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ABSTRACT ADHIKARI, PRAGYA. Mapping QTL Derived From ABSTRACT ADHIKARI, PRAGYA. Mapping QTL Derived from Solanum pimpinellifolium LA3707 for Bacterial Spot Disease Resistance and Fruit Morphology in Tomato (Under the direction of Dr. Dilip R. Panthee and Dr. Chris G. Gunter). Tomato (Solanum lycopersicum L.), is the second most consumed vegetable in the US. The large-scale production of both fresH-market and processing tomatoes is challenged by several diseases including bacterial spot. Bacterial spot disease in tomato is caused by at least four species and four races of Xanthomonas species (Xs) with the distinct geographical distribution. Change in species structure and evolution of new races and strains is a continuous process. In this research, we sought to study the phenotypic and genotypic diversity of Xs strains in NC and characterize the predominant Xs races and species. We then explored the genetic resistance in tomato against the predominant race of Xs in NC. Additionally, we also discerned the genetic basis of diverse shapes, sizes, and color in the cultivated tomato as these traits often determine the market value and culinary purposes of the tomato. To characterize the bacterial spot pathogens, we collected 284 Xs strains from major tomato production regions of western NC. Copper and streptomycin sensitivity tests revealed that over 95% of the Xs strains were copper resistant while 25% and 45% were streptomycin resistant in 2016 and 2015 respectively. DNA fingerprint profiling with BOX repetitive element polymerase chain reaction (BOX-PCR) assay detected four haplotypes among Xs strains. The study on a subset of representative Xs strains (n = 45) identified Xs strains in NC as a single species, X. perforans (Xp) based on highly conserved hrpB7 gene sequences, while virulence of Xs strains on tomato differential cultivars confirmed ~91% and 9% of Xs strains were races T4 and T3, respectively. Additionally, phylogenetic and comparative sequence analysis of six genomic regions (fusA, gapA, gltA, gyrB, lacF, and lepA) suggested that 74% and 13% of Xp strains of NC were similar with tomato races T4 and T3 from Florida, respectively. This suggested that the best bacterial spot management practices in tomato in NC should be implemented with major focus on introducing host resistance against race T4 and by considering the challenges currently posed by the intense use of copper-based bactericides. To identify genomic regions associated with the genetic resistance to race T4, quantitative trait loci (QTL) analysis was performed in an intraspecific recombinant inbred lines (RIL) population NC 10204 at F5 and F6 generations developed from a cross between two elite breeding lines- NC 30P x NC-22L (2008). Experiments were conducted in four environments including two locations over two years. Five major QTL on chromosome 1, 4, 6, 11, and 12, and one minor QTL on chromosome 2 were identified. The QTL on chromosomes 1, 2, 4, and 6 were also validated in an independent inter-specific population developed from the crossing of NC 1CELBR x PI 270443. The QTL on chromosome 6 explained the most substantial phenotypic variance (up to 26%) followed by the QTL on chromosome 1 (up to 23%) and the QTL on chromosome 4 (up to 15%). Since the donor of the resistance associated with these QTL is a released superior breeding line NC 30P, the donor parent and the QTL information will be useful to breed tomato against the bacterial spot disease. The NC 10204 population was also used to identify genomic regions controlling fruit size, shape, and color in tomato using precision phenotyping software Tomato Analyzer (TA). Four fruit size attributes on chromosome 2 explaining up to 20% of the phenotypic variance; three fruit shape attributes on chromosome 10 and 12 explaining up to 25% phenotypic variance; and three- color attributes on chromosome 4, 9, and 6 explaining up to 21% of phenotypic variance were detected. Our study identified novel QTL controlling fruit shape attributes on chromosome 10 and 12, and also confirmed the previously detected genetic loci controlling fruit size and color in inter- specific tomato population. This information will be useful to improve fruit shape in cultivated tomato. © Copyright 2018 by Pragya Adhikari All Rights Reserved Mapping QTL Derived from Solanum pimpinellifolium L3707 for Bacterial Spot Disease Resistance and Fruit Morphology in Tomato by Pragya Adhikari A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Horticultural Science Raleigh, North Carolina 2018 APPROVED BY: _______________________________ _______________________________ Dr. Dilip R. Panthee Dr. Christopher C. Gunter Committee Co-Chair Committee Co-Chair _______________________________ _______________________________ Dr. Frank J. Louws Dr. Hamid Ashrafi Minor Advidor (Plant Pathology) _______________________________ Consuelo Arellano DEDICATION I dedicate this work: In memory of my father Ghanshyam Adhikari, who believed in my ability to be successful in the academic arena when