THE ROLE OF PREZYGOTIC SELF-COMPATIBILITY IN FACILITATING INTERSPECIFIC COMPATIBILITY IN SOLANUM SECTION PETOTA By William Behling A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Breeding, Genetics and Biotechnology – Crop and Soil Sciences – Master of Science 2022 ABSTRACT THE ROLE OF PREZYGOTIC SELF-COMPATIBILITY IN FACILITATING INTERSPECIFIC COMPATIBILITY IN SOLANUM SECTION PETOTA By William Behling Domesticated potato (Solanum tuberosum L.) is the fourth most important food crop worldwide and critical food security crop. The high significance of this crop coupled with high production losses place potato at a high priority for genetic improvement, especially in the face of climate change. Potato breeding is undergoing a seismic shift from a tetraploid outcrossing crop towards a F1 hybrid system using diploid inbred lines as parents. This transformation has also changed how potato breeders access important traits from wild species. Pre- and post- zygotic barriers, such as self-incompatibility (SI) and endosperm failure, inhibit and complicate the use of the more valuable wild species in conventional and diploid breeding schemes. Traditionally plant breeders have employed the SC x SI rule, using self-compatibility (SC) to increase the success of interspecific crosses. In order to evaluate the potential of several different SC factors in facilitating broad interspecific compatibility: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 knockout (KO) lines of S-RNase, HT-B and HT-B + S- RNase in S. tuberosum were evaluated along with the self-compatible species S. verrucosum, and S-locus inhibitor gene (Sli) from S. chacoense. Findings confirm the central role of S-RNase in interspecific pollination barriers and the inconsequential role of HT-B alone. Sli had no effect on interspecific barriers despite its prominent role as a source of SC. Finally, S. verrucosum styles displayed an absence of prezygotic barriers. Further analysis of the role of HT-A, as well as other mechanisms that potentially underlie the SC phenotype in S. verrucosum will further clarify the role of specific genetic factors that regulate interspecific compatibility in Solanum section Petota Copyright by WILLIAM BEHLING 2022 ACKNOWLEDGMENTS I want to thank my adviser Dr. David Douches for his support and guidance during my time at Michigan State. I have been extremely fortunate and I am deeply appreciative of the opportunities that he has given me. I am also very grateful to the members of my guidance committee Dr. Robin Buell and Dr. Amy Iezzoni for taking time to counsel and guide me through this process. I feel very lucky to have arrived just in time as Dr. Robin Buell departs from MSU and Dr. Amy Iezzoni has retired. I would also like to recognize the dedication, skill, and expertise of the members of the Douches Potato Breeding and Genetics Program. I would like to thank Dr. Felix Enciso for his instruction and mentorship in molecular biology and gene exiting. I am very appreciative to Dr. Natalie Kaiser for her guidance and instruction in all aspects of potato propagation, tissue culture, and phenotyping, I wish to thank Dr. Daniel Zarka for being a dependable resource for guidance on experimental design and molecular biology. I would like to thank current graduate student Thilani Jayakody for helping me feel more comfortable in the lab with her friendship and expertise in gene editing and molecular biology. I am grateful to Greg Steere, Matt Zuehlke, Nick Garrity, and Donna Kells for their friendship and for teaching me about the technical aspects of our program. I would like to extend my gratitude to Kate Shaw and Chen Zhang for their support and patient instruction in the field, laboratory and the greenhouse, as well as their friendship which made me feel more at home here in Michigan. I am grateful also for the peer mentorship and friendship of past and present graduate students at Michigan State University Dr. Maher Alsahlany, Dr. Paul Collins, Sarah Lee, and McKena Lipham. iv I would like to thank Dr. Shelley Jansky and Dr. John Bamberg, Project Leader, US Potato Genebank-NRSP-6, for their guidance pertaining to wild species and germplasm resources, and who’s previous research helped inform this thesis. I am very grateful for my family, especially my parents Christine and Richard Behling for their support during my education. Finally, I would like to thank my Grandfathers Rex Behling, Rueben Babcock, and Albert Oman Jr. for instilling a deep and abiding appreciation for the natural world, and helping me cultivate the confidence to pursue my goals. v TABLE OF CONTENTS LIST OF TABLES ..................................................................................................................... viii LIST OF FIGURES ..................................................................................................................... ix CHAPTER 1 .................................................................................................................................. 1 INTRODUCTION......................................................................................................................... 1 Potatoes feed the world ............................................................................................................... 1 The role of diploid potato breeding in the advancement of potato breeding .............................. 1 Importance of wild species and the barriers to their effective use .............................................. 2 The diversity of wild species and their classification ............................................................. 2 Prezygotic interspecific barriers ................................................................................................. 4 The correlation between SC and interspecific compatibility in Solanum verrucosum ........... 5 Sli in interspecific compatibility ............................................................................................. 6 Post-zygotic barriers Endosperm failure and Endosperm Balance Number ............................... 6 Endosperm Balance Number (EBN) ....................................................................................... 7 REFERENCES ............................................................................................................................... 9 CHAPTER 2 ................................................................................................................................ 15 BREAKING BARRIERS: THE ROLE OF PLANT ONTOGENY AND GAMETOPHYTIC SELF-INCOMPATIBILITY FACTORS S-RNASE AND HT-B IN INTERSPECIFIC POLLEN REJECTION IN SOLANUM SECTION PETOTA .............. 15 Abstract ..................................................................................................................................... 15 Objective: .................................................................................................................................. 16 Introduction ............................................................................................................................... 16 Materials and Methods.............................................................................................................. 18 Plant materials ....................................................................................................................... 18 Greenhouse pollination assays .............................................................................................. 18 Stylar squash assays .............................................................................................................. 19 Data collection and analysis.................................................................................................. 20 Results ....................................................................................................................................... 20 The role of S-RNase and HT-B in interspecific pollen rejection.......................................... 20 Effects of plant age on interspecific pollen rejection............................................................ 22 Discussion ................................................................................................................................. 23 APPENDICES .............................................................................................................................. 27 APPENDIX A: Chapter 2 Tables ............................................................................................. 28 APPENDIX B: Chapter 2 Figures ............................................................................................ 29 REFERENCES ............................................................................................................................. 39 CHAPTER 3 ................................................................................................................................ 43 EVALUATION OF SLI AS A GENETIC RESOURCE TO OVERCOME INTERSPECIFIC PREZYGOTIC BARRIERS IN SOLANUM SECTION PETOTA ...... 43 Abstract ..................................................................................................................................... 43 vi Objective: .................................................................................................................................. 43 Introduction ............................................................................................................................... 43 Plant Materials ...................................................................................................................... 45 Greenhouse propagation and pollinations ............................................................................. 46 Stylar squash: assay of pollen tube growth ........................................................................... 46 Data collection and analysis.................................................................................................. 47 Results ....................................................................................................................................... 48 The role of Sli in interspecific pollen rejection .................................................................... 48 Effects of plant ontogeny ...................................................................................................... 49 Discussion ................................................................................................................................. 49 APPENDICES .............................................................................................................................. 52 APPENDIX A: Chapter 3 Tables ............................................................................................. 53 APPENDIX B: Chapter 3 Figures ............................................................................................ 54 REFERENCES ............................................................................................................................. 58 CHAPTER 4 ................................................................................................................................ 62 ASSESSING THE ROLE OF S-RNASE IN SELF_COMPATIBILITY AND INTERSPECIFIC COMPATIBILITY IN SOLANUM VERRUCOSUM ............................ 62 Abstract ..................................................................................................................................... 62 Objective: .................................................................................................................................. 62 Introduction ............................................................................................................................... 63 Materials and methods .............................................................................................................. 64 Plant materials ....................................................................................................................... 64 Greenhouse pollination assays .............................................................................................. 65 Stylar squash assays .............................................................................................................. 66 Pollen tube data analysis ....................................................................................................... 67 Assessment of S-RNase gene expression in Solanum verrucosum .......................................... 67 Amplification and sequencing of S-RNase ........................................................................... 67 S-RNase expression analysis via RT-PCR ........................................................................... 68 Results ....................................................................................................................................... 68 Prezygotic interspecific barriers in S. verrucosum ............................................................... 68 S-RNase in S. verrucosum .................................................................................................... 69 Discussion ................................................................................................................................. 69 APPENDICES .............................................................................................................................. 71 APPENDIX A: Chapter 4 Tables ............................................................................................. 72 APPENDIX B: Chapter 4 Figures ............................................................................................ 73 REFERENCES ............................................................................................................................. 80 CHAPTER 5 ................................................................................................................................ 84 CONCLUSIONS AND FUTURE DIRECTIONS .................................................................... 84 S-RNase and HT in interspecific crosses .................................................................................. 85 Sli and Solanum chacoense....................................................................................................... 86 SC and interspecific compatibility in Solanum verrucosum .................................................... 86 Plant ontogeny .......................................................................................................................... 