Metabolic and genomic analysis of elongated fruit shape in tomato (Solanum lycopersicum) Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Josh Clevenger, B.S. Graduate Program in Horticulture and Crop Science The Ohio State University 2012 Thesis Committee: Esther van der Knaap, Advisor Joe Scheerens Michelle Jones Joshua Blakeslee Copyright by Josh Clevenger 2012 Abstract Tomato fruit shape is an important attribute as its shape informs how the fruit will be marketed and processed. Analysis of how fruit shape is regulated at the molecular level further provides insight into the molecular regulation of early fruit development. We have developed near isogenic lines (NILs) in the wild species (S. pimpinellifolium) background for seven major fruit shape and weight genes, SUN, OVATE, fs8.1, FAS, LC, FW2.2, and FW3.2. Analysis of the SUN, OVATE, and fs8.1 triple NIL shows that all three genes control fruit shape in an additive manner, and SUN exhibits additive-by-additive interaction with OVATE and fs8.1 in controlling fruit shape. All three genes together made a significantly longer fruit. Using an unbiased classification using Elliptic Fourier Coefficients and unsupervised Bayesian clustering we further show that all 27 genotypes produce fruit shapes that can be assigned to six distinct classes, and that membership of these genotypes to these classes reinforces the genetic data. Concurrently, we fine mapped fs8.1 further to a 3 Mb region. It is not fully known how SUN controls fruit elongation at the molecular level. We used metabolite and gene profiling during early fruit development to identify metabolite and gene regulatory networks and to infer the relationship of these networks in round and elongated fruit. We identified distinct metabolite and gene expression regulatory networks, and found that new associations were made in elongated fruit. Analysis of gene expression networks revealed a complex interaction between hormone homeostasis, secondary metabolism, and cell wall modification. These data led us to propose a hypothetical model of the effect of SUN on early fruit development leading to elongated fruit shape. ii Aknowledgements I would like to thank my advisor, Dr. Esther van der Knaap, for providing the enthusiasm and resources which allowed me to complete this thesis, and for especially pushing me to strive for excellence. I accomplished more than I could have expected as a direct result of her guidance. I also am grateful to my committee members, Dr. Joe Scheerens, Dr. Michelle Jones, and Dr. Joshua Blakeslee for listening to my ideas, providing insight, and not minding when I switched directions. A special thanks goes to Dr. Joe Scheerens and Dr. Ann Chanon, who were a great resource and helped me greatly with work that does not appear in this thesis. I would also like to acknowledge Dr. Sophia Visa who inspired me with new ideas every time I spoke with her, and Dr. Gustavo Rodriguez who started the projects that I have worked on and taught me many things about being a researcher. I would also like to thank everyone in the van der Knaap lab for help, advice, and friendly words, especially Shan Wu for her insight and collaboration. Everyone in the lab helped me learn and grow as a researcher. Finally I thank my parents for always supporting my odyssey, my brothers for also being my friends, Amy for being the reason I want to be better, and Elvis for always being happy to see me. iii Vita 2000……………………………………………………………….Hoover High school, North Canton, OH 2009……………………………………………………………….B.S., Biology, Baldwin-Wallace College 2009 – 2010………………………………………… …….…Research Assistant, OARDC, Wooster, OH 2010 – present…….……………………………………..…Graduate Research Associate, Department of Horticulture and Crop Science, The Ohio State University Fields of Study Major Field: Horticulture and Crop Science iv Table of Contents Abstract………………………………………………………………………………………………………………………ii Acknowledgements…………………………………………………………………………………………………….iii Vita……………………………………………………………………………………………………………………………..iv List of Tables……………………………………………………………………………………………………………….vii List of Figures……………………………………………………………………………………………………………..viii Chapter 1: Fruit shape from domestication to industrial agriculture……………………………1 Chapter 2: Development of fruit shape and size NILs and the interaction of SUN, OVATE and fs8.1 on fruit shape…………………….….7 Abstract…………………………………………………………………………………………………………………….….7 Introduction………………………………………………………………………………………………………………….8 Materials and Methods………………………………………………………………………………………………..12 Results………………………………………………………………………………………………………………………….17 Discussion…………………………………………………………………………………………………………………….25 Chapter 3: Metabolic and genomic analysis of SUN on early fruit development: Identification of metabolite and gene regulatory networks…………………….....57 Abstract……………………………………………………………………………………………………………………….57 Introduction…………………………………………………………………………………………………………………58 Materials and Methods………………………………………………………………………………………………..60 Results………………………………………………………………………………………………………………….