THE IDENTIFICATION AND CHARACTERIZATION OF COPY NUMBER VARIANTS IN THE BOVINE GENOME A Dissertation by Ryan N Doan Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Scott Dindot Committee Members, Noah Cohen William Murphy Loren Skow James Womack Intercollegiate Faculty Chair, Craig J. Coates August 2013 Major Subject: Genetics Copyright 2013 Ryan N Doan ABSTRACT Separate domestication events and strong selective pressures have created diverse phenotypes among existing cattle populations; however, the genetic determinants underlying most phenotypes are currently unknown. Bos taurus taurus (Bos taurus) and Bos taurus indicus (Bos indicus) cattle are subspecies of domesticated cattle that are characterized by unique morphological and metabolic traits. Because of their divergence, they are ideal model systems to understand the genetic basis of phenotypic variation. Here, we developed DNA and structural variant maps of cattle genomes representing the Bos taurus and Bos indicus breeds. Using this data, we identified genes under selection and biological processes enriched with functional coding variants between the two subspecies. Furthermore, we examined genetic variation at functional non-coding regions, which were identified through epigenetic profiling of indicative histone- and DNA-methylation modifications. Copy number variants, which were frequently not imputed by flanking or tagged SNPs, represented the largest source of genetic divergence between the subspecies, with almost half of the variants present at coding regions. We identified a number of divergent genes and biological processes between Bos taurus and Bos indicus cattle; however, the extent of functional coding variation was relatively small compared to that of functional non- coding variation. Collectively, our findings suggest that copy number and functional non- coding variants may play an important role in regulating phenotypic variation among cattle breeds and subspecies. ii DEDICATION I dedicate this to my wife, Ashley, for all of her help and support. By always being there when I need her most, she is the most important reason for my successes in life. I also dedicate this to my parents, Neil and Julie, and brother, Chris, who have always supported me throughout my life. Their strong support allowed me to become the person I am today. iii ACKNOWLEDGEMENTS I would like to thank Dr. Scott Dindot for his support and guidance throughout the past four years. The experiences gained during my research will play a key role in my future endeavors. I would also like to thank all of my committee members, Drs. Noah Cohen, William Murphy, Loren Skow, and James Womack for all of their help and guidance during my dissertation project. I would also like to thank all of the people who have been involved in this project who have provided intellectual guidance, access to samples and assistance with experiments. iv TABLE OF CONTENTS Page ABSTRACT ........................................................................................................... ii DEDICATION ........................................................................................................ iii ACKNOWLEDGEMENTS ...................................................................................... iv TABLE OF CONTENTS ......................................................................................... v LIST OF FIGURES ................................................................................................ ix LIST OF TABLES .................................................................................................. xii CHAPTER I INTRODUCTION ......................................................................... 1 Types of Genetic Variation among Individuals.................................................. 1 Mechanism of Structural Variant Formation ..................................................... 3 Mutation Rates of Structural Variants ............................................................... 7 Methods to Identify Structural Variation ............................................................ 7 Detection of Copy Number Variants Using SNP Arrays .................................... 9 Whole-Genome Sequencing ............................................................................ 11 Epigenetic Gene Regulation............................................................................. 13 CNVs and Trait Association ............................................................................. 15 Bovine Evolution .............................................................................................. 16 Importance of Understanding Traits in Cattle ................................................... 17 Bovine Genomics ............................................................................................. 19 Use of Next-Generation Sequencing to Examine Genetic Variation in Cattle ... 21 The Need for a Centralized Variant Database .................................................. 22 Dissertation Strategy for Characterizing CNVs In The Bovine Genome ........................................................................................ 23 CHAPTER II CGH ANALYSIS OF CNVS.......................................................... 25 Introduction .................................................................................................... 25 Methods .................................................................................................... 28 DNA Sample .............................................................................................. 28 Comparative Genomic Hybridization Array Designs ................................... 28 Array Comparative Genomic Hybridization (aCGH) Methods ..................... 29 CNV Confirmation ...................................................................................... 31 Genomic Characterization of CNV Content ................................................ 32 v Analysis of CNV Content in the Bovine Umd3.1 Assembly ......................... 33 Functional Analysis of CNVs ...................................................................... 34 Population Analysis of CNVs ...................................................................... 35 Results .................................................................................................... 36 CGH Array Design...................................................................................... 36 Exome CGH Accuracy and Resolution ....................................................... 40 Genome Distribution of CNVs .................................................................... 45 Umd3.1 Analysis ........................................................................................ 50 Functional Analysis .................................................................................... 52 Population Analysis .................................................................................... 57 Comparative Analysis ................................................................................. 59 Discussion .................................................................................................... 61 CHAPTER III WHOLE GENOME ANALYSIS OF Bos taurus AND Bos indicus COWS ........................................................................................ 63 Introduction .................................................................................................... 63 Methods .................................................................................................... 65 Whole-Genome Sequencing ...................................................................... 65 Sequence Mapping .................................................................................... 66 Variant Detection ........................................................................................ 66 Variant Confirmation................................................................................... 68 Genetic Variant Annotation and Analysis .................................................... 71 Genotyping for Known Mutations................................................................ 72 Evolutionary Analysis ................................................................................. 72 Results .................................................................................................... 72 Genome Sequencing.................................................................................. 72 Variant Identification and Annotation .......................................................... 74 Functional Analysis of Variants .................................................................. 83 Genotyping for Known Mutations................................................................ 85 Evolutionary Analysis ................................................................................. 88 Discussion .................................................................................................... 90 CHAPTER IV VARIATION AT REGULATORY ELEMENTS............................... 95 Introduction .................................................................................................... 95 Methods .................................................................................................... 97 Epigenetic Profiling....................................................................................