: VALIDATION OF QTLS CONTROLLING APHID RESISTANCE IN GLYCINE SOJA 85-32 29 Abstract 30 CHAPTER 3: FINE MAPPING OF THE SOYBEAN APHID RESISTANCE GENES RAG6 AND RAG3C FROM GLYCINE SOJA 85-32 31 Abstract 32 Introduction 33 Materials and Methods 36 Plant materials 36 Soybean aphid resistance bioassay 37 High-throughput DNA isolation with 96-well plates 38 Screening of recombinants and marker development 39 Rag6 validation with a F10:12 residual heterozygous family 41 Assessment of flanking markers of Rag6 and Rag3c in breeding populations 42 Structural variants identification through whole genome exome-capture sequencing of E12901 43 Results 43 Fine mapping delimited Rag6 to a 49-kb interval 43 Fine mapping delimited Rag3c to a 150-kb interval 49 Refined Rag6 region was validated with a F10:12 residual heterozygous family 51 Flanking markers of the refined regions were demonstrated robust in assisting selection for Rag6 and/or Rag3c in breeding populations 53 Structural variant analysis uncovered high-confidence candidate genes 55 Discussion 56 vii APPENDIX REFERENCES 61 76 CHAPTER 4: PYRAMIDING DIFFERENT APHID-RESISTANCE GENES IN ELITE SOYBEAN GERMPLASM TO COMBAT DYNAMIC APHID POPULATIONS 83 Abstract 84 Introduction 85 Materials and methods 89 Plant materials 89 DNA extraction and the Illumina Infinium SoySNP6K iSelect BeadChip genotyping analyses to assess the effectiveness of MAS 91 Evaluation for soybean aphid resistance 92 Results and discussion 93 Data from the Illumina Infinium SoySNP6K iSelect BeadChip verified the successful introgressions of all targeted aphid-resistance genes 93 Indicator lines suggested Biotype 3 and undescribed virulent biotype(s) prevailing in Michigan 95 Lines with rag1c or Rag3d or Rag6 or pyramided Rag genes showed strong and broad resistance 100 Conclusion 104 APPENDIX 106 REFERENCES 108 viii LIST OF TABLES Table 1.1 Soybean aphid biotypes and their virulence to soybean resistance genes 13 Table 1.2 Reported aphid-resistance QTLs in soybean germplasm 14 Table 3.1 Fine mapping populations derived from G. soja 85-32 that were used for screening of recombinants to delimit the locations of Rag6 and Rag3c 37 Table 3.2 Progeny test of Rag6-recombinant lines tested with KASPTM SNP markers 47 Table 3.3 Progeny test of recombinant lines of Rag3c tested with KASPTM SNP markers 50 Table 3.4a Number of plants tested with markers to identify recombination events in the Rag6 and Rag3c intervals each generation and the number of plants selected 62 Table 3.5a Effectiveness of flanking markers in assisting selection for Rag6 and Rag3c in breeding population 130103 and 130170 62 Table 3.6a List of annotated gene models within the interval of Rag6 and structural variants with moderate or high effects 63 Table 3.7a List of annotated gene models within the interval of Rag3c and structural variants with moderate or high effects 64 Table 3.8a Information of SNPs used in the present fine mapping study 66 Table 3.9a SNPs and INDELs discovered from the whole genome exome-capture sequencing of E12901 in the Rag6 and Rag3c fine-mapped regions 71 Table 4.1 Pedigree information for advanced breeding lines integrated with different Rag genes 90 Table 4.2 Aphid damage indices (%) for indicator lines in field trials in Michigan, 2015-2016 95 Table 4.3 Aphid damage indices (%) for advanced breeding lines in field and greenhouse trials in Michigan, 2015-2017 99 ix LIST OF FIGURES Figure 3.1 Graphical representation of chromosome 8 genotypes of critical Rag6 recombinant lines without Rag3c. A Genotypes tested on the Illumina Infinium SoySNP50K iSelect BeadChip array in summer 2013. B Genotypes tested on the Illumina Infinium SoySNP8K iSelect BeadChip array in fall 2014. Colors in bars denote either homozygous genomic regions inherited from the resistant (black) or susceptible (white) parent, or heterozygous regions (gray). Black hatching indicates the previously mapped region of Rag6 (40,047,323 bp - 46,037,031 bp) (Zhang et al. 2017) in A or the narrowed-down region of Rag6 (41,948,645 - 42,338,179 bp) in B. Delimitation analyses are shown with gray lines and black arrows. S represents susceptible phenotype with average DI (%) ranging from 75-100% in the progeny test; MR represents moderate-resistant phenotype with average DI (%) ranging from 37.5-75% in the progeny test; R represents resistant phenotype with average DI (%) ranging from 0-37.5% in the progeny test 46 Figure 3.2 Rag6 validation with a F10:12 residual heterozygous family. A Continuously phenotypic distribution of aphid damage index (%). B Discrete phenotypic distribution of aphid resistance. R means lines with 0-37.5% damage index. H means lines with 37.5-75% damage index. S means lines with 75-100% damage index. C QTL detection of Rag6 using composite interval mapping method. Damage index (%) = å (scale value X no. of plants in the category) / (4 X total no. of plants) X 100 (Mensah et al. 2005) 52 Figure 3.3 Efficiency of marker-assisted selection for Rag6 and/or Rag3c in breeding populations 130103 (A) and 130170 (B). Four distinct genotypes were defined by the presence or absence of the allele from the resistant parent (E12901) for the flanking markers Gm08-15, Gm08-17, Gm16-2, and Gm16-5; - / - represents genotypes carrying susceptible alleles of Rag6 and Rag3c, - / Rag3c represents genotypes carrying susceptible alleles of Rag6 but resistant alleles of Rag3c, Rag6 / - represents genotypes carrying resistant alleles of Rag6 but susceptible alleles of Rag3c and Rag6 / Rag3c represents genotypes carrying resistant alleles of Rag6 and Rag3c. Bars with different letter are significantly different at P < 0.05 54 Figure 4.1 Graphic representation of genomic region(s) of interest for each advanced breeding line. Genomic regions inherited from the original donor(s) of the aphid-resistance gene(s) are presented in black while genomic regions from the susceptible elite background are presented in gray. Targeted aphid-resistance genes with their published genomic locations are listed for each advanced breeding line. Unpublished fine-mapped regions of some Rag genes (including rag1b, rag1c, rag3, rag4) are indicated with rectangle boxes. The genomic locations are according to Glyma.Wm82.a1 on SoyBase (Grant et al. 2010) 94 Figure 4.2 Aphid damage indices (%) of a susceptible check (E00003) and indicator lines used to screen for soybean aphid biotypes in (A) 2015 and (B) 2016 field-cage trials. Bars with same letter(s) are not significantly different at P < 0.05 in each trial 96 x Figure 4.3 Aphid damage indices (%) of a susceptible check (E00003) and the advanced breeding lines with different combinations of aphid-resistance gene(s) in (A) field-cage and greenhouse trials in 2015, (B) field-cage and greenhouse trials in 2016, and (C) a greenhouse trial in 2017. Damage indices from the field-cage trial were presented with gray bars followed by lower-case letters in (A) and (B). Damage indices from the greenhouse trial were presented with black bars followed by upper-case letters in (A) and (B). Within each trial, bars with same letter(s) are not significantly different at P < 0.05 100 Figure 4.4a The genome-wide SNP distribution of the Illumina Infinium SoySNP6K iSelect BeadChip visualized with R 107 xi CHAPTER 1 LITERATURE REVIEW Part of the work presented in this chapter has been accepted in the Book Chapter: Advances in pest-resistant varieties of soybean. Shichen Zhang, Dechun Wang (2017). In H.T. Nguyen (Ed.) Achieving sustainable cultivation of soybeans. Sawston, Cambridge, UK: Burleigh Dodds Science Publishing 1 Soybean and its economic importance The cultivated soybean, Glycine max (L.) Merr., is a legume species native to East Asia, and it has been believed domesticated from the wild ancestor, Glycine soja (Singh 2006). Both G. max and G. soja are annual dicots and belong to the subgenus Soja of the family Leguminosae (Singh 2006). The soybean genome had undergone two rounds of whole genome duplication and a process of diploidization; thus, soybean has been considered as a paleopolyploidy (2n=40) (Shoemaker et al. 1996; Roulin et al. 2013). Soybean was first introduced to North America by a sailor, Samuel Bowen, in 1765 (Hymowitz et al. 1983). Later, soybean has become one of the major crops in the United States as it has multiple uses including animal feed, cooking oil, biofuel, human protein source, etc. In 2016, 83.4 million acres were planted with soybeans in the U.S., ranking the first in world soybean production (4.31 billion bushels) with a total value of $40.94 billion, and 2.03 billion bushels were exported (SoyStats 2016). The soybean aphid and its impacts The soybean aphid, Aphis glycines Matsumura, is an invasive species that originated from China (Wu et al. 2004). Since its first discovery in southern Wisconsin during the summer of 2000 (Alleman et al. 2002), the soybean aphid has aggressively spread to all the major soybean production area in the United States and Canada, and become an economically important pest of soybean (Ragsdale et al. 2011). The soybean aphid is recognized by its black cornicles and pale cauda. It has a heteroecious life cycle with different physical forms and sexual stages (Wu et al. 2004). Female and male aphids 2 mate in the fall and produce eggs to overwinter on the primary host, buckthorn (Frangula alnus). The eggs hatch and develop into wingless fundatrices in the early spring. Later near the blooming stage of buckthorn, these fundatrices asexually reproduce winged alatae that migrate to the secondary host, soybean, during the spring (Wu et al. 2004). After the migration to soybean plants, aphid population build rapidly because of the parthenogenesis and the deformed paedogenesis (Zhang 1988). Due to the rapid unsexual propagation, there are about fifteen generations living on soybean plants during the summer (Wu et al. 2004). They thrive best with temperatures from 22 to 27 ºC during June to August in Michigan. Soybean aphid population densities usually peak during soybean growth stage R3 (the beginning of pod formation) to R5 (the full-size pod) (Ragsdale et al. 2007). The aphid’s stylet feeding removes nutrients and water from soybean plants, resulting in leaf curling, plant wilting and plant death under heavy infestations (Wu et al. 2004); the soybean yield loss caused by aphids’ direct feeding was estimated up to 40% (Ragsdale et al. 2007). Additionally, soybean aphids cause secondary yield loss through transmitting viruses (e.g., Soybean dwarf virus, Soybean mosaic virus, Potato virus, Alfalfa mosaic virus, and Tobacco ring spot virus), which impair soybean growth and yield by causing plant stunting, leaf deformation and reduced pod filling (Hill et al. 2001; Clark &Perry. 2002; Davis et al. 2005). Furthermore, aphids consistently produce honeydew that can cause the growth of sooty mold; excessive sooty mold block soybean plant photosynthesis, leading to additional yield losses (Malumphy, 1997; Lemos Filho and Paiva, 2006). Overall, the economic loss caused by soybean aphids was estimated as $3.6 to $4.9 billion annually in North America (Kim et al. 2008). 3 Soybean aphid management During the growing season of 2003, over 42 million acres of soybean in the north-central U.S. suffered from an outbreak of soybean aphids (Ragsdale et al. 2004). Since then, significant efforts have been made to find solutions to combat soybean aphids; different tactics including chemical, biological and cultural control of aphids have been recommended for protecting soybean yield. Among these tactics, insecticide spray has gained the most popularity in controlling soybean aphids, especially during high outbreak situations. The most commonly applied insecticides in controlling aphids include pyrethroids, neonicotinoids and organophosphates; they are available in the forms of seed treatments or foliar sprays (DiFonzo 2005; Ohnesorg et al. 2009; McCarville and O’Neal 2013). The economic threshold for controlling aphids with insecticides was estimated as 273 aphids per plant to provide a lead-time of seven days before aphid populations reach the economic injury level of 674 aphids per plant (Ragsdale et al. 2007). Due to the rapid establishment of soybean aphids in the U.S., insecticide application on soybean increased from 0.03 million pounds in 2000 to 4.7 million pounds in 2008 (Fernandez-Cornejo et al. 2014). Although insecticide application is effective in protecting soybean yield, this control method increases agricultural input, causes environmental contamination and jeopardizes beneficial insects such as natural enemies and pollinators (Ohnesorg et al. 2009; Lundin et al. 2015). Sometimes, the extensive use of insecticides lead to the appearance of pesticide-resistant insect populations, resurgence of primary pests and secondary pests. Natural enemies such as predators and parasitoids were identified as biological control agents to protect soybean from aphids. Common predators of soybean aphids include the multicolored 4 Asian lady beetle (Harmonia axyridis), insidious flower bug (Orius insidiosus), the lacewing larvae (Chrysopidae sp.) and damsel bugs (Nabidae sp.) (Rutledge et al. 2004). The most common parasitoid of soybean aphids is the parasitic wasp, Aphelinus albipodus, that lays eggs inside soybean aphids, leading to the development of aphid ‘mummies’ (Ragsdale et al. 2011). However, the environmental conditions such as humidity and temperature greatly influence the population size of these natural enemies, leading to inconsistent control of soybean aphids. Host-plant resistance An alternative way of controlling soybean aphids is to employ natural host-plant resistance (HPR) that can provide soybean with economically and environmentally-friendly season-long protection. Thus, HPR is an important component of integrated pest management (IPM), which aims at limiting the usage of pesticides to protect the ecosystem. HPR coupled with biological control is of most interest because they are favorable and compatible in IPM; natural enemies can effectively keep aphid populations in check when the population size is under control by HPR in the early season (Hodgson et al. 2012). There are three categories of HPR, including antibiosis, antixenosis and tolerance (Painter 1958). Antibiosis inhibits the insect biological and/or reproductive process through toxic plant secondary metabolites. Antixenosis morphologically or biochemically deters pests based on a “non-preference” behavior. Tolerance refers to the ability of the plant to maintain its yield under a moderate amount of damage from the pest. As for host-plant resistance against soybean aphids, only antibiosis and antixenosis have been discovered to date and studied in soybean germplasm. 5 Host-plant resistance against soybean aphids have been identified and applied in developing aphid-resistant soybean cultivars (McCarville, 2012). However, one major drawback to employing HPR is the potential lack of its durability, which is usually caused by the emerging virulent aphid biotypes (Kim et al. 2008; Hill et al. 2010; Alt and Ryan-Mahmutagic 2013). In addition to seeking new resistance sources, several methods of sustaining HPR have been practiced, including resistance gene pyramiding, variety mixture and resistance gene deployment based on biotype distribution. Biochemical mechanisms of host-plant resistance against aphids Allelochemicals are secondary plant metabolites that are not essential for plant growth and development; allelochemicals with negative allelopathic effects are known as important for plant defense against herbivory (Stamp 2003). Phytoalexins are antibiotic metabolites that are produced or enriched under biotic stress (Hart et al. 1983). Some flavonoids are important phytoalexins with anti-herbivory effects in Glycine spp. (Burden and Norris 1992). For example, the inducible resistance against Mexican bean beetles (Epilachna varivestis) in PI 227687 was due to increased phenylpropanoid metabolism (total phenolic content) when this accession was challenged by Mexican bean beetles (Chiang et al. 1987). A similar result was reported by Hart et al. (1983) that glyceolin (a type of flavonoid) was functioning as a deterrent to Mexican bean beetles and some other Coleopterans. Coumestrol, another isoflavonoid, was suggested as contributing to antixenosis resistance against Mexican bean beetles in the cultivar “Davis” (Caviness and Walters 1966; Burdern and Norris 1992). Genistein and daidzin can cause greater insect mortality, lower initial larval and pupal weight, reduced growth and elongated larval cycle to southern green stink bugs (Nezara viridula), cabbage loopers (Trichoplusia ni), and 6 velvetbean caterpillars (Anticarsia gemmatalis) (Sharma and Norris 1991; Hoffmann-Campo et al. 2001; Piubelli et al. 2003, 2005). A Chinese soybean cultivar, ‘Zhongdou 27’, has a high isoflavone concentration that help protect soybean from soybean aphid attacks as the major aphid-resistance QTLs found in ‘Zhongdou 27’ were highly associated with high isoflavone content (Meng et al. 2011). Proteinase inhibitors are known as being involved in plant defense to herbivory injury; they are peptides or proteins that inhibit activities of digestive enzymes in the insect gut, thus adversely affecting protein digestion and impeding the growth of insects (Ryan 1990). Broadway and Duffey (1986) reported both soybean trypsin inhibitor and potato proteinase inhibitor significantly reduced the growth of larval beet armyworm (Spodoptera exigua), and larval corn earworm (Helicoverpa zea). The soybean trypsin-chymotrypsin inhibitors induced significant mortality and growth inhibition of the pea aphid (Acyrthosiphon