GENOME-WIDE ANALYSES OF GENES AFFECTING GROWTH, MUSCLE ACCRETION AND FILLET QUALITY TRAITS IN RAINBOW TROUT by Ali Reda Eid Ali A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Molecular Biosciences Middle Tennessee State University August 2019 Dissertation committee: Dr. Mohamed (Moh) Salem, Chair Dr. Anthony L. Farone Dr. Jason Jessen Dr. Mary Farone Dr. Elliot Altman I dedicate this research to my parents, my wife and kids for their unwavering love and endless support throughout this long trip. Without their great selflessness, sacrifices, and prayers, I would not have been able to fulfill and achieve this dream of mine ii ACKNOWLEDGMENTS First of all, I would like to express my deepest respect and indebtedness to my PhD dissertation advisor Dr. Mohamed Salem for his continuous guidance, endless support and encouragement throughout my PhD research work. I was fortunate to have him as my primary advisor who gave me this opportunity to grow as a professional research scholar. He was never wavering in his support, and was always patient and supportive during the more grueling portions of this investigation. My deepest appreciation and respect also go to my PhD dissertation committee members Dr. Anthony Farone, Dr. Elliot Altman, Dr. Jason Jessen, and Dr. Mary Farone for guiding me with their expertise to accomplish this project. I would like to thank Dr. Rafet Al-Tobasei for working together in several projects and sharing scientific ideas. I also want to acknowledge my collaborators Dr. Yniv Palti, and Dr. Timothy D. Leeds from National Center for Cool and Cold Water Aquaculture (NCCCWA)/USDA. I would also like to extend my sincere thanks to my collaborator Dr. Brett Kenney from West Virginia University (WVU). Finally, I would like to express my sincere gratitude to the entire MTSU Molecular Biosciences (MOBI) program and MTSU Biology department for their support and encouragement during my stay as a doctoral student at MTSU. iii ABSTRACT Growth performance is one of the most economic important traits for the aquaculture industry. In addition, fillet quality attributes are among the primary determinants of consumer acceptability. To study the genetic architecture of these traits, phenotypic characterization of whole body weight (WBW), muscle yield, fat content, shear force, moisture, protein content, and whiteness were measured in ~500 fish representing 98 families from a growth-selected line. RNA-Seq was used to sequence the muscle transcriptome of different families exhibiting divergent phenotypes for each trait. In total, 240 and 1,280 differentially expressed (DE) protein-coding genes and long noncoding RNAs (lncRNAs), respectively, were identified in fish families exhibiting contrasting phenotypes. Expression of many DE lncRNAs (n = 229) was positively correlated with overlapping, neighboring or distantly located protein-coding genes (n = 1,030), resulting in 3,392 interactions. Three DE antisense lncRNAs were co-expressed with sense genes known to impact muscle quality traits. A 50K SNP chip has been developed and used across four genome-wide association (GWA) studies to identify quantitative trait loci (QTL) explaining variations in fish growth and fillet quality attributes. In the first study, QTL explaining up to 28.40% of the additive genetic variance for muscle yield were identified; particularly on chromosomes 14 and 16. In the second study, 247 QTL associated with bodyweight gain were identified. Most SNPs affecting muscle yield and bodyweight gain exist in genes that act as major regulators of developmental processes. In the third study, the additive genetic variance for fillet firmness and protein content was investigated where RYR3 harbored most SNPs affecting the two iv traits. In the fourth study, sixty-one common SNPs on chromosomes 19 and 29 affecting the muscle fat and moisture content were identified. SNP-harboring genes, in the common QTL, were mainly involved in lipid metabolic process and cytoskeleton remodeling. Presence of common QTL associated with multiple phenotypes suggests common mechanisms underlying those phenotypes in fish. The present work identified DE genes and genetic markers explaining variations in growth and fillet quality phenotypes in selectively bred trout populations. Such markers could be used for marker-assisted and genomic selection in breeding programs of rainbow trout. v TABLE OF CONTENTS CONTENTS.......................................................................................................... …PAGE LIST OF TABLES ........................................................................................................... ix LIST OF FIGURES ........................................................................................................ xii LIST OF APPENDICES .................................................................................................xv INTRODUCTION..............................................................................................................1 OBJECTIVES ....................................................................................................................9 CHAPTER I: INTEGRATED ANALYSIS OF LNCRNA AND MRNA EXPRESSION IN RAINBOW TROUT FAMILIES SHOWING VARIATION IN MUSCLE GROWTH AND FILLET QUALITY TRAITS ..........................................10 INTRODUCTION .............................................................................................................11 MATERIALS AND METHODS .......................................................................................14 RESULTS AND DISCUSSION ........................................................................................22 CONCLUSIONS................................................................................................................46 REFERENCES ..................................................................................................................48 APPENDICES ...................................................................................................................60 CHAPTER II: GENOME-WIDE ASSOCIATION ANALYSIS WITH A 50K TRANSCRIBED GENE SNP-CHIP IDENTIFIES QTL AFFECTING MUSCLE YIELD IN RAINBOW TROUT .....................................................................................65 INTRODUCTION .............................................................................................................66 MATERIALS AND METHODS .......................................................................................69 RESULTS AND DISCUSSION ........................................................................................76 CONCLUSIONS................................................................................................................92 vi REFERENCES ..................................................................................................................93 APPENDICES .................................................................................................................100 CHAPTER III: GENOME-WIDE ASSOCIATION STUDY IDENTIFIES GENOMIC LOCI AFFECTING FILLET FIRMNESS AND PROTEIN CONTENT IN RAINBOW TROUT .................................................................................................101 INTRODUCTION ...........................................................................................................102 MATERIALS AND METHODS .....................................................................................106 RESULTS AND DISCUSSION ......................................................................................112 CONCLUSIONS..............................................................................................................133 REFERENCES ................................................................................................................134 APPENDICES .................................................................................................................146 CHAPTER IV: IDENTIFICATION OF GENOMIC LOCI ASSOCIATED WITH GROWTH IN RAINBOW TROUT .............................................................................150 INTRODUCTION ...........................................................................................................151 MATERIALS AND METHODS .....................................................................................154 RESULTS AND DISCUSSION ......................................................................................159 CONCLUSIONS..............................................................................................................179 REFERENCES ................................................................................................................180 APPENDICES .................................................................................................................191 CHAPTER V: GENOME-WIDE ASSOCIATION STUDY IDENTIFIES COMMON GENOMIC LOCI ASSOCIATED WITH MUSCLE FAT AND MOISTURE CONTENT IN RAINBOW TROUT ............................................................................193 INTRODUCTION ...........................................................................................................194 vii MATERIALS AND METHODS .....................................................................................198 RESULTS AND DISCUSSION ......................................................................................203 CONCLUSIONS..............................................................................................................224