DECIPHERING THE GENETIC HISTORY OF AN ANCIENT DOG BREED, THE SALUKI, USING Y CHROMOSOME, AUTOSOMAL MICROSATELLITES, AND MITOCHONDRIAL DNA A Thesis Presented to the faculty of the Department of Biological Sciences California State University, Sacramento Submitted in partial satisfaction of the requirements for the degree of MASTER OF SCIENCE in Biological Sciences (Ecology, Evolution, and Conservation) by Kristen D. Ahrens SUMMER 2016 i © 2016 Kristen D. Ahrens ALL RIGHTS RESERVED ii DECIPHERING THE GENETIC HISTORY OF AN ANCIENT DOG BREED, THE SALUKI, USING Y CHROMOSOME, AUTOSOMAL MICROSATELLITES, AND MITOCHONDRIAL DNA A Thesis by Kristen D. Ahrens Approved by: __________________________________, Committee Chair Brett Holland, Ph.D. __________________________________, Second Reader Jamie Kneitel, Ph.D. __________________________________, Third Reader Sarah Brown, Ph.D. ____________________________ Date iii Student: Kristen D. Ahrens I certify that this student has met the requirements for format contained in the University format manual, and that this thesis is suitable for shelving in the Library and credit is to be awarded for the thesis. __________________________, Graduate Coordinator ___________________ Jamie Kneitel, Ph.D. Date Department of Biological Sciences iv Abstract of DECIPHERING THE GENETIC HISTORY OF AN ANCIENT DOG BREED, THE SALUKI, USING Y CHROMOSOME, AUTOSOMAL MICROSATELLITES, AND MITOCHONDRIAL DNA by Kristen D. Ahrens Humans share a unique bond with dogs (Canis lupus familiaris) that is unrivaled by any other domesticated animal. The early history of this relationship remains unclear and detailed genetic study of early dog breeds is limited. Several studies using nuclear markers and small sample sizes have identified the Saluki as one of the oldest, extant dog breeds. This is supported by archaeological data, which age the history of the Saluki in Southwest Asia between 7,000 and 9,000 BP. I used mitochondrial DNA (mtDNA) sequences (hypervariable region I), Y chromosome short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), as well as autosomal microsatellite loci to provide a more comprehensive study of the Saluki’s history. My study compared purebred, “modern” Salukis from the U.S. and South Africa to Salukis of Southwest Asia (Israel and Iran), and additionally compared these populations to feral, village dogs of Southwest Asia and a range of purebred dogs. This design offered insight into effects of breeding practices and the relationship of Salukis to other dog groups. Emphasis was placed on Y chromosome data, which offer an intermediary mutation rate for populations v of this evolutionary timescale. All three molecular markers supported the hypothesis that Southwest Asian Salukis maintain their indigenous ancestry. U.S. Salukis displayed the effects of modern breeding practices at the mtDNA and microsatellite level, but only Y chromosome data confirmed their shared lineage with Salukis of Southwest Asia. Further, Y chromosome analysis showed evidence of considerable genetic isolation of Salukis from all other purebred dogs (72 different breeds) with the exception of the Afghan Hound. Overall, all three markers used in this study demonstrated that the four Saluki populations studied share a unique history and distinct population structure compared to any other breed, and additionally share a long lineage with feral, village dogs of Southwest Asia. Age estimates of two Y chromosome Saluki and village dog haplogroups, between 5,125 ± 2,833 and 6,558 ± 2,078 years, align with the long proclaimed, ancient origin of the Saluki. _______________________, Committee Chair Brett Holland, Ph.D. _______________________ Date vi ACKNOWLEDGEMENTS Foremost, I am extremely grateful to Dr. Sarah Brown for being an ever patient and inspiring mentor from the time I began to work under her guidance as an undergraduate in 2008. You greatly influenced my decision to pursue this degree simply by being an amazing scientist, teacher, and friend. I really cannot thank you enough for making this experience so positive, rewarding, and enjoyable. I would also like to thank Dr. Ben Sacks at UC Davis who took me into his lab as an inexperienced undergraduate eight years ago, and allowed me to continue my work in the Mammalian Ecology and Conservation Lab for this thesis. Thank you for making my formative first experience in a research lab one that solidified my interests, gave me an opportunity for growth and confidence, and ultimately led to my current path. Additional thanks to past and present members of the lab who have been a mentor to me as well, including Mark Statham, Mourad Gabriel, and Greta Wengert. Funding was provided by the Veterinary Genetics Laboratory (UC Davis) and UCOP Pacific Rim Grant. Thank