Bacteria have been the causative agents of major infectious disease pandemics throughout human history. Over the past 4 decades, a combination of changing medical practices, industrialization, and globalisation have led to a number of emergences and re-emergences of bacterial pathogens. The design of rational control programs and bespoke therapies will require an enhanced understanding of the dynamics underpinning the emergence and transmission of pathogenic clones. The recent development of new technologies for sequencing bacterial genomes rapidly and economically has led to a greatly enhanced understanding of the diversity of bacterial populations. This thesis describes the application of whole genome sequencing of 2 bacterial pathogens, Staphylococcus aureus and Legionella pneumophila, in order to understand the dynamics of bacterial infections on different temporal and spatial scales. The first study involves the examination of S. aureus evolution during a chronic infection of a single patient over a period of 26 months revealing differences in antibiotic resistance profiles and virulence factor expression over time. The genetic variation identified correlated with differences in growth rate, haemolytic activity, and antibiotic sensitivity, implying a profound effect on the ecology of S. aureus. Importantly, polymorphisms were identified in global regulators of virulence, with a high frequency of polymorphisms within the SigB locus identified, suggesting this region may be under selection in this patient. The identification of genes under diversifying selection during long-term infection may inform the design of novel therapeutics for the control of refractory chronic infections. Secondly, the emergence and transmission of 3 pandemic lineages derived from S. aureus clonal complex 30 (CC30) were investigated. Independent origins for each pandemic lineage were identified, with striking molecular correlates of hospital- or community-associated pandemics represented by mobile genetic elements, such as bacteriophage and Staphylococcal pathogenicity islands, and non-synonymous mutations affecting antibiotic resistance and virulence. Hospitals in large cities were identified as hubs for the transmission of MRSA to regional health care centres. In addition, comparison of whole genome sequences revealed that at least 3 independent acquisitions of TSST-1 have occurred in CC30, but a single distinct clade of diverse community-associated CC30 strains was responsible for the TSS epidemic of the late 1970s, and for subsequent cases of TSS in the UK and USA. Finally, whole genome sequencing was used as a tool for investigating a recent outbreak of legionellosis in Edinburgh. An unexpectedly high level of genomic diversity was identified among the outbreak strains, with respect to core genome polymorphisms, and accessory genome content. The data indicate that affected individuals may be infected with heterogeneous strains. The findings highlight the complexities in identifying environmental sources and suggest possible differences in pathogenic potential among isolates from a single outbreak. Taken together, the findings demonstrate applications of bacterial genome sequencing leading to enhanced understanding of bacterial pathogen evolution, emergence, and transmission, which may ultimately inform appropriate infection control measures.

This thesis explores the genetic characterization of the mechanisms underlying complex traits in chicken through the use and development of bioinformatics tools. The characterization of quantitative trait loci controlling complex traits has proven to be very challenging. This thesis comprises the study of experimental designs, annotation procedures and functional analyses. These represent some of the main ‘bottlenecks’ involved in the integration of QTLs with the biological interpretation of high-throughput technologies. The thesis begins with an investigation of the bioinformatics tools and procedures available for genome research, briefly reviewing microarray technology and commonly applied experimental designs. A targeted experimental design based on the concept of genetical genomics is then presented and applied in order to study a known functional QTL responsible for chicken body weight. This approach contrasts the gene expression levels of two alternative QTL genotypes, hence narrowing the QTL-phenotype gap, and, giving a direct quantification of the link between the genotypes and the genetic responses. Potential candidate genes responsible for the chicken body weight QTL are identified by using the location of the genes, their expression and biological significance. In order to deal with the multiple sources of information and exploit the data effectively, a systematic approach and a relational database were developed to improve the annotation of the probes of the ARK-Genomics G. gallus 13K v4.0 cDNA array utilized on the experiment. To follow up the investigation of the targeted genetical genomics study, a detailed functional analysis is performed on the dataset. The aim is to identify the downstream effects through the identification of functional variation found in pathways, and secondly to achieve a further characterization of potential candidate genes by using comparative genomics and sequence analyses. Finally the investigation of the body weight QTL syntenic regions and their reported QTLs are presented.

