Graduate Theses, Dissertations, and Problem Reports 2006 Genetic analysis of rainbow trout (Oncorhynchus mykiss): Strain identification via microsatellites and analysis of expressed sequence tags in intestine, liver, kidney, and ovary Amanda B. Stewart West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Stewart, Amanda B., "Genetic analysis of rainbow trout (Oncorhynchus mykiss): Strain identification via microsatellites and analysis of expressed sequence tags in intestine, liver, kidney, and ovary" (2006). Graduate Theses, Dissertations, and Problem Reports. 4272. https://researchrepository.wvu.edu/etd/4272 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Genetic Analysis of Rainbow Trout (Oncorhynchus mykiss): Strain Identification via Microsatellites and Analysis of Expressed Sequence Tags in Intestine, Liver, Kidney, and Ovary Amanda B. Stewart Dissertation submitted to the College of Agriculture, Forestry and Consumer Sciences at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Agricultural Sciences Robert A. Dailey, Ph.D., Chair Kenneth P. Blemings, Ph.D. Patricia M. Mazik, Ph.D. Caird E. Rexroad, III, Ph.D. Daniel Panaccione, Ph.D. Division of Animal and Veterinary Sciences Morgantown, West Virginia 2006 Keywords: Rainbow trout, microsatellite, expressed sequence tags Copyright 2006 Amanda B. Stewart ABSTRACT Genetic Analysis of Rainbow Trout (Oncorhynchus mykiss): Strain Identification via Microsatellites and Analysis of Expressed Sequence Tags in Intestine, Liver, Kidney, and Ovary Amanda B. Stewart Implementation of modern fishing techniques and hatchery technologies necessitates use of genetics in management of fishery stocks. Genetic analysis of rainbow trout, Oncorhynchus mykiss, will assist development of superior strains of a valuable aquacultured species. This study was to (1) develop polymorphic microsatellite markers to examine for variation among rainbow trout strains and develop an identification panel of microsatellites to distinguish individual strains, and (2) to identify novel genes and estimate relative gene expression profiles (expressed sequence tags from rainbow trout liver, intestine, kidney, and ovary non-normalized libraries). Thirty-seven microsatellites were identified by screening 576 clones from TAGA and ATG-repeat enriched libraries. Allele frequencies were used to determine number of alleles per locus, percentage of variable loci, and mean heterozygosity, and calculation of F-statistics using GenePop and Bisosys software. Further analysis of ten individuals of ten strains with 13 markers was produced unique genotypes. Observed heterozygosity over all loci was less than the expected Hardy-Weinberg values. Mean FIS values were high in Wytheville and Ennis strains, suggestive of inbreeding. Between-strain heterogeneity tests were significant (p<0.001) for all pair-wise comparisons of strains, thus each strain is considered unique. Allele frequencies allowed correct assignment of 92% of individuals to strain of origin. Analysis of expressed sequence tags identified 90, 8, 19, 47 previously unknown genes in intestine, liver, kidney, and ovary, respectively. Overall, Wytheville trout appear to be least diverse. Many genes were in more than one tissue, suggesting potential use as positive-controls in PCR-based studies in these tissues. TABLE OF CONTENTS INTRODUCTION ...............................................................................................................1 Rainbow trout and aquaculture ....................................................................................... 2 Origins and classifications of rainbow trout ................................................................... 2 Genetics and aquaculture ................................................................................................ 4 Heritability of production traits ...................................................................................... 5 Negative effects of inbreeding........................................................................................ 7 Initial genetic analyses of salmonids .............................................................................. 9 Common methods of genetic identification.................................................................. 10 Advantages to DNA-based methodologies................................................................... 11 Microsatellites aid identification of quantitative trait loci............................................ 17 Continued use of other marker types ............................................................................ 18 I. Use of Microsatellites for Strain Identification of Rainbow Trout ...............................19 Identification and maintenance of wild, naturalized, and hatchery stocks ................... 22 Identification of food products...................................................................................... 24 Identification of rainbow trout strains........................................................................... 25 Use of F statistics in population studies........................................................................ 25 OBJECTIVE ......................................................................................................................26 MATERIALS AND METHODS.......................................................................................26 Statistical Analyses ....................................................................................................30 RESULTS ..........................................................................................................................31 DISCUSSION....................................................................................................................33 Implications................................................................................................................... 41 II. Gene Identification and Evaluation of Relative Gene Expression in Rainbow Trout (Oncorhynchus mykiss) Intestine, Liver, Kidney, and Ovary Using Expressed Sequence Tags....................................................................................................................................43 INTRODUCTION .............................................................................................................43 MATERIALS AND METHODS.......................................................................................44 Data Analysis.............................................................................................................45 RESULTS ..........................................................................................................................46 DISCUSSION....................................................................................................................49 REFERENCES ..................................................................................................................53 iii LIST of TABLES Table 1. Species of Salmonidae, Fishery and Culture Status ........................................ 101 Table 2. Subspecies of Oncorhynchus mykiss............................................................... 109 Table 3. A Survey of Literature of Genetic Analysis in Salmonids .............................. 110 Table 4. Strains of Rainbow Trout used in Microsatellite Analysis.............................. 128 Table 5. Microsatellites Identified in Rainbow Trout TAGA and ATG Repeat-Enriched Libraries .......................................................................................................................... 129 Table 6. The Variety of Alleles Found at Each Microsatellite Locus in 10 Strains of Rainbow Trout ................................................................................................................ 134 Table 7. Strains of Rainbow Trout with Unique Alleles at Each Locus........................ 135 Table 8. Number of Alleles in Each Rainbow Trout Strain and Average Heterozygosity (H) Within and Among Strains....................................................................................... 136 Table 9. Mean Heterozygosity Within Each Strain of Rainbow Trout.......................... 138 Table 10. FIS Values for Each Locus Within Each Strain of Rainbow Trout*.............. 139 Table 11. FIS, FST, and FIT values for Each Microsatellite Locus .................................. 140 Table 12. Mean Strain FIS Values for Each Strain of Rainbow Trout ........................... 141 Table 13. Individual Rainbow Trout Assigned Incorrectly to Strain of Origin............. 142 Table 14. Genetic Differentiation (Pairwise FST) Between Strains of Rainbow Trout.. 142 Table 15. Genetic Distance