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And Florida (Micropterus Salmoides Floridanus) Largemouth Bass Development and application of diagnostic SNP marker resources for Northern (Micropterus salmoides salmoides) and Florida (Micropterus salmoides floridanus) largemouth bass. By Spencer W. Gowan A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama August, 1st 2015 Keywords: Single Nucleotide Polymorphism, hybridization, Micropterus, bass Copyright 2015 by Spencer W. Gowan Approved by Eric Peatman, Associate Professor; School of Fisheries Aquaculture and Aquatic Sciences Terry Hanson, Professor; School of Fisheries Aquaculture and Aquatic Sciences Steven Sammons, Research Fellow; School of Fisheries Aquaculture and Aquatic Sciences Benjamin Beck, Research Leader; USDA-ARS, Auburn AL Abstract Efforts to improve recreational fisheries have included widespread stocking of Micropterus salmoides floridanus outside its native range of peninsular Florida. Hybridization of Florida bass (M. salmoides floridanus) with Northern largemouth bass (Micropterus salmoides salmoides) has now dramatically expanded beyond a naturally occurring intergrade zone in the southeast U.S. In recent years, there has been growing interest in protecting the genetic integrity of native basses and assessing the impact and nature of M.s. salmoides/M.s. floridanus introgression from the standpoint of hatchery and sport-fishery managers, fish biologists, ecologists and evolutionary biologists. Here, RNA-seq-based sequencing of the transcriptomes of M.s. salmoides, M.s floridanus and their F1 hybrid was conducted and a set of 3674 SNP markers with fixed-allelic differences from 2112 unique genes were identified. A subset of 61 of these markers were then developed into a set of diagnostic multiplex assays and their capacity for assessing integrity and hybridization in hatchery and wild populations of Northern largemouth and Florida bass was evaluated. Use of these markers for population comparisons and hybridization rate evaluations were demonstrated in populations spanning the state of Alabama. Geographic isolation by natural barriers (fall line and drainage basins) were found to lead to variation in introgression level, indicating limited effect of stocking efforts in some locations, while other populations appeared to have had successful introduction of FLMB alleles. ii An attempt was also made to use these markers to assess the effect of FLMB allele introgression on trophy bass populations in Lake Guntersville, AL. Correlation between genotype and size was observed in fish sampled from tournaments. The heavier fish had more FLMB influence and higher heterozygosity. Electrofishing surveys were also conducted to collect size at age data for the Lake Guntersville population and supplement the tournament samples. While growth differences were not apparent between genotype variants within this electrofishing sample, size and genotype differences were observed between fish caught by tournament anglers and fish caught by electrofishing surveys. Some individuals sampled from the tournament bass had higher observed weights than those found within the electrofishing sample. Also significantly higher mean FLMB allele frequency was observed in the tournament samples when compared to electrofishing samples. The availability of this resource, high-quality transcriptomes and a large set of gene- linked SNPs, should continue to greatly facilitate functional and population genomics studies in these key species and allow the identification of traits and processes under selection during introgressive hybridization, as well as facilitate more efficient genetic management of hatchery and stocking programs aimed at enhancing or conserving various populations of largemouth bass. iii Acknowledgments I would like to thank my faculty advisor, Dr. Eric Peatman, for inviting me to work in his lab and for his role as a mentor during my time at Auburn University. I would also like to thank my committee members, Dr. Terry Hanson, Dr. Steve Sammons, and Dr. Benjamin Beck, for their time and guidance on this project. I would like to thank Dr. Chao Li, Dr. Huseyin Kucuktas, and Dr. Milla Kaltenboeck, for their large contributions of time and effort into this project. They played an important role in sample collection, RNA extraction, executing assays on the Sequenom MassARRAY, and analysis. I also must acknowledge the rest of my lab mates who in many different ways contributed to the this project, Ammu Anil, Wilawan Thongda, Dongdong Zhang, and Honggang Zhao. Thank you to Dave Belkoski of Dr. Sammons lab for being a second reader for otoliths. Thanks to the students at the Ireland Center, (Nick Feltz, Lee Grove, and Chris Kemp) for being willing to share and collect extra samples for genetic analysis. Many thanks to Keith Floyd, Phil Ekema, Damon Abernethy, Keith Henderson, Mike Holley, Brian Rinehard and the rest of the ALDCNR biologists and technicians that helped sample Lake Guntersville, as well as collected many of the samples from around Alabama. Thank you as well to GADNR biologists who also contributed samples. ALDCNR and GADNR were responsible for funding the projects I was involved in as a student which allowed me to have this opportunity in the Peatman Lab, and for that I am thankful. A special thank you to my parents Karen and Samuel Gowan and my wife Kelly Gowan for supporting me in my decision to return to school to complete a graduate degree. iv Table of Contents Abstract ......................................................................................................................................... ii Acknowledgments........................................................................................................................ iv List of Tables ............................................................................................................................... vi List of Figures ............................................................................................................................. vii List of Abbreviations ................................................................................................................. viii Introduction ................................................................................................................................. 1 Materials and Methods .............................................................................................................. 10 Marker development ................................................................................................... 10 Statewide population evaluation ................................................................................. 15 Lake Guntersville evaluation ...................................................................................... 19 Results and Discussion ............................................................................................................. 23 Marker development ................................................................................................... 23 Statewide population evaluation ................................................................................. 35 Lake Guntersville evaluation ...................................................................................... 47 Conclusion ................................................................................................................................ 65 References ................................................................................................................................. 69 Appendices ................................................................................................................................ 76 v List of Tables Table 1 ....................................................................................................................................... 23 Table 2 ....................................................................................................................................... 24 Table 3 ....................................................................................................................................... 25 Table 4 ....................................................................................................................................... 26 Table 5 ....................................................................................................................................... 28 Table 6 ....................................................................................................................................... 29 Table 7 ....................................................................................................................................... 32 Table 8 ....................................................................................................................................... 38 Table 9 ....................................................................................................................................... 40 Table 10 ..................................................................................................................................... 40 Table 11 ..................................................................................................................................... 41 Table 12 ..................................................................................................................................... 42 Table 13 ....................................................................................................................................
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