Expanding the Toolbox: SNP Tools for Aquaculture and Conservation Management in the Eastern Oyster (Crassostrea virginica) and the Black Basses (Micropterus spp.) by Wilawan Thongda A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama May 5, 2018 Key words: eastern oyster, black basses, SNP, GBS Copyright 2018 by Wilawan Thongda Approved by Eric Peatman, Chair, Associate Professor of Fisheries, Aquaculture, and Aquatic Sciences William H. Daniels, Associate Professor of Fisheries, Aquaculture, and Aquatic Sciences Charles Y. Chen, Professor of Crop Soil and Environmental Sciences Scott McElroy, Professor of Crop Soil and Environmental Sciences Abstract Single nucleotide polymorphisms (SNPs) are considered as important molecular markers due to their several advantages, including their abundance, distribution in the genome, stability due to low mutation rates, ease of multiplexing, lower cost, amenability to high throughput assays, and low genotyping error rate. The rapid development of technology for SNPs has provided an efficient and cost-effective genetic marker tool for aquaculture and aquatic conservation in recent years for various purposes such as the determination of the population structure, population genomics, traceability, species identification, hybridization rates, and migratory dynamics. Here, relevant aspects of the Eastern oyster (Crassostrea virginica) and black basses (Micropterus spp.), key aquatic species in the southeastern United States, are examined. Culture of the Eastern oyster is rapidly expanding. Combined with their continuing role as an environmental sentinel species and ecological model, this trend necessitates improved molecular tools for breeding and selection, as well as population assessment and genetic conservation. The development and validation of two panels of 58 SNPs for the species are described. Population analyses revealed three distinct populations, based upon FST values and STRUCTURE, among wild oysters sampled from Delaware Bay (1), Northwest Florida (2), Alabama (2), Louisiana (2), and the Texas Gulf Coast (3), consistent with previous microsatellite and mtDNA analyses. In addition, utilizing the developed panels for parentage assignment in cultured oysters resulted in highly accurate parent-offspring pairing (99.37%). The SNP. ii markers could, furthermore, clearly discriminate between hatchery stocks and wild-sourced individuals Black basses are apex predators in North American streams, rivers, and lakes and are important game fishes. Translocation and introductions for angling, accompanied by intrinsically weak genetic barriers, have led to widespread introgressive hybridization and genetic swamping. Species-diagnostic (fixed allele) SNP markers have been utilized successfully in salmonids to monitor hybridization and maintain genetic integrity. Here, similar resources for black basses through initial genotyping-by-sequencing (GBS), followed by extensive validation in additional samples using two panels of 64 SNPs, were developed. Results from >1300 genotyped bass indicated that the developed panels robustly and clearly delineate the majority of species and their hybrids among black basses. iii Acknowledgments As I complete my Ph.D. studies, I am truly grateful to my major academic advisor, Dr. Eric Peatman, for his boldness and patience, as well as for providing this opportunity to me. His advice is very valuable and will be applicable throughout my future career. He has strengthened my critical, logical, and systematic thinking abilities. He allowed me to try whatever I thought was best, as long as he could imagine possible successful results. His attitude toward work makes him one of my role models for scientific work. Every moment when I discuss a project with him is memorable. Aside from Dr. Peatman, I am thankful to my other committee members—Dr. William H. Daniels, Dr. Charles Y. Chen, and Dr. Scott McElroy—for their time, encouragement, suggestions, and comments on my project. My gratitude also extends toward Dr. Chao Li, who served as a senior student and postdoc fellow in Dr. Peatman’s lab from 2012 through 2014. I appreciate his help and friendship. Dr. Li also sometimes acted as a parent who cared for me and was concerned about me. Additionally, I am very happy to thank my fellow lab members, both past and current—Mr. Yupeng Luo, Dr. Xingqiang Wang, Mr. Spencer Gowan, Ms. Ammu Anil, Ms. Taylor Brown, Dr. Dongdong Zhang, Dr. Haitham Mohammed, Mr. Honggang Zhao, and Ms. Lauren Davis—for creating such a nice work environment at all times. I loved to share new information and knowledge, both for work and in personal matters, with each other. Helping each other with our lab work across many projects helped us build strong relationships as colleagues and friends. Furthermore, I would like thank to Ludmilla Kaltenboeck, Dr. Huseyin Kucuktas, and my other lab friends for their help in the laboratory. iv I am thankful to the Royal Thai Government for granting me a scholarship to study in the Ph.D. program at Auburn University. Taking coursework at Auburn University provided me opportunities in the academic area that I truly desired. I am also grateful to Dr. Peatman for providing funding during my last two and a half years so that I could complete the projects in this dissertation. In addition, my life at Auburn University was very happy and memorable; I want to say thank you to all of my friends at Auburn, across the U.S., and in other countries, including Canada, England, Germany, France, and Thailand. Finally, I would like to express my love and thankfulness to my family, who have always stayed beside me and believed in me. Their unconditional love, deep understanding, and continual support and encouragement are the most precious things in my life. v Table of Contents Abstract ........................................................................................................................................ ii Acknowledgments ....................................................................................................................... iv List of Tables .............................................................................................................................. ix List of Figures ............................................................................................................................. xi Chapter I Introduction and Literature Review .............................................................................. 1 Overview ........................................................................................................................... 1 Molecular Markers in Aquaculture and Conservation Genetics: Past and Present .......... 2 SNP Markers ..................................................................................................................... 4 SNP Discovery ................................................................................................................. 6 ESTs ...................................................................................................................... 6 RNA-seq ............................................................................................................... 7 GBS....................................................................................................................... 9 RAD-seq ............................................................................................................. 10 SNP Genotyping Platforms ............................................................................................. 12 TaqMan® platform .............................................................................................. 12 KASPTM genotyping assay .................................................................................. 13 Fluidigm® SNP Type™ Assays ......................................................................... 14 Illumina Golden Gate platform ............................................................................ 15 The Agena MassARRAY .................................................................................... 16 vi All in One Approaches to SNP Genotyping ................................................................... 17 Genotyping-in-Thousands by Sequencing (GT-seq) ...................................................... 19 Eastern Oyster Population Genetics................................................................................ 19 Black Basses ................................................................................................................... 24 Black Bass Diversity ............................................................................................ 24 Hybridization ....................................................................................................... 25 The Use of Molecular Markers in Black Basses ............................................................. 27 Dissertation Overview .................................................................................................... 28 References ....................................................................................................................... 30 Chapter II Development of SNP panels as a new tool to assess the genetic diversity, population structure, and parentage analysis of the eastern oyster (Crassostrea virginica) ......................... 60 Abstract ..........................................................................................................................