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Genetic Variation Within and Among the Suminoe Oyster (Crassostrea Ariakensis) Populations and Stocks As Assessed by Molecular Markers

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Genetic Variation Within and Among the Suminoe Oyster (Crassostrea Ariakensis) Populations and Stocks As Assessed by Molecular Markers W&M ScholarWorks Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects 2003 Genetic Variation Within and among the Suminoe Oyster (Crassostrea ariakensis) Populations and Stocks as Assessed by Molecular Markers Qian Zhang College of William and Mary - Virginia Institute of Marine Science Follow this and additional works at: https://scholarworks.wm.edu/etd Part of the Fresh Water Studies Commons, Genetics Commons, Molecular Biology Commons, and the Oceanography Commons Recommended Citation Zhang, Qian, "Genetic Variation Within and among the Suminoe Oyster (Crassostrea ariakensis) Populations and Stocks as Assessed by Molecular Markers" (2003). Dissertations, Theses, and Masters Projects. Paper 1539617806. https://dx.doi.org/doi:10.25773/v5-nngz-rg29 This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. GENETIC VARIATION WITHIN AND AMONG THE SUMINOE OYSTER (iCRASSOSTREA ARIAKENSIS) POPULATIONS AND STOCKS AS ASSESSED BY MOLECULAR MARKERS A Thesis Presented to The Faculty of the School of Marine Science The College of William and Mary in Virginia In Partial Fulfillment Of the Requirements for the Degree of Master of Science By Qian Zhang 2003 APPROVAL SHEET This thesis is submitted in partial fulfillment of The requirements for the degree of Master of Science Qian Zhang Approved, July 2003 Kimberly S. Reece, Ph.D. Co-Advisor Sj^ndish K. A llel Jr., Ph.D. Co-Advisor A a O —/ s / \ RogepL. Mann, Ph.D. John M. Brubaker, Ph.D. TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................v LIST OF TABLES............................................................................................................. vi LIST OF FIGURES............................................................................................................ ix ABSTRACT.........................................................................................................................x CHAPTER 1. GENERAL INTRODUCTION TO THE SUMINOE OYSTER CRASSOSTRIA ARIAKENSIS .....................................................................1 INTRODUCTION...............................................................................................................2 OBJECTIVES......................................................................................................................7 LITERATURE CITED....................................................................................................... 8 CHAPTER 2. GENETIC VARIATION WITHIN AND AMONG CRASSOSTREA ARIAKENSIS WILD AND HATCHERY SAMPLES, AS REVEALED BY RFLP ANALYSIS OF MITOCHONDRIAL AND NUCLEAR LOCI............................................................................................................13 INTRODUCTION...............................................................................................................14 MATERIAL AND METHODS.......................................................................................... 17 RESULTS............................................................................................................................ 25 DISCUSSION......................................................................................................................34 LITERATURE CITED....................................................................................................... 43 CHAPTER 3. GENETIC VARIATION WITHIN AND AMONG CRASSOSTREA ARIAKENSIS WILD AND HATCHERY SAMPLES, AS REVEALED BY MICROSATELLITE MARKERS......................................................91 INTRODUCTION...............................................................................................................92 MATERIALS AND METHODS........................................................................................94 RESULTS.............................................................................................................................97 DISCUSSION..................................................................................................................... 105 LITERATURE CITED.......................................................................................................113 APPENDIX A .....................................................................................................................148 APPENDIX B.....................................................................................................................158 VITA....................................................................................................................................174 iv ACKNOWLEDGEMENTS I sincerely appreciate the time, guidance, patience, and encouragement my advisors Drs. Kimberly Reece and Standish Allen Jr. provided during my time at VIMS. They gave me much helpful advice over the years and their critical review of this thesis contributed countless suggestions for improvement. I would also like to thank my other committee members, Dr. Roger Mann and Dr. John Brubaker, for their constant support and assistance in the completion of this thesis. Special thanks to everyone in, or associated with, the VIMS Oyster Genetics Laboratory for providing me support and encouragement through all stages of this study. I owe a special thanks to Karen Hudson, who taught me the essentials of laboratory protocols, answered many of my questions and walked me through several procedures during the initial stages of this project. Without her help this work would not have been possible. I am very grateful to Dr. Jan McDowell for her advice and assistance in using ARLEQUIN, RFLPSCAN and GENEPOP software and introducing me to the joys of handling genetic data. Finally, I would like to thank the following individuals and companies for their assistance in collecting specimens: Elizabeth Francis collected Crassostrea ariakensis samples from Japan, Dr. Standish Allen collected C. ariakensis from coastal areas of China, the Taylor United Industry Co. sent samples from Washington states, USA, and Liz Walker facilitated collection of C. ariakensis hemolymph from VIMS hatchery samples. LIST OF TABLES Table Page 1. Sample groups of the Suminoe oyster, used in PCR-RFLP and microsatellite genotyping ................................................................................. 58 2. Primer pairs used for amplification of Crassostrea ariakensis DNA 60 3. Composite haplotypes based on RFLP analysis of the mitochondrial COI gene ..................................................................................................................... 61 4. Percent mean nucleotide sequence divergence based on the mtDNA RFLP analysis ........................................................................................................... 63 5. Tajima’s D and Fu’s F tests to access the deviation from the mutation-drift equilibrium ....................................................................................................... 64 6. Analysis of molecular variance (AMOVA) between the northern (YR and I) and southern (B, C, D) groups based on the mtDNA RFLP analysis ............ 65 7. Hierarchical AMOVA tests based on the mtDNA RFLP analysis .................. 66 8. Analysis of molecular variance (AMOVA) between the northern (YR and I) and southern (C and D) groups based on the mtDNA RFLP analysis ............ 67 9. Population pairwise Fst and significance P-values calculated based on the mitochondrial COI RFLP analysis ................................................................... 68 10. Composite genotypes based on RFLP analysis of the nuclear ITS-1 region .................................................................................................................. 70 vi Hardy-Weinberg equilibrium (HWE) tests based on the nuclear ITS-1 RFLP analysis .............................................................................................................. 72 Percent mean nucleotide sequence divergence based on the nuclear DNA ITS-1 RFLP analysis ......................................................................................... 74 Analysis of molecular variance (AMOVA) between the northern (YR and I) and southern (B, C, D) groups based on the nuclear DNA RFLP analysis ..... 75 Hierarchical AMOVA tests based on the nuclear DNA RFLP analysis ......... 76 Analysis of molecular variance (AMOVA) between the northern (YR and I) and southern (C and D) groups based on the nuclear DNA RFLP analysis.... 77 Population pairwise Fst and significance P- values calculated based on the nuclear ITS-1 RFLP analysis ........................................................................... 78 P-values for pairwise comparisons of allelic and genotypic frequencies based on nuclear the ITS-1 RFLP analysis ....................................................... 79 Microsatellite primers, optimal annealing temperatures (Tm) and product size for locus ucdCg-120, 172 and 199 ............................................................. 122 Number of samples used, number of alleles and genotypes, allele size range, size and frequency at the three microsatellite loci ........................................... 123 Expected and observed heterozygosity, inbreeding coefficient, probability of deviation from Hardy-Weinberg
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