89 REFERENCES ............................................................................................................................. 90 vii LIST OF TABLES Table 2.1. List of potato-related species used as pollen donors.................................................... 28 Table 2.2a. Genotypes used as female parents and respective gene edit ...................................... 28 Table 2.2b. Description of gene edits in female genotypes used .................................................. 28 Table 3.1. List of potato-related species used as females in this study ........................................ 53 Table 4.1. Primers used in this study ............................................................................................ 72 viii LIST OF FIGURES Figure 2.1. Pollen tube measurement description ........................................................................ 29 Figure 2.2. Measurement of the longest pollen tube as well as the pollen tube front with S. bulbocastanum as a pollen donor .................................................................................................. 30 Figure 2.3. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study with S. bulbocastanum as the pollen donor, ........................... 31 Figure 2.4. Measurement of the longest pollen tube as well as the pollen tube front with S. commersonii as a pollen donor ..................................................................................................... 32 Figure 2.5. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study with S. commersonii as the pollen donor, ............................... 33 Figure 2.6. Measurement of the longest pollen tube as well as the pollen tube front with S. jamesii as a pollen donor............................................................................................................... 34 Figure 2.7. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study with S. jamesii as the pollen donor, ........................................ 35 Figure 2.8. Measurement of the longest pollen tube as well as the pollen tube front with S. pinnatisectum as a pollen donor .................................................................................................... 36 Figure 2.9. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study with S. pinnatisectum as the pollen donor, ............................. 37 Figure 2.10. Example images of pollen viability assays .............................................................. 38 Figure 3.1. Measurement of pollen tube front according to female. ............................................ 54 Figure 3.2. Measurement of the longest pollen tube according to female ................................... 55 Figure 3.3. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study according to female................................................................. 56 Figure 3.4. Examples of styles pollinated with 80-1 and M6, ..................................................... 57 Figure 4.1. Measurements of pollen tube front for all pollen donors ......................................... 73 Figure 4.2. Measurements of the longest pollen tube for all pollen donors. ................................ 74 Figure 4.3. Estimated marginal means plotted against the different time-points ........................ 74 ix Figure 4.4. Styles of S. verrucosum SV607845.02 pollinated with each of the four pollen donors. ....................................................................................................................................................... 75 Figure 4.5. Partial sequences of the two S-RNase alleles identified in S. verrucosum ................ 76 Figure 4.6. The two S-RNase alleles of S. verrucosum identified in this study ........................... 77 Figure 4.7. S-RNase expression in S. verrucosum SV607845.02 ................................................ 78 Figure 4.8. UPGMA phylogenetic tree showing the similarity of both SV607845.02 S-RNase alleles to the S-RNase 2 from S. tuberosum and their relation to other known S-RNase alleles within Solanaceae ......................................................................................................................... 79 x CHAPTER 1 INTRODUCTION Potatoes feed the world Cultivated potato, Solanum tuberosum L. Group Tuberosum (2n=4x=48), plays a significant role in the human diet as a primary source of carbohydrates (FAOSTAT, 2019). Potatoes are also the fourth most important food crop worldwide after wheat rice and maize (FAOSTAT, 2019). On a yield per unit area basis, potato is highly productive compared to other staple crops and provides substantially more protein per unit of land area than rice or wheat and is a significant source of carbohydrates, antioxidants and micronutrients (Kaldy, 1972; Brown 2005; Zehara 2012). The United States currently ranks as the world’s fifth largest producer of potatoes where the crop generates 3.9 million dollars over 0.9 million acres (FAOSTAT, 2019; NASS 2020) The role of diploid potato breeding in the advancement of potato breeding The significance of the potato crop, coupled with the high production losses due to both biotic and abiotic forces, place potato as a high priority for genetic improvement, especially in the face of climate change (Raymundo et al., 2018). Nearly all cultivated potatoes are tetraploid (2n=4x=48), which impedes the fixation of desirable alleles in populations and new cultivars. Consequently, there is an effort to develop a F1 hybrid system using diploid inbred lines as parents. This would accelerate the generation of new varieties, and simplify efforts to incorporate better agronomic and abiotic resistance traits into existing germplasm (Jansky et al., 2016). The use of diploid germplasm also simplifies the introgression of traits from wild species that have been major sources for disease resistance and abiotic stress tolerance. 1 Importance of wild species and the barriers to their effective use The diversity of wild species and their classification There are currently 107 recognized species of wild and cultivated potato in Solanum section Petota; with natural ranges extending from the desert southwest of North America to the Southern Cone of South America (Spooner et al., 2014). The classification of this taxonomic group is complicated by differences in ploidy and interspecific hybridization, with taxonomists recognizing as many as 232 species to the current 107 (Hawkes, 1990; Huang et al., 2019; Spooner et al., 2018). The use of DNA analyses to aid classification has elucidated the presence of four main clades, with the first two clades later being recombined to form the larger 1+2 clade after further analysis (Spooner et al., 2018). Another classification system was also created to describe the relationships of sexual compatibility between species by their Endosperm Balance Number (EBN) as proposed by Johnston et al. (1980). In the context of potato breeding both EBN and taxonomic classification play important roles. Different species are assigned an EBN number based on their ability to form normal endosperm when crossed to a species with a “known” EBN number (Johnston et al., 1980). As a general rule only individuals with the same EBN number are able to form viable seeds, and crossing individuals of different EBN numbers results in endosperm failure and aborted seeds (Johnston et al., 1980; Lafon-Placette and Köhler, 2016; Stadler et al., 2021). Of all the wild species available to potato breeders, the species in clade 1+2 which all have an EBN number of one are of special interest because of their high degree of disease resistance, pest resistance, and tuber quality traits (Lara-Cabrera and Spooner, 2004; Jansky and Hamernik, 2009). These “1EBN” species such as S. bulbocastanum, S. pinnatisectum, and S. jamesii are unfortunately difficult for plant breeders to access as all cultivated species have an EBN of two making 2 interspecific hybridization difficult and seed formation exceedingly rare (Jansky and Hamernik, 2009; Johnston and Hanneman, 1982). Wild potato species, especially the 1EBN species, are important sources of robust disease resistance and valuable consumer traits (Bhaskar et al., 2010; Duan et al., 2012; Helgeson et al., 1998). Each species selected as pollen parents for this study have unique value in a potato breeding program. S. bulbocastanum, S. pinnatisectum, and S. jamesii exhibit robust levels of resistance to potato late blight (Karki et al., 2020; Zheng et al., 2020). S. jamesii is also source of Potato Virus Y (PVY) resistance and important long-term tuber storage traits (Singh et al., 1994; Bamberg 2010). The South American species S. commersonii has displayed resistance to bacterial wilt and frost tolerance (Laferriere et al., 1999; Esposito et al., 2019). Each of these four pollen donors have meaningful resistance to Colorado potato beetle (Jansky et al., 2009). Other species used in this study are important in the advancement of diploid breeding and introgression of wild germplasm. S. chacoense (2EBN) represented by the clones M6 and USDA8380-1 are important sources of Sli-based self-compatibility and leptine-mediated Colorado potato beetle (Leptinotarsa decemlineata) host plant resistance, respectively (Kaiser et al., 2020; Jansky et al., 2014). Finally, S. verrucosum (2EBN) is the primary component of bridge crosses and its unilateral incompatibility relationship with cultivated potato has been well documented (Eijlander et al., 2000; Jansky & Hamernik, 2009; Yermishin et al., 2014). Unfortunately, the use of 1EBN species in conventional breeding programs is often impeded by pre- and postzygotic barriers (Novy & Hanneman, 1991; Jansky & Hamernik, 2009). The genetic basis for these interspecific barriers has not been fully explored in Solanum section Petota. Consequently, the contribution of each of these barriers to interspecific incompatibility is not clear. In the closely related Solanum section Lycopersicon, there are several barriers that 3 have been characterized, but these have not been confirmed in Solanum section Petota. Prezygotic barriers such as S-RNase dependent and independent mechanisms not only inhibit interspecific hybridization but also function in gametophytic self-incompatibility (Tovar-Méndez et al., 2014, 2017; Baek et al., 2015). Additionally, postzygotic barriers such as endosperm failure and sterile progeny are major impediments in the use of wild species in conventional breeding (Hermsen and Ramanna, 1976; Lafon-Placette and Köhler, 2016; Städler et al., 2021). The understanding of these barriers is crucial in the effort to introgress wild species into cultivated diploid germplasm. Prezygotic interspecific barriers The prezygotic barrier of gametophytic self-incompatibility (GSI) also presents a significant obstacle to interspecific crosses. The GSI system is controlled by the multiallelic S- locus (Porcher and Lande, 2005). This locus contains a set of tightly linked genes: S-RNase, expressed in the style, and SLF (S-locus F-box), expressed in the pollen (Kubo et al. 2010). SLF proteins recognize specific sets of S-RNase alleles, allowing self vs. non-self-pollen to be recognized. In compatible pollinations, SLF proteins degrade S-RNase proteins allowing the pollen tubes to make it to the ovary. In self-pollinations, SLF proteins do not degrade “self” S- RNase resulting in the cytotoxic activity of S-RNase and pollen tube inhibition. Other components such as HT and the 120kDa glycoprotein facilitate the function of S-RNase, thereby affecting self-incompatibility (Markova et al., 2016; Hua et al., 2008; Baek et al., 2015; Tovar‐ Méndez et al., 2014, 2017). Because HT mediates the action of S-RNase, it also exhibits and influence on interspecific crosses that are inhibited by S-RNase (Baek et al., 2015; Tovar‐ Méndez et al., 2014, 2017). 4 Interspecific crosses in Solanaceae often display unilateral incompatibility or incongruity (UI). UI usually follows the self-incompatible (SI) x self-compatible (SC) rule of Lewis and Crowe (1958). This rule states that SI species × SC species crosses are incompatible, but the reciprocal crosses are compatible (Bedinger et al., 2011; Baek et al., 2015; Murfett et al., 1996). This is because the same factors that affect pollen tube growth to prevent self-pollinations also prevent interspecific hybridization by inhibiting the pollen tubes of other species (Baek et al., 2015; Bedinger et al., 2011). It has been demonstrated that S-RNase partially mediates self- compatibility in Solanum section Petota, but has not been observed to mediate interspecific compatibility as it has in Solanum section Lycopersicon (Tovar-Méndez et al., 2014; Enciso- Rodrigues et al., 2019) Likewise, HT has been demonstrated to mediate S-RNase dependent and independent self and interspecific compatibility in section Lycopersicon but not in Petota (Tovar-Méndez et al., 2014, 2017) It is important to note that not only are section Lycopersicon and Petota close relatives phylogenetically, but that gene sequence and order are conserved between these two taxonomic sections (Bohs & Weese, 2007; Zhu et al., 2008). Because of this, it is likely that there will be similar findings regarding the role of HT and S-RNase in interspecific compatibility in section Petota as has been found in Lycopersicon. The correlation between SC and interspecific compatibility in Solanum verrucosum The interaction between post- and prezygotic barriers confounds and complicates interspecific crosses. S. verrucosum is an important species for creating bridge crosses with important 1EBN species. When used in interspecific crosses, S. verrucosum is used as the female parent as it exhibits unilateral compatibility with self-incompatible species (Dinu et al., 2005). As with Solanum section Lycopersicon, this follows the SI x SC rule since S. verrucosum is a self-compatible species (Tovar-Méndez et al., 2014; Jansky & Hamernik, 2009). Self- 5 compatibility in S. verrucosum has been attributed to the lack of a functional S-RNase protein (Eijlander, 1998). The absence of functional S-RNase alleles prevents the inhibition on pollen tube growth in what would otherwise be incompatible pollinations. While it has been demonstrated that there is no detectable S-RNase at the protein level (Eijlander, 1998), it is unknown if the genes for S-RNase in S. verrucosum are missing, non-functional, or inhibited in some way. The lack of this functional S-RNase protein in S. verrucosum is likely the reason that it is able to serve as a major bridge species in between 1EBN species and cultivated germplasm (Eijlander, 1998). Sli in interspecific compatibility The ability of Sli