pisum) and potato aphid (Macrosiphum euphorbiae) (Rahbé et al. 2003; Azzouz et al. 2005). Potato proteinase inhibitors I and II increased mortality among late instar aphids and reduced production of nymphs in feeding trials of cereal aphid species (Diuraphis noxia, Schizaphis graminum, Rhopalosiphon padi) (Tran et al. 1997). The insecticidal effects of protein inhibitors were demonstrated in several transgenic plants, enhancing host-plant resistance against lepidopteran and coleopteran pests (Hilder et al. 1987; Johnson et al. 1989; De Leo et al. 2001; Falco and Silva-Filho 2003; Lecardonnel et al. 1999; Alfonso-Rubí et al. 2003). Identification of aphid-resistance genes in soybean germplasm Li et al. (2004) first reported three soybean genotypes, ‘Dowling’ (PI 548663), ‘Jackson’ (PI 548657) and PI 200538, confer antibiosis resistance against the Illinois soybean aphid isolate 7 (Hartman et al. 2001). Antibiosis resistance from ‘Dowling’ and ‘Jackson’ were determined to be controlled by a single dominant gene (Rag1/Rag) that was later mapped to the same location between markers Satt435 and Satt463 on LG M/Chr. 7 (Hill et al. 2006a, b; Li et al. 2007). A subsequent genetic allelism test was conducted among 1,000 F2 plants from ‘Dowling’ × ‘Jackson’ and no susceptible plant was observed, suggesting the genes were allelic (Hill et al. 2012). Later, with additional single nucleotide polymorphism (SNP) markers developed, Rag1 was refined to a 115-kb interval and two nucleotide-binding site leucine-rich repeat (NBS-LRR) genes were proposed as the candidates for Rag1 (Kim et al. 2010a) based on the Williams 82 reference genome annotation on SoyBase (Grant et al. 2010). No yield drag was observed with Rag1 (Kim and Diers 2009), and cultivars with Rag1 were commercially released to growers (McCarville, 2012). However, the resistance conferred by Rag1/Rag to the Illinois soybean aphid isolate was reported overcome by an Ohio isolate in 2006 (Kim et al. 2008). Thus, the Illinois isolate was referred to as Biotype 1 and the Ohio isolate was referred to as Biotype 2 (Kim et al. 2008). Mian et al. (2008a, b) discovered three PIs (243540, 567301B and 567324) exhibiting resistance against Biotypes 1 and 2, and mapped a single dominant gene, Rag2, controlling antibiosis resistance in PI 243540 to LG F/Chr.13 between markers Satt334 and Sct_033. In the biotype study by Kim et al. (2008), PI 200538 remained strong antibiosis resistance to Biotype 2; later, a mapping study by Hill et al. (2009) revealed the underlying gene is the same gene as Rag2 (Mian et al. 2008b) since it resides at the same genomic location and confers identical resistance reactions to different biotypes. Fine mapping of Rag2 in PI 200538 delimited it to a 54-kb region with one NBS-LRR gene as the candidate (Kim et al. 2010b). Fox et al. (2014) found that Rag2 8 was significantly associated with resistance in 20 of the 21 F2 populations derived from 21 newly identified aphid-resistant PIs, suggesting Rag2 may be a major aphid resistance source in the USDA soybean germplasm collection. A different QTL conferring antixenosis resistance in PI 567301B was mapped near Rag2 on LG F/Chr. 13. This QTL referred to a different gene (Rag5) in that detached leaves of PI 567301B did not maintain resistance towards aphids while PI 243540 (source of Rag2) did (Michel et al. 2010; Jun et al. 2012). Additionally, a minor QTL providing antixenosis resistance on LG A2/Chr. 8 near marker BARC-063283-18296 was identified in PI 567301B (Jun et al. 2012). Jun et al. (2013) discovered three aphid-resistance QTLs in PI 567324; two major ones (QTL_13_1 and QTL_13_2) were located close to the previously reported Rag2 locus (Mian et al. 2008a; Kim et al. 2010b) and rag4 locus (Zhang et al. 2009) respectively, and a minor one (QTL_6_1) was detected on chromosome 6, where no aphid-resistant gene has been previously reported. The oligogenic resistance from PI 567324 is expected to provide broader and more durable resistance against aphids compared to cultivars with monogenic resistance (Jun et al. 2013). Hill et al. (2010) reported the discovery of Biotype 3 in Indiana, which readily colonized plants with Rag2. Mensah et al. (2005) screened 2,147 soybean accessions from MG 0 to III and identified four MG III accessions (PI 567543C, PI 567597C, PI 567541B and PI 567598B) resistant to mixed aphid biotypes in Michigan. PI 567541B and PI 567598B possess antibiosis resistance while PI 567543C and PI 567597C possess antixenosis resistance. These four PIs were included in the biotype studies by Kim et al. (2008) and Hill et al. (2010), and found resistant to multiple biotypes. Mensah et al. (2008) reported that aphid-resistance in PI 567541B and PI 567598B were both controlled by two major recessive genes. In PI 567541B, one recessive gene 9 was mapped to the Rag1 region and was named rag1c whereas the other recessive gene was mapped to a different region (Satt649-Satt348) from Rag2 on LG F/Chr.13 and was named rag4 (Zhang et al. 2009). The broad antixenosis resistance provided by PI 567543C and PI 567597C were reported controlled by a single partially dominant gene, Rag3 and Rag3e (mapped in close proximity on LG J/Chr.16), respectively (Zhang et al. 2010; Du 2016). Bales et al. (2013) reported the antibiosis resistance in PI 567598B were contributed by two recessive genes (rag1b and rag3) that were mapped to the previously identified Rag1 (Li et al. 2007; Kim et al. 2010a) and Rag3 (Zhang et al. 2010) regions. Interestingly, in addition to Rag3, Rag3e and rag3, more aphid-resistance genes were mapped to this shared genomic region. Zhang et al. (2013) detected a single dominant gene, Rag3b, conferring antibiosis resistance against mixed biotypes of aphids in PI 567537. Du (2016) fine mapped a partially dominant gene, Rag3d (Liu 2010), in PI 567585A, which confers antibiosis resistance against multiple biotypes. According to the Williams 82 reference genome annotation on SoyBase (Grant et al. 2010), this shared region is enriched with NBS-LRR genes. Fine mapping studies have been conducted and delimiting these Rag genes (Rag3, Rag3b, Rag3d, Rag3e and rag3) to similar or different genomic locations (Bales 2013; Du 2016; unpublished data); it is possible that some of these Rag genes are different NBS-LRR genes while others are different alleles of the same NBS-LRR gene(s). Isoflavone may play a role in plant defense against soybean aphids. Meng et al. (2011) mapped two aphid-resistance QTLs to the same locations of QTLs previously reported as associated with high isoflavone content in a Chinese soybean cultivar, ‘Zhongdou 27’. One of the QTLs (qRa_1) 10 was mapped to Satt470 on LG A2/Chr. 8 and explained a large portion of phenotypic variances. The second QTL (qRa_2) was mapped to Satt144 on LG F/Chr.13. The authors suggested that greater isoflavone concentration could help protect soybean from aphid attacks as some members of isoflavones have been reported as having antibiosis effects on some soybean pests. For example, genistein and daidzin are toxic to southern green stink bug, cabbage looper and velvetbean caterpillar (Sharma and Norris 1991; Hoffmann-Campo et al. 2001; Piubelli et al. 2003, 2005). A Brazilian soybean cultivar, IAC-100, has been reported as having high isoflavone (daidzin and genistin) concentrations (Carrao-Panizzil and Kitamura 1995) and antibiotic resistance against stink bugs (Rosseto 1989). In addition to qRa_1 (Meng et al. 2011) and the minor QTL from PI 567301B (Jun et al. 2012), the antixenosis-resistance QTL, [Rag6]_P203, was delimited to a 192-kb interval between SSR_08_75 and SSR_08_88 on LG A2/Chr. 8 in a Chinese soybean line P203 (Xiao et al. 2013). According to the soybean reference genome (Glyma.Wm82.a1v1), five genes are present in this 192-kb interval, of which a gene encoding Ser/Thr protein kinase was proposed as the strongest candidate for [Rag6]_P203 (Xiao et al. 2013). Compared to the NBS-LRR genes of Rag1 and Rag2, this Ser/Thr protein kinase gene was believed conferring a broad resistance to different aphid populations as it recognizes conserved pathogen-associated molecular patterns, playing a major role in the first layer of the plant immune system (Jones and Dangl 2006) Recently, Alt and Ryan-Mahmutagic (2013) reported that a new aphid biotype, Biotype 4, discovered in Wisconsin readily colonized the previously known resistant soybean genotypes, including ‘Dowling’, PI 243540, PI 200538, Rag1/Rag2 pyramided material, PI 567541B and PI 11 567598B. Among the tested genotypes, PI 567543C and PI 567597C remained resistance to Biotype 4 (Alt and Ryan-Mahmutagic 2013). The four reported aphid biotypes (Table 1.1) and unknown virulent biotypes challenge breeders to continually seek new resistance sources. Kim et al. (2014) detected a possible new allele or gene at the Rag1 region in PI 587732, conferring antibiosis resistance against Biotype 2. Nurden et al. (2010) reported that the antixenosis resistance against Biotype 2 in PI 71506 was controlled by a single dominant gene that is distinct from Rag1. This unknown gene need further study to understand its location in the soybean genome and to provide additional resistance. A single dominant gene controlling antibiosis resistance was mapped to an interval different from Rag2, rag4, Rag5 on LG F/Chr. 13 and therefore referred to as a new aphid-resistance gene, R_P746 (Xiao et al. 2014). All the identified aphid-resistance QTLs are summarized in Table 1.2. 12 Table 1.1 Soybean aphid biotypes and their virulence to soybean resistance genes Biotype Aphid-resistance gene and the resistance source Rag Jackson Reference Rag1 Rag2 PIs Rag1 Rag2 Rag3 rag1b, rag3 rag1c, rag4 Rag3e Rag5 PI Dowling PI 567543C PI 567598B 567301B 243540, PI PI Pyramid 200538 567541B 567597C 1 - - - - - - - - - Hill et al. 2004 2 + + - - - - - - - Kim et al. 2008 3 NA -/+* + - - - -/+¶ - NA Hill et al. 2010 4 NA + + + - + + - NA Alt and RyanMahmutagic 2013 + Implies the aphid biotype readily colonize on the soybean plants with the Rag gene – Implies the aphid biotype cannot colonize on the soybean plants with the Rag gene NA represents ‘Not Available’ in the literature * Soybeans with Rag1 were resistant to Biotype 3 in no-choice tests but susceptible to Biotype 3 in choice tests ¶ PI 567541B was moderate resistant to Biotype 3 in choice tests but susceptible to Biotype 3 in no-choice tes 13 Table 1.2 Reported aphid-resistance QTLs in soybean germplasm Gene Source(s) LG/Chr. Flanking markers Rag / Rag1 ‘Jackson’ / ‘Dowling’ M/7 SNPKS9-3 -- SNPKS5 rag1b PI 567598B M/7 Satt567 -- Satt435 rag1c PI 567541B M/7 Satt299 -- Satt435 Rag2 F/13 SNP46169.7 -- SNP21A Rag3 PIs 200538, 243540 PI 567543C J/16 Sat_339 -- Satt414 rag3 PI 567598B J/16 Satt285 -- Satt414 Rag3b PI 567537 J/16 Satt654 -- Sat_399 Rag3d PI 567585A J/16 Rag3e PI 567597C J/16 rag4 PI 567541B F/13 MSUSNP16-44 -MSUSNP16-124 MSUSNP16-13 -MSUSNP16-124 Satt348 -- Satt649 Rag5 PI 567301B F/13 [Rag6]_P203 P203 A2/8 R_P746 P746 F/13 BARCSOYSSR_13_1131 -BARCSOYSSR_13_1148 SSR_08_75--SSR_08_88 BARCSOYSSR_13_1278 -BARCSOYSSR_13_1363 qRa_1 Zhongdou 27 A2/8 Satt470 qRa_2 Zhongdou 27 F/13 Satt144 QTL_6_1 PI 567324 C2/6 BARCSOYSSR_06_0998 QTL_13_1 PI 567324 F/13 BARCSOYSSR_13_1139 QTL_13_2 PI 567324 F/13 Satt649 a Physical position is according to Glyma.Wm82.a1 (Schmutz et al. 2010) b 2 R represents the percentage of phenotypic variation explained by a QTL c NA represents ‘Not Available’ in the literature Physical Position(bp)a 5,608,084 5,492,694 R2b 4,510,477 NA NA Resistance modality Primarily antibiosis Reference(s) 14.035.5% 44.787.7% NA Antibiosis Bales et al. 2013; Mensah 2008 Antibiosis Mensah 2008; Zhang et al. 2009 Antibiosis 74.390.4% 28.445.8% 78.987.4% 93.1% Antixenosis Hill et al. 2009; Kim et al. 2010b; Mian et al. 2008a, b Mensah et al. 2005; Zhang et al. 2010 Antibiosis Bales et al. 2013; Mensah 2008 Antibiosis Zhang et al. 2013 Antibiosis Liu 2010; Du 2016 90% Antixenosis Du 2016 0.9-9.2% Antibiosis Mensah 2008; Zhang et al. 2009 75-91% Antixenosis Jun et al. 2012; Mian et al. 2008a 39,218,719 39,410,489 31,803,199 33,448,866 NA Antixenosis Xiao et al. 2013 NA Antibiosis Xiao et al. 2014 35,187,929 36,462,969 18,713,522 29,274,967 12,953,321 25-35% 7-11% 4.4-11.6% 42.7-70.6 2.1-13.1% Antibiosis Antibiosis Antixenosis Antixenosis Antixenosis Meng et al. 2011 Meng et al. 2011 Jun et al. 2013 Jun et al. 2013 Jun et al. 2013 29,212,318 29,266,469 NA 2,802,418 NA 9,145,723 7,799,265 6,438,676 6,484,276 6,424,067 6,484,676 5,491,250 12,953,321 29,036,526 29,548,875 14 NAc Hill et al. 2006a,b; Li et al. 2007; Kim et al. 2010a Genetic approaches to sustain host-plant resistance against soybean aphids To date, four soybean aphid biotypes have been discovered (Kim et al. 2008; Hill et al. 2010; Alt and Ryan-Mahmutagic 2013) (Table 1.1), and there are likely more unknown virulent biotypes not yet reported. In addition to the continuing discovery of new resistance sources, pyramiding of different Rag genes (Table 1.2) with the assistance of flanking markers could provide cultivars with broader and more durable resistance. Wiarda et al. (2012) investigated aphid development on soybeans with Rag1 alone, Rag2 alone and both genes combined, and discovered soybeans with both genes were more resistant to aphids. A similar investigation also confirmed that pyramiding Rag1 and Rag2 provides yield protection from aphids in North America (McCarville et al. 2014). Therefore, pyramiding different Rag genes, especially with different resistance modalities, has potential to combat diverse and dynamic aphid populations. Gene deployment based on biotype distribution is another effective method to combat different aphid populations geographically. Therefore, knowledge regarding distribution of the different biotypes is important. Cooper et al. (2015) studied the geographic distribution of aphid biotypes at ten locations between 2008 and 2010 and developed a panel of host differentials (indicator lines) to characterize aphid biotypes. According to this study, aphid populations had been diverse and dynamic across the U.S. and Canada. Additionally, PI 567598B and PI 567541B were identified as the most resistant and durable genotypes against aphid populations. The authors inferred the high level of resistance was due to the natural pyramids of two recessive genes in these two accessions. Deploying different Rag gene(s) according to the soybean aphid biotype distribution could avoid genetic vulnerability of a certain resistant cultivar over a large geographic area. 15 Candidate genes for aphid resistance in soybean The candidate genes of Rag1 and Rag2 were identified as NBS-LRR genes (Kim et al. 2010a, b), which are known to play critical roles in host-plant defense against insects or diseases (Marone et al. 2013). NBS-LRR genes were also predicted as candidate genes for aphid-resistance in other crops, including the Mi gene in tomato that confers resistance to potato aphid (Macrosiphum euphorbiae) (Rossi et al. 1998; Kaloshian et al. 2000; Cooper et al. 2004), the Vat gene in melons underlying resistance to the melon aphid (Aphis gossypii) (Villada et al. 2009), the AKR gene against the blue-green aphid (Acyrthosiphon kondoi Shinji) in Medicago truncatula (Klingler et al. 2005), the TTR gene against the spotted alfalfa aphid (Therioaphis trifolii) (Klingler et al. 2007) and the RAP1 gene resistant to the pea aphid (Acyrthosiphon pisum) (Stewart et al. 2009). In addition to NBS-LRR genes as candidates for aphid-resistance genes, a serine/threonine protein kinase encoding gene was predicted as the candidate gene of [Rag6]_P203 (Xiao et al. 2013). As the serine/threonine protein kinase belongs to the family of transmembrane pattern recognition receptors that recognize conserved pathogen-associated molecular patterns, it was believed to play an important role in the first layer in plant immune system that provided broad resistance against different aphid isolates in P203 (Xiao et al. 2013). Studham and MacIntosh (2013) reported that the soybean aphid colonization leads to a decrease of poly-unsaturated fatty acids, which are used by soybean plants for Jasmonic acid (JA) biosynthesis. JA signaling triggered by aphid infestation is known to play a critical role in regulating plant defense (Thompson et al. 2006). Li et al. (2008) also discovered that the direct feeding from soybean aphids partially activates JA-regulated signaling pathways in soybean 16 defense. Additionally, hundreds of transcripts induced by soybean aphids in the susceptible plants were related to hormone signaling pathways, including abscisic acid and ethylene pathways during plant defense (Studham and MacIntosh 2013). Next-gen sequencing technologies applied to soybean genomics study Recently, massively parallel sequencing platforms have become wildly available, which lead to the dramatic reduction in the cost. In 2010, a high-quality soybean reference genome was built by sequencing the cultivar ‘Willams 82’ with whole-genome shutgun sequencing approach (Schmutz et al. 2010). With the availability of the soybean reference genome, SNPs and insertion/deletions have been efficiently identified by aligning the sequencing reads from diverse soybean genotypes to the reference genome. Song et al. (2013) sequenced reduced representation libraries from six cultivated and two wild soybean (G. soja Sieb. et Zucc.) genotypes; a total of 52,041 SNPs identified from this reduced representation sequencing were used to produce the SoySNP50K iSelect BeadChip. A mapping population consisting of 246 recombinant inbred lines were sequenced at an average of 0.19x depth and 109,273 SNPs were identified and used to construct a linkage map; three QTLs were identified as resistant to southern root-knot nematode (Xu et al. 2013). Li et al. (2014) conducted the de novo assembly of seven phylogenetically and geographically representative G. soja accessions and discovered a broader range of NBS LRRgene domain architectures present in the in the G. soja genome than in the G. max genome. In addition to DNA sequencing, transcriptome sequencing also has been applied in soybean genomics studies. Severin et al. (2010) sequenced the transcriptomes of fourteen diverse tissues; the transcripts discovered from this study greatly helped evaluate gene model annotations for the soybean reference genome. Lee et al. (2017) investigated the transcriptome profiles of soybean 17 near-isogenic lines either with the resistant Rag5 allele or the susceptible rag5 allele before and after the infestation with soybean aphid Biotype 2, and discovered three differentially expressed genes near the Rag5 locus as strong candidate genes. Aims of dissertation research G. soja 85-32 was identified as possessing strong resistance against aphids by Yang et al. (2004). Later, the resistance in G. soja 85-32 was initially discovered as being controlled by two QTLs within a bi-parental population (070020) consisting of 140 F3:4 lines (Zhang 2012). The first objective of the present study was to validate these two QTLs in different genetic backgrounds provided by populations 110193 and 110201, and investigate the inheritance manner of these two QTLs with using the F3-derived lines from population 070020. The second objective was to fine map these two QTLs using SNPs discovered from whole genome re-sequencing of the resistant parent (E12901) and the high throughput genome-wide genotyping technology realized by the Illumina Infinium SoySNP50K/8K iSelect BeadChip. The third objective was to identify structural variants in the exons of candidate genes using the whole-genome exome capture sequencing approach. The fourth objective was to develop and evaluate soybean advanced breeding lines pyramided with different aphid-resistance allelic combinations to combat the dynamic aphid populations in Michigan. 