A fundamental goal in biological research is to gain an understanding of the evolutionary processes and genetic elements that drive speciation. Genes responsible for reproductive isolation in young divergent lineages are particularly poorly known. In this thesis, the speciation genetics of carrion (Corvus (corone) corone) and hooded (C. (corone) cornix) crows were studied. These taxa differ strikingly in colouration and meet in a narrow hybrid zone in Europe, yet appear to be very similar genetically. A major component of reproductive isolation is social selection on colour differences. First, we investigated the genetic basis of plumage divergence between carrion and hooded crows using a candidate gene approach. Nucleotide divergence was confirmed to be low, while there was no evidence for any of the sequenced genes to be associated with colour differences. Second, we performed a simulation study to assess the performance of RNA-seq, a relatively novel approach that we later employed ourselves. We asked how variation in transcriptome complexity and bioinformatic workflow affected the accuracy of gene expression profiling. We generally found reassuring robustness and made a number of specific recommendations. Third, we compared the corticosterone stress response of carrion and hooded crows. In accordance with the hypothesis that the degree of melanization and physiological traits are correlated due to pleiotropy, we found a higher stress response in hooded crows, and detected possibly associated gene expression in pituitary. Fourth, we investigated genomic divergence by assembling a hooded crow reference genome followed by whole-genome resequencing of four European population samples. Northern European carrion crows were more similar to hooded crows than to Spanish carrion crows, pointing towards rampant introgression far beyond the hybrid zone. Nevertheless, several narrow genomic regions harboured high between-taxon divergence and were potentially associated with phenotypic traits. Fifth, we compared whole-transcriptome gene expression profiles between crows, focusing on skin with developing feathers. We used a design that allowed to differentiate between taxon-specific, colour-specific and body patterning effects. Widespread underexpression of genes in the melanogenesis pathway was associated with grey colour, and we detected several genes that may contribute to colour divergence in this system.

No description available.

Changes in the structure, content and morphology of chromosomes accumulate over evolutionary time and contribute to cell, developmental and organismal biology. The axolotl (Ambystoma mexicanum) is an important model for studying these changes because: 1) it provides important phylogenetic perspective for reconstructing the evolution of vertebrate genomes and amphibian karyotypes, 2) its genome has evolved to a large size (~10X larger than human) but has maintained gene orders, and 3) it possesses potentially young sex chromosomes that have not undergone extensive differentiation in the structure that is typical of many other vertebrate sex chromosomes (e.g. mammalian XY chromosomes and avian ZW chromosomes). Early chromosomal studies were performed through cytogenetics, but more recent methods involving next generation sequencing and comparative genomics can reveal new information. Due to the large size and inherent complexity of the axolotl genome, multiple approaches are needed to cultivate the genomic and molecular resources essential for expanding its utility in modern scientific inquiries. This dissertation describes our efforts to improve the genomic and molecular resources for the axolotl and other salamanders, with the aim of better understanding the events that have driven the evolution of vertebrate (and amphibian) chromosomes. First, I review our current state of knowledge with respect to genome and karyotype evolution in the amphibians, present a case for studying sex chromosome evolution in the axolotl, and discuss solutions for performing analyses of large vertebrate genomes. In the second chapter, I present a study that resulted in the optimization of methods for the capture and sequencing of individual chromosomes and demonstrate the utility of the approach in improving the existing Ambystoma linkage map and generating targeted assemblies of individual chromosomes. In the third chapter, I present a published work that focuses on using this approach to characterize the two smallest chromosomes and provides an initial characterization of the huge axolotl genome. In the fourth chapter, I present another study that details the development of a dense linkage map for a newt, Notophthalmus viridescens, and its use in comparative analyses, including the discovery of a specific chromosomal fusion event in Ambystoma at the site of a major effect quantitative trait locus for metamorphic timing. I then describe the characterization of the relatively undifferentiated axolotl sex chromosomes, identification of a tiny sex-specific (W-linked) region, and a strong candidate for the axolotl sex-determining gene. Finally, I provide a brief discussion that recapitulates the main findings of each study, their utility in current studies, and future research directions. The research in this dissertation has enriched this important model with genomic and molecular resources that enhance its use in modern scientific research. The information provided from evolutionary studies in axolotl chromosomes shed critical light on vertebrate genome and chromosome evolution, specifically among amphibians, an underrepresented vertebrate clade in genomics, and in homomorphic sex chromosomes, which have been largely unstudied in amphibians.

