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POLYMORPHIC GAIT IN THE HORSE: AN INTERACTION OF GENETICS, MORPHOLOGY, AND BEHAVIOR A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Elizabeth Ann Staiger January 2015 © 2015 Elizabeth Ann Staiger POLYMORPHIC GAIT IN THE HORSE: AN INTERACTION OF GENETICS, MORPHOLOGY, AND BEHAVIOR Elizabeth Ann Staiger, Ph. D. Cornell University 2015 Selection after domestication has primarily focused on performance, conformation and desirable behaviors in the horse, resulting in breeds that are divergent across these traits. An example are the “gaited” breeds, horses with the ability to perform either a lateral or diagonal four- beat gait without a moment of suspension at intermediate speeds, yet varying in overall size and temperament. To investigate the contribution of genetics to these divergent traits, we collected DNA samples, 35 body measurements, gait information, horse discipline, and a behavior survey from 801 gaited horses. Utilizing previously genotyped horses, an across-breed genome-wide association study (GWA) identified three novel candidate regions associated with gait type on ECA1, ECA11, and EC4. A GWA in a single gaited breed, the Tennessee Walking Horse (TWH) identified two additional candidate regions on ECA19 and ECA11. Polymorphisms from whole-genome sequences have identified several SNPs within these candidate regions. We conducted principle component analysis (PCA) on 33 of the body measures for a subset of TWH. A GWA of the first PC, which describes overall size, identified the LCORL locus, which has previously been implicated with size in horses, cattle, and humans. No causal variant has been discovered yet due to extensive linkage disequilibrium (LD) in the region, but LD in the TWH is much lower, improving the resolution capabilities for fine-mapping and variant discovery. To investigate the contribution of genetics to temperament, we utilized factor analysis (FA) on the questionnaire to identify four temperament factors in TWH: neophobia, trainability, hostility, and independence. These four factors account for 64% of the total trait variance. We ran three separate GWAs using the F1-‘neophobia’, F2-‘trainability’, and F3-‘hostility’ scores as the phenotype and identified candidate markers in genes involved with neurodegeneration, steroidogenesis, brain development, and neuronal cell signaling pathways. The results from this work will hopefully lead to future studies to identify the causal variants of locomotion, size, and behavior traits. This will allow for the development of genetic tests to aid horse owners in their breeding and management decisions and help improve horse welfare as horses are selected for appropriate disciplines. BIOGRAPHICAL SKETCH Elizabeth Ann Staiger was born in Portsmouth, Virginia, but spent her formative year in Japan, California, and Maryland. She attended Oklahoma State University and graduated in 2007 with a Bachelor of Science degree in Animal Science and with an Honor’s degree. She continued at Oklahoma State University for a Master of Science degree in Animal Breeding and Genetics under Dr. Raluca Mateescu studying genetic markers for increased milk and meat production in sheep. After completing her Masters in 2009, Ann remained in Dr. Mateescu’s lab as the lab manager. Ann began her doctoral studies in Animal Science at Cornell University in 2010. She pursued her research on the genetics of polymorphic gait in the horse under the direction of Dr. Samantha Brooks. While pursuing her degree, Ann has been the recipient of the Everingham Award, Neal A. Jorgenson Travel Award, and winner of the Equine Science Society Genetics Graduate Student competition. v “Intellectual growth should commence at birth and cease only at death” – Albert Einstein Dedicated to all of the teachers throughout my life. vi ACKNOWLEDGMENTS The inspiration for this research stemmed from my love and involvement with gaited horses, particularly Tennessee Walking Horses, and a curiosity to understand the genetic underpinnings that makes these horses so unique from other breeds. This research would not have been possible without the involvement of several people. I owe a great deal of gratitude to Dr. Samantha Brooks for serving as my committee chair and mentor for the past four and a half years, and for allowing me the freedom to work on a project about which I am truly passionate. I know it has been an adventure, and I can’t thank you enough. Thanks to Dr. Joe Fetcho for help on neurobiology and all of his helpful advice on designing our behavior trials in the horse. Thanks to Dr. Jon Cheetham for providing important insights on the physiology of the horse, especially in regard to changes over time. And a special thank you to Dr. Heather Huson for her advice on performance genetics and for “adopting” me over this past year. This work was definitely a collaborative effort from several members of the equine science community. Thank you to Dr. Rebecca Bellone for the Puerto Rican Paso Fino samples, hosting me on some of my data collection trips, and for all of the career and teaching advice/support she provided. Dr. Nate Sutter provided advice on the measurement data and PCA. Dr. Julia Albright was instrumental in the design of the behavior survey and trial, and oversaw all of the behavior trials. I would also like to thank Dr. Ernest Bailey for all of his advice and support, and graciously hosting me on data collection trips. I could not have survived without the support, friendship, and pet-sitting of my fellow graduate students and friends Kristen Davis, Claire Stephens, Jeremy Allen, and Fernando Migone. A special thanks to my lab mate Dr. Heather Holl for all of the troubleshooting help, discussions, and helping me on data collection trips. I owe a big thank you to the many undergraduates who have worked in the lab and helped with the project over the years. Thanks to Nicole SanGiacomo for entering records into the vii database, Mariya Gugel for genotyping some of the behavior SNPs, Ram Singh for the Marwari samples and genotyping help, and Alexander Thomson, Taylor Baird, Rachel Evanowski, and Janelle Slutsky for help on local farm trips. Special thanks to Chris Posbergh and Lauren Jacobs who were essentially my minions in the lab, but also agreed to go on several out-of-state and overnight trips. I owe a special thanks to all of the horse breeders, owners, and trainers who have submitted samples over the years, and hosted me on my data collection trips. A big thank you to all of the breed organizations that have promoted my research: TWHBEA, RMHA, KMHSA, USIHC, PFHA, NWHA, FOSH, USMMA, ABCCC, and ABCCMM. Without the help of the associations and the owners, I would never have been able to collect so many horses or have the means to carry out the work. I especially want to thank Joe London, Joyce Moyer, Lynn Kelley, Lori Miller, and Laura Patterson. They have been amazing supporters of the study, and have done whatever they could to get me the material I have needed. My deepest gratitude for the financial support my research has received from the Cornell Center for Vertebrate Genomics, FAST, the Dorothy Russell Havemeyer Foundation, the ABCCC, and ABCCMM. And finally, I would like to thank my family for all of their support and love, not to mention their acquiescence when I coerced them into helping on data trips, and for hosting my colleagues and me as we passed through the area on yet another data trip. You’ve truly played an active role in my research. To my parents, my first teachers, I wouldn’t be who and what I am today without your example, encouragement, and sacrifices. I cannot fully express how grateful I am to be your daughter. viii TABLE OF CONTENTS BIOGRAPHICAL SKETCH .......................................................................................................................... v DEDICATION ................................................................................................................................................ vi ACKNOWLEDGMENTS ............................................................................................................................ vii TABLE OF CONTENTS .............................................................................................................................. ix LIST OF FIGURES ....................................................................................................................................... xiii LIST OF TABLES ......................................................................................................................................... xiv CHAPTER 1: INTRODUCTION TO THE DISSERTATION ............................................................. 1 Introduction ........................................................................................................................................................ 2 The Horse as a Model for Complex Traits .................................................................................................... 3 Locomotion ........................................................................................................................................................ 6 The Equine Gait Spectrum .......................................................................................................................... 6 The Innervation of the Central Pattern Generator (CPG) .......................................................................