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Translation from Journal of Jilin Agricultural University (Acta Agriculturae Universitatis Jilinensis). 10:15-17 28 CHAPTER 2 VALIDATION OF QTLS CONTROLLING APHID RESISTANCE IN GLYCINE SOJA 85-32 The work presented in this chapter is part of the final publication: Zhang S, Zhang Z, Bales C, Gu C, DiFonzo C, Li M, Song Q, Cregan P, Yang Z, Wang D (2017) Mapping novel aphid resistance QTL from wild soybean, Glycine soja 85-32. Theor Appl Genet. doi:10.1007/s00122-017-2935-z 29 Abstract The soybean aphid is a major pest of soybean. E08934, derived from the wild soybean Glycine soja 85-32, has shown strong and consistent resistance to soybean aphids. Two major quantitative trait loci (QTLs) were previously detected as significantly associated with aphid resistance in a mapping population (070020) derived from the cross E08934 x E00003 (aphid-susceptible). With using indicator lines, E08934 was demonstrated resistant to all known aphid biotypes. E12901, derived from E08934, was used as the resistant parent to construct two validation populations. The BC1F2 population, 110193, was comprised of 262 individuals derived from E12901 x E00003. The F2 population, 110201, was comprised of 396 individuals derived from the cross between E12901 x E09014 (aphid-susceptible). Both populations were evaluated for aphid resistance at three weeks and four weeks after the initial infestation in the greenhouse trial during Fall, 2012. With SNPs discovered from whole genome resequencing of E12901 or mined from the SoySNP50K iSelect BeadChip, the two aphid-resistance QTLs were successfully validated in populations 110193 and 110201; the QTL confirmed between MSUSNP08-40 and MSUSNP08-4 on chromosome 8 was designated Rag6 whereas the QTL confirmed between MSUSNP16-10 and MSUSNP16-15 on chromosome 16 was designated Rag3c. No significant interaction between Rag6 and Rag3c was detected. A total of 75 F3-derived lines from the mapping population, 070020, were used to determine the gene action of Rag6 or Rag3c. Both QTLs were demonstrated as additive; Rag6 is partially dominant while Rag3c was not determined due to the small sample size of Rag3cheterozygotes in the analysis. The new aphid-resistance gene(s) from the wild soybean G. soja 8532 are valuable in breeding soybeans for aphid resistance. For a full text of this work please go to https://doi.org/10.1007/s00122-017-2935-z. 30 CHAPTER 3 FINE MAPPING OF THE SOYBEAN APHID RESISTANCE GENES RAG6 AND RAG3C FROM GLYCINE SOJA 85-32 The work presented in this chapter is under review: Zhang S, Zhang Z, Wen Z, Gu C, An Y, Bales C, DiFonzo C, Song Q, Wang D (2017). Fine mapping of the aphid resistance genes Rag6 and Rag3c from Glycine soja 85-32. Theor Appl Genet (Under Review) 31 Abstract The soybean aphid, an invasive species, has significantly threatened soybean production in North America since 2001. Host-plant resistance is known as an ideal management strategy for aphids. Two novel aphid-resistance loci, Rag6 and Rag3c, from Glycine soja 85-32, were previously detected in a 10.5 centiMorgan (cM)-interval on chromosome 8 and a 7.5 cM-interval on chromosome 16, respectively. Defining the exact genomic position of these two genes is critical for improving the effectiveness of marker-assisted selection for aphid resistance and for identification of the functional genes. To pinpoint the locations of Rag6 and Rag3c, four populations segregating for Rag6 and Rag3c were used to fine map these two genes. The availability of the Illumina Infinium SoySNP50K/8K iSelect BeadChip, combined with single nucleotide polymorphism (SNP) markers discovered through the whole genome re-sequencing of E12901, facilitated the fine mapping process. Rag6 was refined to a 49-kb interval on chromosome 8 with four candidate genes, including three clustered nucleotide-binding site leucine-rich repeat (NBS-LRR) genes and an amine oxidase encoding gene. Rag3c was refined to a 150-kb interval on chromosome 16 with eleven candidate genes, two of which are a NBSLRR gene and a lipase gene. Moreover, by sequencing the whole genome exome-capture of the resistant source (E12901), structural variants were identified in the exons of the candidate genes of Rag6 and Rag3c. The closely linked SNP markers and the candidate gene information presented in this study will be significant resources for integrating Rag6 and Rag3c into elite cultivars and for future functional genetics studies. 32 Introduction The cultivated soybean, Glycine max (L.) Merr., is widely grown for multiple uses including livestock feed, cooking oil, protein source and biodiesel. However, over the past decade, North American soybean production has been threatened by an invasive species, the soybean aphid (Aphis glycines Matsumura), that originated from Asia (Wu et al. 2004). Since its discovery in the Great Lakes region in 2000 (Hartman et al. 2001), soybean aphid has aggressively spread to all the major soybean production areas in the United States and Canada (Ragsdale et al. 2011). Aphid feeding can lead to up to 40 % yield loss (Ragsdale et al. 2007) by the removal of nutrients and water from soybean plants. This results in stunted plants, reduction in yield components (such as seed number and seed weight), lowered oil production (Beckendorf et al. 2008; Ragsdale et al. 2011) and plant death under heavy infestations (Wu et al. 2004). Virus transmission by aphids also impairs soybean growth and yield by causing plant stunting, leaf deformation and reduced pod-fill (Hill et al. 2001; Clark and Perry 2002). Furthermore, aphids excrete sticky honeydew that can lead to the growth of sooty mold, which may block soybean plant photosynthesis and cause additional yield loss (Malumphy 1997; Lemos Filho and Paiva 2006). After the rapid establishment of soybean aphids in North America, insecticide applications on soybeans increased from 0.03 million pounds in 2000 to 4.7 million pounds in 2008 (FernandezCornejo et al. 2014). The growth of insecticide use not only increased costs of production, but also impacted the ecosystem by removing beneficial insects such as natural enemies and pollinators (Ohnesorg et al. 2009; Lundin et al. 2015). An alternative strategy of controlling aphids is to use the native host-plant resistance existing in soybean germplasm. Host-plant resistance can provide plants with economical, environmentally-friendly, and season-long 33 protection against insects or disease. To date, over thirty G. max plant introductions (PIs) and cultivars have been reported with antibiosis resistance (affecting insect growth, survival, or reproduction) or antixenosis resistance (affecting insect behavior) against aphids in North America (Hill et al. 2004a; Li et al. 2004; Mensah et al. 2005; Hesler et al. 2007; Mian et al. 2008a; Fox et al. 2014; Kim et al. 2014). Usually, choice and no-choice tests are used to distinguish between these two different types of host resistance (Mensah et al. 2005). The aphid-resistance QTLs identified in North America were designated as Rag (Resistance to Aphis glycines) genes. Rag and Rag1 were revealed as a single dominant QTL between Satt463 and Satt435 on chromosome 7 that controls antibiosis resistance to aphids in ‘Dowling’ (PI 548663) and ‘Jackson’ (PI 548657), respectively (Hill et al. 2006a, b; Li et al. 2007). The dominant antibiosis resistance gene Rag2 was detected between Satt334 and Sct_033 on chromosome 13 in PI 200538 and PI 243540 (Kang et al. 2008; Mian et al. 2008b; Hill et al. 2009). Rag5, from PI 567301B, was mapped to the same interval as Rag2, but it confers antixenosis resistance (Jun et al. 2012). Zhang et al. (2010) reported a dominant QTL, Rag3, between Sat_339 and Satt414 on chromosome 16, delivering antixenosis resistance in PI 567543C. Another single dominant QTL that controls antibiosis resistance in PI 567537 was later assigned to the same region as Rag3, and therefore designated Rag3b (Zhang et al. 2013). The antibiosis aphid-resistance in PI 567541B was controlled by two recessive QTLs, rag1c and rag4 (Mensah et al. 2008; Zhang et al. 2009). The recessive rag1c was located between Satt299 and Satt435 on chromosome 7 (Zhang et al. 2009), which is in close proximity with Rag/Rag1 (Li et al. 2007). The recessive rag4 was assigned to an interval between Satt649 and Satt348 on chromosome 13 (Zhang et al. 2009), which is different from the location of Rag2 and Rag5 (Kang et al. 2008; Mian et al. 2008b; Hill et al. 2009; Jun et al. 2012). Bales et al. (2013) mapped 34 two recessive QTLs conferring antibiosis resistance in PI 567598B to the same regions as Rag/Rag1 (Li et al. 2007) and Rag3 (Zhang et al. 2010), and designated these QTLs as rag1b and rag3, respectively. Some of the Rag QTLs clearly overlap physically or are in close proximity. Fine mapping studies or allelism tests of these QTLs are needed to unravel their relationships. Among these Rag QTLs, only Rag1 and Rag2-PI 200538 have been fine mapped thus far (Kim et al. 2010a, b). Rag1 was refined to a 115-kb interval on chromosome 7 with two NBS-LRR genes proposed as the best candidates (Kim et al. 2010a). The fine mapping of Rag2 from PI 200538 delimited it to a 54-kb region on chromosome 13, and again, a NBS-LRR gene was the strongest candidate (Kim et al. 2010b). Kim et al. (2014) detected QTLs controlling antibiosis resistance against aphids in the fine-mapped Rag1 and Rag2-PI 200538 regions (Kim 2010a, b) from PI 587732 that might provide different genes or alleles from Rag1 and Rag2. Additional fine mapping studies or allelism tests are needed to understand the relationships among these Rag QTLs that were mapped to similar genomic regions. It is believed that cultivated soybean (G. max) was domesticated from Glycine soja, a wild annual species native to Asia (Singh 2006). G. soja has been reported as resistant to a wide range of diseases and insects, including soybean aphid (Hill et al. 2004b; Yang et al. 2004). A broader range of NBS R-gene domain architectures were discovered present in the G. soja genome than in the G. max genome after the de novo assembly of seven phylogenetically and geographically representative G. soja accessions (Li et al. 2014). Glycine soja 85-32 was reported as resistant to aphids by Yang et al. (2004). This resistance was later identified as antibiosis delivered by two partially dominant QTLs, Rag6 and Rag3c (Zhang et al. 2017). Rag6 was located at a 1.7 Mbinterval (40.9 – 42.6 Mb, Glyma.Wm82.a2 hereafter) with the mapping population and a 6.0 Mb35 interval (40.0 – 46.0 Mb) with the validation populations on chromosome 8 (Zhang et al. 2017); Rag6 is likely a novel gene as it was mapped to a different interval from any other aphidresistance QTL previously identified on chromosome 8 (Meng et al. 2011; Jun et al. 2012; Xiao et al. 2013). Rag3c was located at a 0.9 Mb interval (6.3 - 7.2 Mb) with the mapping population and a 1.9-Mb interval (6.3 - 8.2 Mb) with the validation populations on chromosome 16 (Zhang et al. 2017); it is within the region of Rag3, Rag3b, and rag3 on chromosome 16 (Zhang et al. 2010, 2013; Bales et al. 2013). Despite good insects/disease resistance genes, G. soja carries undesirable agronomic traits that restrict its direct application in commercial breeding programs. Therefore, fine mapping is needed to develop markers closely linked to Rag6 and Rag3c to assist efficient introgression of aphid-resistance from G. soja 85-32 to cultivated soybeans with minimum negative linkage drags. The objectives of this study were to: (1) fine map Rag6 and Rag3c to identify closely linked markers that could be useful in marker-assisted selection, (2) assess the robustness of these markers in assisting selections for these two genes in breeding populations and (3) identify structural variations within the candidate genes of Rag6 and Rag3c by aligning the whole genome exome-capture sequencing reads of the resistant source (E12901) to the reference genome, Glyma.Wm82.a1 (Schmutz et al. 2010). Materials and Methods Plant materials E12901 is an advanced breeding line derived from G. soja 85-32, and has the same aphid resistance phenotype and genotype (Rag6 + Rag3c) (Zhang et al. 2017). As the resistant parent, E12901 was crossed with three aphid-susceptible parents (E00003, E09014 and E09088) to 36 construct four independent populations (110193, 110201, 110202-1, and 110202-2). All four parents are fully homozygous inbred lines. None of the susceptible parents carries any known aphid-resistance gene. The present fine mapping study started with a total of 1161 BC1F2 and F2 plants from these four fine mapping populations (Table 3.1). Table 3.1 Fine mapping populations derived from G. soja 85-32 that were used for screening of recombinants to delimit the locations of Rag6 and Rag3c Population Female Parent Male Parent Starting Generation Number of Lines 110193 E00003¶ E12901* BC1F2 262 110201 E09014 E12901 F2 396 110202-1 E09088 E12901 F2 321 110202-2 E12901 E09088 F2 182 ¶ E00003 was the recurrent parent for population 110193 * E12901 is the resistant parent that was derived from E00003 X (G. soja 85-32 X Jiyu71) Soybean aphid resistance bioassay Greenhouse trials were performed in the fall of 2012, 2013, 2014 and the spring of 2013 and 2015 in the Plant Science Greenhouse of Michigan State University (MSU) in East Lansing, Michigan. The greenhouse was maintained at 26/15 °C day/night. Sodium vapor lights were applied to supplement light intensity during the day for 14 hours. In the greenhouse trials, eight seeds per line were planted in a plastic pot that was 105 mm in diameter and 125 mm deep. Field trials were conducted on the Agronomy Farm of MSU in East Lansing, Michigan in the summers of 2013, 2014, 2015 and 2016. Fifteen seeds of each line were planted in a single row that was 30 cm long with a row spacing of 60 cm inside a 12.8 x 19.5 m aphid / predator-proof polypropylene cage (Redwood Empire Awning Co., Santa Rosa, CA, USA). Soybean lines from the fine mapping populations were randomly arranged in the greenhouse and field trials without replication. 37 In the greenhouse and field-cage trials, each plant was inoculated at the V2 stage (Fehr and Caviness 1977) with two wingless aphids. As shown in the initial mapping study by Zhang et al. (2017), G. soja 85-32 possesses strong and broad resistance to mixed Michigan biotypes that have overcome the resistance provided by ‘Dowling’, ‘Jackson’, PI 200538 and PI 243540. Therefore, the aphids used to infest plants in the present fine mapping study were mixed biotypes collected from the same locations as in the initial mapping study (Zhang et al. 2017) during the early summer of each testing year. Indicator lines (‘Dowling’ and PI 243540) were included as checks in the field-cage trials and they were readily colonized by mixed Michigan aphids, indicating Michigan aphid populations were primarily comprised of Biotype 3 along with possible Biotype 4 and/or unknown biotype(s). Aphid resistance was visually rated for each plant with a scale of 0 to 4 (with increments of 0.5) when the susceptible checks reached a rating of 3.0 (usually three weeks after the initial infestation); the higher score indicates heavier infestation (Mensah et al. 2005). The fine mapping analysis used an aphid damage index (DI) for each line, calculated as DI (%) = å (rating value x no. of plants in the category) / (4 x total no. of plants) x 100 (Mensah et al. 2005). The DI (%) ranged between 0 for no aphid infestation, and 100 for the most severe aphid damage (Mensah et al. 2005, 2008). High-throughput DNA isolation with 96-well plates Before aphid infestation, tissue samples were collected from a non-expanded trifoliolate leaf of each plant, and placed in 1.0 mL individual wells of 96-well plates (USA Scientific, Irvine, CA). Freeze-dried tissue samples were ground with four 4 mm glass beads (Fisher Scientific, Pittsburgh, PA) per well on a modified paint shaker made by Radia (Plymouth, MN) for 2 minutes. To allow ground tissue settle to the bottom of the wells, plates were centrifuged at 3400 rpm for 15 minutes before opening the plate caps (Greiner Bio-One, Kremsmünster, Austria). A 38 CTAB based DNA extraction buffer (buffer A + buffer B, 200 µL) (Kisha et al. 1997) was added to each well and plates were vortexed for 30 seconds to mix the tissue sample with the buffer. After vortexing, plates were placed in a water-bath set at 65°C for 15 minutes. Plates were centrifuged at 3400 rpm for 1 minute before adding 200 µl chloroform : isoamyl alcohol (24:1) to each well at room temperature. After vortexing the plates slightly to mix the solution, plates were centrifuged at 3400 rpm for 15 minutes. 