A Dissertation entitled Genomic and Microbiomic Architectural Contributions to Aerobic Exercise Capacity by Youjie Zhang Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science _________________________________________ Dr. Bina Joe, Committee Chair _________________________________________ Dr. Lauren Koch, Committee Member ________________________________________ Dr. Jennifer Hill, Committee Member _________________________________________ Dr. Mark Wooten, Committee Member _________________________________________ Dr. Kathryn Eisenmann, Committee Member _________________________________________ Dr. Joshua Park, Committee Member _________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo May 2018 Copyright 2018, Youjie Zhang This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Genomic and Microbiomic Architectural Contributions to Aerobic Exercise Capacity by Youjie Zhang Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo May 2018 The beneficial effect of physical exercise has been well established. Genetic predisposition to low exercise capacity is a strong predictor of morbidity and mortality related to multiple diseases such as hypertension, diabetes, and obesity. Genetic predisposition is being increasingly recognized as predisposition caused by the inherited host and microbiotal genome, ie., the microbiome of the commensal microbes living within the host. The microbiome is shaped by both environment and host genetics, and functions to modulate metabolic phenotypes, behavior, and brain function. While the independent contributions of the genome and the microbiome are investigated, studies to delineate the interplay between the nuclear and the mitochondrial genomes of the host and the microbiome to promote health or disease are limited due to paucity of appropriate models. To address this void, this dissertation describes the construction and characterization of four new inbred rat models. First, to study the genetics of exercise, we developed models iii of health and disease by inbreeding rats divergent in aerobic exercise capacity. These novel inbred strains were developed over 20 generations of inbreeding the selectively bred low and high capacity runner rats and named as LCR/Bj and HCR/Bj respectively. Next, to facilitate studies of the nuclear-mitochondrial genomic interactions, we also developed conplastic animals by “switching” mitochondrial genomes between low and high capacity runner rats, and named them LCR.HCRmt/Bj (LCR rat with mitochondrial genome of the HCR rat) and HCR.LCRmt/Bj (HCR rat with mitochondrial genome of the LCR rat). We screened the whole genomes of the LCR/Bj and HCR/Bj rats by NextGen sequencing and identified over 4.4 million of high quality single nucleotide variants between each of these two inbred strains and the Brown Norway reference genome. Thousands of variants between LCR/Bj and HCR/Bj nuclear DNA (nDNA) are predicted to influence gene structure and protein translation. We also sequenced and compared mitochondrial DNA (mtDNA) with publicly available mtDNA from common inbred strains, and found that LCR/Bj mitochondrial mtDNA was identical to the mtDNA reported from Wistar Kyoto inbred strain, while HCR/Bj mtDNA was identical to the mtDNA reported from Fischer 344 × Brown Norway F1-hybrid strain. This genomic information on the LCR/Bj, HCR/Bj, LCR.HCRmt/Bj, HCR.LCRmt/Bj rat models provides a framework for genetic mapping studies of identify inherited factors for exercise capacity and disease susceptibility, facilitates development of engineered models on homogenous genomic backgrounds for gene structure-function studies, and serve as platforms for function validation of human genome-wide association studies in the research field of exercise. iv To explore the nuclear-mitochondrial genomic interaction in health and disease, we systemically characterized the phenotypes in our inbred and conplastic strains. We show that genetic predisposition to low exercise capacity led to multiple pathophysiological features including increased body weight, metabolic abnormalities, increased accumulation of adipose tissue, high blood pressure, altered cardiac function, depression- like behavior, and deteriorated cognition. We also revealed tissue specific interaction between nDNA and mtDNA in determining metabolism, blood pressure, cardiac function, and long-term memory. Our novel inbred and conplastic strains with detailed genomic characterization provide invaluable resources to comprehensively study the effect of host genome on exercise capacity and complex diseases. To establish the link between host genome and microbiome in determining health and diseases, we did 16S rRNA sequencing and analyzed gut microbiota in fecal samples from our inbred and conplastic animals. Surprisingly, fecal microbial communities of LCR/Bj and HCR/Bj were significant different, which we interpret as being the result of selection of differential host genomes. Of the enriched bacteria we identified, majority are documented as high heritable through human gut microbiotal studies. Correlation analysis further demonstrated that certain gut microbiota closely associated with multiple traits including exercise capacity, body metabolism, heart function, and depression. These findings second the notion that targeting the microbiome could be a novel therapeutic approach to treat common diseases. Moreover, our models with genetic susceptibility to certain disease-associated gut microbiota can fulfill the urgent demand for animal models of translational microbiome research. v This dissertation is dedicated to the memory of my grandfather, Zhaoren Zhang Acknowledgements I would like to express my deepest gratitude to my advisor Dr. Bina Joe for her full support of my Ph.D. study and research, for her patience, endless motivation, enthusiasm, and immense knowledge. Without her help and encouragement this dissertation would not have been written or even finished! Besides my advisor, I would like to thank the rest of my committee members, Dr. Lauren Koch, Dr. Jennifer Hill, Dr. Mark Wooten, Dr. Kathryn Eisenmann, and Dr. Joshua Park for their insightful comments, support, and encouragement. I also thank my past and present lab mates: Dr. Sivarajan Kumarasamy, Dr. Ying Nie, Dr. Resmi Pillai, Dr. Harshal Waghulde, Dr. Xi Cheng, Ms. Blair Mell, Ms. Sarah Galla, Mr. Saroj Chakraborty, and Dr. Jiyoun Yeo, the Department of Laboratory Animal Research (DLAR) staff and the Department of Physiology and Pharmacology, in the University of Toledo College of Medicine and Life Sciences and the College of Graduate Studies for their support and assistance. I would like to thank my parents Anying Zhang and Wenwen Sun, for giving birth to me and supporting me spiritually throughout my life, my relatives, and friends for their constant care and emotional support. Last but not the least, I would like to thank my wife Mengjie Wang, for her encouragement and endless love. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables .................................................................................................................. vii List of Figures .................................................................................................................. viii List of Abbreviations ...........................................................................................................x List of Symbols ................................................................................................................. xii 1 Introduction ..............................................................................................................1 2 Identification of Genetic Variants in Nuclear and Mitochondrial Genomes between Inbred & Conplastic LCR/Bj and HCR/Bj Strains ..............................................18 3 Characterization of Phenotypes in Inbred & Conplastic LCR/Bj and HCR/Bj Strains ……………… .......................................................................................................37 4 Linking the Host Genome and Gut Microbiome in Health and Disease ...............71 5 Summary and Perspective ......................................................................................91 References ..........................................................................................................................95 Appendices .......................................................................................................................113 A Correlation Analysis Results between the Relative Abundance of Gut Microbiota and Multiple Traits ...........................................................................................................113 B Relative Abundance Percentage of Phenotype-Associated Gut Microbiota ........121 vi List of Tables 1.1 Some key milestones