to overcome interspecific barriers was initially reported by Sanetomo et al. (2014). It was also observed that the pollen tubes from Sli carrying pollen donors were able to bypass stylar barriers, while pollen tubes from S. megistacrolobum, S. demissum, and S. phureja stopped growing at or near the stigma of 4x and 2x S. pinnatisectum (Sanetomo et al, 2014). The ability of Sli to overcome all prezygotic stylar barriers, not just those controlling gametophytic self-incompatibility, could be an important factor in accessing the sexually isolated species. Additionally, the widespread use of Sli-based SC in North American diploid potato breeding would mean that most public and private breeders would have access to these methods (Kaiser et al., 2021). The employment of Sli to overcome interspecific barriers could represent and powerful albeit underutilized method to access valuable traits from sexually isolated species. Post-zygotic barriers Endosperm failure and Endosperm Balance Number A significant barrier to interspecific hybridization is endosperm failure. Endosperm regulates the early development of the embryo and is a major food store for the developing seed (Lester & Kang, 1998). In angiosperms, the process of double fertilization occurs when one 6 sperm cell fuses with the egg cell to form an embryo, while a second sperm cell fuses with the polar cell to form the endosperm (Raghavan, 2003). The development and specifically the rate of cellularization of the endosperm must occur in concert with the development of the embryo; otherwise, endosperm failure occurs and embryo growth is arrested (Lafon-Placette and Köhler, 2016). In interspecific and interploidy crosses, the parental dosage between the two parents can be out of balance. This powerful post zygotic barrier maintains the ploidy level and integrity of species. Endosperm Balance Number (EBN) EBN is a measure of "effective ploidy" not actually ploidy (Johnston et al., 1980; Roth et al., 2019; Stadler et al., 2021). Endosperm development is a tightly regulated process, any increase or decrease in expression from the maternal or paternal genomes will result in endosperm failure (Lafon-Placette, and Köhler, 2016; Stadler et al., 2021). Sometimes this difference in expression occurs because of differences in actually ploidy, for example: crossing a tetraploid female to a diploid male of the same species results in endosperm failure (Johnston et al., 1980; Stadler et al., 2021). The extra copy of the female genome results in overexpression of maternally expressed genes, preventing normal seed development, since both parents are of the same species; this is an example of the phenomenon referred to as "triploid block" (Marks, 1966; Stadler et al., 2021). There are different levels of gene expression between species of the same ploidy as well. Because of this, crossing two different species can sometimes have the same outcome as crossing individuals of the same species but different levels of actual ploidy, this is where EBN comes in as a measure of "effective ploidy" (Jansky, and Hamernik, 2009; Lafon- Placette, and Köhler, 2016; Stadler et al., 2021). 7 Many misunderstandings surround EBN and effective ploidy. Among these being: the genetic basis and thereby the phylogenic relationships between different species of the same EBN, and the use of discrete categories to describe post-zygotic barriers. Despite early research by Ehlenfeldt and Hanneman (1988) detailing that genetic control was polygenic, the belief still persists in the potato breeding community that all species of the same EBN are related and intercrossable. The use of discrete categories to classify relationships of interspecific compatibility has limited value and lacks specificity; especially since it is clear that the genetic mechanisms than govern endosperm failure are complex and genome-wide (Ehlenfeldt and Hanneman, 1988; Lafon-Placette, and Köhler, 2016; Stadler et al., 2021). Additionally, individual species of the same EBN are not always closely related (Pritchard, 2005). Therefore, it would be inappropriate to surmise that the genetic mechanisms that result in the categorization of individual North American and South American species as "1EBN" to be the same (Pritchard, 2005). 8 REFERENCES 9 REFERENCES Baek, Y.S., Covey, P.A., Petersen, J.J., Chetelat, R.T., McClure, B. and Bedinger, P.A. (2015), Testing the SI × SC rule: Pollen–pistil interactions in interspecific crosses between members of the tomato clade (Solanum section Lycopersicon, Solanaceae). American Journal of Botany, 102: 302-311. doi:10.3732/ajb.1400484 Bamberg, J. 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(2008) Analysis of 90 Mb of the potato genome reveals conservation of gene structures and order with tomato but divergence in repetitive sequence composition. BMC Genomics 9, 286. doi:10.1186/1471-2164-9-286 14 CHAPTER 2 BREAKING BARRIERS: THE ROLE OF PLANT ONTOGENY AND GAMETOPHYTIC SELF-INCOMPATIBILITY FACTORS S-RNASE AND HT-B IN INTERSPECIFIC POLLEN REJECTION IN SOLANUM SECTION PETOTA Abstract The relationships of interspecific compatibility and incompatibility in Solanum section Petota are complex and intricate. Inquiry into these relationships has elucidated the pleiotropic and redundant function of S-RNase and HT which tandemly and independently mediate both interspecific and intraspecific pollen rejection. The wild species Solanum bulbocastanum, S. commersonii, S. jamesii, and S. pinnatisectum were used as pollen donors to pollinate Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene knockouts of S-RNase and HT-B in the clone DRH195. Analysis of pollen tube growth was used to determine the role of S-RNase and HT-B in prezygotic stylar barriers that inhibit the pollen tube growth of these species. The findings presented here are consistent with previous work conducted in Solanum section Lycopersicon showing that S-RNase plays a central role in interspecific pollen rejection. Statistical analyses also demonstrated that HT-B alone was not a significant factor in these pollinations highlighting the potential function and importance of HT-A and other factors. S. commersonii was unique in comparison to the other pollen donors used as S-RNase did not appear to affect pollen rejection. Intra and interspecific compatibility is also heavily influenced by environmental and ontogenic factors. Although variable gametophytic self-incompatibility responses due to plant and floral age have been previously demonstrated, these findings have not been observed in interspecific pollinations. Analysis of pollen tube length across three time- points indicates that plant age significantly impacts interspecific pollen rejection. 15 Objective: Apply findings from Solanum section Lycopersicon to section Petota by demonstrating that S-RNase and HT play major roles in interspecific compatibility. Knock-outs of S-RNase did increase interspecific compatibility. However, both HT-A and HT-B would need to be knocked out in order to significantly affect interspecific compatibility in these crosses as they serve the same function. These findings are consistent with those in Solanum section Lycopersicon. Introduction Gametophytic self-incompatibility (GSI) is the default condition in diploid cultivated potato and the vast majority of its wild relatives (Carson and Howard 1942; Whalen and Anderson, 1981; Fuji et al. 2016). GSI in Solanaceae is governed by the multiallelic S-locus on chromosome 1, containing S-RNase tightly linked to multiple SLF (S-locus F-box) genes (Enciso-Rodriguez et al. 2019; Kaiser et al., 2021; Kubo et al., 2010; McClure et al., 2011). This GSI system also serves the dual function of preventing interspecific pollinations as well by inhibiting the growth of pollen tubes from other species (Baek et al., 2015; Bedinger et al., 2011). Because of this general overlap in function, interspecific crosses in Solanaceae often display unilateral incompatibility or incongruity (UI). UI usually follows the self-incompatible (SI) x self-compatible (SC) rule reported by Lewis and Crowe (1958) where SC species can only act as the female, as the functional GSI system in the SI species prevents both self and interspecific pollen from reaching the ovary (Bedinger et al., 2011; Baek et al., 2015; Murfett et al,. 1996). While the SI x SC rule proposed by Lewis and Crowe (1958) has been generally helpful, it is an oversimplification and does not fully capture the complex relationships of interspecific compatibility and incompatibility in Solanum. Interspecific pollen rejection by SC species has 16 been observed between members of the tomato and potato clades of Solanum (Baek et al., 2015; Pandey, 1962; Tovar-Méndez et al., 2017). This can be partially explained by the differences in gene presence and function between SC species. S-RNase and other components such as HT which facilitate the function of S-RNase are the primary components of GSI (Markova et al., 2016; Hua et al., 2008; Baek et al., 2015; Tovar‐Méndez et al., 2014, 2017). The lack of functional S-RNase alleles will result in SC and greater acceptance of interspecific pollen (Tovar-Méndez et al., 2017; Enciso-Rodrigues et al., 2019). Additionally, HT has been demonstrated to mediate both SC and S-RNase-independent interspecific compatibility (O’Brien et al., 2002; Tovar-Méndez et al., 2014, 2017). Differences in the response of GSI are also dependent on environmental conditions and plant and floral ontogeny. Environmental conditions such as temperature and humidity significantly impact fruit set even in compatible pollinations (Bienz, 1958; Henderson and LeClerg, 1943). Elevated temperatures and increased floral age has also been observed to significantly weaken the GSI response in S. peruvianum and S. carolinense, respectively (Webb and Williams, 1988; Travers et al., 2004). Because there is a substantial overlap between the mechanisms regulating GSI and interspecific pollen rejection, the impact of temperature and floral age on GSI are relevant to interspecific compatibility as well. It has also been speculated that plant ontogeny plays a role in the strength of the GSI response, independent of floral age (Kaiser, personal communication, 2020). Observational findings have led to the speculation that the GSI response weakens as the plant ages. This has led to the hypothesis that the weakening of GSI in older plants results in greater self-compatibility or that SC is only observable towards the very end of plant maturity. This hypothesis is generally supported by findings in S. carolinense 17 and Nicotiana alata where there is a noticeable deterioration of GSI in older plants (Travers et al., 2004; Liao et al., 2016). Materials and Methods Plant materials The wild species S. bulbocastanum, S. commersonii, S. jamesii, and S. pinnatisectum were used as pollen donors for this study. Multiple plant introduction populations for each species were acquired from the USDA-ARS Potato Germplasm Introduction Station (Sturgeon Bay, WI) and individual genotypes were selected from their respective field-grown plant introduction populations based on their fertility and overall vigor (Table 1). In this study, S- RNase and/or HT-B mediated interspecific pollen rejection was investigated using CRISPR-Cas9 gene knockouts of S. tuberosum clone DRH195 and untransformed controls (Table 2) (Enciso- Rodriguez et al., 2019; Lee et al., 2021). Each genotype was maintained in tissue culture on Murashige and Skoog Basal Medium with Vitamins and Sucrose (PhytoTech Labs) and Phyto Agar (Research Products International) prepared with DI water and balanced with 1M HCL and 8N NaOH to a pH of 5.8, and cultured in growth chambers with 16-h-light/8-h-dark photoperiod at 22°C and average light intensity of 200 μmoles m-2s-1. Greenhouse pollination assays In October of 2020, two individuals from each genotype were transferred directly from tissue culture to 14L (3.8 gallon) plastic pots filled with Suremix Perlite peat and perlite soilless medium (Michigan Grower Products INC.). For the duration of the experiments, greenhouse conditions were maintained at 20° C with a 16-hr photoperiod under Philips GreenPower light- emitting diode (LED) DR/W-MB lights (Philips Lighting Holding B.V., Netherlands). In order to validate the male fertility of the pollen donors, pollen from 5-6 anthers was collected directly 18 onto a glass slide and immediately stained with acetocarmine-glycerol as described by Ordoñez (2014), covered with a cover slip, and sealed using clear nail polish. These slides were stored at room temperature in the dark and visualized the same day using a Leica DM750 binocular microscope (Leica Microsystems, Germany) with the associated Leica imaging software at 10x and 40x magnification. A minimum of 100 pollen grains were used to calculate the percentage of viable pollen. Stained, turgid pollen was classified as viable while any grains that were shriveled, unstained, or unusually large or small were classified as unviable (Figure 2.10). Stylar squash assays For the evaluation of stylar barriers in interspecific crosses, 6-8 newly opened flowers (within 24 hours after anthesis) from each of the female genotypes were carefully emasculated and pollinated with fresh pollen collected on a glass slide directly from the pollen donor. Styles were collected 48 hours post-pollination, by removing the remaining petals and sepals and storing the remaining intact style, ovary, and receptacle in 1.5mL microcentrifuge tube containing a 3:1 ethanol/acetic acid fixation solution. These styles were then kept in the dark at room temperature for at least 24 hours. Styles and ovaries were then softened using an 8N NaOH solution at 60C for 1 hour. Samples were then rinsed three times with distilled water and stained with 0.1% aniline blue in 0.1N K3PO4 keeping them in dark conditions with light shaking. Styles with attached ovaries were then placed on glass slides, gently squashed under a coverslip, and sealed with nail polish for subsequent visualization. Samples were visualized using a Nikon Eclipse Ni-U upright microscope (Nikon Instruments Inc., Melville, NY, United States) with a SOLA light engine (Lumencor, Beaverton, OR, United States), and photographed with the attached ANDOR Zyla sCMOS camera (Oxford Instruments, United Kingdom) and NIS- 19 Elements BR 5.02 software. Images of each stylar sample were stitched together using Image Composite Editor 2.0 software. Pollen tube growth measurements were made using ImageJ 1.53e software (Schneider et al., 2012). Measurements of pollen tube growth were calculated as a proportion of the total length of the style due to the variation of total