FINE MAPPING AND CANDIDATE GENE CHARACTERIZATION OF THE BACTERIAL SPOT RESISTANCE GENE bs6 IN PEPPER By REBECCA LAUREN WENTE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2018 © 2018 Rebecca Lauren Wente To Jeffrey and Janet Wente ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Samuel Hutton, for challenging and inspiring me to be a better researcher and plant scientist. His talent for plant breeding and knowledge of the tomato industry inspired me to work more resourcefully and obtain results that can be directly useful for growers. Thank you to Dr. Hugh Smith for providing guidance with my project even after it deviated away from entomology. I sincerely appreciate the work of Dr. Jeffrey Jones and Jerry Minsavage for developing robust research on Xanthomonas and for facilitating the disease screens. Thank you to the tomato breeding laboratory at the Gulf Coast Research and Education Center. Tim Davis, Nathan Brown, Dolly Cummings, and Judith Lopez supported many of the technical portions of this work including help with seed extraction, sowing, and plant care. Jose Diaz, Kazuyo Ueda, and Keri Druffel were helpful with marker sampling, high resolution melting analysis, and sequencing. Thank you to Dr. Upinder Gill and Dr. Reza Shekasteband for assistance with primer design, sequencing, and gene expression studies. Dr. Hutton's graduate students, Jasmine Lopez, Leticia Kumar, Edgar Sierra, and especially Jessica Chitwood-Brown became great friends and helped with statistical analysis, professional development, and coursework. Thank you to my Disney family at The Land for helping me find my path in plant breeding and for being wonderful friends. Finally, a huge thank you to Zachary Naylor, Jeffrey Wente, Janet Wente, and Ryan Wente for providing continual motivation and encouragement. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 6 LIST OF FIGURES .......................................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................. 8 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION .................................................................................................... 12 Tomato Industry ...................................................................................................... 12 Bacterial Spot ......................................................................................................... 12 Bacterial Spot Management .................................................................................... 14 Bacterial Resistance in Pepper ............................................................................... 15 Tomato Resistance ................................................................................................. 17 Transgenic Tomatoes ............................................................................................. 19 Gene Editing Tomatoes .......................................................................................... 19 bs6 Mapping ........................................................................................................... 20 2 bs6 FINE MAPPING AND CANDIDATE GENE IDENTIFICATION ......................... 22 Objectives ............................................................................................................... 22 Materials and Methods............................................................................................ 22 Results .................................................................................................................... 29 3 CONCLUSIONS ..................................................................................................... 50 APPENDIX A NUCLEIC ACID SEQUENCE OF ECW AND 60R IN EACH OF THE GENES IN THE FINE MAPPED INTERVAL. ............................................................................ 54 B TOMATO GENES RECIPROCALLY BLASTED BACK TO THE TOMATO CODING SEQUENCE ............................................................................................ 56 LIST OF REFERENCES ............................................................................................... 57 BIOGRAPHICAL SKETCH ............................................................................................ 64 5 LIST OF TABLES Table page 2-1 High resolution melting (HRM) markers, cleaved amplified polymorphic sequence (CAPS) markers, and SNP markers detected by sequencing used for fine mapping the bs6 locus on chromosome six of pepper. ........................... 36 2-2 Primers designed to amplify the eight genes in the 666 Kb bs6 fine mapped interval for sequencing, along with their corresponding annealing temperatures. ..................................................................................................... 37 2-3 Sequencing primers designed to shorten the distal portion of the 620 Kb interval. ............................................................................................................... 38 2-4 Primers designed to amplify portions of two housekeeping genes and of five bs6 candidate genes, along with their corresponding annealing temperatures and amplicon sizes. ............................................................................................ 39 2-5 Translated amino acid sequences of ECW and 60R for each of the 5 genes in the 473 Kb fine mapped interval. .................................................................... 40 2-6 Comparison of the 13 predicted genes in the 1.2 Mb fine mapped interval of the pepper genome to the predicted tomato genes. ........................................... 41 2-7 Comparison of the five candidate genes in the 473 Kb interval to the similar genes in the pepper genome. ............................................................................. 42 2-8 Tomato genes syntenic to the five candidate pepper genes and their homology to other genes in the tomato genome................................................. 43 6 LIST OF FIGURES Figure page 2-1 Numbers of F2 plants scored as resistant (R) or susceptible (S) to bacterial spot race 6 and genotypes of these plants at the bs6 locus on chromosome 6, where Bs6/Bs6 represents homozygous for the susceptible Early Calwonder allele, Bs6/bs6 represents heterozygous, and bs6/bs6 represents homozygous for the resistant 60R allele. ............................................................ 44 2-2 Disease response to Xanthomonas euvesicatoria race 6 for parents, Early Calwonder (ECW) and 60R, and recombinant inbred lines (RILs) differing for portions of the chromosome 6 introgression from 60R. At each marker, the 60R allele genotype is indicated by “+” and by gray shading, and the ECW allele genotype is indicated by a “-“ and no shading. The resistant phenotype is indicated by an “R”, while susceptibility is indicated by a “S”. ......................... 45 2-3 High resolution genetic map of and gene annotations within the bs6 region of pepper chromosome 6. Chromosomal diagrams of the region show the extent of 60R (black shading) and Early Calwonder (light gray) DNA present in the six most informative recombinant inbred lines (RILs); medium gray shading represents segments that of undetermined genotype. Disease response (S susceptible, R resistant) of each RIL is described to the right of each chromosomal diagram. Gene predictions are based on the pepper genome release 1.55. ......................................................................................... 46 2-4 Expression analysis of four candidate bs6 genes. Average ΔCt values for treatment and genotype interaction were assessed using ANOVA, and mean separation is based on post-hoc Tukey's HSD. .................................................. 47 2-5 Gene expression analysis for candidate genes which was analyzed by calculating the average ΔCt values for the genotype by time interaction where significance was assessed using ANOVA and post-hoc Tukey's HSD. ... 48 2-6 Syntenic gene comparisons using blastn between tomato and pepper in the 1.2 Mb fine mapped interval with an e-value threshold of 1e-10......................... 49 7 LIST OF ABBREVIATIONS 60R Resistant parent containing bs6 gene ANOVA Analysis of Variance avr gene Avirulence Gene BLAST Basic Local Alignment Search Tool CAPS Cleaved Amplified Polymorphic Sequence cDNA Complementary Deoxyribose Nucleic Acid CFU Colony Forming Units CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CTAB Cetyl Trimethylammonium Bromide DNA Deoxyribose Nucleic Acid dNTP Deoxynucleotide ECW Early Calwonder, susceptible parent GBS Genotyping By Sequencing HR Hypersensitive Reaction HRM High Resolution Melting HPLC water High Performance Liquid Chromatography grade water MAS Marker Assisted Selection PCR Polymerase Chain Reaction qPCR Quantitative Polymerase Chain Reaction qRT-PCR Quantitative Real-Time Polymerase Chain Reaction QTL Quantitative Trait Loci R gene Resistance Gene RIL(s) Recombinant Inbred Line(s) 8 RNA Ribose Nucleic Acid SAR Systemic Acquired Resistance SNP(s) Single