Genomic Approaches to Identify Important Traits in Avian Species Bhuwan Khatri University of Arkansas, Fayetteville
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University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2018 Genomic Approaches to Identify Important Traits in Avian Species Bhuwan Khatri University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Bioinformatics Commons, Large or Food Animal and Equine Medicine Commons, and the Poultry or Avian Science Commons Recommended Citation Khatri, Bhuwan, "Genomic Approaches to Identify Important Traits in Avian Species" (2018). Theses and Dissertations. 2901. http://scholarworks.uark.edu/etd/2901 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Genomic Approaches to Identify Important Traits in Avian Species A dissertation submitted in partial fulfillment of the requirements for degree of Doctor of Philosophy in Poultry Science by Bhuwan Khatri Tribhuvan University Bachelor in Science in Microbiology, 2005 Tribhuvan University Master in Science in Medical Microbiology, 2008 August 2018 University of Arkansas This dissertation is approved for recommendation to the Graduate Council. ________________________ Byungwhi Caleb Kong, Ph.D. Dissertation Director ____________________ ____________________ Walter G. Bottje, Ph.D. Young M. Kown, Ph.D. Committee Member Committee Member ___________________________ Charles F. Rosenkrans Jr., Ph.D. Committee Member ABSTRACT ABSTRACT This dissertation focusses on identifying different molecular markers that have impact on overall poultry production. Chapter one reviews microRNA (miRNA), copy number variation (CNV) and single nucleotide polymorphism (SNP) as markers suggested in different avian species by various studies. It reviews modern genomic approaches that are employed for next generation sequencing data analysis and verification. Chapter two seeks to identify and validate the muscle specific miRNAs in the breast muscle of modern broilers and its foundational chicken line. Small RNA sequencing was performed to identify differentially expressed mature miRNAs in the breast muscles of these two chicken lines. Results showed that nine different mature miRNAs were differentially expressed (DE) in the breast muscle of modern broilers compared to foundational chicken lines. Target genes of DE miRNAs were involved in MAP/ERK1/2, calcium signaling, axonal guidance signaling and NRF2-mediated oxidative response pathways suggesting their roles in muscle growth and development. Chapter three is focused on identifying and validating copy number variation in the whole genome of two divergently selected high and low stress quail lines. Whole genome sequencing was performed, and data were analyzed for copy number variation detection in genome of the quail lines. Results showed the unique sets of copy number variable regions and genes in the genomes of high stress and low stress birds. Importantly, these genes were involved in development of nervous/endocrine systems, and humoral/cell-mediated immune responses suggesting that they could be potential biomarkers for understanding effects of stress in the well-being and growth performance of avian species and other animals. Chapter four focuses on identifying SNPs in whole genome of Arkansas Progressor (AP) and Regressor (AR) chicken lines selected for tumor progression and tumor regression upon v-src oncogene induction. Whole genome sequencing was performed, SNPs were analyzed and validated using allele-specific PCR. Results showed the unique sets of SNPs in AP and AR lines. Based on the functional studies, the candidate SNPs were associated with ubiquitylation, and PI3K and NF-kB signaling pathways, suggesting their role in tumor regression in AR chickens. ACKNOWLEDGMENTS First and foremost, I would like to thank my advisor Dr. Byungwhi Caleb Kong for not only providing me golden opportunity to work in his lab but also for his continuous guidance and persistent support throughout my PhD. His tremendous mentorship helped me grow as an independent researcher. His continuous support has helped me to overcome many hurdles during my researches and finally come up with this dissertation successfully. I am very thankful to grow under his research expertise and knowledge which will be invaluable resources to me in future as I continue chasing my research goals. I am equally thankful to my committee members: Dr. Walter G. Bottje, Dr. Young Min Kwon and Dr. Charles F. Rosenkrans Jr. for their directions, support and guidance in my research. I will always remember and appreciate their invaluable inputs. Further, I would like to thank Stephanie S. Shouse for helping me in the lab for the research. I am also indebted to all my friends and staffs of Department of Poultry Science who directly or indirectly helped me during my study period. Finally, and the most importantly, I would like to thank my wife Lakhi Maya Devi Khatri for her support and encouragement for everything I do. We would like to welcome our PhD daughter in our life. I thank to my sister, brothers, nephews and niece for the support of any kind that helped me to achieve this degree. DEDICATION I would like to dedicate this dissertation to my mom and dad. I will always remember them for their unwavering support and encouragement that has helped me to travel this far in my research career chasing my goals. TABLE OF CONTENTS CHAPTER 1 ..........................................................................................................................1 1.1 INTRODUCTION .....................................................................................................1 1.2 MICRO RNA, TRANSCRIPTOMICS AND REGULATORY FUNCTIONS .........2 1.3 MICRORNA TARGETS PREDICTION ..................................................................4 1.4 MIRNA REGULATING MUSCLE GROWTH IN CHICKEN……………………5 1.5 WHOLE GENOME RE-SEQUENCING AND GENETIC VARIATION IN CHICKENS......................................................................................................................6 1.6 APPLICATION OF NEXT GENERATION SEQUENCING ..................................7 1.7 VARIANTS DETECTION IN WHOLE GENOME RESEQUENCING DATA .....8 1.8 GENETIC VARIATION IN CHICKENS .................................................................9 1.9 SINGLE NUCLEOTIDE POLYMORPHISM ..........................................................10 1.10 COPY NUMBER VARIATION..............................................................................11 1.11 PHENOTYPIC VARIATION DUE TO CNV ........................................................14 1.12 OBJECTIVES ..........................................................................................................17 REFERENCES ......................................................................................................................18 CHAPTER 2 ..........................................................................................................................26 2.1 ABSTRACT ...............................................................................................................27 2.2 INTRODUCTION .....................................................................................................29 2.3 METHODS ................................................................................................................31 2.3.1 ETHICS STATEMENT .................................................................................31 2.3.2 SAMPLES......................................................................................................31 2.3.3 MICRORNA SEQUENCING AND DATA ANALYSIS .............................31 2.3.4 HIERARCHICAL CLUSTERING ................................................................32 2.3.5 TARGET PREDICTION OF DE MIRNA ....................................................32 2.3.6 INGENUITY PATHWAY ANALYSIS ........................................................32 2.3.7 SMALL RNA PURIFICATION, CDNA SYNTHESIS AND QUANTITATIVE REAL TIME PCR (QPCR) .............................................33 2.4 RESULTS ..................................................................................................................34 2.4.1 MICRORNA PROFILING IN PEM AND BPR CHICKENS BY DEEP SEQUENCING ..............................................................................................34 2.4.2 DIFFERENTIALLY EXPRESSED miRNAs IN PEM AND BPR ...............34 2.4.3 TARGET PREDICTION AND NETWORK ANALYSIS............................35 2.5 DISCUSSION ............................................................................................................36 2.6 CONCLUSION ..........................................................................................................40 REFERENCES ......................................................................................................................41 APPENDIX ............................................................................................................................45 CHAPTER 3 ..........................................................................................................................49 3.1 ABSTRACT ..............................................................................................................50 3.2 BACKGROUND ......................................................................................................52 3.3 MATERIALS AND METHODS ..............................................................................55