style length with a single genotype. Pollinations were made at three discrete time points, separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. Data collection and analysis Measurements of the pollen tube front, or the point where the majority of the pollen tubes stop, the longest pollen tube, and the total length of the style were collected using ImageJ software (see Figure 2.1 for example of measurement methods). From these measurements, distance from the surface of the stigma to the pollen tube front and length of the longest pollen tube were calculated based on the total length of the style in which they were measured. Significant differences between means (α = 0.05) from replicated measurements where calculated using analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) using “R” software version 4.0.4. Results The role of S-RNase and HT-B in interspecific pollen rejection S-RNase and HT-B play important roles in GSI by preventing self-pollination and function as primary components of S-RNase dependent and independent rejection of interspecific pollen in Solanum (Tovar-Méndez et al., 2014, 2017; Baek et al., 2015). In this study, the use of stylar squash assays were employed to visualize the growth and inhibition of 20 pollen tubes within the style. Results from statistical analysis of pollen tube growth are consistent with previous findings by Tovar-Méndez et al (2014) showing that S-RNase plays a central role in interspecific pollen rejection. This is readily observable in the majority of pollen tubes as well as the longest pollen tubes from S. bulbocastanum (Figures 2.2-2.3) and the longest pollen tubes for S. pinnatisectum (Figure 2.8). Pollen tubes from these two species were able to penetrate significantly further down the styles in S-RNase and S-RNase/HT-B knockouts. Additionally when S. bulbocastanum and S. pinnatisectum were the pollen donors, the longest pollen tubes were nearly able to traverse the entire length of the style in S-RNase knockouts (Figures 2.2 & 2.8). With S. jamesii (Figures 2.6-2.7) and S. commersonii (Figures 2.4-2.5) as the pollen donors the effect of S-RNase was not statistically significant, although the inhibitory effect of S-RNase was still evident for S. jamesii (Figure 2.6). Results from the same analyses demonstrate that HT-B alone is not a significant factor in the inhibition of pollen tubes from these species. No statistically significant difference was observed between the ability of pollen tubes to penetrate the styles of wild-type plants compared to the HT-B knock-out line. Likewise, the differences between the S-RNase knock-out and the S- RNase/HT-B knock-out were insignificant; showing that knocking out both S-RNase and HT-B compared to S-RNase alone offered no advantage to pollen tubes attempting to traverse the style. For example, pollen tubes from S. jamesii were generally arrested closer to the stigma in the HT- B knock-out compared to the wild type DRH195 (Figure 6). These results do not contradict the findings from Tovar-Méndez et al (2014, 2017) that showed HT proteins played a significant role in interspecific pollen rejection. Due to the vicissitudes of gene editing, knock-outs of HT-A were not available for this study. Since HT-A remains functional and active with its expression in DRH195 shown by RT-PCR and cDNA sequencing in all of the female genotypes used, it can be 21 concluded that either: 1) HT-A plays a greater role in interspecific pollen rejection compared to HT-B; 2) there is a significant overlap in gene function between HT-A and HT-B such that the loss of HT-B is not significant; and/or 3) HT in general is not a significant component of interspecific pollen rejection in these combinations. The contrast between S. commersonii and the other pollen donors used in this study highlights the diversity in interspecific pollen rejection systems. S. commersonii was unique as a pollen donor as both S-RNase and HT-B did not appear to affect the rejection of pollen in DRH195. As discussed previously, since HT-A is still functional, HT-A alone may mediate the S- RNase/HT-B independent rejection of S. commersonii pollen. If this is the case, this would be consistent with the findings of Tovar-Méndez et al (2017). The rejection or allowance of interspecific pollen is entirely dependent on the specific combination of species used as parents (Baek et al., 2015). This is also the case with the role of certain genes in interspecific pollen rejection. HT-A, HT-B, S-RNase and other factors such as the 120 kDa arabinogalactan protein or farnesyl pyrophosphate synthase (FPS2) may play significant or inconsequential roles during the rejection of interspecific pollen; and these roles are entirely dependent on the specific species and genotypes present in these interactions (Bedinger et al, 2011; Baek et al, 2015; Qin and Chetelat, 2021). Effects of plant age on interspecific pollen rejection It has been speculated that plant age plays a role in the GSI response in S. tuberosum and its hybrids. It has been hypothesized that in greenhouse pollinated plants, the GSI response to pollen weakens as a plant ages. Resulting in the hypothesis that the weakening of GSI with age would result in greater self-compatibility in older plants. Outside of Solanum section Petota, this deterioration of GSI with plant age, as well as the weakening of the GSI response, has been 22 observed in S. carolinense and Nicotiana alata (Travers et al., 2004; Liao et al., 2016). In order to evaluate the effect of plant age alone, pollinations were only made on newly opened flowers, within 24 hours of anthesis. Pollinations were made at three discrete time-points, with the first pollination occurring at the initial onset of flowering and each time-point being separated by 30 days. Different conclusions can be drawn from the analysis of pollen tube front and longest pollen tube measurements. Examination of the pollen tube front, the majority of pollen tubes were able to penetrate further down the style in older plants, however, this is not consistent. In contrast, analysis of the longest pollen tubes yielded different results. Individual pollen tubes were able to penetrate significantly further down the style at the third time-point in almost every case, dependent on the pollen donor and the female used. When S. bulbocastanum (Figure 2.3) or S. jamesii (Figure 2.7) were the pollen donors, the highest mean values for the longest pollen tubes were always found in the styles collected at the oldest plant stage. With S. commersonii as a pollen donor, the mean values for the longest pollen tubes were always among the lowest at the first time point (Figure 2.5). For S. pinnatisectum, pollen tubes always traveled further down the style in older plants with the exception of the HT-B knock-out where this trend was reversed (Figure 2.9). Amongst all the pollen donors, individual pollen tubes penetrated significantly further down the style at the third time-point compared to the first; the only exception being the combination of S. pinnatisectum as the pollen donor and the HT-B knock-out being the female (Figure 2.9). Discussion The relationships of interspecific compatibility and incompatibility in Solanum section Petota are complex and intricate. Inquiry into these relationships has elucidated the pleiotropic 23 and redundant function of S-RNase and HT which tandemly and independently mediate both interspecific and intraspecific pollen rejection (Baek et al., 2015; Tovar-Méndez et al., 2014, 2017). The outcome of interspecific pollinations is specific to the individual species involved and direction of the cross due to the direct and indirect involvement of multiple factors such as HT and S-RNase, 120 kDa, Cullin1 (CUL1), and farnesyl pyrophosphate synthase (FPS2) (Bedinger et al., 2011; Li and Chetelat, 2015; McCormick, 2018; Tovar-Méndez et al., 2017). This recent advancement partially explains the inconsistencies to the SI x SC rule found by Pandey (1962) and Dionne (1961) where interspecific pollen was rejected by the SC species S. polyadenium. The findings presented here are consistent with previous work conducted in Solanum section Lycopersicon showing that S-RNase plays a central role in interspecific pollen rejection (Baek et al., 2015; Tovar-Méndez et al., 2014). Statistical analyses also demonstrated that HT-B alone is not a significant factor in these pollinations. These results do not contradict the findings from Tovar-Méndez et al. (2014, 2017) where HT proteins played a significant role in interspecific pollen rejection. Since HT-A remained functional in all of the female genotypes used these findings demonstrate the overlap in gene function between HT-A and HT-B, the greater importance of HT-A, and/or that HT is not a significant factor in these combinations implicating other mechanisms (Baek et al, 2015; Qin and Chetelat, 2021). In comparison to the other species used as pollen donors, S. commersonii was unique as both S-RNase and HT-B did not appear to affect the rejection of pollen in DRH195. Since the rejection or allowance of interspecific pollen is entirely dependent on the specific combination of species used as parents, HT, S-RNase and other factors may play significant or inconsequential roles during the rejection of interspecific pollen (Bedinger et al, 2011; Baek et al., 2015). In this instance HT-A alone may mediate the S-RNase independent rejection of S. commersonii pollen in 24 the HT-B/S-RNase knock-out. If this is the case, this would be consistent with the findings of Tovar-Méndez et al (2017). Intra- and interspecific compatibility is also heavily influenced by environmental and ontogenic factors such as temperature, humidity, flower age, and plant age (Bienz, 1958; Henderson and LeClerg, 1943; Travers et al., 2004; Liao et al., 2016; Webb and Williams, 1988). Although variable GSI responses due to plant and floral age have been demonstrated in S. carolinense and N. alata, these findings have not been observed in interspecific pollinations (Travers et al., 2004; Liao et al., 2016). In order to examine the effect of plant age on interspecific pollen tube growth, pollinations were made only on newly opened flowers, within 24 hours of anthesis, at three discrete time-points. The first time-point for pollinations occurred at the initial onset of flowering, with each of the time-points being separated by 30 days. The results from statistical analyses indicate that plant age does have a significant impact on interspecific pollen rejection, consistent with the findings published by Travers et al., (2004) and Liao et al., (2016). Analysis of the longest pollen tubes revealed that individual pollen tubes were able to travel significantly further down the style at the third time-point in almost every case. Amongst all the pollen donors, individual pollen tubes penetrated significantly further down the style at the third time-point compared to the first; the only exception being the combination of S. pinnatisectum as the pollen donor and the HT-B knock-out being the female where this trend was reversed. Across all females and pollen donors, the third time point yielded the only cases where pollen tubes were nearly able to travel completely down the style. Future assessment of the role of HT-A in interspecific pollen rejection is essential to inform our understanding of interspecific pollination barriers. Efforts are being made to make these gene edits and further assessment will be made after the edits are confirmed. There is still a 25 lot to understand about plant ontogeny and the degradation of interspecific and GSI barriers with age. Since all of the plants in this study were planted at the same time, it is unknown whether the age of the pollen donor or the female parent are more or less responsible for the break-down in prezygotic barriers. Further inquiry into these questions will aid in the identification of the causative mechanisms. 26 APPENDICES 27 APPENDIX A: Chapter 2 Tables Pollen Donors ID Species PI# SBGG505-A S. bulbocastanum 275197 Scmm320266-02 S. commersonii 320266 SPGG544-A S. pinnatisectum 275232 SJGG520-A S. jamesii 592417 Table 2.1. List of potato-related species used as pollen donors. PI# indicates the source plant introduction. Female Genotypes Line Gene Edit DRH195 WT (wild type) DRH195.158 S-RNase DRH195.121_009 HT-B DRH195.124_001 S-RNase/HT-B Table 2.2a. Genotypes used as female parents and respective gene edit. Line Edit sgRNA-HTB1 PAM DRH195 5' ATGGCAAATATTTTGCTTATATTTTCTTTGGTTCTTATGATTATATCATCAGAAGTTATTGCAAGGGAGATAGTTGAGCCTTCACTTCCATTGCTTGAGG 3' 121_009 -15(bp) 5' ATGGCAAATATTTTGCTTATATTTTCTTTGGTTCTTATGATTATATCATCAGAAGTTATTG---------------AGCCTTCACTTCCATTGCTTGAGG 3' 124_001 -6(bp) 5' ATGGCAAATATTTTGCTTATATTTTCTTTGGTTCTTATGATTATATCATCAGAAG------CAAGGGAGATAGTTGAGCCTTCACTTCCATTGCTTGAGG 3' Line Edit sgRNA-1 PAM DRH195 5' ATGGGGATTTCGACAAATTGCAACTGGTATTAACATGGCCACCATCATTT 3' DRH195.158 -1(bp) 5' ATGGGGATTTCGACAAATTGCAACTGGTATT-ACATGGCCACCATCATTT 3' 124_001 -2(bp) 5' ATGGGGATTTCGACAAATTGCAACTGGTAT--ACATGGCCACCATCATTT 3' Table 2.2b. Description of gene edits in female genotypes used. The first alignment depicts HT- B gene edits for lines 121_009 and 124_001 compared to the wild-type DRH195 sequence. Line 121_009 has a 15bp deletion flanking the PAM (protospacer adjacent motif) sequence while line 124_001 shows a 6bp deletion upstream from the PAM sequence. The second alignment depicts the gene edits in S-RNase for DRH195.158 and 124_001 which have 1bp and 2bp deletions respectively, upstream from the PAM sequence. The single guide RNA sequences are also shown: sgRNA-HTB1 (single guide RNA targeting the first exon of the HT-B gene), sgRNA-1 (single guide RNA targeting the first exon of the S-RNase gene. 28 APPENDIX B: Chapter 2 Figures Figure 2.1. Pollen tube measurement description (style of DRH195 with S. pinnatisectum as pollen donor). Total length of the style was measured along the midline of the style from the stigma surface to the base of the style, following the curvature if present (depicted by the red line). The pollen tube front was measured along the midline, at the point where the majority of the pollen tubes stopped (Blue line). The measurement would be taken at the point where the blue and red lines intersect. The longest pollen tube (identified by the green arrow) was measured along the midline and would be taken where the green and red line intersect. 