GENOMIC CAUSES AND CONSEQUENCES OF THE EVOLUTION OF SELF-FERTILIZATION IN THE FLOWERING PLANT GENERA CAPSELLA AND COLLINSIA by Khaled Hazzouri A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Graduate Department of Ecology and Evolutionary Biology University of Toronto © Copyright by Khaled Hazzouri 2012 GENOMIC CAUSES AND CONSEQUENCES OF THE EVOLUTION OF SELF-FERTILIZATION IN THE FLOWERING PLANT GENERA CAPSELLA AND COLLINSIA Khaled Hazzouri Doctor of Philosophy Department of Ecology and Evolutionary Biology University of Toronto 2012 Abstract The shift in mating system from outcrossing to selfing is associated with many evolutionary changes including reduced flower size and changes in sex allocation, leading to a suite of morphological characteristics known as the selfing syndrome. Furthermore, the evolution of selfing is expected to have important effects on genetic variation and the efficacy of natural selection. However, the underlying genomic causes of morphological evolution and the extent of relaxed selection remain unresolved. In this thesis I use new genomic approaches to investigate the genetic basis of floral evolution as well as the consequences of the evolution of selfing in the genus Capsella (Brassicaceae), in which the highly selfing C. rubella evolved recently from the self-incompatible, obligately outcrossing C. grandiflora. Quantitative trait locus (QTL) mapping results suggest that few loci with major effects on multiple floral phenotypes underlie the evolution of the selfing syndrome. Patterns of neutral diversity in QTL regions from both resequencing and next-generation transcriptome sequencing suggest an important role for positive directional selection in the evolution of the selfing syndrome. Combined with the identification of differentially expressed genes, the signals of positive selection provide candidate regions for identifying the causal evolutionary changes. Analysis of whole- transcriptome polymorphism patterns shows a significant increase in the ratio of unique ii nonsynonymous to synonymous polymorphism in C. rubella, consistent with a genome-wide relaxation of selection. To investigate the evolution of selfing in the genus Collinsia, I combined transcriptome sequencing with Sanger resequencing of multiple populations of the large-flowered Collinsia linearis with its small flowered, more highly selfing sister species Collinsia rattanii. Patterns of nucleotide diversity suggest the recent divergence of C. rattanii associated with a severe population bottleneck. C. rattanii showed an increased proportion of nonsynonymous polymorphism, consistent with a relaxation of natural selection following the population bottleneck and shift to selfing. In general, my results using combined genome-wide polymorphism, QTL mapping and gene expression data allow for a powerful approach to investigate the targets of adaptive evolution following the shift to selfing, and highlight the potential for relaxation of selection associated with this transition. iii ACKNOWLEDGEMENTS I would like to express my deep and sincere gratitude to my supervisor, Dr. Stephen Wright, whose expertise, understanding and patience, added so much experience to me as a graduate. Without his vast knowledge in the field, this thesis would not appear in its present form. I would like to thank him for his continuous support. I would like to thank my committee members, Dr. Asher Cutter, Dr. Aneil Agrawal for the assistance they provided at all levels of the research project. Finally, I would like to thank Dr. Kermit Ritland from the University of British Columbia (UBC) for taking time out to serve as my external examiner. I would like to thank Dr. Tanja Slotte, who I learned a lot from her when she was a postdoctoral fellow in the Wright lab. Particularly, I would like to thank Dr. Peter Andolfatto from the University of Princeton, for his willingness to have me in his lab to learn and perform the multiplex genotyping shotgun sequencing (MSG) protocol. I would like to thank Adrian platte from McGill University for all the help in programming skills. I would like to thank Rob Ness and Juan Escobar, who was there to help with their expertise in the field and taking the time to explain things. I would like to thank my colleagues in the Wright lab for our interesting debates and exchanges of knowledge, which helped enrich my experience. I would like to thank Bruce and Andrew at the University of Toronto, greenhouse, for all the help provided in terms of advices about handling and taking care of plants. Finally, I would like to thank my precious family. In particular, I would like to dedicate this thesis to my Dad and Mom who waited long time and supported me through my entire life abroad. With their