100 µL of supernatant from each well was transferred to a new 0.5 mL 96-well plate (USA Scientific, scientific, Irvine, CA). 200 µL of chilled (-20 °C) ethanol (95%) was added to each well to precipitate DNA. After the centrifugation at 3400 rpm for 15 minutes, plates were quickly drained and 100 µL of roomtemperature 70% ethanol was added to each well to wash the DNA pellets. After centrifuging the plates at 3400 rpm for 5 minutes, plates were quickly drained and air-dried for 30-45 minutes under the fume hood. DNA pellets were re-hydrated overnight with 100 µL of ddH2O in a 4 °C refrigerator. DNA concentration of each sample was determined with a ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA) and was normalized to 20-150 ng/ µL for Taqmanâ or KASPTM SNP genotyping reactions. Screening of recombinants and marker development To be conservative, the BC1F2 and F2 recombinants-screening started with markers flanking the larger intervals of Rag6 (Gm08-3, Gm08-28) and Rag3c (Gm16-1, Gm16-9) suggested by the validation populations in the initial mapping study (Zhang et al. 2017). The Taqmanâ and KASPTM SNP genotyping reactions were performed as described by Zhang et al. (2014) and Zhang et al. (2017). Although no interaction has been detected between Rag6 and Rag3c (Zhang et al. 2017), it was found that the presence of one could confound the recombinant analysis of the 39 other. Therefore, F2 individuals with recombination events in the Rag6 region but with the susceptible genotype of Rag3c were selected as Rag6-recombinants; vice versa, F2 individuals with recombination events in the Rag3c region but with the susceptible genotype of Rag6 were selected as Rag3c-recombinants. F2 individuals with the heterozygous genotype of Rag6 or Rag3c, but with the susceptible genotype of the other Rag QTL, were also selected to be screened for new recombination events of interest in their progenies. A total of 116 F2 individuals (including recombinants and heterozygotes of interest) were selected and their progenies (2,479 F3 recombinant plants) were genotyped and phenotyped in the 2013 spring greenhouse trial (Table 3.4a). Additionally, genomic DNA of nine critical Rag6-recombinants were isolated with a modified CTAB protocol (Kisha et al. 1997), and genotyped with the Illumina Infinium SoySNP50K iSelect BeadChip (Illumina, San Diego, USA) (Song et al. 2013). Starting with the F2:3 generation, the screening for recombinants of Rag6 or Rag3c at each generation were conducted separately (Table 3.4a). By comparing phenotypic data with genotypic data of each recombinant line, the genomic intervals of Rag6 and Rag3c were gradually delimited over generations. At each generation, lines were screened with markers flanking the newly refined regions of Rag6 and Rag3c (Table 3.4a). Recombinant and heterozygous lines of interest were then tested with additional SNP markers (Table 3.8a) that were within the newly refined regions of interest. These SNPs were discovered from whole genome re-sequencing data of E12901 (Bales 2013) or mined from the Illumina Infinium SoySNP50K iSelect BeadChip. Flanking sequences of these SNPs were obtained from the soybean reference genome, Glyma.Wm82.a1(Schmutz et al. 2010). Progenies of each line were assayed for aphid resistance in the next season's greenhouse or field-cage trial. At the F8 generation, genomic DNA of twelve critical Rag6-recombinant lines were isolated with the 40 modified CTAB protocol (Kisha et al. 1997), and genotyped with the customized Illumina Infinium SoySNP8K iSelect BeadChip that is a subset of ~ 7000 SNPs from SoySNP50K with additional SNPs discovered from the whole genome-resequencing of aphid-resistant soybean genotypes, including E12901 (Bales 2013). In the delimitation analyses of Rag6 and Rag3c, genetic associations between a segregating genetic marker and the aphid-resistance phenotypic data of recombinant lines were analyzed using the PROC GLM function in SAS9.4 (SAS Institute, Cary NC). Rag6 validation with a F10:12 residual heterozygous family A F10 residual heterozygous line of Rag6 was identified in the 2015 field-cage trial and developed into a F10:12 residual heterozygous family. Genomic DNA of each line in this family was isolated with the 96-well plate CTAB protocol described earlier. The whole family was genotyped with eleven SNP markers (Table 3.8a) that cover ± 1Mb of the fine-mapped region of Rag6 in spring 2016. A total of 201 F10:12 lines from this residual heterozygous family were evaluated for aphid resistance (DI, %) in the 2016 field-cage trial. Phenotypic and genotypic data of each line from this family were input into the QTL Cartographer V2.5 (Wang et al. 2012) using physical positions obtained from Glyma.Wm82.a2 (Grant et al. 2010) as the map positions of each SNP marker; the forward and backward regression method was used in the composite interval mapping analysis. The LOD threshold was statistically determined from 1000 permutations at a significance level of 0.05. The physical map and the LOD plot were drawn with MapChart 2.2 (Voorrips. 2002). 41 Assessment of flanking markers of Rag6 and Rag3c in breeding populations To assess the effectiveness of flanking markers in assisting selections for soybean lines integrated with Rag6 and/or Rag3c, two breeding populations (130103 and 130170) with a shared genetic background that is different from that of any fine mapping population were evaluated for aphid resistance in the 2016 field-cage trial and genotyped with two markers flanking the fine-mapped Rag6 or Rag3c. Breeding population 130103, consisting of 156 F2:3 individuals, was derived from a cross between E13918 (carrying Rag6 and Rag3c) and E07051 (possessing soybean cyst nematode resistance, phytophthora root and stem rot resistance, high yielding traits). Breeding population 130170, consisting of 502 F2:3 individuals, was derived from a cross between E14902 (carrying Rag6 and Rag3c) and E07051. E13918 and E14902 are two homozygous sibling lines from the cross between E09088 and E12901. E07051 is an advanced homozygous breeding line derived from IA3017 (Bilyeu et al. 2006) x Loda (Nickell et al. 2001). The phenotyping and genotyping process of these two breeding populations were the same as described earlier. Individuals in each breeding population were classified into different genotypic groups based on the alleles of the flanking markers. Distinct genotypes were defined by the presence or absence of the allele from E12901 for flanking markers of Rag6/Rag3c. Genotypes with corresponding aphid resistance phenotype data were analyzed with one-way analysis of variance (ANOVA) and paired-wise comparisons using the PROC GLM function in SAS9.4 (SAS Institute, Cary NC). 42 Structural variants identification through whole genome exome-capture sequencing of E12901 Leaf tissue was collected from young soybean seedlings of E12901, and DNA isolation was performed with the modified CTAB method (Kisha et al. 1997). Fragmented DNA with a peak of 150 to 200 bp long was used to prepare a DNA library with an Illumina TruSeq kit. The library was then hybridized with the NimbleGen SeqCap oligo pool (Roche NimbleGen, Madison, WI) designed to capture and enrich targeted DNA fragments, according to Glyma.Wm82.a1v1 (Schmutz et al. 2010). The enriched DNA fragments were amplified and sequenced on Illumina HiSeq for 2 x 100bp paired end reads. Sequence reads for each sample were quality-trimmed and then mapped to the reference genome Glyma.Wm82.a1 (Schmutz et al. 2010). After sequence reads were mapped to the reference genome, SNPs and insertion/deletions (INDELs) were called using the probabilistic model with CLC Genomics Workbench 7.02, then further filtered with a minimum frequency of 20 %, a minimum average base quality 30. For heterozygous SNP/INDEL, a minimum coverage was 6. The rest had a minimum coverage of 3. Variant annotation was performed using snpEff v3.3 (Cingolani et al. 2012). Results Fine mapping delimited Rag6 to a 49-kb interval At the F2:3 generation, nine critical Rag6 recombinant lines were identified with KASPTM and/or Taqmanâ single SNP genotyping assays and further genotyped with the Illumina Infinium SoySNP50K iSelect BeadChip (Figure 3.1A). Progeny tests of these nine recombinant lines were conducted in the 2013 field-cage trial and some of them were retested in the following seasons (Figure 3.1A). As shown in Figure 3.1A, the associations between the susceptible phenotype and 43 the susceptible genotype of five recombinant lines, R6-1 to R6-5, defined the bottommost border of Rag6 as marker Gm08-9. This conclusion was supported by four aphid-resistant lines, R6-6 to R6-9, with resistant genotype above marker Gm08-9 (Figure 3.1A). Additionally, nineteen recombinant lines (R6-10 to R6-28) were identified with twelve SNP markers at the Rag6 region (Table 3.2). The strong associations between marker Gm08-16 and the segregating phenotypes in the progenies of lines R6-13 and R6-14 suggested Rag6 was to the right of marker Gm08-12 (Table 3.2). In addition, no significant association was observed between the segregating marker Gm08-12 and the susceptible phenotype of line R6-12 (Table 3.2), supporting that the left border of Rag6 was at marker Gm08-12. The significant association between the segregation of aphid resistance and the tested segregating marker in each of the six lines (R6-19, R6-22, R6-23, R6-24, R6-27 and R6-28) defined the right border of Rag6 as marker Gm08-19 (Table 3.2). Therefore, Rag6 was delimited to a 390-kb interval between markers Gm08-12 and Gm08-19 (41,948,645 - 42,338,179 bp, Glyma.Wm82.a2, hereafter, unless otherwise stated) on chromosome 8. This conclusion was supported by ten additional fixed recombinant lines shown with arrows in Table 3.2. To further refine Rag6, a total of twelve high-generation (F8) critical recombinant lines were genotyped with the customized Illumina Infinium SoySNP8K iSelect BeadChip in fall, 2014. Phenotype-genotype associations of five recombinant lines, R6-29 to R6-33, suggested Rag6 was between markers Gm08-6 and Gm08-20 (41,402,338 - 42,448,802 bp), which verified the delimited 390-kb interval of Rag6 (41,948,645 - 42,338,179 bp) (Figure 3.1B). The susceptible phenotype and the susceptible genotype of line R6-34 suggested Rag6 was below marker Gm0818 (Figure 3.1B). Additionally, four lines (R6-35 to R6-38) showed consistent resistance against aphids across 2014 and 2015, and had resistant genotypes below marker Gm08-17. Moreover, 44 resistant lines R6-39 and R6-40 had the resistant genotypes above marker Gm08-14 (Figure 3.1B). Therefore, Rag6 was further defined to a 100-kb interval between markers Gm08-14 and Gm08-17 (42,095,417 – 42,195,720 bp) on chromosome 8. This refined 100-kb interval of Rag6 was supported by the associations between the genotype and phenotype of eight additional recombinant lines, R6-41 to R6-48, listed in Table 3.2. Out of 1295 Rag6-recombiant and -heterozygous lines, only one line (R6-49) was found as having a recombination event in this 100-kb region. Its progenies were heavily colonized by aphids and there was no association between the susceptible phenotype and the segregating marker Gm0815 (P = 0.65, R2 = 0.03), indicating Rag6 is on the right side of marker Gm08-15 (Table 3.2). Therefore, Rag6 was delimited to a 49-kb interval between makers Gm08-15 and Gm08-17 (42,146,252 - 42,195,720 bp) on chromosome 8. 45 Figure 3.1 Graphical representation of chromosome 8 genotypes of critical Rag6 recombinant lines without Rag3c. A Genotypes tested on the Illumina Infinium SoySNP50K iSelect BeadChip array in summer 2013. B Genotypes tested on the Illumina Infinium SoySNP8K iSelect BeadChip array in fall 2014. Colors in bars denote either homozygous genomic regions inherited from the resistant (black) or susceptible (white) parent, or heterozygous regions (gray). Black hatching indicates the previously mapped region of Rag6 (40,047,323 bp - 46,037,031 bp) (Zhang et al. 2017) in A or the narrowed-down region of Rag6 (41,948,645 42,338,179 bp) in B. Delimitation analyses are shown with gray lines and black arrows. S represents susceptible phenotype with average DI (%) ranging from 75-100% in the progeny test; MR represents moderate-resistant phenotype with average DI (%) ranging from 37.5-75% in the progeny test; R represents resistant phenotype with average DI (%) ranging from 0-37.5% in the progeny test 46 Table 3.2 Progeny test of Rag6-recombinant lines tested with KASPTM SNP markers KASPTM SNP markers (Gm- ) and physical positions (Mb)a Pop Line Gen 08-11 08-12 08-14 08-15 08-16 08-17 08-19 08-21 08-24 08-25 08-26 08-27 Progeny test 2014 2015 2015 2015 Markerh Pr > Fi R2j 41.716 41.949 42.095 42.146 42.160 42.195 42.338 42.522 43.391 44.129 45.398 45.913 summer fall spring summer 110- number 202-2 R6-10 F5:6 rb® sc s s s s s s s s s s Se 202-1 R6-11 F3:4 s s s s s s s s s s s S ¬r 202-1 R6-12 F5:6 hd s s s s s s s s s s S 08-12 0.1570 0.25 h® 193 R6-13 F5:6 r h h h h h h h Segf 08-16 <0.0001 0.85 r® ¬r 202-2 R6-14 F5:6 s h h h h h h h Seg 08-16 0.0013 0.7 s® ¬r 193 R6-15 F3:4 r® s s s s s s s s s r S ¬r 202-1 R6-16 F4:5 r r r r r r r r r r s Rg R ¬s 202-2 R6-17 F3:4 s s s s s s s s s r r S ¬r 202-2 R6-18 F3:4 r r r r r r r r r s s R R ¬s 202-2 R6-19 F5:6 s h h h h h r r Seg 08-21 <0.0001 0.9 s® ¬r 202-2 R6-20 F4:5 s s s s s s s s r r r S S ¬r 193 R6-21 F4:5 r r r r r r r r s s s R R ¬s 202-1 R6-22 F5:6 h h h h h h h h r r r Seg 08-21 0.0003 0.80 ¬r 202-2 R6-23 F5:6 h h h h h h h h r r r Seg 08-21 0.0072 0.91 ¬r 202-1 R6-24 F5:6 h h h h h h h h s s s Seg 08-21 <0.0001 0.91 ¬s 202-2 R6-25 F5:6 s s s s s s s s r r r S S ¬r 202-2 R6-26 F5:6 r r r r r r s s s s s R R R R ¬s 202-1 R6-27 F5:6 h h h h h r r r r r Seg 08-16 <0.0001 0.82 ¬r 202-1 R6-28 F4:5 h h h h h s s s s s Seg 08-16 <0.0001 0.97 ¬s 193 R6-41 F8:9 s r r r r R R s® 193 R6-42 F7:8 s r r r r R R R s® 202-2 R6-43 F6:7 r s r r r r R R R s® 202-1 R6-44 F7:8 r r s s s s S S S r® 202-2 R6-45 F7:8 r r s s s s S S r® 202-1 R6-46 F7:8 s s s s r S S S ¬r 202-1 R6-47 F5:6 r r r r r s R R R ¬s 202-2 R6-48 F5:6 r r r r r s R R R ¬s 202-1 R6-49 F4:10 h h h s s s s s s s s S S S 08-15 0.65 0.03 h® 193 R6-50 F8:10 s s h h h s r r r r Seg 08-17 0.0003 0.84 s® ¬s a Physical positions of markers according to Glyma.Wm82.a2 on SoyBase (Grant et al. 2010) b r represents alleles from the resistant parent c s represents alleles from the susceptible parent d h represents heterozygous alleles 47 Table 3.2 (cont’d) e S represents susceptible phenotype with average DI (%) ranging from 75-100% f Seg represents segregating phenotypes g R represents resistant phenotype with average DI (%) ranging from 0-37.5% h Marker used in F-test i Significance level of the marker-trait association j 2 R value of the marker-trait association 48 Fine mapping delimited Rag3c to a 150-kb interval At the F2:3 generation, three lines (R3c-1 to R3c-3) were identified as having recombinant events in the Rag3c region (Table 3.3). A strong association between the segregation of aphid resistance and marker Gm16-7 was observed for each of these three lines (Table 3.3), indicating Rag3c resides to the left of marker Gm16-8. According to the associations between the susceptible genotype and susceptible phenotype of three recombinant lines (R3c-4 to R3c-6), Rag3c was further delimited to the left of marker Gm16-6. This was supported by resistant lines R3c-7 and R3c-8 with the resistant genotype to the left of marker Gm16-6. Furthermore, recombinant line R3c-9 delimited Rag3c to the left of marker Gm16-5 in that a strong association was observed between marker Gm16-4 and the phenotypes of the progenies (P = 0.0020, R2 = 0.75). The association between the resistant phenotype and the resistant genotype in each of the lines (R3c10 to R3c-15) suggested the same right border, marker Gm16-5, for Rag3c. Additionally, six resistant lines, R3c-16 to R3c-21, suggested Rag3c resides to the right of marker Gm16-2. The left border of Rag3c was further pushed to marker Gm16-3 by the phenotype-genotype association of line R3c-22. Therefore, Rag3c was refined to a 150-kb interval between markers Gm16-3 and Gm16-5 (6,621,540 - 6,771,675 bp) on chromosome 16. 