29 Figure 2.2. Measurement of the longest pollen tube as well as the pollen tube front with S. bulbocastanum as a pollen donor. Blue bars depict the estimated marginal pollen tube means (emmean) as a proportion of total style length at 48h post pollination. Female knock-outs and wild type DRH195 (S. tuberosum) genotypes are depicted on the left vertical axis, and time- points or rounds are depicted on the right vertical axis. Pollinations were made at three discrete time points (rounds), separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. 30 Figure 2.3. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study with S. bulbocastanum as the pollen donor, showing effect of plant age on interspecific pollen tube inhibition. The vertical axis depicts the estimated marginal means (Linear prediction) of the longest measured pollen tube as a proportion of the total length of the style. The horizontal axis plots the three pollination time-points (rounds) used in the study with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse with subsequent pollination rounds separated by 30 days. 31 Figure 2.4. Measurement of the longest pollen tube as well as the pollen tube front with S. commersonii as a pollen donor. Blue bars depict the estimated marginal pollen tube means (emmean) as a proportion of total style length at 48h post pollination. Female knock-outs and wild type DRH195 (S. tuberosum) genotypes are depicted on the left vertical axis, and time- points or rounds are depicted on the right vertical axis. Pollinations were made at three discrete time points (rounds), separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. 32 Figure 2.5. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study with S. commersonii as the pollen donor, showing effect of plant age on interspecific pollen tube inhibition. The vertical axis depicts the estimated marginal means (Linear prediction) of the longest measured pollen tube as a proportion of the total length of the style. The horizontal axis plots the three pollination time-points (rounds) used in the study with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse with subsequent pollination rounds separated by 30 days. 33 Figure 2.6. Measurement of the longest pollen tube as well as the pollen tube front with S. jamesii as a pollen donor. Blue bars depict the estimated marginal pollen tube means (emmean) as a proportion of total style length at 48h post pollination. Female knock-outs and wild type DRH195 (S. tuberosum) genotypes are depicted on the left vertical axis, and time-points or rounds are depicted on the right vertical axis. Pollinations were made at three discrete time points (rounds), separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. 34 Figure 2.7. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study with S. jamesii as the pollen donor, showing effect of plant age on interspecific pollen tube inhibition. The vertical axis depicts the estimated marginal means (Linear prediction) of the longest measured pollen tube as a proportion of the total length of the style. The horizontal axis plots the three pollination time-points (rounds) used in the study with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse with subsequent pollination rounds separated by 30 days. 35 Figure 2.8. Measurement of the longest pollen tube as well as the pollen tube front with S. pinnatisectum as a pollen donor. Blue bars depict the estimated marginal pollen tube means (emmean) as a proportion of total style length at 48h post pollination. Female knock-outs and wild type DRH195 (S. tuberosum) genotypes are depicted on the left vertical axis, and time- points or rounds are depicted on the right vertical axis. Pollinations were made at three discrete time points (rounds), separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. 36 Figure 2.9. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study with S. pinnatisectum as the pollen donor, showing effect of plant age on interspecific pollen tube inhibition. The vertical axis depicts the estimated marginal means (Linear prediction) of the longest measured pollen tube as a proportion of the total length of the style. The horizontal axis plots the three pollination time-points (rounds) used in the study with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse with subsequent pollination rounds separated by 30 days. 37 Figure 2.10. Example images of pollen viability assays using acetocarmine-glycerol as described by Ordoñez (2014). Starting in the upper left-hand corner and moving in a clockwise are pollen grains from each of the four pollen donors: S. bulbocastanum (SBGG505-A), S. commersonii (Scmm320266-02), S. jamesii (SJGG520-A), and S. pinnatisectum (SPGG544-A). 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E., & Stephenson, A. G. (2004). Plasticity in the self‐ incompatibility system of Solanum carolinense. Plant Species Biology, 19(3), 127-135. Whalen, M. D., & Anderson, G. J. (1981). Distribution of gametophytic self‐incompatibility and infrageneric classification in Solanum. Taxon, 30(4), 761-767. Webb, M. C., & Williams, E. G. (1988). Effects of temperature, light, nutrients and carbon dioxide on the strength of the self-incompatibility response in detached flowers of Lycopersicon peruvianum. Annals of botany, 61(4), 395-404. 42 CHAPTER 3 EVALUATION OF SLI AS A GENETIC RESOURCE TO OVERCOME INTERSPECIFIC PREZYGOTIC BARRIERS IN SOLANUM SECTION PETOTA Abstract The ability of Sli to overcome interspecific barriers was reported by Sanetomo et al. (2014). Because Sli based self-compatibility (SC) is widespread in North American diploid germplasm, the use of Sli to overcome interspecific barriers could represent a both underutilized and widely accessible resource. Statistical analysis of pollen tube growth in four wild species S. pinnatisectum, S. bulbocastanum, S. commersonii, and S. jamesii demonstrated that there was no practical significance between pollen donors carrying Sli and pollen donors without Sli. These findings directly contradict previous findings of Sanetomo et al. (2014) using the same S. pinnatisectum PI (275232). These inconsistencies are discussed and can be largely explained by differences in pollen donors used. Objective: Demonstrate the ability of Sli to overcome prezygotic barriers in interspecific pollinations. Despite previous research claiming otherwise, Sli was found to be an insignificant factor in these interspecific crosses. Introduction The 107 wild species of potato represent a rich and diverse source of disease resistance and tuber quality traits for cultivated potato (Spooner and Bamberg, 1994; Jansky and Hamernik, 2009). However, in the century long history of potato breeding the enormous value of wild species traits has been realized only occasionally (Bethke et al., 2017). Among all the species characterized, the diploid 1EBN species are of particular interest as they exhibit exceptional resistance to economically important pests such as early blight (Alternaria solani), Colorado 43 potato beetle (Leptinotarsa decemlineata), and late blight (Phytophthora infestans) (Jansky et al., 2008, 2009; Zlesak and Thill 2004; Zheng et al., 2020). However, due to prezygotic stylar barriers and differences in effective ploidy between 1EBN species and cultivated germplasm, accessing the traits from these species is extraordinarily difficult (Novy and Hanneman, 1991; Jansky and Hamernik, 2009; Städler et al., 2021). If Sli is able to overcome barriers between diploid cultivated germplasm and the 1EBN species, it would be an enormous breakthrough. The ability of Sli to overcome interspecific barriers was reported by Sanetomo et al. (2014). Using inbred 2x pollen donors carrying Sli, 388 total pollinations were made with 4x S. pinnatisectum (PI275232) yielding nine 3x hybrid seedlings. It was also observed that the pollen tubes from Sli carrying pollen donors were able to bypass stylar barriers, while pollen tubes from S. megistacrolobum, S. demissum, and S. phureja stopped growing at or near the stigma of 4x and 2x S. pinnatisectum (Sanetomo et al., 2014). The ability of Sli to overcome all prezygotic stylar barriers and not just those controlling gametophytic self-incompatibility could be an important factor in accessing the sexually isolated Mexican diploid species (Sanetomo et al., 2014). Additionally, the widespread use of Sli-based self-compatibility (SC) in North American diploid potato breeding would mean that most public and private breeders would have access to these methods (Kaiser et al., 2021). Currently, North American diploid potato breeding largely relies on the dominant SI inhibitor gene Sli on chromosome 12 as a source of SC (Hosaka and Hanneman 1998a, b; Kaiser et al., 2021). The inbred S. chacoense clone M6 is currently a primary source of Sli based SC, and an alternative source to the other S. chacoense Sli donor clones that harbor recessive deleterious and lethal alleles linked to Sli (Endelman et al., 2019; Jansky et al., 2014; Kaiser et al., 2021). The employment of Sli to overcome interspecific barriers could represent a powerful 44 albeit underutilized method to access valuable traits from sexually isolated species. In order to better capture the value of Sli, this study was designed to confirm the findings of Sanetomo et al. (2014) and determine if these findings can be applied to other Solanum species. Relationships of interspecific compatibility and incompatibility in Solanum are complex and are mediated by many different characterized and uncharacterized factors (Bedinger et al., 2011; Pandey, 1962; Tovar‐Méndez et al., 2017). To eliminate variables associated with other species, only S. chacoense pollen donors were selected for this study. Materials and Methods Plant Materials The S. chacoense clone USDA8380-1 (PI 458310, 80-1) and the S. chacoense self- compatible inbred line M6 were selected as pollen donors selected for this study (Jansky et al., 2014). The M6 clone was selected as it is a homozygous, well characterized, and widely used source of Sli based SC (Jansky et al., 2014; Kaiser et al., 2021). In order to reduce confounding variables the self-incompatible clone 80-1 was selected as a control as it is largely homozygous, and also the same species as M6 (Kaiser et al., 2020). Individual clones were selected as representatives of genetically valuable wild species (S. bulbocastanum, S. commersonii, S. jamesii, and S. pinnatisectum) and were used as females. These female clones were selected from multiple plant introduction populations of each species acquired from the USDA-ARS Potato Germplasm Introduction Station (Sturgeon Bay, WI) with individual genotypes selected from their respective field-grown plant introduction populations based on their fertility and overall vigor (Table 1). Each genotype was maintained in tissue culture on Murashige and Skoog Basal Medium with Vitamins and Sucrose (PhytoTech Labs) and Phyto Agar (Research Products International) prepared with DI water and balanced with 1M HCL and 8N NaOH to a pH of 5.8, 45 and cultured in growth chambers with 16-h-light/8-h-dark photoperiod at 22°C and average light intensity of 200 μmoles m-2s-1. Greenhouse propagation and pollinations In October of 2020, two individuals from each genotype were transferred directly from tissue culture to 14L (3.8 gallon) plastic pots with filled with Suremix Perlite peat and perlite soilless medium (Michigan Grower Products INC.). For the duration of the experiments greenhouse conditions were maintained at 20°C with a 16-hr photoperiod under Philips GreenPower light-emitting diode (LED) DR/W-MB lights (Philips Lighting Holding B.V., Netherlands). The pollen donors 80-1 and M6 are known to exhibit high levels of male fertility, pollen from these individuals displayed high percentage of pollen stainability as well as high percentages of in vitro and in vivo pollen tube growth (Alsahlany et al., 2021; Jansky et al., 2014). Male fertility was validated by collecting pollen from 5-6 anthers directly onto a glass slide and immediately stained with acetocarmine-glycerol as described by Ordoñez (2014), covered with a cover slip, and sealed using clear nail polish. These slides were stored at room temperature in the dark and visualized the same day using a Leica DM750 binocular microscope (Leica Microsystems, Germany) and the associated Leica imaging software at 10x and 40x magnification. A minimum of 100 pollen grains were then used to calculate the percentage of viable pollen. Stained, turgid pollen was classified as viable while any grains that were shriveled, unstained, or unusually large or small were classified as unviable. Stylar squash: assay of pollen tube growth For the evaluation of stylar barriers in interspecific crosses, 6-8 newly opened flowers (within 24 hours after anthesis) from each of the female genotypes were carefully emasculated 46 and pollinated with fresh pollen collected on a glass slide directly from the pollen donor. Styles were collected 48 hours post-pollination, by removing the remaining petals and sepals and storing the remaining intact style, ovary and receptacle in 1.5mL microcentrifuge tube containing a 3:1 ethanol/acetic acid fixation solution. These styles were then kept in the dark at room temperature for at least 24 hours. Styles and ovaries were then softened using an 8N NaOH solution at 60°C for 1 hour. Samples were then rinsed three times with distilled water and stained with 0.1% aniline blue in 0.1N K3PO4 keeping them in dark conditions with light shaking. Styles with attached ovaries were then placed on glass slides, gently squashed under a coverslip, and sealed with nail polish for subsequent visualization. Samples were visualized using a Nikon Eclipse Ni-U upright microscope (Nikon Instruments Inc., Melville, NY, United States) with a SOLA light engine (Lumencor, Beaverton, OR, United States), and photographed with the attached ANDOR Zyla sCMOS camera (Oxford Instruments, United Kingdom) and NIS- Elements BR 5.02 software. Images of each stylar sample were stitched together using Image Composite Editor 2.0 software. Pollen tube growth measurements were made using ImageJ 1.53e software (Schneider et al., 2012). Due to the variation of style length with a single genotype, measurements of pollen tube growth were calculated as a proportion of the total length of the style. Pollinations were made at three discrete time points, separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. Data collection and analysis Measurements of the pollen tube front, or the point where the majority of the pollen tubes stop, the longest pollen tube, and the total length of the style were collected using ImageJ software. From these measurements, distance from the surface of the stigma to the pollen tube 47 front and length of the longest pollen tube were calculated based on the total length of the style in which they were measured. Significant differences between means (P<0.05) from replicated measurements where calculated using analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) using “R” software version 4.0.4 (R Foundation for Statistical Computing, 2012, available at www.r-project.org). Results The role of Sli in interspecific pollen rejection A statistically significant difference was observed between Sli +/+ and Sli -/- pollen donors in the styles of S. bulbocastanum and to a lesser extent S. commersonii, with pollen tubes of the Sli+/+ clone M6 traveling further down the style (Figures 3.1 and 3.2). However, findings from the same analysis failed to find any statistical significance between pollen donors in the styles of S. jamesii or S. pinnatisectum, which is inconsistent with previous findings (Figures 3.1 and 3.2). Pollen tubes from M6 generally traveled further down the style in all of the four species compared to pollen tubes from the self-incompatible Sli -/- clone 80-1 (Figure 3.2). However, even the statistically significant differences between pollen donors failed to have any practical significance and did not change the outcome of these interspecific pollinations. In the styles of S. commersonii and S. bulbocastanum, the difference between where the majority of the pollen tubes stopped was not significant in the first time point. In the subsequent time-points, the growth of the majority of pollen tubes from 80-1 was arrested closer to the stigma while the majority of pollen tubes from M6 penetrated much further down the style. As noted above, this difference lacked significance as the longest pollen tubes from both parents were able to traverse the entirety of the style in S. commersonii, and all pollen tubes were arrested in the first third of the style in S. bulbocastanum (Figure 3.4). 