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Symbiosis is the close and protracted interaction between organisms. The molecular interactions that occur during symbiosis are complex with multiple barriers that must be overcome. Many Gram-negative, host-associated bacteria use a type III secretion system to mediate associations with their eukaryotic hosts. This secretion system is a specialized apparatus for the injection of type III effector proteins directly into host cells, which in the case of plant pathogens, are collectively necessary to modulate host defense. The type III secretion system is not a mechanism exclusive to pathogens, however, as many strains of commensal Pseudomonas fluorescens and mutualistic rhizobia demonstrably require a type III secretion system to interact with their host plants. The work presented in this thesis describes genome-enabled approaches for characterizing type III effector genes across the range of plant symbiosis. Using high-throughput sequencing technology, draft genome sequences were generated for the plant pathogen, Xanthomonas hortorum pv. carotae M081, the plant commensal, Pseudomonas fluorescens WH6, and six strains from the plant mutualists Sinorhizobium fredii and Bradyrhizobium japonicum. Analyses of the draft genome sequences and publicly available finished sequences contributed insights into mechanisms of host-association and to increasing the inventory of type III effector sequences as well as developing methods directly applicable for agriculture. Finally, characterization of the genetic diversity of type III effectors from rhizobia shows that collections of type III effectors of mutualists are static, with little diversity in content and sequence variation. This represents the first comprehensive cataloging of type III effector from species of mutualistic bacteria and the first to provide evidence for purifying selection of this important class of genes. / Graduation date: 2012

This research project presents a comprehensive functional analysis of a complex prokaryotic signal transduction system and the mechanisms underlying its evolution. The chemotaxis system regulates motility in prokaryotes and is their most complex signal transduction system. The system has been extensively characterized experimentally, but recent studies have created new questions about the function and origin of this system. Comparative genomics analyses are well-suited for studying the chemotaxis system since it is present in taxonomically diverse organisms. The first aim of this project is to understand the evolutionary history of the chemotaxis system that has resulted in the diversity of chemotaxis systems that have been experimentally. The results reveal three functional families of chemotaxis systems that regulate flagellar motility, type IV pili motility, and non-motility outputs. The flagellar family shows extensive diversity with 10 conserved classes that have variable accessory proteins, and these classes show a co-evolutionary relationship with flagella. The second aim of this project is to analyze the molecular evolution of chemotaxis system components and utilize that information to predict the contact sites involved in protein-protein interactions. The analysis supports that there is evolutionary pressure at the amino acid sequence level to maintain protein-protein interactions. From this observation, a method to predict the contact sites of protein-protein interactions from sequence information alone was developed and validated by experimental and structural information.

The human genome contains ~1.5% coding sequence, with the remaining 98.5% being non-coding. The functional potential of the majority of this non-coding sequence remains unknown. Much of this non-coding sequence is derived from transposable element (TE) sequences. These TE sequences contain their own regulatory information, e.g. promoter and transcription factor binding sites. Given the large number of these sequences, over 4 million in the human genome, it would be expected that the regulatory information that they contain would affect the expression of nearby genes. This dissertation describes research that characterizes that alternation of and contribution to the human transcriptome by non-coding elements, including TE sequences.

Sexual selection has long been a working explanation for the elaboration of appreciable traits in plants and animals, but the idea that it is an equally potent agent of change at the level of individual molecules is relatively recent. Indications that genes associated with reproductive biology evolve especially rapidly planted this notion, but many details about the genomics of sex remain elusive. Numerous studies have characterized rapid sequence and expression divergence of sex-related molecules, but few if any have demonstrated convincingly that these patterns exist as a result of sexual selection. This dissertation describes several genome-scale studies related to reproduction and the sexes in teleost fishes, a group of animals underexploited in regard to this topic. Using commercial microarrays I measured the extent of sexually dimorphic gene expression in the zebrafish, Danio rerio. Sex-biased patterns of gene expression in this species are similar to those described in other animals. A number of genes expressed at high levels in ovaries and testes relative to the body were identified as a product of the study, and these data may be useful for future studies of reproductive genes in Danio fishes. In a second study, the recent advent of high throughput cDNA pyrosequencing was leveraged to characterize the relationships between tissue-, sex-, and species-specific expression patterns of genes and rates of sequence evolution in swordtail fishes (Xiphophorus). I discovered ample evidence for expression biases of all three types, and a generally positive but idiosyncratic relationship between the magnitude of expression bias and rates of protein-coding sequence evolution. Pyrosequencing of cDNA was also used to explore the possibility that postcopulatory sexual selection drives the rapid evolution of male pregnancy genes, a novel class of reproductive molecules unique to syngnathid fishes (seahorses and pipefishes). Genes differentially expressed in the male brooding tissues as a function of pregnancy status evolve more rapidly at the amino acid level than genes exhibiting static expression. Brooding tissue genes expressed during male pregnancy have evolved especially rapidly in polyandrous lineages, a finding that supports the hypothesized relationship between postcopulatory sexual selection and the adaptive evolution of reproductive molecules.