49 Table 3.3 Progeny test of recombinant lines of Rag3c tested with KASPTM SNP markers KASPTM SNP markers (Gm- ) and physical positions (Mb)a Progeny test Pop Line 2013 2013 2014 2014 Gen 16-1 16-2 16-3 16-4 16-5 16-6 16-7 16-8 16-9 110- number 6.314 6.618 6.622 6.657 6.772 6.871 6.884 7.229 8.208 summer fall summer fall b e 2 c h 193 R3c-1 F2:3 h h h h h h s Seg (P<0.0001, R =0.96, 16-7)* ¬s h 193 R3c-2 F2:3 h h h h h h ¬s rd Seg (P=0.0022, R2=0.74, 16-7)* h 193 R3c-3 F2:3 h h h h h h s Seg (P=0.0027, R2=0.91, 16-7)* ¬s s 193 R3c-4 F3:4 s s s s r r r Sf S ¬r F3:4 s 193 R3c-5 s s s s r r S S ¬r F s 3:4 193 R3c-6 s s s s ¬r r r r S S r 202-1 R3c-7 F7:8 r r r r s s s Rg R ¬s r 202-1 R3c-8 F6:7 r r r r s s r R R ¬s 2 R3c-9 h 193 F2:3 h h h s s s s Seg (P=0.0020, R =0.75, 16-4)* ¬s r 193 R3c-10 F2:3 r r r ¬s s s s s R R MR r 193 R3c-11 F3:4 r r r s s s MR R R ¬s R3c-12 r 202-1 F5:6 r r r s s s s MR R ¬s r 202-1 R3c-13 F6:7 r r r s s s s MR R ¬s R3c-14 r 202-1 F4:5 r r r ¬s s s s s MR R r 202-1 R3c-15 F4:5 r r r s s s s MR R ¬s 202-1 R3c-16 F3:4 s r r r r r MR R R s® R3c-17 202-1 F5:6 s r r r r r r MR R s® 202-1 R3c-18 F6:7 s s® r r r r r r MR R 202-1 R3c-19 F6:7 s r r r r r r MR R s® R3c-20 202-1 F6:7 s r r r r r r R R s® 202-1 R3c-21 F6:7 s r r r r r r MR MR s® s® 202-2 R3c-22 F5:6 s s r r r r r r MR R a Physical positions of markers according to Glyma.Wm82.a2 on SoyBase (Grant et al. 2010) b h represents heterozygous alleles c s represents alleles from the susceptible parent d r represents alleles from the resistant parent e Seg represents segregating phenotypes f S represents susceptible phenotype with DI (%) ranging from 75-100% g R represents resistant phenotype with DI (%) ranging from 0-37.5% h MR represents moderate resistant phenotype with DI (%) ranging from 37.5-75% *The association between marker Gm16-7/16-4 and the segregation of aphid resistance in the progenies was significant 50 2015 spring 2015 summer R MRh R R MR MR R MR R R R R R R R R MR R R R R R R R Refined Rag6 region was validated with a F10:12 residual heterozygous family A F10 line, R6-50, with a 243-kb heterozygous interval (42,095,417 - 42,338,179 bp) was identified in the 2015 field-cage trial (Table 3.2) and developed into a F10:11 residual heterozygous family in spring, 2016. A total of 201 F10:12 lines of this family were tested with aphids in the 2016 field-cage trial. Aphid-resistance phenotype of the family distributed normally (Figure 3.2A). As suggested in the initial mapping study, Rag6 has an additive effect (Zhang et al. 2017). When grouping lines with a damage index of 0 - 37.5% as resistant, 37.5 - 75% as moderate resistant, and 75 - 100% as susceptible, these three categories followed a 1:2:1 segregation ratio according to the chi-square test, X2 (2, N = 201) = 1.481, P = 0.4768 (Figure 3.2B). The entire family was genotyped with eleven SNP markers covering ± 1 Mb of the finemapped Rag6 region. As shown in Figure 3.2C, a significant peak (LOD = 33.4 while LOD threshold was 1.7 from 1000 permutations at a significance level of 0.05) was detected between markers Gm08-15 and Gm08-19 (42,146,252 - 42,338,179 bp), which validated the fine-mapped region of Rag6 (42,146,252 - 42,195,720 bp). The phenotypic variance explained by this QTL peak was 67.6 %. The additive effect provided by Rag6 was -18.6, indicating the Rag6 allele helps reduce the aphid damage index (%) by 18.6 %. 51 Figure 3.2 Rag6 validation with a F10:12 residual heterozygous family. A Continuously phenotypic distribution of aphid damage index (%). B Discrete phenotypic distribution of aphid resistance. R means lines with 0-37.5% damage index. H means lines with 37.5-75% damage index. S means lines with 75-100% damage index. C QTL detection of Rag6 using composite interval mapping method. Marker positions (Mb) are physical positions according to Glyma.Wm82.a2 on SoyBase (Grant et al. 2010). Damage index (%) = å (scale value X no. of plants in the category) / (4 X total no. of plants) X 100 (Mensah et al. 2005) 52 Figure 3.2 (cont’d) Flanking markers of the refined regions were demonstrated robust in assisting selection for Rag6 and/or Rag3c in breeding populations Breeding population 130103, consisting of 156 F2:3 individuals, and breeding population 130170, consisting of 502 F2:3 individuals, were genotyped with KASPTM SNP markers that flank Rag6 and Rag3c fine-mapped regions, including Gm08-15 (42,146,252 bp), Gm08-17 (42,195,720 bp), Gm16-2 (6,617,689 bp) and Gm16-5 (6,771,675 bp). A total of 328 individuals were grouped into four distinct homozygous genotypes by the presence or absence of the allele from E12901 for these flanking markers (Table 3.5a). Individuals with ambiguous or missing genotype data were excluded from the analysis. From the one-way ANOVA and pair-wise comparison analyses of these four genotype groups, the LSMEANs of the rating score of each genotype group were significantly different (P <0.05), with -/- having the highest damage score 53 and Rag6/Rag3c having the lowest damage score in each breeding population (Figure 3.3A, B). Between the genotypes with only one of the Rag genes from G. soja 85-32, genotypes with Rag6 showed significantly lower aphid damage than genotypes with Rag3c, which is consistent with the observation of Rag6 conferring a stronger resistance in the previous initial mapping study (Zhang et al. 2017). The strong associations between the flanking markers (Gm08-15, Gm08-17, Gm16-2, and Gm16-5) and aphid damage indices demonstrated the robustness of these markers in assisting selections of Rag6 and/or Rag3c under different genetic backgrounds. Figure 3.3 Efficiency of marker-assisted selection for Rag6 and/or Rag3c in breeding populations 130103 (A) and 130170 (B). Four distinct genotypes were defined by the presence or absence of the allele from the resistant parent (E12901) for the flanking markers Gm08-15, Gm08-17, Gm16-2, and Gm16-5; - / - represents genotypes carrying susceptible alleles of Rag6 and Rag3c, - / Rag3c represents genotypes carrying susceptible alleles of Rag6 but resistant alleles of Rag3c, Rag6 / - represents genotypes carrying resistant alleles of Rag6 but susceptible alleles of Rag3c and Rag6 / Rag3c represents genotypes carrying resistant alleles of Rag6 and Rag3c. Bars with different letter are significantly different at P < 0.05 54 Figure 3.3 (cont’d) Structural variant analysis uncovered high-confidence candidate genes According to Glyma.Wm82.a2.v1 on SoyBase (Grant et al. 2010), four candidate genes are present in the 49-kb interval (42,146,252 - 42,195,720 bp) of Rag6, including three clustered NBS-LRR genes (Glyma.08g303500, Glyma.08g303600, and Glyma.08g303700) and one amine oxidase gene (Glyma.08g303800). DNA sequence variations were detected within these candidate genes by comparing the reads from the whole genome exome-capture sequencing of E12901 to the reference genome Glyma.Wm82.a1(Schmutz et al. 2010). Variants with moderate to high effects are listed in Table 3.6a. Two frame shifts caused by deletions, along with a codon deletion and a non-synonymous coding change, were detected in Glyma.08g303500. Due to the absence/unavailability of Glyma.08g303600 in Glyma.Wm82.a1v1, which was used to design the bait sequences for whole genome exome pull-down of soybean, the DNA sequence variants of Glyma.08g303600 are unknown. A total of six non-synonymous coding changes with 55 moderate effects were detected in Glyma.08g303700. Besides a few synonymous coding changes, no variations in the exons of Glyma.08g303800 were detected that can change the protein product. As shown in Table 3.7a, eleven candidate genes are present in the 150-kb interval (6,621,540 6,771,675 bp) of Rag3c based on Glyma.Wm82.a2.v1 (Grant et al. 2010). Of these eleven candidate genes, there is only one NBS-LRR gene (Glyma.16g066800); however, no variations were detected in the exons of this gene. A total of thirteen variations, including a frame shift, were detected in the exons of Glyma.16g066900, which is annotated as encoding lipase. Additionally, a few structural variations were discovered in five other candidate genes (Glyma.16g067000, Glyma.16g067200, Glyma.16g067500, Glyma.16g067800 and Glyma.16g068100) that are unclear in defense mechanisms (Table 3.7a). A total of 185 SNPs and INDELs in Rag6 and Rag3c fine-mapped regions are summarized in Table 3.9. Discussion In this study, two aphid-resistance genes, Rag6 and Rag3c, were fine-mapped to a 49-kb interval (42,146,252 - 42,195,720 bp) and 150-kb interval (6,621,540 - 6,771,675 bp) on chromosome 8 and chromosome 16, respectively. The availability of the Illumina Infinium SoySNP50K/8K iSelect BeadChip, combined with the SNPs discovered through the whole genome re-sequencing of E12901, facilitated the fine mapping process and the development of robust SNP markers in assisting selections of Rag6 and/or Rag3c. Detailed information of all the SNP markers used in this study are summarized in Table 3.8a. The antibiosis resistance gene, Rag6 from G. soja 85-32, discovered by Zhang et al. (2017) was refined to a 49-kb interval in the present study that does not overlap with any other aphid- 56 resistance loci identified on chromosome 8 (Meng et al. 2011; Jun et al. 2012; Xiao et al. 2013). Therefore, Rag6 is a novel gene that can provide additional resistance against soybean aphids. Although multiple aphid-resistance genes (Rag3, Rag3b, Rag3c, and rag3) were detected in a shared region (Zhang et al. 2010, 2013; Bales et al. 2013; Zhang et al. 2017), Rag3c confers a lower aphid-resistance level and is located at a different fine-mapped region than the others (Unpublished dissertations by Zhang 2012, Bales 2013, and Du 2016). According to the genome annotation of Williams 82, this shared region is enriched with NBS-LRR genes; it may explain why multiple aphid-resistance genes were mapped to this region. By mapping the reads from the whole genome exome-capture sequencing of E12901 (derived from G. soja 85-32) to the aphid-susceptible reference genome, Glyma.Wm82.a1.v1, DNA sequence variations were detected within the regions of interest. Multiple structural variants (two frame shifts, two deletions and a non-synonymous coding change) were detected in Glyma.08g303500 that is highly homologous with the gene AT5G45520.1 encoding LRR family protein in Arabidopsis thaliana (Zybailov et al. 2008). These structural variants might change the final protein product of Glyma.08g303500 and lead to the resistance phenotype. It is also possible that Glyma.08g303500 is a susceptibility gene with NBS-LRR domains as a few NBSLRR genes in plants have been reported conferring sensitivity to pathogens (Lorang et al. 2007; Nagy and Bennetzen 2008; Faris et al. 2010); the disrupted protein produced by Glyma.08g303500 in G. soja 85-32 might lose the function of sensitivity to aphids. The homologous gene (AT5G46450.1) of Glyma.08g303600 has been reported as encoding disease resistance proteins (TIR-NBS-LRR family) that confer resistance against cabbage leaf curl virus and Pseudomonas syringae pv. tomato in Arabidopsis (Mohr et al. 2007; Ascencio-Ibáñez et al. 2008). Tan et al. (2007) also reported that the expression level of AT5G46450.1 was altered after 57 flagellin or salicylic acid treatment. Due to the absence/unavailability of Glyma.08g303600 in Glyma.Wm82.a1v1, which was used to design the bait sequence for exome-capture of E12901, structural variants within this gene are unknown. However, possible variants may exist in Glyma.08g303600 and result in the aphid-resistance provided by Rag6. The Arabidopsis gene, AT5G17680.1, is highly homologous to Glyma.08g303700 and was predicted as encoding disease resistance protein (TIR-NBS-LRR family). Interestingly, Yu et al. (2015) reported that eight out of ten NBS-LRR genes in the fine-mapped region of the Asian soybean rust resistance gene Rpp2 are highly homologous with AT5G17680.1. This suggests Glyma.08g303700 might play a role in defense to soybean aphid and its multiple non-synonymous coding changes in G. soja 85-32 may lead to the resistance phenotype. The homolog of Glyma.08g303800 (which encodes amine oxidase) in A. thaliana is AT2G43020.1 with multiple molecular functions, including reducing reactive oxygen species production and increasing defense gene expression (Ascencio-Ibáñez et al. 2008; Sagor et al. 2016). No effective variations in the exons of Glyma.08g303800 were detected, however, structural variation(s) might occur at the promoter or UTR regions that can change the expression of this gene. Therefore, the three NBS-LRR genes (Glyma.08g303500, Glyma.08g303600 and Glyma.08g303700) and the amine oxidase gene (Glyma.08g303800) present in the Rag6 interval are important for future investigations. The best candidate genes for Rag3c are Glyma.16g066800 and Glyma.16g066900 that encode NBS-LRR protein kinase and lipase, respectively. Although no structural variants were detected in the exons of Glyma.16g066800, variation(s) may exist in the promoter or UTR region(s) and lead to the moderate resistance. Multiple structural variants, including a frame shift, were detected within Glyma.16g066900, of which the homolog gene in Arabidopsis is the lipaseencoding gene AT1G53920.1 (Oh et al. 2005). Interestingly, most members in the lipase gene 58 family are required for full local and systemic resistance against pathogen infection in plants (Kumar and Klessig 2003; Jakab et al. 2003; Oh et al. 2005). Jasmonic acids (JAs) are well known as lipid-based hormone signals that are critical for plant defense against herbivory (Paré and Tumlinson 1999). Studham and MacIntosh (2013) reported that soybean aphid colonization leads to a decrease of poly-unsaturated fatty acids, which is used by soybean plants for JA biosynthesis. JA signaling triggered by aphid infestation plays a critical role in regulating plant defense (Thompson et al. 2006). Li et al. (2008) also suggested that aphid-feeding partially activates JA-regulated signaling pathways in soybean defense. Thus, besides the NSB-LRR gene (Glyma.16g066800), it is possible that the moderate resistance of Rag3c is contributed by the lipase gene, Glyma.16g066900. It is well-known that NBS-LRR genes are present in clusters as the result of gene duplication and recombination (Martin et al. 2003; Leister 2004). Clusters of NBS-LRR genes may confer resistance to different pathogens or to different races of the same pathogen (Leister 2004). Three NBS-LRR genes are clustered within the 49kb-interval of Rag6 (Glyma.08g303500, Glyma.08g303600 and Glyma.08g303700) and all share the core sequence information as tandem repeats. In addition to the possibility of one or two NBS-LRR gene(s) governing Rag6 resistance, it is possible that all three NBS-LRR genes are needed for the Rag6-mediated resistance. It is also possible that the durable resistance of Rag6 is achieved by different NBSLRR genes conquering different soybean aphid biotypes. Interestingly, a QTL resistant to Sclerotinia sclerotiorum was mapped in PI 391589B near marker Satt209 (42,190,808 bp) (Guo et al. 2008), which is in close proximity to the NBS-LRR cluster of Rag6; however, PI 391589B is susceptible to soybean aphids (Mensah et al. 2005). According to the field data, G. soja 85-32 is also resistant to S. sclerotiorum (unpublished data). This NBS-LRR gene cluster in G. soja 85- 59 32 may confer resistance to both soybean aphids and S. sclerotiorum. Markers closely linked to this NBS-LRR cluster (e.g. Gm08-15, Gm08-17 and Satt209) can be screened for associations with resistance against S. sclerotiorum in a segregating population derived from G. soja 85-32 to test this hypothesis in the future. Detected DNA structural variants (Table 3.6a and Table 3.7a) within the fine-mapped regions of Rag6 and Rag3c may explain the resistance phenotype delivered by G. soja 85-32. However, candidate gene predictions for Rag6 and Rag3c are based on the reference genome, Williams 82, which is susceptible to soybean aphids. Therefore, the resistance phenotype might be attributed to a different copy number of the NBS-LRR gene(s) or some unknown gene(s) exclusively existing within the fine-mapped regions of Rag6 and Rag3c in the G. soja 85-32 genome. De novo assembly of longer reads from deep re-sequencing (e.g. PacBio) of G. soja 85-32 would be helpful to validate the structure variants detected in this study and uncover possible gene(s) or copy number variance exclusively present in the regions of interest in the G. soja 85-32 genome. The breakdown of aphid-resistance genes by emerging new biotypes in North America (Hill et al. 2004a; Kim et al. 2008; Hill et al. 2010; Alt and Ryan-Mahmutagic 2013) increases the need to identify new resistance genes and the need of gene pyramiding in commercial soybean varieties to achieve durable resistance to aphids. The closely-linked SNP markers identified in the present study were proved robust in assisting selections for Rag6 and/or Rag3c under different genetic backgrounds. They will facilitate marker-assisted selections for aphid resistance from G. soja 85-32 and gene pyramiding with minimum negative linkage drags. Additionally, the predicted candidate genes and the identified structural variations would help expedite future functional studies and map-based cloning efforts. 