ABSTRACT COUCH, CHARLENE REESE. Microsatellite DNA marker-assisted selective breeding of striped bass. (Under the direction of Dr. Craig. V. Sullivan). Although the hybrid striped bass (HSB; female white bass, Morone chrysops x male striped bass, M. saxatilis) supports the fourth most valuable form of finfish aquaculture in the United States, neither parental species has been genetically improved. Expansion of the HSB industry is limited by culture inefficiencies associated with reliance on wild broodstock for annual fingerling production. Domestication and selective breeding are expected to increase production efficiency and to promote market expansion. Resource limitations currently prohibit the individual rearing of multiple larval families for striped bass performance testing, necessitating a breeding program that is based on communal rearing of progeny groups with molecular markers as genetic tags for offspring identification. This dissertation research addresses fundamental questions relevant to selective breeding of the male parental species of the HSB, the striped bass, including: (1) Evaluation of genetic variation within a captive striped bass broodstock population; (2) Examination of the feasibility of communal rearing protocols based on microsatellite markers for progeny identification during performance evaluations of striped bass; (3) Assessment of paternal variation in performance traits of striped bass at both research and commercial scale throughout the HSB production cycle. Examination of three captive striped bass broodstock strains using three highly variable microsatellite markers revealed that the broodstock population contains moderately high genetic diversity, with an average allelic richness of 13.7 alleles per locus and an average observed heterozygosity of 0.84. Crosses among the three differentiated strains should provide a valuable starting point for establishing a highly variable base population for selective breeding. Twenty-four experimental families were produced from captive, genotyped broodstock for communal evaluations of progeny survival and performance. Parentage was determined by microsatellite genotyping at six loci and more than 99% of progeny were attributable to a single sire-dam pair at each production phase and in all rearing environments. Application of large-scale communal rearing trials based on microsatellite markers for progeny identification should be a viable approach in a selective breeding program for striped bass. There was limited evidence of family effects on early growth or survival to 35 days of age; however, significant paternal effects on growth performance, body shape, and carcass traits were detected at later culture stages and variation in antimicrobial peptide activity, a measure of innate disease resistance, differed by strain within the research ponds. In general, progeny of domesticated Santee:Chesapeake sires out-performed those of other strains both at research-scale and in the commercial tank. In research ponds, performance of fish as yearlings (Phase II) allowed prediction of performance at Phase III (18-20 months of age). Performance in research ponds also was predictive of Phase III performance in the commercial tank. Results from performance evaluations provide evidence of genetic variation in economically important traits which may be exploited for selective breeding of striped bass. This research provides fundamental information needed to accelerate selective breeding and to increase production efficiency for the hybrid striped bass industry. MICROSATELLITE DNA MARKER-ASSISTED SELECTIVE BREEDING OF STRIPED BASS by CHARLENE REESE COUCH 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 ZOOLOGY Raleigh 2006 APPROVED BY: ____________________________________ ____________________________________ (Ronald G. Hodson) (Trudy F.C. Mackay) ____________________________________ ____________________________________ (Kenneth H. Pollock) (Craig V. Sullivan) Chair of Advisory Committee DEDICATION This work is dedicated to the good people of the United States hybrid striped bass industry and to the memory of my grandmother, Callie Ensley Reese. ii BIOGRAPHY Charlene Reese Couch was born on June 28, 1967 in Gastonia, NC and raised on a small farm in Kings Mountain, NC by her parents, Robert and Joyce Ledford. Charlene graduated from Kings Mountain Senior High School and pursued a Bachelor of Science degree in Biology at the University of North Carolina-Wilmington where she was first exposed to scientific research when offered an opportunity to study mammalian zoogeography on North Carolina’s barrier islands. Shortly after graduation in 1989, Charlene married Paul Couch and moved to Bethlehem, PA in