I was just four years old. “You left your six years old daughter and couldn’t see her long journey to this stage, but I am sure you would have been very proud of your little girl”. To my mother, Laxmi Sharma Adhikari; grandfather, Dwarika P. Adhikari; brother, Shishir Adhikari; and my aunt, Shova Adhikari, without their guidance, love, and support, I would not have reached where I stand today. To my husband, Niraj Rayamajhi, who always had confidence in me and offered me encouragement and support in all my endeavors. “Finally, the time has come to be with you”. ii BIOGRAPHY Pragya Adhikari was born in Nawalparasi, Nepal. She pursued her primary and secondary education in Nepal. She completed her undergraduate degree in Agricultural Science with an elective in plant breeding and genetics, from the Institute of Agriculture and Animal Science, Tribhuvan University, Nepal in 2011. She served as a research assistant in Nepal Agriculture Research Council after earning her bacHelor’s degree for 10 months, where she was involved in a rice and wheat breeding program. Then, she came to the United States to pursue her graduate degrees. She earned her Master’s degree of Science in Plant Science from Missouri State University, Springfield, Missouri in December 2014. In 2015, she joined North Carolina State University as a graduate research and teaching assistant in the Department of Horticultural Science to pursue her Doctor of Philosophy in Horticultural Science with a major in plant breeding and a minor in plant pathology. iii ACKNOWLEDGMENTS I wish to express my sincere appreciation to my advisor Dr. Dilip R. Panthee for accepting me as his graduate student, and for his guidance, support (both professionally and personally), advice, and encouragement throughout my graduate career, and prompt edits on the manuscripts. I am fortunate to have Dr. Frank J. Louws in my committee as a minor advisor, which lead to a minor degree in plant pathology. Thank you, Dr. Louws, for providing me with wealth of experiences and knowledges in plant pathology by giving me opportunities to learn through a major project and expose myself to a different laboratory outside of the University in Florida. I owe mucH of my doctoral learning in these experiences that you were not required to do but did it anyway for my learning. I cannot fully express my gratitude for your time and guidance in the laboratory work, your encouraging and motivating words during stressful points, and your constant support throughout my study period. I am also grateful to my co-chair Dr. Christopher C. Gunter, and my committee members Dr. Hamid Ashrafi and Dr. Consuelo Arellano for their suggestions and guidance during the research projects. Thank you, Dr. Consuelo, for assisting me with tHe data analysis, and thank you for your patience and every SAS code that you emailed me, especially since I was located off-campus at the research station. I would also like to thank my extension mentor for mentoring me in the extension activities. I gratefully acknowledge the funding sources- National Science Foundation grant IOS- 1546625 to Dr. Dilip R. Panthee; and Department of Horticultural Science, who supported my graduate study and research projects. I would like to thank all those who helped me in various ways to complete my Ph.D. projects: • Dr. Jeff Jones (University of Florida) for providing the lab space, tomato differentials, and control bacteria to characterize the race and species structure of bacterial strains of NC causing bacterial spot disease in tomato; and Dr. Sujan Timilsina (University of iv Florida) for assisting me to conduct experiments at the University of Florida and helping me with the phylogenetic analysis of the bacterial strains. • Dr. David Ritchie for providing the control bacteria and protocol for copper and streptomycin sensitivity test of bacterial strains. • Craig Mauney for helping me to coordinate with tomato growers to identify fields for the collection of symptomatic leaf samples. • Thomas Ingram and Kerstin Bieler for helping me collect the symptomatic leaf samples and isolating the bacteria. I appreciate the help from Thomas in teaching me to use various software. • Dr. Ralph A. Dean for providing me lab space in Raleigh; and Dr. Yeon Yee Oh for her guidance and suggestions in the lab work. • Pradeep Neupane for his suggestions and help to troubleshoot the PCR problems. • Dr. Tika B. Adhikari for his valuable suggestions in finishing up my bacterial projects and constructive comments in the manuscripts. • Ann Piotrowski for her support in the field research and Amelia Heintz-Botz for helping me with the fruit scanning. • Mario De Jesus Velasco Alvarado and Navin Shrestha for helping me to record the disease data. • Dr. Zianbo Zhang for his help in the data analysis. • Field crews of the Mountain Horticultural Crops Research and Extension Center (MHCREC) and Piedmont Research Station (PRS) for preparing the field, tomato planting, and taking good care of the field throughout the seasons.
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