60 APPENDIX 61 APPENDIX Table 3.4a Number of plants tested with markers to identify recombination events in the Rag6 and Rag3c intervals each generation and the number of plants selected Rag6 Rag3c Gen Tested plantsa Flanking markers Selected plantsb Tested plantsa Flanking markers Selected plantsb F2 1161c Gm08-3, Gm08-28 58/9 1161c Gm16-1, Gm16-9 36/13 F3 1533 Gm08-11, Gm08-27 124/80 946 Gm16-1, Gm16-8 18/75 F4 1780 Gm08-12, Gm08-26 21/10 797 Gm16-2, Gm16-7 25/5 F5 1277 Gm08-12, Gm08-25 44/159 673 Gm16-2, Gm16-6 96/67 F6 1570 Gm08-12, Gm08-24 111/156 1140 Gm16-2, Gm16-5 89/58 F7 2522 Gm08-14, Gm08-21 93/55 1388 Gm16-2, Gm16-5 76/9 F8 1428 Gm08-14, Gm08-19 72/81 694 Gm16-3, Gm16-5 76/6 F9 1209 Gm08-14, Gm08-17 115/34 708 Gm16-3, Gm16-5 92/23 F10 1295 Gm08-15, Gm08-16 1 911 Gm16-3, Gm16-5 a At higher generations, among the tested plants, many of them shared pedigree because they were derived from same heterozygous recombinants. Additionally, some of the tested plants were recombinants that were carried over from the previous season to confirm their phenotype and genotype b Selected plants included recombinants and heterozygotes, indicated by recombinants/heterozygotes. Some recombinant selections were carried over to the following seasons to confirm their phenotype and genotype c Recombinants screening for Rag6 and Rag3c started with 1161 F2 plants. Starting with the F3 generation, screening for recombinants of Rag6 and Rag3c were separated as no interaction had been detected between Rag6 and Rag3c Table 3.5a Effectiveness of flanking markers in assisting selection for Rag6 and Rag3c in breeding population 130103 and 130170 Flanking markersa Pop Genotype No. of lines Mean Standard error Rag6 130103 130170 Gm08-15 Gm08-17 Gm16-2 Gm16-5 -/- 21 - - - - 2.98 0.12 -/Rag3c 15 - - + + 2.25 0.15 Rag6/- 18 + + - - 1.75 0.13 0.11 Rag6/Rag3c 27 + + + + 1.17 -/- 59 - - - - 3.01 0.08 -/Rag3c 68 - - + + 1.94 0.07 Rag6/- 64 + + - - 1.58 0.08 + 0.79 0.08 Rag6/Rag3c a Rag3c 56 + + + + Implies allele from E12901; - Implies allele from the susceptible parent 62 Table 3.6a List of annotated gene models within the interval of Rag6 and structural variants with moderate or high effects Gene model Physical Gene model Physical Physical Glyma.Wm82. E12901 DNA sequence Annotation A.thaliana homolog (a2.v1) position of (a1.v1) position of position of a1 variations (a2.v1) (annotation) gene (bp) gene (bp) variant (bp) (a2.v1) (a1.v1) (a1.v1) Glyma.08g303500 42150074 - Glyma08g41550 41508240 41509151 ACGT A Codon deletion LRR-containing AT5G45520.1 (LRR 42151723 41509192 protein family protein) Glyma.08g303500 42150074 - Glyma08g41550 41508240 41509156 GA G Frame shift* LRR-containing AT5G45520.1 (LRR 42151723 41509192 protein family protein) Glyma.08g303500 42150074 - Glyma08g41550 41508240 41509158 TACCCAAT T Deletion, frame shift* LRR-containing AT5G45520.1 (LRR 42151723 41509192 A protein family protein) Glyma.08g303500 42150074 - Glyma08g41550 41508240 41509173 G T Non-synonymous LRR-containing AT5G45520.1 (LRR 42151723 41509192 coding change protein family protein) Glyma.08g303600 42160184 LRR-containing AT5G46450.1(disease 42161884 protein resistance protein, TIRNBS-LRR family) Glyma.08g303700 42162347 - Glyma08g41560 41520515 41521312 TG GA Non-synonymous LRR-containing AT5G17680.1 (disease 42167295 41525323 coding change protein resistance protein, TIRNBS-LRR family) Glyma.08g303700 42162347 - Glyma08g41560 41520515 41521322 C T Non-synonymous LRR-containing AT5G17680.1 (disease 42167295 41525323 coding change protein resistance protein, TIRNBS-LRR family) Glyma.08g303700 42162347 - Glyma08g41560 4152051541521326 C A Non-synonymous LRR-containing AT5G17680.1 (disease 42167295 41525323 coding change protein resistance protein, TIRNBS-LRR family) Glyma.08g303700 42162347 - Glyma08g41560 41520515 41521466 G T Non-synonymous LRR-containing AT5G17680.1 (disease 42167295 41525323 coding change protein resistance protein, TIRNBS-LRR family) Glyma.08g303700 42162347 - Glyma08g41560 41520515 41523569 T A Non-synonymous LRR-containing AT5G17680.1 (disease 42167295 41525323 coding change protein resistance protein, TIRNBS-LRR family) Glyma.08g303700 42162347 - Glyma08g41560 41520515 41523617 A C Non-synonymous LRR-containing AT5G17680.1 (disease 42167295 41525323 coding change protein resistance protein, TIRNBS-LRR family) Glyma.08g303800 42174238 - Glyma08g41570 41532406 Amine oxidase AT2G43020.1 42179859 41538027 (polyamine oxidase 2) * Mutations with high effects Pull-down sequences /primer sequences were designed based on Glyma.Wm82.a1v1 63 Table 3.7a List of annotated gene models within the interval of Rag3c and structural variants with moderate or high effects Gene model (a2.v1) Physical Gene model (a1.v1) Physical Physical Glyma.W E12901 DNA sequence Annotation (a2.v1) position of position of position of m82.a1 variations gene (bp) gene (bp) mutation (bp) (a2.v1) (a1.v1) (a1.v1) Glyma.16g066700 6620330 - Glyma16g07200 6473613 Ubiquitin 6621988 6475030 Glyma.16g066800 6627025 6628243 6636803 6639454 Glyma16g07220 - - - 6490128 A G Non-synonymous GDSL-like coding change Lipase/Acylhydrolase Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6490135 T G Non-synonymous GDSL-like coding change Lipase/Acylhydrolase Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6490341 G A Non-synonymous GDSL-like coding change Lipase/Acylhydrolase Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6491630 A C Non-synonymous GDSL-like coding change Lipase/Acylhydrolase Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6492005 G A Non-synonymous GDSL-like coding change Lipase/Acylhydrolase Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6492389 G T Non-synonymous GDSL-like coding change Lipase/Acylhydrolase Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6492399 T G Non-synonymous GDSL-like coding change Lipase/Acylhydrolase Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6492448 A C Non-synonymous GDSL-like coding change Lipase/Acylhydrolase Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6492455 G GTT Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6492460 C T Glyma.16g066900 Glyma16g07230 6480168 6481544 6490023 6492571 64 - Frame shift* LRR protein kinase GDSL-like Lipase/Acylhydrolase Non-synonymous GDSL-like coding change Lipase/Acylhydrolase A. thaliana homolog (annotation) AT5G14360.1 (Ubiquitin-like superfamily protein) AT1G58190.2 (LRR) AT1G53920.1 (GDSL-motif lipase 5) AT1G53920.1 (GDSL-motif lipase 5) AT1G53920.1 (GDSL-motif lipase 5) AT1G53920.1 (GDSL-motif lipase 5) AT1G53920.1 (GDSL-motif lipase 5) AT1G53920.1 (GDSL-motif lipase 5) AT1G53920.1 (GDSL-motif lipase 5) AT1G53920.1 (GDSL-motif lipase 5) AT1G53920.1 (GDSL-motif lipase 5) AT1G53920.1 (GDSL-motif lipase 5) Table 3.7a (cont’d) Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6492464 G Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6492509 CTTA Glyma.16g066900 6636803 6639454 Glyma16g07230 6490023 6492571 6492539 A Glyma.16g067000 6640311 6646267 6658305 6665699 Glyma16g07240 6493587 6499049 6511457 6518819 6498906 G 6511512 T Glyma.16g067200 6658305 6665699 Glyma16g07260 6511457 6518819 6511797 A Glyma.16g067500 6679009 6683608 Glyma16g07280 6532136 6536360 6536221 T Glyma.16g067700 6699818 6704992 Glyma16g07300 6552432 6557313 - - Glyma.16g067800 6715004 6726073 6729704 6731321 6733543 6736390 Glyma16g07330 6574306 6578855 - 6574345 A - - Glyma16g07350 6586743 6589581 - - 6761573 6765861 6761573 6765861 6761573 6765861 Glyma16g07360 6614785 6619065 6614785 6619065 6614785 6619065 6614967 G 6616052 T 6616627 T Glyma.16g067200 Glyma.16g067900 Glyma.16g068000 Glyma.16g068100 Glyma.16g068100 Glyma.16g068100 Glyma16g07260 - Glyma16g07360 Glyma16g07360 *DNA sequence variations with high effects Pull-down sequences /primer sequences were designed based on Glyma.Wm82.a1v1 65 T Non-synonymous GDSL-like AT1G53920.1 coding change Lipase/Acylhydrolase (GDSL-motif lipase 5) C Codon deletion GDSL-like AT1G53920.1 Lipase/Acylhydrolase (GDSL-motif lipase 5) T Non-synonymous GDSL-like AT1G53920.1 coding change Lipase/Acylhydrolase (GDSL-motif lipase 5) GGAA Codon insertion CCT motif AT5G14370.1 (CCT motif family protein) C Non-synonymous Kub3-prov protein AT3G03420.1 coding change (Ku70-binding family protein) T Non-synonymous Kub3-prov protein AT3G03420.1 coding change (Ku70-binding family protein) A Non-synonymous alpha/beta hydrolase AT5G14390.1 coding change (alpha/betaHydrolases superfamily protein) Mediator complex AT3G01680.1 subunit 28 (Mediator complex subunit ) G Non-synonymous Unknown protein AT5G40600.1 coding change (unknown protein) Methyltransferase AT3G01660.1 (methyltransferase) Translation factor AT1G07930.1 (translation elongation factor) T Non-synonymous Cytochrome P450 AT5G14400.1 coding change (cytochrome P450) G Non-synonymous Cytochrome P450 AT5G14400.1 coding change (cytochrome P450) C Non-synonymous Cytochrome P450 AT5G14400.1 coding change (cytochrome P450) Table 3.8a Information of SNPs used in the present fine mapping study Coded Original Name Assay type Reference Position (bp)a Position (bp)b Name Gm08-1 Gm08_38277532_T_C SoySNP50K 38277532 38909869 Gm08-2 Gm08_39004896_A_G - SoySNP50K 39004896 Flanking sequence (201bp or 100 bp)c - 39638822 Gm08-3 MSUSNP08-40 KASP Gm08-4 MSUSNP08-44 KASPTM SNP discovery 40020445 Gm08-5 Gm08_40289000_C_T - SoySNP50K 40289000 Gm08-6 Gm08_40766548_G_T - SoySNP50K 40766548 40047323 5’GACAAGAAGCAACGAATTCCTCAAATTCAAACATC TTAATGCAATCAATGCTTCCAATCAACCGGAGTTAAT ACACTTGATTAGGAGCGGACGATATTTA[G/T]CAAAA CAAAACTGCAGATGGGAGACAAAGTGACAGATCCCA GTAGCTGAAGATGACACAAAATTCCATACAGAAGCA TGCAAAAGTTAATCAGTCAAATG -3’ 40648794 5’ATCCGCGCGAAGCGTGCCTGAATCCTACCAAATGC GGATCAACTGAAACCTAAGGGGATCATCTACATCCC TACCCTATGACTACTGTGCTCAAGTATAA[C/T]GCAA ACGCGGATCAGCCAAATGCATACGCGGATCAACTAT CCTAAGCACTACACAAAAACCCCACAATGGAAACGC AAACGACATCGAGGGAGAAGAGTG-3’ 40925463 41402338 - Gm08-7 Gm08_40801297_C_T - SoySNP50K 40801297 41437087 - Gm08-8 Gm08_40884892_A_G - SoySNP50K 40884892 41520716 - Gm08-9 Gm08_40995409_T_C - SoySNP50K 40995409 41640233 - Gm08-10 Gm08_41061846_G_A - SoySNP50K 41061846 41705040 Gm08-11 MSUSNP08-49 KASPTM SNP discovery 41072605 Gm08-12 MSUSNP08-50 KASPTM SNP discovery 41305451 TM SNP discovery d 39410860 66 41715799 5’AAAGTAGGGACATTGGCCGAATAGCCTACAACACA CGATACACGAACTGGGAAAAAATAACTTTAAATTGC ACAATAATGTAATGCAGTTTTTCTAAAAT[G/C]TATTT GATTAATTTTGAAAATTAAGCTATTATTATAACCTTA ATGTGCTTACAAACTCATTGATTTGCTTGATATATAA GTTTAAGTTCGACTGATGAAA-3’ 41948645 5’AATTGCTTTCAAGTAGTTGTCGGACCAATTGTGTAA GGAACATAACCAAATAACAACATTGTCCTTAGGACA TATAATGACCATTTGCCAATGTGCATTG[C/T]ATTGGA GAATATTAAGTTATTATAATGCATACATTATTTAAAT TGATTTGTTCAGTTTTACTTACTCATTCAAGTAGGTTC TTAGGTAAACATCTCTATG-3’ Table 3.8a (cont’d) Gm08-13 MSUSNP08-96 KASPTM SoySNP50K 41419491 Gm08-14 MSUSNP08-97 KASPTM SoySNP50K 41453586 Gm08-15 MSUSNP08-100 KASPTM SoySNP50K 41504420 Gm08-16 MSUSNP08-51 KASPTM SNP discovery 41518390 Gm08-17 MSUSNP08-101 KASPTM SoySNP50K 41553888 Gm08-18 Gm08_41650869_A_C - SoySNP50K 41650869 SNP discovery 41696347 SoySNP50K 41807810 Gm08-19 MSUSNP08-52 Gm08-20 Gm08_41807810_A_G KASP - TM 67 42061322 5’TTGTTAGAGATGTCCATATTTAGTGTCTGACCGTGC CTCAAACAAGATTGCTTATGAAGGAGGAGAATTGAG GGAAACAAAAACAAATAAGCCTTATTTT[A/G]TTGAG CTAGTGACCTATGCATATTGTCAGCTAGCCAAACCAT ATGTTGTTGGTTGCAACCTATATACCTAACTCTTGGT CTAAGTGGCTCGTCATTGAAG-3’ 42095417 5’ATATTTTCAAAATCTATTCATTCTTGTAATTTTTTTA AGAAATTAACCCATTTGTGTAAATTTGCCAACATTTG AAGATATAGAAGCAATTTTAAATTTT[T/C]TCTGTAGT AAATAATATTTTATGGGCTTTGACTTTTGCAGGAAGG GTAAATTAAATAAATTATGAAAAGGAAAATGTTAGT GATACATCCCATTTTGGTT-3’ 42146252 5’TCATTTAATTACAAAAACCTCATCATTTTTTTAAAA CTTTATTTATTTATAAATAATAATTCTTTTTAAATTAA TCTACGAAAAATGGGATGTTACACCT[T/C]CACCCTT GGATTCTCCCTTCTCAACCTTGTGCTATGCCTGCCTCC CTCACTTTGCGGATCGTGAGCCACGTGTCCCTTTCTT CTAACAGAATTTCGTGCC-3’ 42160222 5’GAGGGGTAGAGGGTGTCACATGAGTGAAGTTTCAT ACCGGTTAAGTAATCACAAGAAGATTATCATTTCTGC TGGCATGAAATCTTCCTCCTCTTTGATG[T/C]ATGTAG AAGTTGTTCCCACTCAATGGATTCACATTCAAAAATT TCTTTGCCACTATTGCTGACCAGCTTCAATCCTGATG TTGTGGCATATACTGGGAAG-3’ 42195720 5’TAGTGGTGAGCACGAGTCGGTTTGAGAAAAAAATC CTAATCCGATAAAAACCGACCACTGAAAAATATGGC CCATCACTGTCTACTGTCCGTTTAAGTGT[G/T]GAGAT TGAATCGAGTAAGGTGGGTGGGTTATTTGGGATCAT CTTCATTCTCGACAACCACAAGAGGCTCTAAGTTCTG GTCCTGCACTAGTGTGCATCAT-3’ 42292701 42338179 5’GGCATATGACTCGGTGTCGTGGGATTTTCTGTTATA TATGTTGAAGAGAACAGGCTTTAGTTCTAAATGGAT AAGGTGGATGGAAGGGTGTTTGAATTCT[G/A]TCTCA ATTTCAGTCTTGGTAAATGGCAGCCCCACAACGGAG TTCATACCTCAAAGGGGTCTTAGACAAGAGGACCCT TTAGCTCCATTTTTATTCAATGT-3’ 42448802 - Table 3.8a (cont’d) Gm08-21 MSUSNP08-53 KASPTM SNP discovery 41880642 Gm08-22 MSUSNP08-54 KASPTM SNP discovery 41984985 Gm08-23 MSUSNP08-71 KASPTM SoySNP50K 42050788 Gm08-24 MSUSNP08-69 KASPTM SoySNP50K 42563620 Gm08-25 MSUSNP08-67 KASPTM SoySNP50K 43293884 Gm08-26 MSUSNP08-65 KASPTM SoySNP50K 44373623 68 42521634 5’CATGGCAAAAATATTTTAAAGATATATAATCTTGT GTATTTTTTTCTTACAATATAAATTAATATGGATATA ATAAATTATGATTGGTCACTTAAATAGA[C/T]AGACA TAAAAAATGGATGAAATTTATTAGGTTGTTGAAACC CACTTTGATAAAATCTATGGATTGGGCTTTAAATTTT AAAATTGAATCAATGGGAGCTA-3’ 42636560 5’CAGAGGATCAATTTTTGGGTTATTTTGGGTTGTTTT ATGAAATTCAATTCCATTCTTGTGTTTTTAATCATGG ATTGATTGTGTTTGACGGATCAATTGG[C/T]GTCCCAA TGCGAAATTGTTTTGAAATTGGTATGTTTTTGTGTTA AGTATGAATCCTAGGAATTAGGTTTTTTTTTTCTTCTA TTTAGTGTGAATTGTTGA-3’ 42702363 5’GAGAGGACAGGAAAAGCATTTCCTTGGTAAGTCTA ATACAATGTTTCCATATACTTTTCAAGTCCAAGGAAC ATTAGTTAGCGCAAAAATTACTAATCTA[T/G]AGTCA CACTCTAACCACAATTTTGTCCAGCCCACTGAATGGG CATATTCAATTGCAGGATTGAATTCGGTCCTAACATT TTAGAGGCTTCAAGCATAAAA-3’ 43390745 5’TTCATACAGCTCGTACAATTAGCAACAGTATAGCTT CATTTTTTCTTTTAAAATTTTAAGTTTAAATTTTCTGT ACGATTAGTGAGGGTAGCTGGTATTG[T/C]TCACCAT CCTGACAAACTCCTCAGCACTAACCTCACGATCATCT GCATATTTTACAGCCAAAAAACAGATATAACATCTC ACAATGATGAAAATAAATTA-3’ 44128863 5’TACCGCGAAGGATGAGATTAATCCTTAATCGCTAC CACTATATAAAAACTCGTAAATACAACTCTCACTTTT GCAACTGTTTACAACAATAATAAGTTAA[T/C]AACTC GCGTAGCGCAAGGAACAGCATAAACGACGCTGCGCT GAACAACGACGGTGTCAGACAAACGGTGCCGGTTGG TGGGGCCGCCACGCTATTACGCC-3’ 45398297 5’TTTCATAAGCCAGTAAGATAAATGTCTTCCTTCCAT GAATTATGTTTTCGTTGCAACCAAATGCCAAAGGTAC AAGGGAGAAGGGAAAGGGAAGGCCAGT[G/A]TAGGG AAATGAGGAGAGGTAATTGTGTCACTTTACAGTTTTT TGAAAAATCTGGGGGAGAAAGGAAAACAAGGAGAT GAGAATAGAAATTCCCTGAAAAA-3’ Table 3.8a (cont’d) Gm08-27 MSUSNP08-64 KASPTM SoySNP50K 45060561 Gm08-28 MSUSNP08-4 TaqMan® SoySNP6K 45189358 Gm16-1 MSUSNP16-10 KASPTM SoySNP50K 6262227 Gm16-2 MSUSNP16-47 KASPTM SoySNP50K 6470812 Gm16-3 Gm16_6474663_A_G - SoySNP50K 6474663 TM SoySNP50K 6510537 Gm16-4 MSUSNP16-127 KASP Gm16-5 MSUSNP16-178 KASPTM SoySNP50K 6624879 Gm16-6 MSUSNP16-180 KASPTM SoySNP50K 6716691 69 45913059 5’CGCGGCACCACCACCACCCATCGAACCCCTCTGCT GAACCCTCCAGAAGGACGCGCTGCGGGAGCGCGCGT GGGATTGGAATAAGGGTGAGGCTTGGAGC[C/T]TGGG GTGTGTGGAACACGCGACTGGGATTGAGGCCATTAA GAAGGGAGGGGGGGTGTCGGCGGCGATGACCGTGA AGGCGCTGCCGGTGAGGTTAGGGAG-3’ 46037031 5’GCAACAAGATTAGAAGGCCTAACTCTTTAAAAACA GTCCCCAACCCCTTCGGCGGGAGGGCGACGCGGGGC TCACGAGGGCATCTTCCAAGGGAGGAAGG[T/C]GCGT AGAGTCGCCACCAACGTTTATTCGAGGAAAACGTCG GAAAAACCGGAAAGGTGTGGTCTACGGACTTTAAGC GTGAAAGGTTCGGGAGTTG-3’ 6314120 5’CCCATGATGTCATGAGGTGTAAACTTGTTAAGACA TATCAAACTTAGGGTTTAAGTTAAC[C/T]AGATCCGA AAAAGCTGCCACTATAGTGCCTTCTCTTTGAGTATGT GGTAATTATTGATTG-3’ 6617689 5’GATACAAAATAAAGTAAATTATGAGTACATACACA TGCTTAGATCTAAAAACAATCAATATAAAATGTCAC ATATATGAAAACATGTTTGATATTGTAAA[G/A]TTAC ATAAATCAAACTTCTAAGACTAAATTTTCAATCTACA ATCTCCCTCTTTTTGGTTTTTGAGAATGCCAAATCAA AATGATGTGTATTGATGTTTTC-3’ 6621540 6657416 5’CTCCAAGACTAGACGAACTCTTCAAGCTTTTCTCCA ACTCCAAAACTCACTAAAAAACCTCACAAAATCAAT AACTTTTCTCTACTTGGTACTAGTAGCT[A/G]GTGTGA AATGAGCAATGGTTGAGGCTCTATTTGCAGGGGCAG ATGAAGGTCCTAGAAGGTGTTGCCTGAAGCTTGGTCT AGGGAAGATGGCAAGGATGGC-3’ 6771675 5’GATGTATCTTGTGTGGTGGCGGTGGTGGCCCAAGG CCGCGGTGTGTCGCGTGACTGCGTGAGTCGTGTCCAC GGTGAGGAGAAGAAGATGAGAAGAAATG[C/T]TGTA AGAGGAGAAGAATAAAGCAAGGTACTAGTCCTTAAA GTGGTACTAGTCCAATGGTTCTTAAAGTGAAAAAGA AAAAATCCAACGTACTAAAACTAA-3’ 6871009 5’GAGAATTATTACTCTGCGAGGGCCTTGAACAACTG TTGCTTTATAAAGTGCAGTAGTTCCTGGATAGACCGC CAAAACATGTTTTCCAGGAGGGAAATCA[G/A]GAGC Table 3.8a (cont’d) Gm16-7 MSUSNP16-137 KASPTM SoySNP50K 6729421 6883739 Gm16-8 MSUSNP16-85 KASPTM SoySNP50K 7070805 7228568 Gm16-9 MSUSNP16-15 TaqMan® SoySNP50K 8051585 8208418 a GCTAGAAGGATCATTACTCTTGGGGAAAGGAATGAT ATTTGCCATGGGAAGCTTGTATTGTCTGTGAATGCAG CAGGAGAAACCAATGAAAATAAA-3’ 5’ATTGATATGCTTTGTTAATTATGGTGGTACAGAAAT CTCGCACTTTGGTTGTTGTTGTTGTTGCCTCTCCTTTT CCCATTCGTGTATGTGTTTTTTTTGG[G/A]TTCCTTAT AATTGAAGCCACGTATTAGGTTGTGTAGTACCATGTT TCATGTTTTTGTTTGTTGGTACTTGATAAAAAAAAAA AAAAAAGTGAAGAGGGAG-3’ 5’ATGCAAGGGAAGCAGCTGCAAGAGATGCAAGGGA TGCAAAGGTGGAGGCGAGAGATGTAAAGAGAACAA CAGTGACAGCAACAACCGCAACCGCATGAAC[G/A]T GATGAGTATTAATGTGTTGTTATGAACTTATGATGTT GGTTTATGTGGGGAAATAAATGATGTATGTACCTCTT CTTGCCTATGTAGTAGGTTTGGGTG-3’ 5’TCCGTTTCATGTGTTTCACAATATCCTTATACTTAG AGCTATCAAAATGGGTCAGCCCGG[T/C]CTACATGGG CTGACCCGCAACGGGTTGAGCTAAAAGTGGGCTAGT CCAGCTCGGCTCACT-3’ Position is according to Glyma.Wm82.a1 (Schmutz et al. 2010) Position is according to Glyma.Wm82.a2 assembly on SoyBase (Grant et al. 2010) c Flanking seuquences were mined from Glyma.Wm82.a1 (Schmutz et al. 2010) d SNP was discovered by mapping the reads from the whole genome re-sequencing data of E12901 to Glyma.Wm82.a1 (Schmutz et al. 2010) b 70 Table 3.9a SNPs and INDELs discovered from the whole genome exome-capture sequencing of E12901 in the Rag6 and Rag3c fine-mapped regions Chromosome Physical position(bp)* Glyma.Wm82.a1 E12901 Gm08 41504561 T C Gm08 41504624 A T Gm08 41509151 ACGT A Gm08 41509156 GA G Gm08 41509158 TACCCAATA T Gm08 41509173 G T Gm08 41511432 TTGTTAA T Gm08 41519031 T C Gm08 41519484 T C Gm08 41519770 T C Gm08 41520615 T A Gm08 41521312 TG GA Gm08 41521322 C T Gm08 41521326 C A Gm08 41521441 C T Gm08 41521466 G T Gm08 41521554 A ATCT Gm08 41521643 G A Gm08 41523569 T A Gm08 41523617 A C Gm08 41523772 C T Gm08 41523786 T C Gm08 41532427 T C Gm08 41532432 C A Gm08 41532513 C T Gm08 41532697 C T Gm08 41532888 G A Gm08 41534025 G A Gm08 41537628 T CA Gm08 41537717 C T Gm08 41537978 A G Gm08 41538072 C T Gm08 41538087 C A Gm08 41540630 TACAA T Gm08 41540636 T C Gm08 41540664 T C 71 Table 3.9a (cont’d) Gm08 41540678 T A Gm08 41540683 A G Gm08 41541175 C T Gm08 41541256 T C Gm08 41541285 T C Gm08 41541311 A T Gm08 41541351 C T Gm08 41541399 G A Gm08 41541404 A T Gm08 41541407 A G Gm08 41541422 T C Gm08 41541439 C T Gm08 41541511 G A Gm08 41541532 T C Gm08 41541582 C G Gm08 41541954 T C Gm08 41541985 A G Gm08 41542026 T G Gm08 41542052 G A Gm08 41542064 GCG AAA Gm08 41542075 G A Gm08 41542109 G A Gm08 41542116 C T Gm08 41542126 G A Gm08 41542136 GA AG Gm08 41542145 G A Gm08 41542160 C T Gm08 41542210 G A Gm08 41542212 TG CA Gm08 41542215 C T Gm08 41542218 C T Gm08 41542227 G A Gm08 41542245 T C Gm16 6474663 A G Gm16 6474803 T A Gm16 6477224 T C Gm16 6477552 C T Gm16 6477668 G A Gm16 6477685 GG AA 72 Table 3.9a (cont’d) Gm16 6481071 C A Gm16 6481140 A T Gm16 6481246 T A Gm16 6481575 G GGGA Gm16 6481665 A T Gm16 6482551 A G Gm16 6490128 A G Gm16 6490135 T G Gm16 6490137 T A Gm16 6490341 G A Gm16 6490348 G GAAT Gm16 6490379 G T Gm16 6490388 CTA C Gm16 6490599 A G Gm16 6490605 T C Gm16 6490624 C T Gm16 6491249 C T Gm16 6491575 T C Gm16 6491630 A C Gm16 6491696 C T Gm16 6491831 G T Gm16 6491856 C A Gm16 6492005 G A Gm16 6492038 G T Gm16 6492047 T C Gm16 6492050 G A Gm16 6492339 G A Gm16 6492344 G C Gm16 6492356 C T Gm16 6492358 A G Gm16 6492371 TG CC Gm16 6492381 AC GA Gm16 6492389 G T Gm16 6492399 T G Gm16 6492448 A C Gm16 6492455 G GTT Gm16 6492460 C T Gm16 6492464 G T Gm16 6492509 CTTA C 73 Table 3.9a (cont’d) Gm16 6492539 A T Gm16 6496577 A C Gm16 6496643 C A Gm16 6496661 T C Gm16 6498489 A C Gm16 6498906 G GGAA Gm16 6507406 G A Gm16 6507450 C CTT Gm16 6507475 C T Gm16 6507486 C T Gm16 6507567 A T Gm16 6507754 G A Gm16 6511407 GA G Gm16 6511512 T C Gm16 6511797 A T Gm16 6511816 T A Gm16 6511904 TC T Gm16 6511906 TC T Gm16 6511911 C T Gm16 6512694 A G Gm16 6512944 AT A Gm16 6525101 GC G Gm16 6534261 C T Gm16 6534270 A G Gm16 6536028 G A Gm16 6536221 T A Gm16 6542809 T C Gm16 6542838 T C Gm16 6548053 C T Gm16 6552391 T C Gm16 6552513 A AT Gm16 6552525 G A Gm16 6552621 T G Gm16 6552765 A T Gm16 6555297 A G Gm16 6568373 T A Gm16 6571017 C T Gm16 6574259 T A Gm16 6574345 A G 74 Table 3.9a (cont’d) Gm16 6582889 C T Gm16 6582898 C T Gm16 6582958 T C Gm16 6583157 TTA T Gm16 6583190 A C Gm16 6583209 G T Gm16 6583975 C G Gm16 6583992 CTTAAA C Gm16 6584029 C T Gm16 6584194 C A Gm16 6584419 C T Gm16 6584471 G A Gm16 6584477 G A Gm16 6587151 G C Gm16 6587505 G A Gm16 6587643 T A Gm16 6588425 G C Gm16 6589456 A T Gm16 6589499 GT G Gm16 6589507 T A Gm16 6589532 C G Gm16 6606049 G T Gm16 6609767 G A Gm16 6610055 A G Gm16 6614967 G T Gm16 6615640 A G Gm16 6616052 T G Gm16 6616122 G T Gm16 6616627 T C Gm16 6617276 A ACT Gm16 6623320 C T Gm16 6627491 TA T * Physical position is according to Glyma.Wm82.a1 (Schmutz et al. 2010) 75 REFERENCES 76 REFERENCES Alt J, Ryan-Mahmutagic M (2013) Soybean aphid biotype 4 identified. 