you to individuals who aided in sample collection: M. Okon, L-A. Crooke, S. Jafari, Wyatt Delfino and The Veterinary Genetics Dog Research group. I am grateful to Dr. Brett Holland who had confidence in my abilities and offered much encouragement throughout. Thank you for helping me to always remain curious, think critically, and ask all of the questions – you’ve been influential since my time in vii your courses as an undergraduate. Thank you for helping me hone my skills as a writer of science and for building my confidence, and especially for always doing so with a superb sense of humor. Thank you to Dr. Jamie Kneitel who has also helped me become a more effective writer and has additionally expanded my knowledge in so many other areas of ecology beyond my own microcosm. Thank you for reading this thesis and providing valuable feedback. I owe much gratitude to my family for always supporting my endeavors. Thank you to my parents, Greg and Brenda Ahrens who have nourished my curiosity in the sciences since I was a child. Thank you for always believing I am the very best, whether or not there is evidence to support this. Thank you for every year of love and support. Thank you to my grandpa who has passed, but still seems ever present in my life as he has shaped the woman I am today. To some of my favorite humans – Tatiana Biton, Samantha Buchanan, Kenny Muñoz, Rachel Robles, and Daniel Wintz – your friendship and years of support mean everything to me. You’ve helped keep me grounded and sane, and provided me with so much love and laughter. I am especially appreciative to you for making sure I got outside and maintained some balance in my life. All of my gratitude and love to each of you! Extra thanks to Daniel and his “Harvard Super Computer” for helping run some of the week long statistical programs that my sad, layman’s computer could not handle. viii Lastly, no study of dogs would be proper or complete without thanking my own canine companion, Indie. Thanks for being my adventure buddy, keeping me smiling, and reminding me not to take life so seriously. You are a ridiculous gem of a mutt, and I’m sure you have the DNA to prove it. ix TABLE OF CONTENTS Page Acknowledgements ................................................................................................................. vii List of Tables ........................................................................................................................... xi List of Figures ......................................................................................................................... xii INTRODUCTION ………………...………………...………………………………………. 1 MATERIALS AND METHODS .............................................................................................. 7 Samples…. ........................................................................................................................ 7 Laboratory Procedures ...................................................................................................... 9 Data Analysis .................................................................................................................. 10 RESULTS ............................................................................................................................... 13 Mitochondrial DNA ........................................................................................................ 13 Y Chromosome ............................................................................................................... 13 Autosomal Microsatellites .............................................................................................. 18 DISCUSSION ......................................................................................................................... 23 Appendix A. The Domestication of the Dog ........................................................................ 28 Literature Cited ....................................................................................................................... 35 x LIST OF TABLES Table Page 1. Sample sizes used for data analyses of mtDNA region I (HVI), Y-chromosome STRs and SNPs, and autosomal microsatellites …………………………………...………8 2. Previously published mtDNA sequences accessed from GenBank included in present study………………………………………..………………………………………. 11 3. Diversity of 476 bp fragment of mitochondrial DNA, hypervariable region I, per population ............................................ ………….…………………………………. 15 4. Results based on 24 autosomal microsatellite loci …………………….… .......... ………. 22 xi LIST OF FIGURES Figure Page 1. Universally occurring haplotypes shared among Salukis and other dogs ........................... 14 2. Y chromosome network created