ABSTRACT EHRENREICH, IAN MICHAEL. the Genetics Of
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ABSTRACT EHRENREICH, IAN MICHAEL. The Genetics of Phenotypic Variation in Arabidopsis thaliana. (Under the direction of Michael Purugganan.) All organisms exhibit substantial quantitative trait variation within populations. Such variation is important because it can affect fitness and serve as the substrate for adaptive evolution. Identifying the quantitative trait genes (QTGs) responsible for phenotypic variation is necessary to understand the mechanisms that generate trait variation and to determine the historical action of natural selection on quantitative traits and QTGs. However, in most complex organisms, the genetic mapping of QTGs is difficult and presently not feasible to do systematically at a gene-level resolution. Model organisms that are both tractable in the laboratory and complex developmentally can serve as trial systems for developing broadly applicable methods for QTG mapping. Using the plant genetic model Arabidopsis thaliana, I have attempted to map QTGs for ecologically-significant quantitative traits – shoot branching and flowering time – through a combination of forward and reverse genetic methods. Three main research projects are reported here: i) candidate gene association mapping and linkage mapping of shoot branching; ii) regulatory network-wide candidate gene association mapping of flowering time; and iii) a survey of intra- and interspecific genetic variation at nearly half of the microRNAs (miRNAs) and their binding sites in the genome. These studies have identified strong candidate QTGs for traits that are determinants of A. thaliana fitness in the wild. I synthesize my results with those of other researchers in this area to highlight the achievements, future promise, and looming challenges for statistical genetics in terms of elucidating the genetic basis of trait variation. The Genetics of Phenotypic Variation in Arabidopsis thaliana by Ian Michael Ehrenreich A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Genetics Raleigh, North Carolina 2008 APPROVED BY: ____________________________ ____________________________ Michael D. Purugganan Stephanie E. Curtis Chair of Advisory Committee Co-Chair of Advisory Committee ____________________________ ____________________________ Gregory C. Gibson Robert G. Franks BIOGRAPHY Ian Ehrenreich was born and raised in Sacramento, CA. Ian attended Stanford University (Class of 2002) in Stanford, CA prior to becoming a student at NCState. It was at Stanford that Ian became interested in evolutionary genetics. Ian took Bill Durham’s course entitled ‘Darwin, Evolution, and the Galapagos,’ which culminated in a trip to the Galapagos. Retracing the voyage of the HMS Beagle inspired Ian to study evolution and led him to join Brendan Bohannan’s lab to do an honors thesis on the experimental evolution of drug- resistant bacteria. Upon graduating, Ian switched to studying biochemical adaptation in butterflies in Ward Watt’s lab at Stanford and the Rocky Mountain Biological Laboratory. After leaving the butterflies, but prior to starting at NCState, Ian worked on the ecophysiology of marine cyanobacteria with John Waterbury and Eric Webb at the Woods Hole Oceanographic Institution in Woods Hole, MA. At NCState, Ian did his research on the molecular population genetics of complex trait variation in plants with Michael Purugganan, who actually moved to New York University in New York City. Ian followed Michael to NYU to finish his PhD research. Ian next plans to study the genetic basis of gene expression and complex trait variation in a variety of organisms with Leonid Kruglyak at Princeton University. ii ACKNOWLEDGMENTS Numerous people have contributed to my graduate education. I would especially like to thank my research advisor Michael Purugganan for providing me with the autonomy and support necessary to develop not only as a student, but also as a scientist and more generally as a person. Michael’s emphasis on striking a balance between career and personal life, with a prioritization on the personal life, has left an indelible mark on me and has had a strong, positive impact on my happiness. I would also like to thank my teachers and mentors throughout graduate school, in particular my past and present committee members Philip Awadalla, Stephanie Curtis, Robert Franks, and Greg Gibson. Altogether, these professors were very influential in shaping my thoughts on science and helped me to find my interests in genetics. I also owe a debt of gratitude to numerous friends both at NCState and NYU, and to my labmates in the Purugganan lab, all of who helped make the process of graduate school more enjoyable. I would especially like to thank Ana Caicedo, my postdoc mentor in the lab during my first couple years, and my two undergraduate apprentices – Lucy Chou and Phillip Stafford. Lastly, I must thank my family – my wife Hannah, mother Veronica, and brothers Kevan and Ryan – for their love and support throughout graduate school. iii TABLE OF CONTENTS Page LIST OF TABLES……..