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Theor Appl Genet. doi:10.1007/s00122-017-2935-z 82 CHAPTER 4 PYRAMIDING DIFFERENT APHID-RESISTANCE GENES IN ELITE SOYBEAN GERMPLASM TO COMBAT DYNAMIC APHID POPULATIONS The work presented in this chapter has been submitted: Zhang S, Wen Z, DiFonzo C, Song Q, Wang D (2017) Pyramiding different aphid-resistance genes in elite soybean germplasm to combat dynamic aphid populations. Molecular Breeding (Submitted) 83 Abstract The soybean aphid, an invasive species, has posed a significant threat to soybean production in North America since 2001. Use of resistant cultivars is an effective tactic to protect soybean yield. However, the variability and dynamics of aphid populations could limit the effectiveness of host-resistance gene(s). Gene pyramiding is a promising way to sustain host-plant resistance. The objectives of this study were to determine the prevalent aphid biotypes in Michigan, and to assess the effectiveness of different combinations of aphid-resistance genes. A total of eleven soybean genotypes with known resistance gene(s) were used as indicator lines. Based on their responses, Biotype 3 was a major component of Michigan aphid populations collected during 2015 – 2016. The different performance of Rag-‘Jackson’ and Rag1-‘Dowling’ along with the break-down of resistance in plant introductions (PIs) 567301B and 567324 may be explained by the presence of Biotype 3 or an unknown virulent biotype establishing in Michigan. With the assistance of flanking markers, twelve advanced breeding lines carrying different aphidresistance gene(s) were developed and evaluated for effectiveness in five trials across 2015 to 2017. Lines with rag1c, Rag3d, Rag6, Rag3c+Rag6, rag1b+rag3, rag1c+rag4, rag1c+rag3+rag4, rag1c+Rag2+rag3+rag4 and rag1b+rag1c+rag3+rag4 demonstrated strong and consistent resistance. Due to the variability of virulent aphid populations, different combinations of Rag genes may perform differently across geographic regions. However, advanced breeding lines pyramided with three or four Rag genes likely will provide broader and more durable resistance to diverse and dynamic aphid populations. 84 Introduction Soybean [Glycine max (L.) Merr.] is one of the most important crops in North America because of its multiple uses as an animal feed, cooking oil, biofuel, and human protein source. In 2016, the U.S. ranked the first in world soybean production (11.73 million metric tons) with 5.52 million metric tons exported (SoyStats 2016). However, soybean production in North America has been threatened by the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), an invasive species native to Asia (Wu et al. 2004). Soybean aphid has aggressively dispersed to all major soybean producing areas in the U.S. and Canada (Ragsdale et al. 2011) since its discovery in southern Wisconsin in 2000 (Alleman et al. 2002). The direct aphid stylet-feeding on plant sap is the most prominent damage that can cause up to 40% soybean yield loss (Ragsdale et al. 2007). Under heavy infestations, soybean foliage can be stunted, wrinkled, distorted, and wilted; yield components, such as seed size and number, are also reduced (Wu et al. 2004). Transmissions of plant viruses by soybean aphids lead to further yield loss in soybean production (Hill et al. 2001; Clark and Perry, 2002). In addition, honeydew secreted by aphids promotes growth of sooty mold on leaves, impairing soybean photosynthesis by blocking sunlight and causing additional yield loss (Malumphy, 1997; Lemos Filho and Paiva, 2006). Currently, insecticides are wildly used to manage soybean aphids. However, this control method increases production cost, the risk of environmental contamination and the mortality of beneficial insects (e.g., natural enemies and pollinators) (Ohnesorg et al. 2009; Lundin et al. 85 2015). The formation of insecticide resistance in soybean aphid populations is also an increasing concern. A more cost-effective and environmentally friendly way to managing soybean aphids is to utilize the native host-plant resistance present in soybean germplasm. Extensive screening of different soybean germplasm pools has identified ~30 plant introductions (PIs) and cultivars with antibiosis (affecting insect biology or reproduction) or antixenosis (non-preference) resistance (Hill et al. 2004; Li et al. 2004; Yang et al. 2004; Mensah et al. 2005; Hesler et al. 2007; Mian et al. 2008a; Fox et al. 2014). Despite the high number of PIs and cultivars identified as resistant to soybean aphid, many share the same resistance genes or alleles, this due in part to the genetic bottleneck of soybean germplasm used in North America (Hyten et al. 2006). Aphid resistance QTLs identified in North America are designated as Rag (Resistance to Aphis glycines); different resistance alleles have been uncovered at six loci, Rag1 to Rag6. The dominant antibiosis-resistant Rag1/Rag (Hill et al. 2006a, b; Li et al. 2007), the recessive antibiosis-resistant rag1c (Zhang et al. 2009) and rag1b (Bales et al. 2013) were mapped to chromosome 7 between markers Satt463 and Satt567. Additionally, Rag1 was fine-mapped to a 115-kb interval between markers SNPKS9-3 and SNPKS5 (Kim et al. 2010a). The dominant Rag2 (Mian et al. 2008b; Hill et al. 2009) and Rag5 (Jun et al. 2012) were mapped to a genomic region between Satt334 and Sct_033 on chromosome 13, but they confer different resistance modality (antibiosis vs. antixenosis) (Michel et al. 2010; Jun et al. 2012). Rag2 later was refined to a 54-kb interval between markers SNP46169.7 and SNP21A (Kim et al. 2010b). Aphid resistance in 20 PIs is associated with Rag2, indicating Rag2 may be a major aphid-resistance source in the USDA soybean germplasm collection (Fox et al. 2014). The recessive antibiosis rag4 was mapped to a different location 86 (between Satt649 and Satt348) on chromosome 13 (Zhang et al. 2009). Jun et al. (2013) identified two major QTLs (QTL_13_1 and QTL_13_2) near Rag2 and rag4, and a minor QTL (QTL_6_1) on chromosome 6; these three QTLs suggested PI 567324 has oligogenic antixenosis resistance to soybean aphids. Five aphid-resistance QTLs/alleles were detected in a region between markers Satt285 and Satt654 on chromosome16, and designated Rag3 (antixenosis), Rag3b (antibiosis), rag3 (antibiosis), Rag3c (antibiosis), Rag3d (antibiosis) and Rag3e (antixenosis) (Zhang et al. 2010, 2013; Bales et al. 2013; Du 2016; Zhang et al. 2017a). Additionally, Rag3c was delimited to a 150-kb interval between markers Gm16_6474663_A_G and MSUSNP16-178 (Zhang et al. 2017b). The antibiosis-resistance gene Rag6 was refined to a 49-kb interval between markers MSUSNP08-100 and MSUSNP08-101 on chromosome 8 (Zhang et al. 2017a, b). The biggest concern of employing host-plant resistance is the breakdown of single resistance genes by virulent biotypes. To date, four different soybean aphid biotypes have been discovered in North America. Biotype 1 is avirulent to all Rag genes (Hill et al. 2004). Biotype 2 can reproduce on soybean plants with Rag1 (Kim et al. 2008). Biotype 3 readily colonizes soybeans with Rag2; it also reproduces on soybeans with Rag1 in choice tests (Hill et al. 2010). A recent multi-year study reported that the occurrence of soybean aphid biotypes was highly variable across both locations and years in the Midwestern U.S. (Cooper et al. 2015). The variability and dynamics of aphid populations could limit the durability of effectiveness of a single resistance gene. In this study, PI 567541B (a natural pyramid of rag1c/rag4) and PI 567598B (a natural pyramid of rag1b/rag3) demonstrated the widest spectrum of resistance to aphids across locations and years (Cooper et al. 2015). Similarly, other studies showed that soybean lines with 87 artificial pyramids of Rag1/Rag2 had significantly lower aphid colonization than lines with the Rag1 or Rag2 gene alone (Wiarda et al. 2012; McCarville et al. 2014). However, Alt and RyanMahmutagic (2013) reported a new soybean aphid biotype, Biotype 4, capable of colonizing PI 567541B, PI 567598B and soybean lines with the pyramid of Rag1/Rag2. There are likely more virulent biotypes not yet discovered. Therefore, integrating cultivars with multiple resistance genes, particularly with different modes of action, is important to achieve a broader and more durable resistance against different aphid populations. The Soybean Breeding and Genetics Program at Michigan State University (MSU) has identified seven soybean accessions carrying resistant alleles at four resistance loci, including Rag1, Rag3, Rag4, and Rag6 (Bales et al. 2013; Zhang et al. 2009, 2010, 2013; Zhang et al. 2017a; Du, 2016). Zhang et al. (2017b) refined Rag6 to a 49-kb interval between markers MSU08-100 and MSUSNP08-101, and Rag3c to a 150-kb interval between markers Gm16_6474663_A_G and MSUSNP16-178. Fine mapping studies of five other aphid-resistance QTLs (rag1b, rag1c, rag3, Rag3d and rag4) refined their genomic locations and identified closely linked SNP markers (Unpublished data). With the assistance of these SNP markers, a pool of improved soybean germplasm with different combinations of aphid-resistance genes was developed. The objectives of this study were to 1) assess the introgression of aphid-resistance gene(s) using the Illumina Infinium SoySNP6K iSelect BeadChip, 2) determine the prevalence of soybean aphid biotypes in Michigan, 3) assess the effectiveness of different Rag gene combinations against Michigan aphid populations. 88 Materials and methods Plant materials A total of eleven resistant soybean genotypes, including ‘Jackson’, LD05-16060 (Rag1‘Dowling’), PI 243540, PI 567543C, PI 567585A, PI 567597C, PI 567598B, PI 567541B, PI 567301B, E08934 (derived from G. soja 85-32) (Zhang et al. 2017a), and PI 567324, were used as indicator lines to screen for aphid biotypes in field-cage trials during the summers of 2015 and 2016. LD05-16060 was an advanced breeding line carrying the Rag1 gene from ‘Dowling’ and was developed by Dr. Brian Diers at University of Illinois Urbana-Champaign (UIUC). In total, twelve advanced breeding lines (Table 4.1) carrying different Rag gene(s) were developed through marker-assisted selection (MAS) with markers flanking the initial-mapped or fine-mapped regions (Li et al. 2007; Hill et al. 2009; Kim et al. 2010a, b; Zhang et al. 2017a, b; Unpublished data). LD05-16657a with Rag1 and LD08-12430a with Rag2 were developed by Dr. Brian Diers at UIUC while ‘E’ lines were developed at MSU in East Lansing, Michigan with different combinations of rag1b, rag1c, Rag2, Rag3c, Rag3d, rag3, rag4, Rag6 (Hill et al. 2009; Zhang et al. 2009; Bales et al. 2013; Du, 2016; Zhang et al. 2017a) (Table 1). E00003 has been consistently susceptible to Michigan aphids over the years (Zhang et al. 2017a, b), and it served as a susceptible check in this study. 89 Table 4.1 Pedigree information for advanced breeding lines integrated with different Rag genes Indicator Line Rag gene(s) Pedigree information E00003 none C95001 (AP1995) x C94043 (PIO 9281) LD05-16657a Rag1 Dwight (3) x (Dowling x Loda) E14922 rag1c [E00003 x (SDX00R-039-42 x PI 567541B)] x E00003 LD08-12430a Rag2 LD02-4485(2) x (Ina x PI 200538) E11950 rag3 (Titan x PI 567598B) x LD05-16060 E12904 Rag3d (Skylla x PI567585A) x Skylla E14923 Rag6 (Skylla x LD01-7323) x [E00003 x (Jiyu 71 x G.soja 85-32)] E14912 rag1b, rag3 [LD01-5907 x (Titan x PI 567598B)] x LD02-4485 E13369 rag1c, rag4 E07051 x {[E00003 x (SDX00R-039-42 x PI 567541B] x E00003)} E14902 Rag3c, Rag6 (Skylla x LD01-7323) x [E00003 x (Jiyu 71 x G.soja 85-32)] E13901 rag1c, rag3, rag4 {(Skylla x PI 567598B) x [Skylla x (SDX00R-039-42 x PI 567541B)]} x E07051 E13903 rag1c, Rag2, rag3, rag4 {[Skylla x PI 567598B] x [Skylla x (SDX00R-039-42 x PI 567541B)]} x LD08-12430a E14919 rag1b, rag1c, rag3, rag4 [E00003 x (SDX00R-039-42 x PI 567541B)] x [LD01-5907 x (Titan x PI 567598B)] * Donor of each Rag gene was indicated with an underline 90 DNA extraction and the Illumina Infinium SoySNP6K iSelect BeadChip genotyping analyses to assess the effectiveness of MAS Leaf tissue was collected from a seedling of each advanced breeding line. Genomic DNA from each sample was extracted using the modified CTAB protocol described by Kisha et al. (1997), and genotyped using the Illumina Infinium SoySNP6K iSelect BeadChip (Illumina, San Diego, CA), which consists of 5,403 single nucleotide polymorphisms (SNPs) selected from the Illumina Infinium SoySNP50K iSelect BeadChip (Song et al. 2013). The genome-wide SNP distribution of the Illumina Infinium SoySNP6K iSelect BeadChip was visualized with R (R Development Core Team 2016) (Figure 4.4a). Genotypes were called using the program GenomeStudio (1.9.4 version, Illumina, San Diego, CA). Each SNP was coded based on the standard codes for nucleotides derived from the International Union of Pure and Applied Chemistry. The quality of each SNP was checked as previously reported (Yan et al. 2010). SNPs with call rate <80% across all samples were removed from the dataset. The genome-wide SNP data of each advanced breeding line was compared to that of the original aphid-resistancegene(s) donor, mined from the public SoySNP50K iSelect BeadChip data on SoyBase (Grant et al. 2010) except for E12901. Graphic representation of genomic regions of interest from each sample were drawn with the program FlapJack (Milne et al. 2010). SNP markers that are monomorphic between the original donor line and the elite parental line were filtered. At each SNP of the advanced breeding line, the allele same as that of the original donor was assigned with the black color, and the alternative allele was assigned with the gray color. 