order for Paul to pursue seminary education. During this time, she became interested in genetics and returned to school in 1995 in Richmond, VA, obtaining a Master of Science in Biology in 1998 from Virginia Commonwealth University with an emphasis in population genetics. After applying to the graduate program at North Carolina State University, Charlene was offered a position in Dr. Craig Sullivan’s laboratory in the Department of Zoology where she was able to integrate her interests in molecular biology and field work by using microsatellite markers to support development of a striped bass breeding program. After completion of her doctoral education, Charlene will further her training in quantitative and population genetics through a postdoctoral position in the laboratory of Dr. Trudy Mackay in the Department of Genetics. It is Charlene’s hope that the sum of these experiences and training will enable her to develop a career in which she can use molecular methods to address issues relevant to local and regional agriculture, preferably in the area of aquaculture genetics. iii ACKNOWLEDGMENTS Completion of this dissertation research would not have been possible without the energetic participation and support of many individuals. It has been both an honor and a pleasure to have worked alongside them. I would first like to thank Dr. Craig Sullivan for his creativity and support as an advisor and for his commitment to a thriving hybrid striped bass industry. His vision of using modern molecular technologies for improvement of striped bass allowed me the opportunity to exercise my interests in genetics, agriculture and field biology in a novel project, and I am grateful to him for friendship, guidance and thorough training during the course of this research. The project would not have been possible without his many long hours in the hatchery over two spawning seasons. I also greatly appreciate the efforts of the members of my Graduate Advisory Committee, Dr. Trudy Mackay, Dr. Kenneth Pollock, and Dr. Ron Hodson, who provided much-needed advice in the planning of this project, spent a great deal of time assisting with data analysis, and offered valuable insight into the history and current realities of the hybrid striped bass industry. The field portion of these studies was largely carried out at the Pamlico Aquaculture Field Laboratory (PAFL) in Aurora, NC. Many thanks are owed to the staff of PAFL, including Dr. Andy McGinty, Michael Hopper and Robert Clark for spawning fish, for teaching me how to grow a striped bass, and for their enthusiastic involvement in the project. Wade Gereats was tireless in sampling and grading fish, and Blake Martin devoted half a winter to harvesting fish and recording data. Blake also saved my life by fishing me iv out of a very cold pond, for which I am very grateful. Dr. Beth Chiddick cheerfully aided with striped bass spawning activities in Spring 2000. The generous support of the hybrid striped bass industry was critical to the success of this project. In particular, Dr. James Carlberg, President of Kent SeaTech Corporation, and Dr. Mark Westerman, Director of Molecular Biology at Kent SeaTech, assisted me in obtaining a Sea Grant Industry Fellowship to support this research. The company not only provided matching funds for a doctoral fellowship but also dedicated commercial tanks for rearing striped bass families under intensive production conditions. Kent SeaTech also hired a professional fish filleter from a seafood processing company in order that we might collect data on fillet yield from hundreds of striped bass. Dr. Westerman and Jason Stannard provided training in microsatellite marker enrichment methods, facilitated work at the Mecca, CA farm, sampled hundreds of fish, and repeatedly served as terrific hosts in San Diego. Steve Mitchell helped to coordinate Phase III sampling, spent a long three days gutting fish and provided production data for striped bass. Dr. Vaughn Ostland, John Creek, Alex Ma, Herñand Nuñez, Kim Nguyen and Greg Swartz all took part in the Phase III sampling effort. At Keo Fish Farms, Mike Freeze dedicated a commercial fingerling production pond for rearing striped bass Phase I fingerlings and ensured the success of this project by producing fingerlings to stock the research ponds and commercial tanks. Both Mike Freeze and Mike Clark provided assistance in receiving fry from NC and sampling fingerlings. Keo Fish Farms’ trucks carried fingerling striped bass from Arkansas to California and North Carolina in May 2001 and then transported broodstock from California to North Carolina in Spring 2002. Lee Brothers of Carolina Fisheries in Aurora, NC, v donated feed for Phase II-III pond rearing trials