48 As with S. bulbocastanum and S. commersonii, Sli presence or absence did not have a significant impact on interspecific pollen-tube inhibition in the styles of S. jamesii and S. pinnatisectum. Pollen tubes from M6 generally grew further down the style in S. pinnatisectum with the opposite occurring for S. jamesii (Figures 3.1 and 3.2). No pollen tubes from either pollen donor entered the ovary in S. jamesii (Figure 3.2), while the majority of pollen tubes from both pollen donors traversed the entire style in S. pinnatisectum (Figure 4). Pollinations of S. pinnatisectum by both M6 and 80-1 also formed fruit although all the seeds were aborted. Effects of plant ontogeny It is clear that plant maturity plays a role in the interspecific pollen-tube rejection response in all the species evaluated in this study (Figure 3.3). In S. pinnatisectum and S. jamesii, no statistical difference was observed between pollen tube measurements taken at different time- points. Despite this, it is clear that pollen tubes from both parents were more likely to travel further down the style at the third time-point compared to the first time-point. This trend aligns with the results observed in S. commersonii that is statistically significant (Figure 3.3). Analysis of pollen tubes in S. bulbocastanum styles displayed dynamic genotype by environmental effects. At the first time-point, pollen-tubes from 80-1 penetrated further down the style compared to M6. At the second and third time-points, pollen-tube growth of 80-1 was arrested closer to the stigma, while M6 pollen-tubes traveled significantly further down the style (Figure 3.3). Discussion Although the difference between pollen donors was statistically significant in the styles of S. commersonii and S. bulbocastanum, the differences between pollen donors lacked any practical significance. The presence or absence of Sli did not change the outcome of any of the pollinations made. Additionally, no significant difference was observed in pollinations made 49 with S. pinnatisectum, which is inconsistent with the findings of Sanetomo et al. (2014) despite the use of the same S. pinnatisectum PI (273232). This study suggests that Sli is not a significant factor in interspecific pollen rejection in these pollinations. The lack of consistent findings between these experiments and those of Sanetomo et al. (2014) is likely attributable to some unknown factors in S. chacoense besides Sli. The pollinations conducted in this study demonstrated that the clones M6 and 80-1 were comparable in their ability to bypass stylar barriers, or in their uniform inhibition in the styles of S. bulbocastanum. Unlike the pollen donors used by Sanetomo et al. (2014), these pollen donors were from a single species, S. chacoense while the pollen donors used by Sanetomo et al. (2014) came from interspecific crosses between S. chacoense and S. phureja. The difference between pollen donors is the likely source of inconsistencies as shown in the introduction of unknown genetic variables from S. phureja presenting a confounding variable in the studies carried out by Sanetomo et al. (2014). The ability to form viable seeds cannot be attributed to the presence or absence of Sli either, as postulated by Sanetomo et al. (2014). Hosaka and Hanneman (1998a, b), observed that Sli inhibits S-gene expression in the pollen, thus overcoming the prezygotic GSI barrier. Recent findings have identified Sli as a non-S-locus F box protein capable of interacting with a diverse array of S-RNase alleles (Eggers et al.,2021; Ma et al.. 2021). Failure to produce viable seed after fertilization is a post-zygotic barrier that is exhibited commonly as endosperm failure. These two interspecific barriers are independent even though the outcome is the same. Because the genetic and molecular mechanisms surrounding endosperm failure and effective ploidy are complex and poorly characterized it would be inappropriate to conclude that a genetic factor effecting pre- 50 zygotic stylar barriers also plays a role in post-zygotic barriers without significant substantial evidence. The effect of plant ontogeny plays a fascinating role in interspecific pollen rejection. No statistical significance was observed between pollen tube measurements taken at different time points was found in S. pinnatisectum or S. jamesii. However, it is clear that pollen tubes from both parents were more likely to travel further down the style at the third time-point compared to the first time-point. The differences between the growth pollen tubes in the styles of S. bulbocastanum over time also show the dynamic nature of prezygotic barriers over time. Although Sli played no significant role in these pollinations, it is possible that S. chacoense as a pollen donor is better able to bypass stylar barriers in EBN1 species such as S. pinnatisectum compared to S. megistacrolobum, S. demissum, and S. phureja (S. tuberosum group phureja) as reported by Sanetomo et al. (2014). Consequently, S. chacoense may be a valuable resource in accessing these EBN1 species regardless of Sli status. Currently, other methods such as bridge crossing with S. verrucosum are more reliable and feasible in their ability access EBN1 species (Jansky, and Hamernik; 2009). Unlike the methods proposed by Sanetomo et al. (2014) and Carputo et al. (1997), the use of S. verrucosum bridge crosses is also more accessible for most plant breeders as it does not require ploidy manipulation, interploidy crosses, or the use of advanced techniques (Jansky, and Hamernik; 2009). 51 APPENDICES 52 APPENDIX A: Chapter 3 Tables Female Genotypes Male Genotypes ID Species PI# ID Species PI# SBGG505-A S. bulbocastanum 275197 M6 S. chacoense (Jansky et Scmm320266-02 S. commersonii 320266 al., 2014) SPGG544-A S. pinnatisectum 275232 USDA8380-1 S. chacoense 458310 SJGG520-A S. jamesii 592417 Table 3.1. List of potato-related species used as females in this study. PI number indicates the source plant introduction. 53 APPENDIX B: Chapter 3 Figures Figure 3.1. Measurement of pollen tube front according to female. Starting from the top left and moving clockwise: S. bulbocastanum, S. commersonii, S. pinnatisectum, and S. jamesii. Blue bars depict the marginal pollen tube means (emmean) as a proportion of total style length at 48h post pollination. Female genotypes are depicted on the left vertical axis, and time-points or rounds are depicted on the right vertical axis. Pollinations were made at three discrete time points (rounds), separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. 54 Figure 3.2. Measurement of the longest pollen tube according to female. Starting from the top left and moving clockwise: S. bulbocastanum, S. commersonii, S. pinnatisectum, and S. jamesii. Blue bars depict the marginal pollen tube means (emmean) as a proportion of total style length at 48h post pollination. Female genotypes are depicted on the left vertical axis, and time-points or rounds are depicted on the right vertical axis. Pollinations were made at three discrete time points (rounds), separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. 55 Figure 3.3. Estimated marginal means (Linear prediction), plotted against the different time- points (rounds) used in this study according to female. Starting from the top left and moving clockwise: S. bulbocastanum, S. commersonii, S. pinnatisectum, and S. jamesii showing the effect of plant age on interspecific pollen tube inhibition. The vertical axis depicts the estimated marginal means (Linear prediction) of the longest measured pollen tube as a proportion of the total length of the style. The horizontal axis plots the three pollination time-points (rounds) used in the study with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse with subsequent pollination rounds separated by 30 days. 56 S. pinnatisectum x S. pinnatisectum x S. bulbocastanum x S. bulbocastanum x M6 80-1 M6 80-1 Figure 3.4. Examples of styles pollinated with 80-1 and M6, showing no difference between pollen donors despite the presence of Sli in M6. Styles of S. pinnatisectum are displayed in the two images on the left side, and styles from S. bulbocastanum are displayed in the two images on the right side. Pollen tubes from both pollen donors are uniform in their ability to penetrate the full length of the styles in S. pinnatisectum. In S. bulbocastanum, pollen tubes from both pollen donors are arrested in the first third of the style. 57 REFERENCES 58 REFERENCES Alsahlany, M., Enciso-Rodriguez, F., Lopez-Cruz, M. et al. 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Breakdown of a hybridization barrier between Solanum pinnatisectum Dunal and potato using the S locus inhibitor gene (Sli). Euphytica 197, 119–132. https://doi.org/10.1007/s10681-013-1057-1 Spooner, D.M., Bamberg, J.B. (1994). Potato genetic resources: Sources of resistance and systematics. American Potato Journal 71, 325–337. https://doi.org/10.1007/BF02849059 60 Städler, T., Florez-Rueda, A. M., & Roth, M. (2021). A revival of effective ploidy: the asymmetry of parental roles in endosperm-based hybridization barriers. Current Opinion in Plant Biology, 61, 102015. https://doi.org/10.1016/j.pbi.2021.102015 Tovar‐Méndez, A., Lu, L. and McClure, B. (2017), HT proteins contribute to S‐RNase‐ independent pollen rejection in Solanum. Plant J, 89: 718-729. doi:10.1111/tpj.13416 Zheng, J., Duan, S., Armstrong, M. R., Duan, Y., Xu, J., Chen, X., ... & Li, G. (2020). New Findings on the Resistance Mechanism of an Elite Diploid Wild Potato Species JAM1-4 in Response to a Super Race Strain of Phytophthora infestans. Phytopathology, 110(8), 1375-1387. https://doi.org/10.1094/PHYTO-09-19-0331-R Zlesak, D. C., & Thill, C. A. (2004). Foliar resistance to Phytophthora infestans (Mont.) de Bary (US-8) in 13 Mexican and South American Solarium species having EBNs of 1, 2, and 4 and implications for breeding. American journal of potato research, 81(6), 421-429. https://doi.org/10.1007/BF02870203 61 CHAPTER 4 ASSESSING THE ROLE OF S-RNASE IN SELF_COMPATIBILITY AND INTERSPECIFIC COMPATIBILITY IN SOLANUM VERRUCOSUM Abstract Solanum verrucosum (2x=2n=24 (2EBN)) is a unique wild potato species originating from central Mexico. Primary traits of interest include self-compatibility (SC) and the ability to act a parent in as a bridge crosses to access traits from genetically isolated 1EBN wild species. The postzygotic barrier of endosperm failure is a large gap that separates cultivated potato from the valuable traits in the 1EBN wild species. The use of S. verrucosum bridge crosses is a simple and valuable technique that offers direct access to these trait as bridge crosses using S. verrucosum require less time and resources compared to other methods used to access valuable traits from 1EBN species despite its low efficiency. In order to increase the efficiency of this method, characterization of interspecific barriers and S-RNase expression was necessary. Stylar pollen tube growth from four 1EBN pollen donors were used to visualize prezygotic barriers; no significant stylar barriers were observed. The use of primers designed to amplify conserved regions of S-RNase were able to capture two partial S-RNase alleles; however, RT-PCR expression analysis indicated that these were not expressed. Further inquiry into the SC and interspecific compatibility mechanisms in S. verrucosum is required to further characterize these mechanisms. Objective: Demonstrate and validate that self- and broad interspecific compatibility in S. verrucosum is primarily rooted in its lack of functional S-RNase. Although genomic S-RNase sequences were discovered and sequenced, not expression was detected. 