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Gene Mapping of Morphological Traits in Chickens Gene Mapping of Morphological Traits in Chickens Jingyi Li Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Animal and Poultry Sciences Paul B. Siegel, Chair Leif Andersson Elizabeth Ruth Gilbert D. Phillip Sponenberg March 23, 2017 Blacksburg, Virginia Keywords: Linkage mapping, feather patterns, feathered legs, polydactyly, chickens Copyright 2017, Jingyi Li Gene Mapping of Morphological Traits in Chickens Jingyi Li Abstract (Public) Chickens, one of the major protein sources in diets for humans, have a long cultural, sport and religious history since their initial domestication during the neolithic period. Darwin wrote of the importance of variation, which today we see for example in size of body, length of shank, number of toes, distribution of feathers, comb types, and plumage color patterns resulting in a plethora of breeds of chickens that differ in appearance. Some of these traits are “simply” inherited, which in the molecular era facilitates the study of relationships between DNA sequences and phenotypes. This dissertation focuses on identification of differences in DNA sequences among chickens responsible for these “simply” inherited phenotypes. The 12 phenotypes that were studied included 6 plumage color patterns (Pattern, Columbian, Melanotic, mottling, Blue, and chocolate), 2 forms of feathered-legs, polydactyly, dark brown eggshell color, vulture hock, and creeper. Designed were ten 3-generation populations to produce 1,880 chickens. An additional 339 DNA samples from other populations were included. Of the 12 phenotypes, 8 involved genotyping of pooled DNA samples, a cost-effective initial screen to target DNA sequences. This was followed by genotyping individual samples in 5 of the more promising studies. Candidate genes identified as associated with these 5 phenotypes underwent further studies which identified differences in DNA sequences associated with 4 of them (mottling, feathered-leg, Blue, and chocolate). These findings provide insights of how DNA sequences contribute to the phenotypic appearance of animals. ii Gene Mapping of Morphological Traits in Chickens Jingyi Li Abstract (Academic) Chickens exhibit considerable variation in morphological traits, with some populations having undergone intensive selection for uniqueness and uniformity. These populations are a source of experimental material to study the genetics of morphological traits. An important first step in such studies is to map the genes and the causal mutations that influence these traits. This research focused on gene mapping of 12 morphological traits including 4 intra-feather color patterns (Pattern, Columbian, Melanotic, and mottling), 2 inter-feather color patterns (Blue and chocolate), 2 forms of feathered-legs, polydactyly, dark brown eggshell color, vulture hock, and creeper. Ten backcross and/or F2 populations were designed to produce 1,880 individuals. An additional 339 DNA samples from other populations were included. The procedures for gene mapping were: 1. Pooling of DNA samples of backcross or F2 individuals based on their shared phenotypes, followed by microarray assays for genotyping, a cost-effective initial screen for the candidate genomic regions, 2. Linkage mapping to narrow the range of candidate genes, 3. Sequencing to identify the candidate mutations, 4. Diagnostic tests to confirm the association between the candidate mutation and the phenotype. Of the 12 traits studied, 3 (mottling, Blue, and chocolate) made progress into step 4. Complexities due to genomic context, modifiers, and environmental factors precluded step 4 for the first form of the feathered-leg gene, step 3 for the mapping of Melanotic, and earlier stages for the mapping of Pattern, Columbian, dark brown egg, vulture hock, and the second form of feathered-leg. These findings provide insights of the complexity of how background iii genome can influence the phenotypic expression of single genes (gene × genetic background interactions) and an understanding of cellular and molecular mechanisms involved in morphogenesis. iv Acknowledgements Dr. Paul Siegel In my hardest time during the past 4 years, you have rescued me in many ways. By accepting me as your graduate student and treating me with respect and kindness like that of your own son, I have become a better person and scientist. My vision was broadened by your many life experiences, and by default, you have encouraged me to explore other topics outside of my field of study. Dr. Leif Andersson I appreciate all the assistance you have provided, including advising and partial funding for my projects. Most of our communication has been through email, which in my view should have been inefficient, but