REPETITIVE DNA AND NUCLEAR INTEGRANTS OF ORGANELLAR DNA SHAPE THE EVOLUTION OF COCCIDIAN GENOMES by SIVARANJANI NAMASIVAYAM (Under the Direction of Jessica C. Kissinger) ABSTRACT The greatest diversity in eukaryotic genomes is observed in the deep branching protist phyla. The protist phylum Apicomplexa consists of at least 5,000 species of mostly obligate intracellular parasites, many of medical and veterinary importance. A number of apicomplexan genomes have been sequenced, providing us with a rich resource for comparative evolutionary studies. Apicomplexans have reductive genomes, yet, they show immense genome diversity and innovation. This diversity is captured in the parasites of the coccidian lineage. Sarcocystis neurona has the largest sequenced apicomplexan genome at >125 Mb, twice as large as the next largest genome from Toxoplasma gondii, the highly-successful zoonotic pathogen. We find that repeats are responsible for a large S. neurona genome, however the out-group parasite Eimeria while repetitive has a ~55 Mb genome. The in-group parasites, T. gondii, Neospora caninum and Hammondia hammondi are repeat-poor. Instead, they contain unprecedented levels of organellar DNA insertions; NUM/PTs. While repeats shape the genomes of the early-branching coccidians, our study suggests NUM/PTs to be the significant drivers of genome evolution in later-branching coccidians. INDEX WORDS: Apicomplexa, evolution, NUMTs, NUPTs, T. gondii, S. neurona REPETITIVE DNA AND NUCLEAR INTEGRANTS OF ORGANELLAR DNA SHAPE THE EVOLUTION OF COCCIDIAN GENOMES by SIVARANJANI NAMASIVAYAM B. TECH, Vellore Institute of Technology, India, 2008 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2015 © 2015 Sivaranjani Namasivayam All Rights Reserved REPETITIVE DNA AND NUCLEAR INTEGRANTS OF ORGANELLAR DNA SHAPE THE EVOLUTION OF COCCIDIAN GENOMES by SIVARANJANI NAMASIVAYAM Major Professor: Jessica C. Kissinger Committee: Jeffrey L. Bennetzen Michael J. McEachern Boris Striepen Chung-Jui Tsai Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia August 2015 ACKNOWLEDGEMENTS I would like to thank my advisor Jessie and my committee members Jeff, Mike, CJ and Boris for providing useful suggestions and feedback about my research. Committee meetings were always productive and encouraging. My committee helped me to think critically about my work, which has helped me grow as a scientist. Jessie has been an excellent mentor. She has encouraged me to think independently and look at the big picture always keeping the question I am trying to answer in mind. She has sent me to a number of conferences. Presenting at these meetings and interacting with other scientists has helped me grow in confidence. I also want to thank her for greatly improving my scientific writing and presentation skills. She has always encouraged my interests and supported me to attend the Biology of Parasitism course. Attending this course has truly been a rewarding experience. The Kissinger lab has been a positive environment to work in. Everyone is willing to help and share his or her knowledge. I must thank the past members of the lab from whose work and advice I have greatly benefitted. I also want to thank the Genetics Department and members of the CTEGD, particularly the Striepen lab. It has been a fruitful and enjoyable graduate school journey. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ........................................................................................................... iv LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ....................................................................................................................... ix CHAPTERS 1. INTRODUCTION AND LITERATURE REVIEW .................................................................. 1 The Apicomplexa ........................................................................................................................ 1 Purpose of this study ................................................................................................................... 2 Evolution of eukaryotic genomes ............................................................................................... 3 Nuclear integrants of mitochondrial origin (NUMTs) ................................................................ 8 Evolution of the apicomplexan genome ................................................................................... 10 The mitochondrial genome of Toxoplasma gondii ................................................................... 20 Organization of this dissertation ............................................................................................... 25 References ................................................................................................................................. 26 2. EVOLUTIONARY FATE AND CONSEQUENCE OF >11,000 NUCLEAR- INTERGRATED ORGANELLAR DNAS IN THE ZOONOTIC PARASITE, TOXOPLASMA GONDII ......................................................................................................................................... 54 Abstract ..................................................................................................................................... 55 Introduction ............................................................................................................................... 56 Materials and Methods .............................................................................................................. 58 v Results ....................................................................................................................................... 63 Discussion ................................................................................................................................. 71 References ................................................................................................................................. 75 3. NUCLEAR SEQUENCES OF MITOCHONDRIAL ORIGIN GENERATE STRAIN- SPECIFIC DIFFERENCES IN THE GENOME OF TOXOPLASMA GONDII .......................... 99 Abstract ................................................................................................................................... 100 Introduction ............................................................................................................................. 101 Materials and Methods ............................................................................................................ 103 Results and Discussion ........................................................................................................... 110 Conclusions ............................................................................................................................. 124 References ............................................................................................................................... 125 4. INSIGHTS INTO THE ABNORMALLY LARGE GENOME OF THE APICOMPLEXAN PARASITE SARCOCYSTIS NEURONA .................................................................................... 153 Abstract ................................................................................................................................... 154 Introduction ............................................................................................................................. 155 Materials and Methods ............................................................................................................ 158 Results and Discussion ........................................................................................................... 165 Conclusions ............................................................................................................................. 175 References ............................................................................................................................... 177 5. DISCUSSION AND FUTURE DIRECTIONS ...................................................................... 206 References ............................................................................................................................... 211 6. APPENDICES ........................................................................................................................ 212 vi LIST OF TABLES Page Table 1.1. Summary of sequenced apicomplexan genomes ......................................................... 50 Table 1.2. Primers used in PCR analysis of the T. gondii mt genome .......................................... 51 Table 1.3. Genomic and EST reads representing different arrangements of mtDNA elements ... 52 Table 1.4. Comparison of T. gondii and N. caninum mtDNA elements ....................................... 53 Table 2.1. NUM/PTs in apicomplexan genomes and other eukaryotes ........................................ 89 Table 2.2. Characteristics of the 23 mtDNA elements in T. gondii and N. caninum .................... 90 Table 2.3. Genomic and EST reads representing different observed arrangements of mtDNA elements ................................................................................................................................ 92 Table 2.4. Annotation of the 3,369 bp strain-specific NUMT in