62 Introduction The wild species Solanum verrucosum (2x=2n=24 (2EBN)) is unique among the wild species of potato for several reasons. Geographically speaking, S. verrucosum is an anomaly as it is closely related to species from southern South America and only distantly related to the other wild species in central Mexico where it is found (Lara-Cabrera and Spooner, 2004; Spooner et al., 2004; Huang et al., 2018). S. verrucosum is also unusual as it is self-compatible unlike the vast majority of species within Solanum section Petota (Abdalla, and Hermsen, 1973; Spooner et al., 2014). Of primary interest to potato breeders is the ability of S. verrucosum to act as a bridging species to access genetically isolated wild species (Hermsen and Ramanna, 1976; Hamernik et al., 2001). Although a large portion of diploid wild potato species are intercrossable, there are important barriers that prevent the use of certain species. When two species used in interspecific crosses have different levels of “effective ploidy” or EBN, these crosses are rarely successful (Stadler et al., 2021: Johnston et al., 1980). Interspecific crosses of two diploid species of differing EBN usually results in endosperm failure which represents a strong postzygotic barrier to interspecific hybridization (Johnston et al., 1980; Lafon-Placette, and Köhler, 2016). This is a significant barrier to the genetic improvement of potato as diploid and tetraploid cultivated potatoes have an EBN of two and four respectively while the Mexican diploid species which have exceptional disease and pest resistance traits have an EBN of one (Jansky and Hamernik, 2009). Because of this difference in effective ploidy, introgressing traits from these species is exceedingly difficult. The postzygotic barrier of endosperm failure is a large gap that separates cultivated potato from the valuable traits in the 1EBN wild species. The use of S. verrucosum bridge 63 crosses are a simple and valuable technique that offers direct access to these traits (Jansky and Hamernik, 2009: Yermishin et al., 2014). Although this technique has relatively low efficiency, it is still less time and resource intensive than previous attempts to access valuable traits from 1EBN species using complex strategies of ploidy manipulation, somatic fusion, embryo rescue, and transgenesis (Carputo et al., 1997, Helgeson et al., 1998; Orbegozo et al., 2016; Valkonen et al., 1995). The ability of S. verrucosum to form interspecific hybrids with species of differing EBN is partially rooted in its lack of a functional S-RNase protein (Eijlander, 1998). The absence of functional S-RNase protein prevents the inhibition on pollen tube growth in what would otherwise be incompatible pollinations. While it is known that there is no detectable S-RNase at the protein level (Eijlander, 1998), it is unknown if the genes for S-RNase in S. verrucosum are missing, non-functional, or inhibited in some way. The lack of a functional S-RNase protein in S. verrucosum is likely the reason it serves as the major bridge species in between EBN1 species and cultivated germplasm. Endosperm failure is the main barrier that impedes the use of certain species. Prezygotic barriers (e.g. S-RNase and/or HT proteins) conceal relationships that might otherwise be compatible. Materials and methods Plant materials The wild species S. bulbocastanum, S. commersonii, S. jamesii, and S. pinnatisectum were used as pollen donors for this study. Multiple plant introduction populations for each species were acquired from the USDA-ARS Potato Germplasm Introduction Station (Sturgeon Bay, WI) and individual genotypes were selected from their respective field-grown plant introduction populations based on their fertility and overall vigor (Table 1). In this study 64 interspecific compatibility in S. verrucosum was investigated using the clone SV607845.02. This S. verrucosum clone (Table 1) was selected based on its previous ability to create interspecific hybrid offspring with S. bulbocastanum. Each genotype was maintained in tissue culture on Murashige and Skoog Basal Medium with Vitamins and Sucrose (PhytoTech Labs) and Phyto Agar (Research Products International) prepared with DI water and balanced with 1N HCL and 8N NaOH to a pH of 5.8 and cultured in growth chambers with 16-h-light/8-h-dark photoperiod at 22°C and average light intensity of 200 μmoles m-2s-1. Greenhouse pollination assays In October of 2020, two individuals from each genotype were transferred directly from tissue culture to 14L (3.8 gallon) plastic pots with filled with Suremix Perlite peat and perlite soilless medium (Michigan Grower Products INC.). For the duration of the experiments greenhouse conditions were maintained at 20°C with a 16-hr photoperiod under Philips GreenPower light-emitting diode (LED) DR/W-MB lights (Philips Lighting Holding B.V., Netherlands). In order to validate the male fertility of the pollen donors, pollen from 5-6 anthers was collected directly onto a glass slide and immediately stained with acetocarmine-glycerol as described by Ordoñez (2014), covered with a cover slip, and sealed using clear nail polish. These slides were stored at room temperature in the dark and visualized the same day using a Leica DM750 binocular microscope (Leica Microsystems, Germany) and the associated Leica imaging software at 10x and 40x magnification. A minimum of 100 pollen grains were then used to calculate the percentage of viable pollen. Stained, turgid pollen was classified as viable while any grains that were shriveled, unstained, or unusually large or small were classified as unviable. 65 Stylar squash assays For the evaluation of stylar barriers in interspecific crosses, 6-8 newly opened flowers (within 24 hours after anthesis) from each of the female genotypes were carefully emasculated and pollinated with fresh pollen collected on a glass slide directly from the pollen donor. Styles were collected 48 hours post-pollination, by removing the remaining petals and sepals and storing the remaining intact style, ovary and receptacle in 1.5mL microcentrifuge tube containing a 3:1 ethanol/acetic acid fixation solution. These styles were then kept in the dark at room temperature for at least 24 hours. Styles and ovaries were then softened using an 8N NaOH solution at 60°C for 1 hour. Samples were then rinsed three times with distilled water and stained with 0.1% aniline blue in 0.1N K3PO4 keeping them in dark conditions with light shaking. Styles with attached ovaries were then placed on glass slides, gently squashed under a coverslip, and sealed with nail polish for subsequent visualization. Samples were visualized using a Nikon Eclipse Ni-U upright microscope (Nikon Instruments Inc., Melville, NY, United States) with a SOLA light engine (Lumencor, Beaverton, OR, United States), and photographed with the attached ANDOR Zyla sCMOS camera (Oxford Instruments, United Kingdom) and NIS-Elements BR 5.02 software. Images of each stylar sample were stitched together using Image Composite Editor 2.0 software. Pollen tube growth measurements were made using ImageJ 1.53e software (Schneider et al., 2012). Measurements of pollen tube growth were calculated as a proportion of the total length of the style due to the variation of total style length with a single genotype. Pollinations were made at three discrete time points, separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. 66 Pollen tube data analysis Measurements of the pollen tube front, or the point where the majority of the pollen tubes stop, the longest pollen tube, and the total length of the style were collected using ImageJ software (Figure 1 for an example of measurement methods). From these measurements, distance from the surface of the stigma to the pollen tube front and length of the longest pollen tube were calculated based on the total length of the style in which they were measured. Significant differences between means (α = 0.05) from replicated measurements where calculated using analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) using “R” software version 4.0.4 Assessment of S-RNase gene expression in Solanum verrucosum Amplification and sequencing of S-RNase DNA was isolated from young leaves of SV607845.02 using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Two sets of primers were used to capture the S-RNase sequences of both alleles. The SP3 primer set designed by Ye et al. (2018) was only able to capture one allele sequence (Table 1). A second set of primers were designed using reported S-RNase sequences from the Solanaceae family. For this, a T-BLASTN was performed against the M6 genome assembly using BLAST v2.2.31 (Altschul et al., 1990) with default parameters. Primers were designed from the conserved S-RNase regions using the top blast hit (Table 1). Both alleles were amplified using the following thermocycler conditions: one initial cycle of denaturation for 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 56°C, 1 min at 72°C and then a final extension of 5 min at 72°C. Amplicons were gel purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and then cloned into the pGEM T-Easy cloning vector (Promega, Madison, WI, United States) and transformed into DH5α competent cells (Thermo Fisher, 67 Carlsbad, CA, United States). A total of 22 colonies were Sanger sequenced and sequences were aligned manually using BioEdit version 7.2.5 (Hall, 1999). S-RNase expression analysis via RT-PCR A total of 100 styles were collected from newly opened flowers of S. verrucosum clone SV607845.02 and RNA was isolated using the Qiagen RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RNA Samples were then treated using Turbo DNase (Thermo Fisher Scientific, Waltham, MA, USA) to remove DNA contamination. S-RNase expression was assessed using the SuperScript III First-Strand Synthesis system for RT-PCR (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA and cDNA generated from the RT-PCR were then amplified using the aforementioned primer sets and thermocycler schedule. Resultant PCR products were then visualized on 1% agarose genes and assessed for the presence or absence of the bands correlating with the respective primer sets. Results Prezygotic interspecific barriers in S. verrucosum Pollen tubes from all of the pollen donors except for S. bulbocastanum were able to easily travel down the full length of the S. verrucosum style (Figures 4.1 and 4.2). The effect of plant age was minimal in these pollinations with the longest pollen tubes from S. jamesii and S. pinnatisectum present in the ovary at 48h post pollination in all three time-points (Figure 4.2). The majority of pollen tubes from S. bulbocastanum did not penetrate the full length of the style at any of the time-points, and the majority of pollen tubes for S. commersonii and S. jamesii only reached the ovary in the second and third time-points (Figure 4.1). Overall, no significant stylar barriers were observed and pollen tubes from all four pollen donors were observed in the vicinity 68 of the ovules. Fruit formation was observed for all four pollen donor species and hybrid progeny between S. verrucosum and S. bulbocastanum and S. commersonii were recovered. S-RNase in S. verrucosum Two S-RNase alleles were amplified and partially sequenced (Figure 4.5) despite the absence of S-RNase at the protein level as reported by Eijlander et al., (2000). Results from RT- PCR indicated a lack of S-RNase expression (Figure 4.7). Discussion The use of S. verrucosum as a bridge to access sexually isolated 1EBN species has been demonstrated several times (Jansky and Hamernik, 2009; Yermishin et al., 2014). Despite its recent and growing use, our understanding of this method is poor. If the EBN hypothesis is correct, no hybrids should result from interspecific crosses between S. verrucosum and any 1EBN species (Johnston et al., 1980). However, diploid interspecific hybrids are obtainable, making this bridging method a valuable technique to access EBN1 species. Demonstrations of the S. verrucosum bridging technique by Jansky and Hamernik, (2009) and Yermishin et al. (2014) have a low efficiency. Only a few seeds are produced after a large number of pollinations, and often, hybrid progeny lack sufficient fertility for further use (Hermsen and Ramanna, 1976; Jansky and Hamernik, 2009; Yermishin et al., 2014). The use of mentor pollen (Yermishin et al., 2014) and careful or no emasculation (Bamberg, 2020) may increase seed set, but only to a marginal degree. Certain S. verrucosum clones may also vary in their ability to form fertile inter-EBN hybrids, and current research to identify elite clones is ongoing. The use of S. verrucosum bridge crosses remains a viable option despite its low efficiency rate, as it is less labor and resource intensive compared to other methods such as embryo rescue, complex ploidy manipulations, and somatic fusion (Ramon and Hanneman, 69 2002; Carputo et a., 1997; Polzerová et al., 2011; Jansky and Hamernik, 2009). Nevertheless, improving the efficiency of this method is necessary for it to gain greater use. Prezygotic stylar barriers are not an important barrier in these pollinations as demonstrated in this study (Figure 4). The lack of prezygotic stylar barriers in S. verrucosum is comparable to S. lycopersicum (Baek et al., 2015). The lack of interspecific stylar barriers is a very valuable phenotype which allows S. lycopersicum to not only be SC, thus allowing the development of inbreds and F1 hybrids, but also serve as a parent for wide interspecific crosses allowing the introgression of valuable traits from genetically distant wild relatives (Baek et al., 2015; Canady et al., 2005: Ji et al., 2004). The lack of SC and interspecific incompatibility in S. tuberosum is a significant barrier to the effective use of wild species; and the ability to replicate the phenotype observed in S. verrucosum and S. lycopersicum within S. tuberosum would be a significant advancement in potato breeding (Novy and Hanneman, 1991; Enciso-Rodriguez et al., 2019). Understanding the causative genetic mechanisms behind SC and interspecific compatibility in S. verrucosum is necessary to replicate this phenotype. Additionally, stylar barriers are not the only significant factors barring the use of wild potato species in potato breeding. Therefore, further inquiry and manipulation of the genetic mechanisms underlying effective ploidy and endosperm balance number are likely to result in the largest increases in efficiency. 70 APPENDICES 71 APPENDIX A: Chapter 4 Tables Primer Sequence (5'-3') Purpose Amplifying partial sequence of SP3 F: GGGGAAACTGGAAAATGGTT allele 1 R: ATGTGAATGTGTTCAGCGAAA Amplifying partial sequence of M6 F: ACGATTCACGGGCTTTGG allele 2 R: AATATTGATAGAGCCATAAGGGTTGT Table 4.1. Primers used in this study 72 APPENDIX B: Chapter 4 Figures Figure 4.1. Measurements of pollen tube front for all pollen donors. Blue bars depict the marginal pollen tube means (emmean) as a proportion of total style length at 48h post pollination. Female genotypes are depicted on the left vertical axis, and time-points or rounds are depicted on the right vertical axis. Pollinations were made at three discrete time points (rounds), separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. Pollen donor species are represented by the following three letter species codes: S. bulbocastanum (blb), S. commersonii (cmm), S. jamesii (jam), and S. pinnatisectum (pnt). 