to my surprise, it was very beneficial to me. I came to realize that you trusted me enough not to micro-manage me, which coincidentally helped me to build my confidence in research. Therefore, I decided to have an academic career for the rest of life. Dr. Elizabeth Gilbert I remember the day we met for my interview in Beijing more than 4 years ago. I appreciate your kindness, and without your help, I may not have been accepted by Virginia Tech. Moreover, I am grateful for your suggestions regarding my research. Dr. Phillip Sponenberg Your inputs in my research projects were insightful. Your broad knowledge of chickens and other species developed my thought process and will benefit my current and future academic careers. v Dr. Ben Dorshorst I am grateful for the opportunity that you afforded me. I have gained hands on experience in the poultry genetics laboratory and in establishing mapping populations for my research. Thank you for accepting me as your first graduate student and also for all your input in our lab and my research. Members of my research group Thanks to Dez-Ann Sutherland, Michelle Jambui, Christa Honaker, and undergraduate students in our lab. Thanks to John Thomas. You directly contributed to my projects, such as DNA isolation, pooling of DNA samples, and husbandry of chickens. I also want to thank you for establishing long lasting friendships, making the past 4 years such a wonderful time. Researchers and breeders who provided the samples Chickens, blood samples, and DNA samples involved in this dissertation were provided by multiple researchers and chicken breeders. Without your help, these projects would not be possible. My family, Mei Liu, Jie Li, and Sijia Xie Thanks to my parents and girlfriend who supported me. Special thanks to my mother who spent almost an entire year in Blacksburg, which alleviated my homesickness. vi Table of contents Abstract (Public)…………………………………………………………………………..ii Abstract (Academic)……………………………………………………………………...iii Acknowledgements………………………………………………………………………..v List of tables……………………………………………………………………………..xii List of figures……………………………………………………………………………xiv Chapter 1. Introduction and literature review……………………………………………..1 Introduction………………………………………………………………………………..1 Literature review of plumage color patterns…………………………………………1 Tables and figures…………………………………………………………………..10 References…………………………………………………………………………..11 Chapter 2. General materials and methods………………………………………………13 Animals……………………………………………………………………………..13 Genotyping of DNA pools………………………………………………………….13 Linkage mapping and diagnostic test ………………………………………………14 Individual genotyping using 600K SNP array……………………………………...15 PCR and sequencing………………………………………………………………..16 Bioinformatics analysis……………………………………………………………..18 Tables and figures…………………………………………………………………..19 References…………………………………………………………………………..26 Chapter 3. Pattern (Pg) gene mapping…………………………………………………...28 Abstract……………………………………………………………………………..28 Introduction…………………………………………………………………………28 vii Materials and methods……………………………………………………………29 Results and discussion……………………………………………………………...30 Tables and figures…………………………………………………………………..32 References…………………………………………………………………………..35 Chapter 4. Columbian (Co) gene mapping………………………………………………36 Abstract……………………………………………………………………………..36 Introduction…………………………………………………………………………36 Materials and methods……………………………………………………………37 Results and discussion……………………………………………………………...38 Tables and figures…………………………………………………………………..41 References…………………………………………………………………………..42 Chapter 5. Melanotic (Ml) gene mapping………………………………………………..43 Abstract……………………………………………………………………………..43 Introduction…………………………………………………………………………43 Materials and methods……………………………………………………………44 Results and discussion……………………………………………………………...45 Tables and figures…………………………………………………………………..48 References…………………………………………………………………………..50 Chapter 6. Mottling (mo) gene mapping…………………………………………………51 Abstract……………………………………………………………………………..51 Introduction…………………………………………………………………………51 Materials and methods……………………………………………………………54 Results………………………………………………………………………………55 viii Discussion…………………………………………………………………………..58 Tables and figures…………………………………………………………………..62 References…………………………………………………………………………..66 Chapter 7. Feathered-leg in chickens is associated with a SNP on chromosome 15 and a region on chromosome 12…………………………………………………………..68 Authors……………………………………………………………………………...68 Affiliations………………………………………………………………………….68 Summary……………………………………………………………………………68 Introduction…………………………………………………………………………69 Materials and methods……………………………………………………………...71 Results………………………………………………………………………………73 Discussion…………………………………………………………………………..75 Acknowledgements…………………………………………………………………78 References…………………………………………………………………………..78
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