Comparative genomics provides a tool to investigate large biological datasets, i.e. genomic datasets. In my thesis I focused on inferring patterns of selection in coding and non-coding regions of avian genomes. Until recently, large comparative studies on selection were mainly restricted to model species with sequenced genomes. This limitation has been overcome with advances in sequencing technologies and it is now possible to gather large genomic data sets for non-model species.  Next-generation sequencing data was used to study patterns of nucleotide substitutions and from this we inferred how selection has acted in the genomes of 10 non-model bird species. In general, we found evidence for a negative correlation between neutral substitution rate and chromosome size in birds. In a follow up study, we investigated two closely related bird species, to study expression levels in different tissues and pattern of selection. We found that between 2% and 18% of all genes were differentially expressed between the two species. We showed that non-coding regions adjacent to genes are under evolutionary constraint in birds, which suggests that noncoding DNA plays an important functional role in the genome. Regions downstream to genes (3’) showed particularly high level of constraint. The level of constraint in these regions was not correlated to the length of untranslated regions, which suggests that other causes play also a role in sequence conservation. We compared the rate of nonsynonymous substitutions to the rate of synonymous substitutions in order to infer levels of selection in protein-coding sequences. Synonymous substitutions are often assumed to evolve neutrally. We studied synonymous substitutions by estimating constraint on 4-fold degenerate sites of avian genes and found significant evolutionary constraint on this category of sites (between 24% and 43%). These results call for a reappraisal of synonymous substitution rates being used as neutral standards in molecular evolutionary analysis (e.g. the dN/dS ratio to infer positive selection). Finally, the problem of sequencing errors in next-generation sequencing data was investigated. We developed a program that removes erroneous bases from the reads. We showed that low coverage sequencing projects and large genome sequencing projects will especially gain from trimming erroneous reads.

Cancer development is driven by somatic genome alterations, ranging from single point mutations to larger structural variants (SV) affecting kilobases to megabases of one or more chromosomes. Studies of somatic rearrangement have previously been limited by a paucity of whole genome sequencing data, and a lack of methods for comprehensive structural classification and downstream analysis. The ICGC project on the Pan-Cancer Analysis of Whole Genomes provides an unprecedented opportunity to analyse somatic SVs at base-pair resolution in more than 2500 samples from 30 common cancer types. In this thesis, I build on a recently developed SV classification pipeline to present a census of rearrangement across the pan-cancer cohort, including chromoplexy, replicative two-jumps, and templated insertions connecting as many as eight distant loci. By identifying the precise structure of individual breakpoint junctions and separating out complex clusters, the classification scheme empowers detailed exploration of all simple SV properties and signatures. After illustrating the various SV classes and their frequency across cancer types and samples, Chapter 2 focuses on structural properties including event size and breakpoint homology. Then, in Chapter 3, I consider the SV distribution across the genome, and show patterns of association with various genome properties. Upon examination of rearrangement hotspot loci, I describe tissue-specific fragile site deletion patterns, and a variety of SV profiles around known cancer genes, including recurrent templated insertion cycles affecting TERT and RB1. Turning to co-occurring alteration patterns, Chapter 4 introduces the Hierarchical Dirichlet Process as a non-parametric Bayesian model of mutational signatures. After developing methods for consensus signature extraction, I detour to the domain of single nucleotide variants to test the HDP method on real and simulated data, and to illustrate its utility for simultaneous signature discovery and matching. Finally, I return to the PCAWG SV dataset, and extract SV signatures delineated by structural class, size, and replication timing. In Chapter 5, I move on to the complex SV clusters (largely set aside throughout Chapters 2—4) , and develop an improved breakpoint clustering method to subdivide the complex rearrangement landscape. I propose a raft of summary metrics for groups of five or more breakpoint junctions, and explore their utility for preliminary classification of chromothripsis and other complex phenomena. This comprehensive study of somatic genome rearrangement provides detailed insight into SV patterns and properties across event classes, genome regions, samples, and cancer types. To extrapolate from the progress made in this thesis, Chapter 6 suggests future strategies for addressing unanswered questions about complex SV mechanisms, annotation of functional consequences, and selection analysis to discover novel drivers of the cancer phenotype.