……………………………………………………...…………vi LIST OF FIGURES………..……………………………………………….…………....vii CHAPTER ONE: The molecular genetic basis of adaptation…………………………….1 Abstract……………………………………….………………………… ………...3 The nature of adaptations…………………….…………………………………….3 Identifying plant adaptive genes…………….……………………………………..5 Contemporary studies of adaptation in plants.……………………………………10 Discussion…………….……………….………………………………………….19 Conclusion………………………………………………………………………..21 Acknowledgments…………………….…………………………..………………22 References…………………………….…………………………………………..23 CHAPTER TWO: The genetic architecture of shoot branching in Arabidopsis thaliana: A comparative assessment of candidate gene associations vs. quantitative trait locus mapping………………………….………………………………………….31 Abstract………………………………….…………………………..…………….33 Introduction……………………………….……………………………………….33 Materials and methods…………………….………………………………………36 Results and discussion………………….….……………………..……………….39 Acknowledgments………………………….……………………………………..48 References………………………………….……………………………………..49 CHAPTER THREE: Network-wide candidate gene association mapping in Arabidopsis thaliana…………………………………….…………………………………..…67 Abstract…………………………………….……………………………………..69 Introduction……………………………………………………………………….69 Results and discussion.………………………………………………………..….72 Materials and methods…………………………………………………………....82 Acknowledgments………………………………………………………………...85 iv References…………………………………………………………………….....86 CHAPTER FOUR: Sequence Variation of MiRNAs and Their Binding Sites in Arabidopsis thaliana...........................................................................................106 Abstract.………………………………………………………………….…….108 Introduction…………………………………………………………………….108 Results………………………………………………………………….………112 Discussion……………………………………………………………….……..117 Materials and methods………………..………………………………………..120 Acknowledgments…………………………………………………..…………122 References…………………………………………………………………..…123 CHAPTER FIVE: Conclusion………………………………………………………….136 Abstract……………………………………………………………….….…….137 The genetic basis of adaptation in complex traits……………………….……..137 Reverse Genetics and the Mapping of Flowering Time and Shoot Branching QTGs in A. thaliana……………………………………………………………139 New Resources and Technologies Give Rise to New Ways of Conducting Quantitative Genetics in A. thaliana…………………………………………..142 References……………………………………………………………………..145 v LIST OF TABLES Page CHAPTER TWO Table 1: Genes included in this study……………………………………………..54 Table 2: Genetic variation at the sequenced genes…..............................................55 Table 3: Nominal P-values for mixed model association tests…………………....56 Table 4: Epistatic QTLs detected in the Ler × Col RILs….………...………….....57 Table 5: Epistatic QTLs detected in the Cvi × Ler RILs….………...………….....58 Table S1: Broad-sense heritabilities (H2) for branching traits in 96 accession association mapping panel…………………………….....………………66 CHAPTER THREE Table 1: Numbers of nominally significant background loci by model………......89 Table 2: Candidate gene core associations……………… ....…………………….90 Table 3: Counts of nominally significant loci per trait……………………………91 Table 4: Counts of empirically significant loci per trait…………………………..92 Table 5: Comparison of associations in accessions and HSRILs………………...93 Table 6: Locations of replicated htSNPs and polymorphisms that are in LD with them…………………………………………………………………94 Table S1: Genes included in this study..................................…………….............100 CHAPTER FOUR Table 1: MiRNAs and targets included in this study............................................128 vi LIST OF FIGURES Page CHAPTER TWO Figure 1: Maximum parsimony gene genealogies of the 36 candidate genes……..59 Figure 2: Trait distributions for the 96 accessions used for association mapping...61 Figure 3: Linkage disequilibrium (LD) across the MAX2 and MAX3 regions…….62 Figure 4: Decay of LD in the MAX2 and MAX3 genomic regions….………….....63 Figure 5: Trait associations across the MAX2 and MAX3 regions...………………64 Figure 6: Genomic map of candidate gene associations and QTLs in the Ler × Col and Cvi × Ler RILs...........……………………………………………….65 CHAPTER THREE Figure1: Population structure in the genotyped accessions......……..…………….95 Figure 2: Haplotype sharing across the unique genotypes ....…………………….96 Figure 3: Cumulative density functions (cdfs) for the 2010 Background loci using several alternative models………………………………………………..97 Figure 4: Associations across all haplotypes……………………..……………….98