………66 v Discussion……………………………………………………………………………………………………………………72 References………………………………………………………………………………………………………………….108 Appendix A: Relative metabolite accumulation in early fruit development………………..118 Appendix B: RNA seq mapping statistics…………………………………………………………………….124 Appendix C: Correlation among gene expression replicates……………………………………….127 Appendix D: Log fold change GRN patterns………………………………………………………………..129 vi List of Tables Table 2.1. NIL introgressions…………………………………………………………………………………………31 Table 2.2. Markers used for construction of NILs………………………………………………………….32 Table 2.3. Comparison of floral organ traits among NILs…………………………………………..…37 Table 2.4. Comparison of fruit shape among NILs………………………………………………………..38 Table 2.5. Comparison of fruit shape characteristics and weight among NILs……………...39 Table 2.6. Interaction of SUN, OVATE, and fs8.1 on fruit shape index………………………….41 Table 2.7. Epistatic interaction of SUN and OVATE on fruit shape index………………………44 Table 2.8. Epistatic interaction of SUN and fs8.1 on fruit shape index…………………………45 Table 2.9. Membership in fruit shape classes related to genotype………………………………47 Table 2.10. Comparison of fruit characteristics among FAS, FW2.2, and fs8.1 NILs………48 Table 2.11. Gene action of SUN, OVATE, fs8.1, FAS, and FW2.2…………………………………..51 Table 2.12. Annotated genes expressed in flowers in the fs8.1 region…………………………53 Table 2.13. Progeny test of fs8.1 recombinants……………………………………………………………56 Table 3.1: New metabolite correlations in elongated fruit...............................................87 Table 3.5: Differentially expressed genes by tissue type..................................................89 Table 3.3: Ontology of members in the SUN network in septum tissue...........................92 Table 3.4: Ontology of members in the DEFL1 network in septum tissue……………....……94 vii List of Figures Figure 2.1. Pedigree of SUN + OVATE + fs8.1 NILs............................................................30 Figure 2.2. SUN, OVATE, and fs8.1 NILS............................................................................34 Figure 2.3. Representation of the intogressions of SUN, OVATE, and fs8.1 NILs..............35 Figure 2.4. Representation of the intogressions of FAS, FW2.2, FW3.2, and LC NILs.......36 Figure 2.5. Development of fruit elongation...................................................................40 Figure 2.6. Interaction plots of SUN, OVATE, and fs8.1 on fruit shape index..................42 Figure 2.7. 3d interaction plot of SUN, OVATE, and fs8.1 on fruit shape index...............43 Figure 2.8. Fruit shape classes determined by morphometric evaluation and elliptic fourier coefficients.........................................................................................46 Figure 2.9. Interaction plots on weight of FAS, FW2.2 and fs8.1......................................49 Figure 2.10. 3d interaction plot of fs8.1, FAS, and FW2.2................................................50 Figure 2.11. Fine-mapping of fs8.1...................................................................................52 Figure3.1. MRN1……………………………………………………………………………………………………….…80 Figure3.2. MRN2……………………………………………………………………………………………………….…81 Figure3.3. MRN3……………………………………………………………………………………………………….…82 Figure3.4. MRN4……………………………………………………………………………………………………….…83 Figure3.5. MRN5……………………………………………………………………………………………………….…84 Figure3.6. MRN6……………………………………………………………………………………………………….…85 Figure 3.7: Metabolite changes in elongated fruit………………………………………………………..86 Figure 3.8: Metabolite regulatory networks with new associations in elongated fruit….88 Figure 3.9: Gene expression network in septum tissue containing SUN……………………….93 Figure 3.10: Gene expression network in septum tissue containing DEFL1…………………..95 viii Figure 3.11: Gene regulatory network (GRN) regulating photosynthesis……………………..97 Figure 3.12: GRN 46………………………………………………………………………………………………..…..98 Figure 3.13: GRN 5……………………………………………………………………………………………………….99 Figure 3.14: GRN 9…………………………………………………………………………………………………….100 Figure 3.15: GRN 52…………………………………………………………………………………………………..101 Figure 3.16: Over representation analysis of differentially expressed genes………………102 Figure 3.17: gene – metabolite correlations……………………………………………………………….104 Figure 3.18: gene – metabolite network………………………………………………………………..…..105 Figure 3.19: gene – metabolite network with close ups of gene hubs…………………………106 Figure 3.20: GRN