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Larina Carroll Thesis February 9 2018 FINAL for SUBMISSION

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Larina Carroll Thesis February 9 2018 FINAL for SUBMISSION Functional Genomics Studies of Atlantic Salmon (Salmo salar) Development, and Sac Fry Responses to Chronic Incremental Hyperthermia By: Larina A. Carroll A thesis submitted to the Department of Biology and the School of Graduate Studies in partial fulfilment of the requirements for the degree of Master of Science Ocean Sciences Centre and Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada ABSTRACT Functional genomics techniques, including the 32K cGRASP microarray and real-time quantitative polymerase chain reaction (QPCR), were used to study gene expression in a single Atlantic salmon cohort: 1) during embryonic and early sac fry development; and 2) after sac fry were exposed to chronic incremental hyperthermia (water temperature increased by 1 °C every 24 hours, from 7.4 to 21.4 °C). The first study focused on the transcript expression of four microarray-identified paralogous γM-crystallin genes as well as four paralogous α-sHSP genes. The four α-sHSPs, as well as cryGM4-like and cryGM3-like, were shown by QPCR as higher expressed at hatch or post-hatch stage relative to eye-up stage (although the degree of induction varied between paralogues). This research provides possible evidence of divergent transcript expression (i.e., regulation) of duplicated genes, suggesting that some of the paralogues studied may have diverged functionally. These results provide insight into the evolutionary relationships between these genes, and may provide evidence of neofunctionalization following GD events. The second study focused on four microarray-identified genes of interest (trypsin-1 precursor, chymotrypsin b, ferritin middle subunit, and ubiquitin) as well as the four paralogous α-sHSP genes. The transcripts encoding trypsin-1 precursor, ferritin middle subunit, and ubiquitin, as well as α-sHSPs 1, 3 and 4, were shown by QPCR as responsive to the incremental hyperthermia conditions. This study identified molecular biomarkers that may be useful for studying adaptation of early life stage salmon under potentially stressful conditions (e.g., warming climate). ii ACKNOWLEDGEMENTS I thank my supervisor, Dr. Matthew Rise, for his expertise, support, and encouragement throughout the course of this research. In addition, I would like to thank the staff of the Marine Institute’s Aquaculture Facility, particularly Jason Nichols and Keith Rideout, who provided instruction in, and assistance with, gamete stripping and fertilization, and gave advice on aquaculture procedures during the rearing stages. Terry Bungay provided assistance in setting up the rearing tanks, advice during rearing, and technical training on the LaMotte SMART 2 Colorimeter (LaMotte Company, Chestertown, MD). Dr. Laura Halfyard also assisted with training on the colorimeter. Dr. Lourdes Peña-Castillo consulted on bioinformatics procedures. Dr. Marije Booman and Dr. Tiago Hori provided assistance and advice on functional genomics lab procedures. Charles Feng provided technical training and assisted with primer design, sequence alignment and molecular phylogenetics. Danny Ings and Charles Feng aided in, and advised on, statistical analysis of the QPCR data. Dr. Albert Caballero Solares advised on and conducted the statistical analysis of the water quality and mortality data in Chapter 3 of this thesis. I thank Dr. Harry Murray for his time, insight, and advice during the writing of this thesis. I acknowledge my supervisory committee, Dr. Matthew Rise, Dr. Paul Snelgrove, and Dr. Kurt Gamperl (Department of Ocean Sciences, MUN), who provided knowledgeable expertise, guidance, and feedback. This research was supported by an NSERC Discovery Grant to Dr. Matthew Rise. iii TABLE OF CONTENTS ABSTRACT..................................................................................................................ii ACKNOWLEDGEMENTS........................................................................................iii TABLE OF CONTENTS.............................................................................................iv LIST OF TABLES.......................................................................................................ix LIST OF FIGURES...................................................................................................xiii LIST OF ABBREVIATIONS AND SYMBOLS.......................................................xvi CO-AUTHORSHIP STATEMENT.............................................................................xx 1. GENERAL INTRODUCTION………………….………………………….......1 1.1 ATLANTIC SALMON (SALMO SALAR) LIFE HISTORY.....................1 1.2 FISH EARLY LIFE STAGE GENE EXPRESSION IN RESPONSE TO TEMPERATURE CHANGES...............................................................2 1.3 WHOLE GENOME DUPLICATION EVENTS........................................4 1.4 FUNCTIONAL GENOMICS TOOLS.........................................................6 1.5 RESEARCH OBJECTIVES.........................................................................7 1.6 REFERENCES..............................................................................................9 2. FUNCTIONAL GENOMICS STUDY OF ATLANTIC SALMON DEVELOPMENT SURROUNDING THE HATCH EVENT........................18 2.1 ABSTRACT.................................................................................................18 iv 2.2 INTRODUCTION.......................................................................................18 2.3 METHODS...................................................................................................20 2.3.1 Gamete stripping and fertilization....................................................20 2.3.2 Water quality......................................................................................22 2.3.3 Sampling.............................................................................................23 2.3.4 RNA extraction and purification......................................................24 2.3.5 Microarray hybridization and data acquisition.............................27 2.3.6 Microarray data analysis..................................................................30 2.3.7 In silico identification of Atlantic salmon α-sHSP sequences........32 2.3.8 Sequence analysis...............................................................................32 2.3.9 QPCR primer design and quality testing........................................34 2.3.10 cDNA synthesis and QPCR assays of selected genes of interest....36 2.4 RESULTS.....................................................................................................39 2.4.1 Experimental paradigm and water quality monitoring at the MI..................................................................................................39 2.4.2 Microarray hybridization using the 32K cGRASP salmonid array platform, and identification of Atlantic salmon γM-crystallin transcripts....................................................................42 v 2.4.3 Characterization of four paralogous Atlantic salmon γM-crystallin contiguous sequences...................................................59 2.4.4 Transcript expression of four putative γM-crystallin paralogues during development...........................................................................64 2.4.5 Characterization of four paralogous Atlantic salmon α-sHSP contiguous sequences..........................................................................64 2.4.6 Transcript expression of four α-sHSP paralogues during developmental stages surrounding the hatch event.........................73 2.5 DISCUSSION...............................................................................................76 2.5.1 γM crystallin paralogues: evidence of gene duplication and divergence, molecular phylogenetics, and functional significance.........................................................................................76 2.5.2 α-sHSP paralogues: evidence of gene duplication and divergence, molecular phylogenetics, and functional significance.........................................................................................81 2.5.3 Other evidence of gene duplication and divergence in fish...........86 2.6 CONCLUSIONS..........................................................................................87 2.7 REFERENCES............................................................................................89 2.8 WEB REFERENCES................................................................................102 vi 3. FUNCTIONAL GENOMICS STUDY OF ATLANTIC SALMON SAC FRY RESPONSES TO INCREMENTAL INCREASES IN WATER TEMPERATURE..............................................................................................104 3.1 ABSTRACT...............................................................................................104 3.2 INTRODUCTION.....................................................................................105 3.3 METHODS.................................................................................................109 3.3.1 Experimental design, water quality, and sampling procedure....109 3.3.2 RNA extraction and purification....................................................113 3.3.3 Microarray hybridization and data acquisition............................114 3.3.4 Microarray data analysis................................................................116 3.3.5 QPCR primer design and quality testing......................................116 3.3.6 cDNA synthesis and QPCR
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