91 Evaluation for soybean aphid resistance Indicator lines and the advanced breeding lines were evaluated in choice tests in field-cage trials (Mensah et al. 2005) during the summers of 2015 and 2016. All the lines were planted in a randomized complete block design with three replications in a 12.2 x 18.3 m aphid- and predator-proof polypropylene cage (Redwood Empire Awning Co., Santa Rosa, CA) on the Agronomy Farm of MSU, East Lansing, Michigan. In each replication, fifteen seeds from each line were planted in a single 60 cm long plot with 60 cm row spacing. The advanced breeding lines were also evaluated in the greenhouse choice-tests (Mensah et al. 2005) in the Plant Sciences greenhouse at MSU during Fall 2015, Spring 2016 and Spring 2017. Eight seeds from each line were planted in a 125-mm deep, 105-mm diameter plastic pot. All the lines were arranged in a randomized complete block design with three replications. The greenhouse was maintained at 26/15 ºC day/night with supplemental light (14 hours/day) provided by sodium vapor lights. Soybean aphids were collected from multiple locations across Michigan in the early summer of each testing year, and maintained on susceptible soybean plants (E00003) in field-cages or the greenhouse. In each trial, each plant was artificially infested with two wingless aphids at the soybean V2 stage (Fehr and Caviness, 1977). Each plant was visually rated for aphid resistance using a 0-4 scale (Mensah et al. 2005) when the susceptible check reached rating of 3.0 (usually three weeks after the initial infestation). Criteria of the 0-4 scale are as follows: 0 = no aphids; 0.5 = fewer than 10 aphids; 1 = 11-100 aphids; 1.5 = 101-150 aphids; 2 = 151-300 aphids; 2.5 = 92 301-500 aphids; 3 = 501-800 aphids, leaves and stems are covered with aphids, leaves appear slightly curly and shiny; 3.5 = more than 800 aphids, the plant appears stunted with curled yellow leaves, the plant is covered with few cast skins, no sooty mold; 4 = more than 800 aphids, the plant appears stunted with severely curled yellow leaves, the plant is covered with cast skins and sooty mold (Mensah et al. 2005). A damage index (DI) for each replication of each line was calculated as DI (%) = å (rating value x no. of plants in the category) / (4 x total no. of plants) x 100 (Mensah et al. 2005). The DI ranged from 0% (no infestation) to 100% (most severe infestation). In each trial, the average DI of each line from three replications were analyzed with one-way analysis of variance (ANOVA) at a significance level of 0.05 followed by paired-wise comparisons using the PROC GLM function in SAS 9.4 (SAS Institute, Cary, NC). Lines with DI less than 37.5% were considered as aphid-resistant (Zhang et al. 2017 a, b). Results and discussion Data from the Illumina Infinium SoySNP6K iSelect BeadChip verified the successful introgressions of all targeted aphid-resistance genes The advanced breeding lines were visualized as graphical genotypes where genomic regions inherited from the original donor are indicated with the black color whereas genomic regions from the elite germplasm are presented in gray color (Figure 4.1). Targeted aphid-resistance genes with their published genomic locations (Glyma.Wm82.a1) were listed for each advanced breeding line. Unpublished fine-mapped regions of some Rag genes (including rag1b, rag1c, rag3, rag4) were indicated with rectangle boxes. When inspecting the regions of interest, all targeted aphid-resistance genes were successfully integrated into these advanced breeding lines, 93 which verified the different Rag gene combination in each of the advanced breeding lines. The original genome-wide SNP data of each advanced breeding line along with E12901 (the donor of Rag6 and Rag3c) were presented in Table 4.4a (an electronic supplementary file). Figure 4.1 Graphic representation of genomic region(s) of interest for each advanced breeding line. Genomic regions inherited from the original donor(s) of the aphid-resistance gene(s) are presented in black while genomic regions from the susceptible elite background are presented in gray. Targeted aphid-resistance genes with their published genomic locations are listed for each advanced breeding line. Unpublished fine-mapped regions of some Rag genes (including rag1b, rag1c, rag3, rag4) are indicated with rectangle boxes. The genomic locations are according to Glyma.Wm82.a1 on SoyBase (Grant et al. 2010) 94 Indicator lines suggested Biotype 3 and undescribed virulent biotype(s) prevailing in Michigan In both the 2015 and 2016 field-cage trials, LD05-16060 (Rag1), PI 243540, PI 567301B and PI 567324 were heavily colonized by aphids collected from Michigan fields and their DIs (ranging from 61.7 to 79.2%) were not significantly different from the susceptible check, E00003 (DIs ~ 79.2 to 83.3%) (Figure 4.2 and Table 4.2). PI 567585A was moderately resistant in 2016 (DI of 43.3%), although it performed better in 2015 (Figure 4.2 and Table 4.2). The remaining soybean genotypes, including ‘Jackson’ showed strong resistance (DIs ranging from 12.5 to 33%) to the same aphid populations in both field trials (Figure 4.2 and Table 4.2). Table 4.2 Aphid damage indices (%) for indicator lines in field trials in Michigan, 2015-2016 Mean soybean aphid damage index (%)* Line Rag genes Field 2015 Field 2016 E00003 None 83.3a 79.2a LD05-16060 Rag1 66.7a 79.2a PI 243540 Rag2 79.2a 61.7ab PI 567301B Rag5 + QTL_8 75a 68.3a PI 567324 Rag2' + rag4' + QTL_6_1 75a 70.8a Jackson Rag 33.3b 12.5d PI 567541B rag1c +rag4 25bc 25cd PI 567543C Rag3 20.8bc 20.8d PI 567597C Rag3e 20.8bc 16.7d PI 567585A Rag3d 20.8bc 43.3bc E08934 Rag6+Rag3c 16.7bc 16.7d PI 567598B rag1b+rag3 12.5c 16.7d * DI (%) followed by same letter(s) are not significantly different at P < 0.05 in each trial 95 Figure 4.2 Aphid damage indices (%) of a susceptible check (E00003) and indicator lines used to screen for soybean aphid biotypes in (A) 2015 and (B) 2016 field-cage trials. Bars with same letter(s) are not significantly different at P < 0.05 in each trial 96 ‘Dowling’(Rag1) and ‘Jackson’(Rag) were reported as overcome by Biotype 2 in both choice and no-choice tests (Kim et al. 2008). Biotype 3 aphids readily colonized Rag2 soybeans in choice and no-choice tests as well as Rag1 soybeans in choice tests (Hill et al. 2010). Alt and Ryan-Mahmutagic (2013) discovered a new biotype, Biotype 4, capable of colonizing PI 567541B and PI 567598B. In our study, the Rag1 (LD05-16060) and Rag2 (PI 243540) lines were readily colonized by aphids; in contrast, the Rag line (‘Jackson’), PI 567541B and PI 567598B maintained strong resistance. This suggests that Biotype 3 aphids were a major component of the collected aphid populations in Michigan during 2015 and 2016. The response of ‘Jackson’ to Biotypes 3 or 4 is unknown as it was not included in the previous aphid biotype studies by Hill et al. (2010) and Alt and Ryan-Mahmutagic (2013). In our study, ‘Jackson’ performed differently than LD05-16060 (carrying Rag1-‘Dowling’) in both years; it showed a strong resistance in 2015 and a very strong resistance in 2016 whereas LD05-16060 was consistently as susceptible as E00003. In a regional investigation conducted by Cooper et al. (2015), ‘Jackson’ was characterized as resistant in multiple states (SD, IA, MI and OH) whereas ‘Dowling’ was susceptible in all ten participating states in the year of 2010. Zhang et al. (2017a) also observed that ‘Jackson’ was resistant whereas ‘Dowling’ was susceptible in Michigan during 2010. Combining the evidences from Cooper et al. (2015) and Zhang et al. (2017a), the different reactions of these two varieties to aphid populations in some years (2010, 2015, and 2016) suggested that Rag and Rag1 themselves are different, despite being mapped to a similar genomic region (Li et al. 2007). They could be allelic at a same locus or different QTLs located closely. ‘Jackson’ showed strong resistance to aphid populations that were primarily Biotype 3 in our field trials during 2015 and 2016, which suggests Biotype 3 is likely not able to overcome 97 the resistance in ‘Jackson’. Further study on the response of ‘Jackson’ to Biotype 3 is needed to exam this hypothesis. It is also possible that the different performance of Rag1 and Rag in the present study was due to an undescribed aphid biotype capable of colonizing Rag1 but not Rag soybeans. Single clones of Michigan aphids will be tested on ‘Dowling’ and ‘Jackson’ to explore this possibility. Mian et al. (2008a) reported that PI 567301B had strong antixenosis resistance to Biotypes 1 and 2, controlled by a major QTL (Rag5) and a minor QTL on chromosome 8 (Jun et al. 2012). Similarly, Mian et al. (2008a) reported that PI 567324 showed moderate antixenosis resistance to Biotype 1 and strong resistance to Biotype 2, contributed by QTL13_1 mapped closely to Rag2, QTL13_2 mapped closely to rag4 and a minor QTL_6_1 on chromosome 6 (Jun et al. 2013). Jun et al. (2013) suggested that the oligogenic resistance in PI 563724 would provide broader and more durable aphid resistance compared to lines with a single aphid resistance gene. However, in our field trials during 2015 and 2016, both PI 567301B and PI 563724 were heavily colonized by aphids. Although the reaction of these PIs to other biotypes has not been tested, their high damage indices (ranging from 68.3 to 75%) in our study could be explained by their susceptibility to Biotype 3 aphids which appeared to predominate the aphid population in 2015 and 2016; it also could be due to an undescribed virulent biotype in Michigan. PI 567301B and PI 563724 will be tested with Biotype 3 and/or single clones isolated from Michigan aphid populations to further investigate the hypotheses. 98 Table 4.3 Aphid damage indices (%) for advanced breeding lines in field and greenhouse trials in Michigan, 2015-2017 Mean soybean aphid damage index (%)* Line Rag genes E00003 Field 2015 Greenhouse 2015 Field 2016 Greenhouse 2016 Greenhouse 2017 None 83.3a 68.5A 79.2a 75A 70.8a LD05-16657a Rag1 66.8b 70A 70.5ab 72.8A 68.3a LD08-12430a Rag2 83.3a 73.5A 76.7a 75A 67.5a E11950 rag3 42.2c 12.5B 60.0b 25B 13.5c E14923 Rag6 22.2de 19.5B 23.9c 23.6BC 36.0b E12904 Rag3d 27.4d 12.5B 12.5c 12.5C 12.5c E14922 rag1c 12.5e 12.5B 12.5c 12.5C 21.7c E14902 Rag3c + Rag6 12.5e 12.5B 12.5c 12.5C 13.3c E14912 rag1b+rag3 20.8de 12.5B 12.5c 12.5C 16.7c E13369 rag1c+rag4 12.5e 14.1B 12.5c 12.5C 15.8c E13901 rag1c+rag3+rag4 14.1e 19.8B 12.5c 12.5C 12.5c E13903 rag1c+Rag2+rag3+rag4 14.2e 15.6B 12.5c 13.3C 16.7c 13.3c 13.3C 18.2c E14919 rag1b+rag1c+rag3+rag4 12.5e 13.0B * DI (%) followed by same letter(s) are not significantly different at P < 0.05 in each trial 99 Lines with rag1c or Rag3d or Rag6 or pyramided Rag genes showed strong and broad resistance Several soybean lines with a single aphid-resistance gene were readily colonized by aphids in our study (Figure 4.3). LD05-16657a with Rag1 and LD08-12430a with Rag2 had severe aphid damages (DI ~ 66.8 to 88.3%) in all trials across 2015 – 2017 (Table 4.3), which was consistent with the performance of indicator lines, LD05-16060 (Rag1) and PI 243540 (Rag2). E11950 with rag3 showed strong resistance in all the greenhouse trials but had moderate aphid damages (DI ~ 42.2 to 60%) in the field trials (Table 4.3), whereas the original donor, PI 567598B, had very strong resistance in the field trials (DI ~ 12.5 to 16.7%) (Table 4.2). PI 567598B also had the lowest frequency (18%) of aphid colonization across eleven locations during 2008-2010 (Cooper et al. 2015). Combining the results from Cooper et al. (2015) and the present study, the pyramid of rag1b/rag3 is critical to provide soybean with broad and durable resistance. Figure 4.3 Aphid damage indices (%) of a susceptible check (E00003) and the advanced breeding lines with different combinations of aphid-resistance gene(s) in (A) field-cage and greenhouse trials in 2015, (B) field-cage and greenhouse trials in 2016, and (C) a greenhouse trial in 2017. Damage indices from the field-cage trial were presented with gray bars followed by lower-case letters in (A) and (B). Damage indices from the greenhouse trial were presented with black bars followed by upper-case letters in (A) and (B). Within each trial, bars with same letter(s) are not significantly different at P < 0.05 100 Figure 4.3 (cont’d) E14923 with Rag6 alone was highly resistant (DI ~ 19.5 to 23.9%) to aphids across all trials during 2015 - 2016. However, its damage index (36.0%) in 2017 greenhouse trial was slightly below the resistance threshold (DI ~ 37.5%), and it was statistically greater than those of the remaining resistant lines (Figure 4.3C and Table 4.3). The original donor, E08934 (Rag6 + Rag3c), and the advanced breeding line, E14902 (Rag6 + Rag3c), exhibited very strong and 101 consistent resistance (DI ~ 12.5 to 16.7%) across all trials (Tables 4.2 and 4.3). Collectively, Rag6 alone offers a strong resistance, however, the pyramid of Rag6/Rag3c provides a stronger and more durable resistance. E12904 with Rag3d appears to have a more consistent strong resistance compared to its original donor, PI 567585A. It displayed a strong resistance (DI ~ 12.5 to 27.4 %) across all five trials during 2015 - 2017 (Figure 4.3 and Table 4.3). However, PI 567585A had moderate aphid damage (DI ~ 43.3%) in 2016 field trial even though it had a lower damage index (20.8%) in 2015 field trial (Figure 4.2 and Table 4.2). The consistent strong resistance effect of Rag3d in E12904 may be attributed to the elite genetic background; some background gene(s) may upregulate the expression of Rag3d. Across all five trials during 2015-2017, E14922 with rag1c showed a consistent strong resistance (DI ~ 12.5% to 21.7%) whereas LD05-16657a with Rag1 was consistently susceptible (DI ~ 66.8 to 72.8%) (Figure 4.3 and Table 4.3). The strong resistance provided by rag1c alone suggested that rag1c is a different gene or allele from Rag1 even though they were mapped in close proximity (Li et al. 2007; Zhang et al. 2009; Kim et al. 2010a). This conclusion is consistent with the genotypic evidence collected by Zhang et al. (2009); the band patterns of SSR markers flanking rag1c were distinctive between PI 567541B and ‘Dowling’. Among the resistant soybean genotypes tested by Cooper et al. (2015), PI 567541B and PI 567598B demonstrated the widest spectrum of resistance to aphid populations across North America during 2008-2010; the broad resistance was deduced contributed by the natural 102 pyramids of two resistance genes in these two PIs. However, PI 567541B and PI 567598B were later found fully colonized by Biotype 4 (Alt and Ryan-Mahmutagic, 2013). In our study, E14912 (rag1b+rag3 from PI 567598B) and E13369 (rag1c+rag4 from PI 567541B) showed very strong resistance across 2015 to 2017 (Figure 4.3 and Table 4.3), however, their resistance might be limited in geographic regions that have a higher pressure of Biotype 4 or other undescribed virulent biotypes. rag1c and rag3 are the two major genes controlling aphid resistance in PI 567541B and PI 567598B, respectively (Zhang et al. 2009; Bales et al. 2013). Additionally, Chandrasena et al. (2015) detected a significant additive x additive interaction between rag1c and rag3, contributing up to 24% of the phenotypic variation in aphid resistance. To achieve a broader and more durable resistance, additional aphid-resistance gene(s) were pyramided with rag1c+rag3. Advanced breeding line E13901 was pyramided with three aphid-resistance genes, including rag1c, rag3 and rag4. Compared to E13901, E13903 has one more aphid-resistance gene, Rag2, to provide additional resistance. E14919 has all four genes from PI 567541B and PI 567598. All these advanced breeding lines (E13901, E13903 and E14919) pyramided with multiple aphidresistance genes had very strong and consistent resistance to aphid populations in Michigan across 2015-2017 (Figure 4.3 and Table 4.3), and they are expected to be strong and durable when combating diverse and dynamic aphid populations across geographic regions. 103 Conclusion The utilization of host-plant resistance is an effective way to control soybean aphids. However, the aphid resistance provided by Rag1 soybeans, PI 243540 (Rag2), PI 567301B (Rag5) and PI 567324 (Rag2’+rag4’+QTL_6_1) was overcome by aphids in our field trials during 2015 and 2016. The high damage indices of PI 567301B and PI 567324 could be explained by their susceptibility to Biotype 3 aphids which appeared to be prevalent in our field trials. In contrast to the susceptibility of Rag1 soybeans, ‘Jackson’ maintained strong resistance in the field trials during 2015 and 2016. Coupled with the similar evidences from Cooper et al. (2015) and Zhang et al. (2017a), Rag1 and Rag are likely different loci or alleles, which may be distinguished by Biotype 3. In addition, it is possible that an undescribed virulent biotype prevalent in our field trials caused the susceptibility of PI 567301B and PI 567324 and the different responses from Rag1 soybeans and ‘Jackson’. Biotype 3 and single isolates of Michigan aphids will be tested on these soybean genotypes to further exam the hypotheses. Advanced breeding lines with single aphid-resistance genes, such as rag1c, Rag3d and Rag6 showed very strong resistance to Biotype 3 across trials during 2015 - 2017. The strong resistance provided by rag1c suggested that it is a different locus or allele from Rag1 even though they were mapped closely. According to a regional study by Cooper et al. (2015), soybean aphids have a high degree of virulence diversity in North America, which means the effectiveness of a single aphid resistance gene is likely limited by soybean aphid virulence variability. 104 Advanced breeding lines pyramided with two aphid-resistance genes, such as rag1b+rag3, rag1c+rag4, and Rag3c+Rag6 demonstrated strong resistance in Michigan. Although Biotype 3 dominated in our trials, there is variability in soybean aphid populations from year-to-year across the Midwest, and undescribed biotypes are likely yet to be identified. Lines with multiple Rag genes, such as rag1c+rag3+rag4, rag1c+Rag2+rag3+rag4 and rag1b+rag1c+rag3+rag4, likely will provide broader and more durable resistance to diverse and dynamic aphid populations. The advanced breeding lines with different combinations of Rag genes developed in this study are significant resources for breeders to develop varieties to combat different aphid populations across many geographies. 105 APPENDIX 106 APPENDIX Figure 4.4a The genome-wide SNP distribution of the Illumina Infinium SoySNP6K iSelect BeadChip visualized with R 107 REFERENCES 108 REFERENCES Alleman RJ, Grau CR, Hogg DB (2002) Soybean aphid host range and virus transmission efficiency. In: Proceedings of Wisconsin Fertilizer, Aglime, and Pest Management Conference. Madison, WI Alt J, Ryan-Mahmutagic M (2013) Soybean aphid biotype 4 identified. Crop Sci 53(4):14911495 Bales C, Zhang G, Liu M, Mensah C, Gu C, Song Q, Hyten D, Cregan P, Wang D (2013) Mapping soybean aphid resistance genes in PI 567598B. Theor Appl Genet 126:2081-2091 Chandrasena D, Wang Y, Bales C, Yuan J, Gu C, Wang D (2015) Pyramiding 3, 1b, 4, and 1c aphid-resistant genes in soybean germplasm. 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