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Abstract Cash, Christine Bradish ABSTRACT CASH, CHRISTINE BRADISH. (Under the direction of Dr. Gina Fernandez and Dr. Penelope Perkins-Veazie). Black and red raspberries are high value crops in great demand with health-conscious consumers. However, both crops lack up-to-date genetic resources that could be used to more rapidly develop cultivars for production in diverse climates. Black raspberry (Rubus occidentalis, 2n=2x=14) breeding and cultivation in the United States has been stagnated for the last 75 years due to lack of elite germplasm and adapted, disease resistant cultivars. Commercial red raspberry (Rubus idaeus, 2n=2x=14) production has been hindered in the southeast where cultivars are maladapted to the warm climate and fluctuating winter temperatures. Breeding goals for black and red raspberry have been centered around heat tolerance for over 50 years at North Carolina State University. Efforts are in in place to use applied and molecular breeding techniques to collect comprehensive phenotypic data and develop molecular tools for future black and red raspberry improvement. A series of studies were done to utilize and build on this framework. A linkage map was constructed (Chapter 2) and heat tolerance in black raspberry explored (Chapter 3), heritability of plant and fruit traits between diverse crosses of Rubus breeding germplasm was documented (Chapter 4), and diversity in Rubus germplasm characterized using traditional pedigree and molecular techniques (Chapter 5). Two mapping populations, ORUS 4304 (192 progeny) and ORUS 4305 (115 progeny), that segregate for aphid resistance were planted in 2012 at the Sandhills Research Station in Jackson Springs, North Carolina. These populations share a common wild germplasm parent selected for heat tolerance and fungal resistance in the Piedmont Region (NC 84-10-3). Between fall 2012 and summer 2015, phenotypic measures of vigor, fruit and plant traits, and heat tolerance were evaluated. Using single nucleotide polymorphisms and transferable microsatellite markers from black and red raspberry, a linkage map for ORUS 4304 was constructed. The maternal and paternal maps each contain seven linkage groups, with a total of 484 GBS SNP markers and 22 microsatellite markers, and was compared to the existing map for ORUS 4305. Based on phenotypic data collected over the three years in NC, three clones were selected that had heat tolerance, good fruit quality, and high vigor. These will be used in future breeding efforts as parental material. In addition, molecular and genomic tools developed for black raspberry in OR and NC will be used to identify quantitative trait loci (QTL) and screen germplasm for important traits such as heat tolerance, winter hardiness, and aphid resistance. Introgression of wild species such as R. parvifolius has been crucial for the integration of heat tolerance and disease resistance in raspberry germplasm, yet there is less of an understanding the inheritance of traits in this species and intricacies of gene function at the molecular level. An incomplete diallel consisting of thirteen crosses between nine parents in four Rubus species was made and evaluated for a number of plant and fruit traits. Higher general combining ability (GCA) was observed for leaflet number and fruit color among female parents, and for fruit shape and spine color among male parents. Specific combining ability (SCA) was significant for fruit shape and cane color. Narrow-sense heritability ranged from 0.00-1.00; broad-sense heritability ranged from 0.21-1.00. Cane color and fruit shape had low heritability, spine density and spine color were moderately heritable, and leaflet number and fruit color were highly heritable, indicating varying degrees of genetic and environmental influence on the genetic control of each trait. In the future, we expect ‘genetic fingerprinting’, to become increasingly valuable for a breeding program for plant and nursery mix-ups, patenting and parental identification. Using a six microsatellite fingerprinting panel for Rubus, 226 selections and cultivars from the NCSU program were genotyped. A pedigree analysis tracing all 647 selections to 66 founding clones was performed in addition to the calculation of inbreeding coefficients, genetic contribution, and coefficients of relationship. Genetic distance calculations by traditional pedigree and molecular marker analysis were positively correlated. DNA fingerprinting and pedigree analysis will be catalogued for easy reference for patents, parent selections and trueness-to-type testing. Studies of Linkage Mapping, Trait Heritability, and Pedigrees for Breeding Improvement of Southeastern Black and Red Raspberry by Christine Bradish Cash 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 2016 APPROVED BY: _______________________________ _______________________________ Dr. Gina E. Fernandez Dr. Penelope Perkins-Veazie Committee Co-chair Committee Co-chair _______________________________ _______________________________ Dr. Susana Milla-Lewis Dr. Bode Olukolu Biotechnology Minor Representative _______________________________ Dr. Nahla Bassil ii DEDICATION In thanks and honor of Dr. Kneeland Nesius, who to this day is my favorite professor, taught me and many other students about plants with minimal resources, and truly inspired us to be better scientists. In memory of my undergraduate thesis advisor, Dr. Timothy Motley, who still had much to give to the plant world and who I know is dearly missed at ODU. Finally, a dedication to my father, who is the same stubborn person as me, but always encourages me to aim higher. I would have never gotten this far if you hadn’t taken the time to help me, teach me, and give me practical advice. iii BIOGRAPHY Christine is originally from Smithfield, Virginia. She holds a B.S. from Old Dominion University in Norfolk, VA with a major in Biology and a minor in Applied Mathematics. She moved to Raleigh, NC in 2010 to pursue graduate degrees in horticulture. She discovered plant breeding as a career option in her M.S., and the rest is…history! iv ACKNOWLEDGMENTS …I realize I have been in this PhD program (and graduate school) for a significant amount of time, and that in that time I have met some amazing people that have taught me some amazing things, and have supported me in times when I have really needed it. I’m not sure I would have ever made it this far without any or all of you, and I know a line in this dissertation is hardly a thanks, but it is what I can offer you for now. Hopefully one day I can repay you suitably. Also, if I forget to thank you here in text, I’m terribly sorry. I don’t want to miss anyone, but sometimes the mind goes astray, especially when you’re writing your dissertation! In no particular order: • Gina, my advisor, who has been exceedingly patient through five years of questions, crises, etc. I know it may sometimes be lost, but I am still to this day overwhelmingly thankful to have had the opportunity to attend graduate school and to work with you on such a great crop like raspberries. Your willingness to include me in extension and outreach has been a wonderful opportunity for me to grow in both the academic and professional sense. With your help, my time at NC State has truly been more than school, and I thank you for preparing me practically for the future. • Penny, who obviously saw potential in me as her first graduate student, and hopefully still does as I finish under her (though she has had many come and go in the time I have still been here!). Throughout the course of graduate school, you have provided opportunities for me to present my research in places where I would never have been able v to travel otherwise and have given me the chance to expand my knowledge and learn how to network. I was very thankful to have the resources provided in your lab, and I have learned a lot from your perspectives. • Susana, thank you for serving on my committee. I admire your personal strength and your breeding program, and am glad to have had the opportunity to work with you and your students. Thank you for taking the time to help me and teach me • Bode, thank you much for agreeing to step in so close to the end. Your knowledge of GBS, linkage mapping, and markers in general has been exceedingly helpful to me as I have been ‘putting all the pieces’ together for the 4304 linkage map in these last couple of months. I have great faith that you will produce excellent research and develop genetic resources for the sweetpotato, and will be looking for your successes in the future! • Nahla, I am exceedingly glad that I met you at the beginning of my PhD. You are one of the kindest people I have ever met, and you made me realize that I was capable of doing things that were previously daunting in the molecular genetics lab. I want you to know how much I appreciate your patience whenever things got difficult for me in the lab, and that I was equally as frustrated as you must have felt that my progress was so stalled. Thank you for working with me, for providing me with positive feedback, and for making me a part of the black raspberry project. • To the faculty and staff of Horticultural Science at NCSU, I can’t thank you enough. You have been mentors and teachers, and I owe all of you my gratitude for your support. All of you have helped me in some way, whether it be with technical issues, general vi questions, or being there to encourage me and provide positive reinforcement. I am taking away a positive spirit from this program in addition to an excellent education, and it is because of the time we shared together.
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