{Replace with the Title of Your Dissertation}
Total Page：16
File Type：pdf, Size：1020Kb
Population genomics of the legume symbionts Sinorhizobium meliloti and S. medicae A Dissertation SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Brendan James Epstein IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Peter Tiffin, Michael Sadowsky November 2013 © Brendan Epstein 2013 Acknowledgements First, I am extremely grateful to my advisors, Peter Tiffin and Michael Sadowsky, for their advice, encouragement, and patience. Peter and Mike were both a great pleasure to work with and were very generous with their time and resources. Their consistent support has been tremendously helpful through all the ups and downs of grad school. I also thank my committee members, Georgiana May, Kate VandenBosch, and Dave Moeller. I truly appreciate your constructive feedback, the letters of recommendation you've written, and the time and energy you've committed to making me a better scientist. My friends and colleagues in the Tiffin and Sadowsky labs have helped me in many ways: teaching me how to program computers, saving me from my own clumsiness in the lab, planting and nodule counting, and, most of all, showing me kindness. I am especially grateful to Stephanie Erlandson, Masayuki Sugawara, Antoine Branca, John Stanton-Geddes, Mohammed Yakub, Jeremy Yoder, Matt Hamilton, Betsy Martinez- Vaz, Tim Paape, Stephen Keller, and Ping Wang. I would also like to thank the many members of the Center for Community Genetics at the U of M for interesting and useful discussions. I would like to highlight critical contributions from several people: Joann Mudge, Andrew Farmer, and Arvind Bharti from the National Center for Genome Resource provided sequencing and bioinformatics support. Masayuki Sugawara assisted with data analysis and DNA extractions. Ping Wang also helped with DNA extractions, and Shawn Davison helped collect nodule count data for Chapter 3. Katy Heath, Betsy Martinez- Vaz, and Mohamed Aouani provided strains for sequencing. I was lucky to have access to great resources. Once again, my advisors were very generous with funding, including both stipends and research support. I've also had support from the Plant Biology Graduate Program and the Microbial and Plant Genome Institute at the U of M in the form of travel money and fellowships. The Minnesota Supercomputing Institute provided computing resources for much of this work, and i National Science Foundation grants (082005 and IOS-1237993) to Nevin Young and my advisors ultimately provided much of my funding. Finally, I would like to thank my family for their love and support. Without my parents' encouragement, enthusiasm, and confidence in me over many years, I would never have been able to get this far. Thank you. ii Abstract The nitrogen-fixing mutualism between legumes and rhizobia is ecologically and agriculturally important and is a model for the molecular genetics of plant microbe interactions and the evolution of mutualism. The goal of this research was to investigate the evolutionary forces shaping genetic diversity in two species of rhizobia, Sinorhizobium meliloti and S. medicae by integrating population genetic tools, experimental evolution, and whole-genome sequencing. In Chapter 1, I characterize the diversity and divergence of S. meliloti and S. medicae and ask how selection and horizontal gene transfer (HGT) have shaped nucleotide variation. I find limited evidence for HGT between S. meliloti and S. medicae, indicating that recombination with closely related species does not have much impact on nucleotide diversity in Sinorhizobium spp. and does not prevent species from diverging. I also find that the targets of strong positive selection are different in the two species, suggesting that S. meliloti and S. medicae may be subject to different selective pressures in nature. The goal of Chapter 2 was to examine gene content and copy number variation. While I find that S. meliloti and S. medicae both have extensive variation in content and copy number, most of this variation seems to be deleterious. This suggests that the large size of bacterial pangenomes is due, in part, to many short-lived, deleterious gains and losses of genes rather than adaptation. Finally, in Chapter 3, I use experimental evolution to tests for costs of mutualism. I do not find clear evidence of costs, but I do identify a mutation in the purM gene that may affect host range. Overall, this contributes to our understanding of both the evolution of rhizobia, and the evolutionary forces shaping variation in prokaryotes. iii Table of Contents Acknowledgements...............................................................................................................i Abstract...............................................................................................................................iii Table of Contents.................................................................................................................iv List of Tables……………………………………………………………………………...vi List of Figures……………………………………………………………………………vii Introduction..........................................................................................................................1 Chapter 1 Population genomics of nucleotide variation in Sinorhizobium meliloti and S. medicae............................................................................................................................4 Summary..............................................................................................................................5 Introduction..........................................................................................................................6 Methods................................................................................................................................8 Results................................................................................................................................12 Discussion..........................................................................................................................18 Tables and Figures.............................................................................................................25 Supplemental Tables and Figures......................................................................................33 Chapter 2 Selection on structural variation in S. meliloti and S. medicae..................60 Summary............................................................................................................................61 Introduction........................................................................................................................61 Methods..............................................................................................................................64 Results................................................................................................................................74 Discussion..........................................................................................................................80 Tables and Figures.............................................................................................................88 Supplemental Figures and Tables.....................................................................................100 Chapter 3 Experimental evolution of rhizobial host range........................................105 Summary..........................................................................................................................106 Introduction......................................................................................................................106 Methods............................................................................................................................109 Results..............................................................................................................................113 Discussion........................................................................................................................116 Tables and Figures...........................................................................................................122 iv Concluding Remarks........................................................................................................130 Bibliography……………………………………………………………………………134 v List of Tables Chapter 1 Table 1-1. Shared polymorphisms and fixed differences. 25 Table 1-2. Diversity statistics and SNP counts. 26 Table S1-1. PCR conditions for SNP validation. 33 Table S1-2. Sampling information for S. medicae strains. 37 Table S1-2. Sampling information for S. meliloti strains. 39 Table S1-4. List of horizontally transferred regions. 42 Table S1-5. List of targets of strong positive selection. 44 Chapter 2 Table 2-1. Counts of duplicated windows at different levels of filtering. 87 Table 2-2. Repeatability of duplication calls. 88 Table 2-3. Diversity statistics for structural variants. 89 Table 2-4. Mean Ka, Ks, and Ka:Ks values. 90 Table 2-5. Distribution of fitness effects of non-synonymous mutations. 91 Table 2-6. Mean segregating frequency of variants. 92 Table S2-1. “Heterozygous” base calls in duplicated regions for S. meliloti. 99 Table S2-2. “Heterozygous” base calls in duplicated regions for S. medicae. 101 Chapter 3 Table 3-1. Strains sequenced. 121 Table 3-2. Nodule counts for mutant strain. 123 Table 3-3. Point mutations and small indels. 124 Table 3-4. Copy number mutations.