73 Figure 4.2. Measurements of the longest pollen tube for all pollen donors. Blue bars depict the marginal pollen tube means (emmean) as a proportion of total style length at 48h post pollination. Female genotypes are depicted on the left vertical axis, and time-points or rounds are depicted on the right vertical axis. Pollinations were made at three discrete time points (rounds), separated by 30 days, with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse. Pollen donor species are represented by the following three letter species codes: S. bulbocastanum (blb), S. commersonii (cmm), S. jamesii (jam), and S. pinnatisectum (pnt). Figure 4.3. Estimated marginal means plotted against the different time-points (rounds) used in this study for all pollen donors showing effect of plant age on interspecific pollen tube inhibition. The vertical axis depicts the estimated marginal means of the longest measured pollen tube as a proportion of the total length of the style. The horizontal axis plots the three pollination time- points (rounds) used in the study with the first time point being approximately six weeks after plants were transferred from tissue culture to the greenhouse with subsequent pollination rounds separated by 30 days. Pollen donor species are represented by the following three letter species codes: S. bulbocastanum (blb), S. commersonii (cmm), S. jamesii (jam), and S. pinnatisectum (pnt). 74 S. bulbocastanum S. commersonii S. jamesii S. pinnatisectum Figure 4.4. Styles of S. verrucosum SV607845.02 pollinated with each of the four pollen donors. Pollen tubes are visible at the base of the style and penetrating the ovary at 48h post pollination showing an absence of interspecific stylar barriers in these pollinations. 75 >Solanum_verrucosum_S-RNase_SV607845.02_1 GGGGAAACTGGAAAATGGTTAAACCACAACTCACATCAGCTCTCTTCATTGTGCTTT TTGCTCTTTCTCCCGCTTATGGGGATTTCGATTCCCTCCAACTGGTATTAACATGGCC AGCATCATTTTGCCATGTTAATGATTGTGTGCGAATAGCTCCAAAAAACTTCACGAT TCACGGGCTTTGGCCGAATAAAGAGGGAACGGTGCTGCAGAACTGCAAGCCAAAAC CTAAGTATGTTAATTTCAAGGTAAGCAATAGCATTTTTTTAGAGCCCGCTTTTCCGCT CAGTTCAATTTACTTGAAAGATTCTTTTCGAAATGCTTACAGGATAAGATGTTCAAC GATCTTGACAAACACTGGATTCAGTTGAAGTTTGATGAAGATTATGGTGAAAAGGA ACAACCTTTATGGCTCTATCAATATTTTAAGCATGGATCTTGTTGTCAGAAAATGTAC AACCAAAACACGTATTTTAGTCTAGCCTTGCGCTTAAAAGACAGGTTTGATCTTCTG AGAACTCTCCAAATACATCATATTTTTCCTGGATCAAGTTATACATTTAAGAAAATC TTTGATGCCGTCAAGACAGCTACTCAAATGGATCCTGACCTTAAGTGTACTAAAGGA GTACCGGAACTATATGAAATAGGCATATGTTTCACCCCAAATGCAGATGCTCTGATT CCATGTCGTCAAAGTAATACATGCGATAGGACAGGAAAAATCTTTTTTCGCTGAACA ACTTCACAT >Solanum_verrucosum_S-RNase_SV607845.02_2 ACGATTCACGGGCTTTGGCCGGATAAAGAGGGAACGGTGCTGCAGAACTGCAAGCC AAAACCTAAGTATGTTAATTTCAAGGATAAGATGTTCAACGATCTTGACAAACACTG GATTCAGTTGAAGTTTGATGAAGATTATGGTGAAAAGGAACAACCTTTATGGCTCTA TCAATATT Figure 4.5. Partial sequences of the two S-RNase alleles identified in S. verrucosum. Allele SV607845.02_1 was captured using the SP3 primers designed by Ye et al. (2018). The SV607845.02_2 allele was capture using primers designed from conserved S-RNase regions. 76 Figure 4.6. The two S-RNase alleles of S. verrucosum identified in this study aligned to the genome region of the publicly available assembly GCA_900185275.1 available through NCBI. 77 Ladder C WT cDNA RNA 258bp S-RNase 178bp Actin Figure 4.7. S-RNase expression in S. verrucosum SV607845.02. No S-RNase expression was observed after RT-PCR. Total RNA was isolated from 100 styles collected from newly opened flowers within 24 hours after anthesis. Samples were DNase treated before using RT-PCR to assess expression of S-RNase 78 Figure 4.8. UPGMA phylogenetic tree showing the similarity of both SV607845.02 S-RNase alleles to the S-RNase 2 from S. tuberosum and their relation to other known S-RNase alleles within Solanaceae. 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American journal of potato research, 91(6), 610-617. https://doi.org/10.1007/s12230-014-9385-9 83 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS The past decade has seen a renewed interest in diploid potato breeding as current and past breeding efforts at the tetraploid level have been plagued by lengthy breeding cycles, excessive heterozygosity, and inbreeding depression which impedes the fixation of desirable alleles (Lindhout et al., 2011; Stokstad, 2019). Genetic improvement of potato at the diploid level would accelerate the generation of new varieties, and simplify efforts to incorporate and stack valuable agronomic and abiotic resistance traits into existing germplasm (Jansky et al., 2016; Kaiser et al., 2021). The use of diploid germplasm also simplifies the introgression of traits from wild species that have been major sources for disease resistance and abiotic stress tolerance. Because previous attempts to introgress wild species traits have focused on incorporating traits into tetraploid potato germplasm; the movement towards a diploid breeding system has reignited interest in understanding how to best access the genetically isolated 1EBN wild species without complex ploidy manipulation, somatic fusion, or embryo rescue that have previously been required (Carputo et al., 1997; Ramon and Hanneman, 2002; Polzerová et al., 2011; Bethke et al., 2017). Unfortunately, due to prezygotic stylar barriers and differences in effective ploidy between 1EBN species and cultivated germplasm, accessing the traits from these species is extraordinarily difficult (Novy and Hanneman, 1991; Jansky and Hamernik, 2009; Städler et al., 2021). By better characterizing the prezygotic barriers between 1EBN species and cultivated diploids, further research can better understand the significant postzygotic barriers that inhibit the use of these valuable species. 84 S-RNase and HT in interspecific crosses The relationships of interspecific compatibility and incompatibility in Solanum section Petota are complex and intricate. Inquiry into these relationships has elucidated the pleiotropic and redundant function of S-RNase and HT which tandemly and independently mediate both interspecific and intraspecific pollen rejection (Baek et al., 2015; Tovar-Méndez et al., 2014, 2017). The outcome of interspecific pollinations is specific to the individual species involved and direction of the cross due to the direct and indirect involvement of multiple factors such as HT and S-RNase, 120 kDa, Cullin1 (CUL1), and farnesyl pyrophosphate synthase (FPS2) (Bedinger et al., 2011; Li and Chetelat, 2015; McCormick, 2018; Tovar-Méndez et al., 2017). The findings presented here are consistent with previous work conducted in Solanum section Lycopersicon showing that S-RNase plays a central role in interspecific pollen rejection (Baek et al., 2015; Tovar-Méndez et al., 2014). Statistical analyses also demonstrated that HT-B alone is not a significant factor in these pollinations. These results do not contradict the findings from Tovar-Méndez et al (2014, 2017) where HT proteins played a significant role in interspecific pollen rejection. Since HT-A remained functional in all of the female genotypes used, these findings demonstrate the overlap in gene function between HT-A and HT-B, the greater importance of HT-A, or that HT is not a significant factor in these combinations implicating other mechanisms (Baek et al, 2015; Qin and Chetelat, 2021). Therefore, there is a need to generate gene knock-outs of HT-A to further elucidate the function of HT in these pollinations as HT-A alone may mediate the S-RNase independent rejection of interspecific pollen. Future assessment of the role of HT-A in interspecific pollen rejection will inform our understanding of interspecific pollination barriers. Efforts are being made to make these gene edits and further assessment will be made after the edits are confirmed. 85 Sli and Solanum chacoense This study draws the clear conclusions that Sli is not a significant factor in interspecific pollen rejection in these pollinations. The presence or absence of Sli did not change the outcome of any of the pollinations made, which is inconsistent with the previous findings of Sanetomo et al. (2014) despite the use of the same S. pinnatisectum PI (273232). These inconsistencies can be attributed to unknown factors in S. chacoense besides Sli. The pollen donors used in the assessments made by Sanetomo et al. (2014) came from interspecific crosses between S. chacoense and S. phureja, while the pollen donors used in these assessments were of one species, S. chacoense. The difference between pollen donors is the likely source of inconsistencies; the introduction of unknown genetic variables from S. phureja represents a confounding variable in the studies carried out by Sanetomo et al. (2014). Although Sli played no significant role in these pollinations, it is possible that S. chacoense as a pollen donor is better able to bypass stylar barriers in EBN1 species such as S. pinnatisectum compared to S. megistacrolobum, S. demissum, and S. phureja (S. tuberosum Group Phureja) as reported by Sanetomo et al. (2014). Consequently, S. chacoense may be a valuable resource in accessing these EBN1 species regardless of Sli status. The limitations and possibilities associated with Sli with be more apparent when the genetic mechanisms behind Sli can be clearly identified. Further testing of a diverse panel of S. chacoense pollen donors would also clarify if pollen from these species is accepted to a greater degree in interspecific pollinations. SC and interspecific compatibility in Solanum verrucosum Currently, bridge crossing with S. verrucosum is among the most reliable and feasible methods to access EBN1 species (Jansky, and Hamernik; 2009). Unlike the methods proposed by 86 Sanetomo et al. (2014), Ramon and Hanneman (2002), Polzerová et al. (2011), and Carputo et al. (1997), the use of S. verrucosum bridge crosses is more accessible for most plant breeders as it does not require ploidy manipulation, interploidy crosses, or the use of advanced techniques such as somatic fusion or embryo rescue (Jansky and Hamernik, 2009). However, although the use of S. verrucosum as a bridge to access sexually isolated 1EBN species is has been demonstrated previously, our understanding of this method is poor (Jansky and Hamernik, 2009; Yermishin et al., 2014). Technically if the EBN hypothesis is correct, no hybrids should result from interspecific crosses between S. verrucosum and any 1EBN species (Johnston et al., 1980). The ability to produce hybrids between S. verrucosum and 1EBN species highlights our limited understanding of effectively ploidy and endosperm balance number. Recent research by Stadler et al. (2021) and others has demonstrated the complex dosage-sensitive nature of endosperm development and failure (Baek et al., 2016; Roth et al 2017; Coughlan et al., 2020). The use of discrete categories, such as endosperm balance numbers, is inconsistent with our current understanding of effective ploidy. The multigenic mechanisms that underpin effective ploidy are complex and genome wide (Ehlenfeldt and Hanneman, 1988; Lafon-Placette, and Köhler, 2016; Stadler et al., 2021). Additionally, individual species of the same EBN are not always closely related (Pritchard, 2005). Therefore, caution should be applied when using EBN to predict the outcome of interspecific crosses as it represents an outdated understanding of effective ploidy with demonstrable flaws. The ability of S. verrucosum to form interspecific hybrids with species of differing EBN is likely partially rooted in its lack of a functional S-RNase (Eijlander, 1998). The absence of functional S-RNase allows interspecific pollen tube growth in what would otherwise be incompatible pollinations. While it is known that there is no detectable S-RNase at the protein 87 level (Eijlander, 1998), the use of primers targeted to conserved regions of S-RNase was able to locate S-RNase in the genome. These alleles for S-RNase in S. verrucosum are likely non- functional or inhibited in some way as no expression was ever observed. Due to the absence of S- RNase, and possibly other factors, prezygotic stylar barriers are not an important barrier in interspecific pollinations when S. verrucosum is the female parent. Non-functional genes for HT and other factors may further allow interspecific pollen to penetrate the style. Further analysis into the promotor regions of S-RNase and into other factors such as HT in S. verrucosum will clarify the causative genetic mechanisms allowing S. verrucosum to act as a receptive parent to self and interspecific pollen. Although more resource and time efficient than other techniques, demonstrations of the S. verrucosum bridging technique by Jansky and Hamernik (2009) and Yermishin et al. (2014) however have shown a low efficiency. Only a few seeds are produced after a large number of pollinations, and often hybrid progeny lack sufficient fertility for further use (Hermsen and Ramanna, 1976; Jansky and Hamernik, 2009; Yermishin et al., 2014). The use of mentor pollen (Yermishin et al., 2014) and careful or no emasculation (Bamberg, 2020) may increase seed set, but only to a marginal degree. The use of S. verrucosum bridge crosses remain a viable option despite its low efficiency rate, as it is less labor and resource intensive compared to other methods used (Jansky and Hamernik, 2009). Nevertheless, improving the efficiency of this method is necessary for it to gain greater use. Therefore, further inquiry and manipulation of the genetic mechanisms underlying effective ploidy and endosperm balance number are likely to result in the largest increases in efficiency. 88 Plant ontogeny Intra- and inter-specific compatibility is heavily influenced by environmental and ontogenic factors such as temperature, humidity, flower age, and plant age (Bienz, 1958; Henderson and LeClerg, 1943; Travers et al., 2004; Liao et al., 2016; Webb and Williams, 1988). Although variable GSI responses due to plant and floral age have been demonstrated in S. carolinense and N. alata, until now these findings have not been observed in interspecific pollinations (Travers et al., 2004; Liao et al., 2016). Results from statistical analysis indicate that plant age does have a significant impact on interspecific pollen rejection, consistent with the findings published by Travers et al. (2004) and Liao et al. (2016). There is still a lot to learn about plant ontogeny and the degradation of interspecific and GSI barriers with age. Since all of the plants in this study were planted at the same time it is unknown whether the age of the pollen donor or the female parent are more or less responsible for the break-down in prezygotic barriers. Further experiments that focus on the age of the pollen donor and female parent separately will aid in the identification of the causative mechanisms and the overall implications of plant age in breeding programs. 89 REFERENCES 90 REFERENCES Baek, Y.S., Covey, P.A., Petersen, J.J., Chetelat, R.T., McClure, B. and Bedinger, P.A. (2015), Testing the SI × SC rule: Pollen–pistil interactions in interspecific crosses between members of the tomato clade (Solanum section Lycopersicon, Solanaceae). 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