sacrificial love and encouragement, I would not have finished this thesis. iv TABLE OF CONTENTS Abstract……………………………………………………………………………………………ii Acknowledgements……………………………………………………………………………….iv Table of Contents…………………………………………………………………………………vi List of Tables…………………………………………………………………………………….xii List of Figures…………………………………………………………………………………...xiv CHAPTER ONE: GENERAL INTRODUCTION………………………………………………..1 Causes and consequences of mating system evolution……………………………………....1 Capsella as a study system…………………………………………………………………..8 Collinsia as a study system…………………………………………………………………..9 Research objectives…………………………………………………………………………10 CHAPTER TWO: GENETIC ARCHITECTURE AND ADAPTIVE SIGNIFICANCE OF THE SELGING SYNDROME IN CAPSELLA………………………………………………………..13 Summary……………………………………………………………………………………13 Introduction…………………………………………………………………………………13 Materials and Methods……………………………………………………………………...17 Results………………………………………………………………………………………26 Discussion…………………………………………………………………………………..32 Conclusions…………………………………………………………………………………38 CHAPTER THREE: EVOLUTIONARY GENOMICS OF MATING SYSTEM IN CAPSELLA.....................................................................................................................................50 Summary……………………………………………………………………………………50 Introduction…………………………………………………………………………………51 v Materials and Methods……………………………………………………………………...56 Results………………………………………………………………………………………62 Discussion…………………………………………………………………………………..67 CHAPTER FOUR: COMPARATIVE POPULATION GENOMICS IN TWO COLLINSIA SPECIES WITH CONTRASTING MATING SYSTEM………………………………………..89 Summary……………………………………………………………………………………89 Introduction…………………………………………………………………………………90 Materials and Methods……………………………………………………………………...94 Results……………………………………………………………………………………..101 Discussion…………………………………………………………………………………106 CONCLUSION…………………………………………………………………………………121 LITERATURE CITED…………………………………………………………………………122 vi LIST OF TABLES Table 2.1. Means and standard deviations for vegetative, floral and reproductive traits in C. rubella and C. grandiflora……………………………………………………………………….41 Table 2.2. List of significant QTL, including 1.5-LOD and 2-LOD confidence intervals and effect size estimates……………………………………………………………………………...42 Table 2.3. Population genetics of narrow QTL regions compared to the rest of the genome…...44 Table 3.1. Sampling locations of C. grandiflora and C. rubella………………………………...71 Table 3.2. Summary of the Capsella rubella genome assembly and annotation………………...72 Table 3.3. Summary of the initial ordering of de novo assembly of different contigs…………..73 Table 3.4. Genome-wide polymorphism summary statistics…………………………………….76 Table 3.5. Down-regulated genes in Capsella rubella after enrichment analysis using David bioinformatics……………………………………………………………………………………77 Table 3.6. Up-regulated genes in Capsella rubella after enrichment analysis using David bioinformatics……………………………………………………………………………………78 Table 3.7. Estimates of the distribution of fitness effects (DFE) of amino acid mutations that are unique to C. grandiflora and C. rubella………………………………………………………….79 Table 4.1. Population samples used for Collinsia rattanii and Collinsia linearis……………...111 Table 4.2. Summary of the de novo assembly of C. linearis and C. rattanii………………………………………………………......................................................112 Table 4.3. Pairwise comparisons of synonymous polymorphisms (unique, shared and fixed differences) between Collinsia linearis and Collinsia rattanii…………………………………113 vii Table 4.4. Codon preference and GC bias from pairwise comparison at synonymous sites that differ between C. linearis and C. rattanii……………………………………………………....114 viii LIST OF FIGURES Figure 2.1. Schematic showing floral measurements……………………………………………45 Figure 2.2. Histograms of representative floral and reproductive traits in the F2, F1 as well as in C. grandiflora and C. rubella……………………………………………………………………46 Figure 2.3. Character correlations in the F2 population…………………………………………47 Figure 2.4. Capsella linkage map and 1.5-LOD confidence intervals…………………………...48 Figure 2.5. Fine-mapping of QTL for self-incompatibility on linkage group seven……………………………………………………………………………………………..49 Figure 3.1. Comparative genome mapping of A. lyrata, C. rubella and S. parvula……………..80 Figure 3.2. A sliding window of synonymous diversity in C. grandiflora and C. rubella……………………………………………………………………………………………81 Figure 3.3. Proportion of nonsynonymous relative to synonymous polymorphism over the different frequency class (1-5) as well as site frequency spectra for synonymous and nonsynonymous in both Capsella grandiflora and Capsella rubella……………………………………………………………………………………………82 Figure 3.4. Plot of the normalized mean expression versus ln fold change for the difference in C. grandiflora compared to