Cotton is a crop with a large global economic impact as well as a large, complex genome. Most industrial cotton production is from two tetraploid species (Gossypium hirsutum L. and Gossypium barbadense L.) which contain two subgenomes, specifically the AT and DT subgenomes. The DT subgenome is nearly half the size of the AT subgenome in tetraploid cotton and is closely related to an extant D-genome Gossypium species, G. raimondii Ulbr. Characterization of the structural variants present in diploid D-genome should provide greater insight into the evolution of the DT subgenome in the tetraploid cotton. Bionano (BNG) optical mapping uses patterns of fluorescent labels inserted at specific endonuclease sites to create physical maps of the genomes which can then be examined for structural variation. To develop optical maps in G. raimondii, we first developed a de novo PacBio long read sequence assembly of G. raimondii. This sequence assembly consisted of 2,379 contigs, an average contig length of 413 Kb and a contig N50 of 4.9 Mb. Using BNG technology, we developed two optical maps of the diploid D genome of G. raimondii. One was created using the Nt.BssSI endonuclease and one with the Nt.BspQI endonuclease. Using the BNG optical maps, the PacBio assembly was hybrid scaffolded into 100 scaffolds (+ 5 unscaffolded contigs) with an average scaffold length of 7.5 Mb and a scaffold N50 of 13.1 Mb. A comparison between the Nt. BssSI BNG optical map and the two sequence assemblies identified 3,195 structural variants. These were used to validate the accuracy of the reference sequence of G. raimondii and structural variants were used to create a new phylogeny of nine major cotton species.

Sex and meiosis are ubiquitous in eukaryotes as the primary mode of reproduction. This suggests that despite the theoretical energetic advantages of asexual reproduction, organisms capable of sexual reproduction are at a much greater long-term evolutionary advantage. Rotifers, a group of microinvertebrates, offer unique opportunities to examine the evolution of sex due to their extensive proliferation, successful adaptation to a wide variety of ecological niches, and the diversity of reproductive modes represented in the group. The cyclically parthenogenetic monogonont rotifers have overcome constraints on the loss of sexual reproduction in order to frequently transition between sexual and asexual generations, making them a powerful system with which to address the maintenance of sex in animals. Obligately asexual bdelloid rotifers appear to have thrived without sex for tens of millions of years, a period of time much longer than expected given the hypothesized advantages of sexual reproduction. However, the molecular nature of sex and parthenogenesis is poorly understood in any rotifer species. To expand our knowledge of the molecular mechanisms of monogonont reproduction, we sequenced genomes of two distantly related species, Brachionus calyciflorus and Brachionus manjavacas and identified over 80 homologs for genes involved in meiotic processes. Several of these genes have undergone duplication events specific to the monogonont lineage, including genes with known roles in regulation of cell cycle transitions during meiosis. In addition, global gene expression patterns were determined using obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of B. calyciflorus. Quantitative comparison of expression between these strains revealed differentially expressed genes specific to sexual and asexual reproduction in this species, including genes related to dormancy/resting egg formation, meiosis, and hormone signaling pathways that are thought to be involved in the induction of sexual reproduction in monogononts. Finally, we analyzed gene expression in bdelloid rotifers for evidence of sexual reproduction or the utilization of meiotic genes under conditions inducing high levels of recombination. Through this work, we have established molecular markers for sexuality and asexuality in monogonont rotifers, and used these markers to evaluate reproduction in bdelloids. The data generated specifically allows for more informed analyses of the evolution of cyclical parthenogenesis and rotifer reproduction. Furthermore, this work extends the use of monogononts as a model system for addressing broader questions regarding the evolution of sexual reproduction.

Existing methods to systematically characterize sequence-intrinsic activity of promoters are limited by relatively low throughput and the length of sequences that could be tested. Here we present Survey of Regulatory Elements (SuRE), a method to assay more than a billion DNA fragments in parallel for their ability to drive transcription autonomously. In SuRE, a plasmid library is constructed of random genomic fragments upstream of a barcode and decoded by paired-end sequencing. This library is transfected into cells and transcribed barcodes are quantified in the RNA by high-throughput sequencing. By computationally analyzing the resulting data using generalized linear models, we succeed in delineating subregions within promoters that are relevant for their activity on a genomic scale, and making accurate predictions of expression levels that can be used to inform minimal promoter reporter construct design. We also show how our approach can be extended to analyze the differential impact of single-nucleotide polymorphisms (SNPs) on gene expression.

<p></p><p>Sorghum (<i>Sorghum bicolor) </i>is a well-known agronomic crop of global importance. The demand for sorghum as a food crop makes it the fifth most important cereal in the world. The grain of sorghum is utilized for food and feed, whereas the sorghum biomass may have many other uses such as for fodder, bioenergy or even for construction. Globally, sorghum is consumed as a food crop and used for home construction primarily in the developing world. The grain and biomass yield of sorghum is drastically reduced by the parasitic plant <i>Striga hermonthica </i>which is endemic to Sub-Saharan Africa. To date, only one sorghum gene, <i>LGS1</i>, has been characterized as a genetic mechanism that reduces <i>S. hermonthica</i> parasitism by altering the strigolactone composition of the host root exudates which results in a reduction of the parasites ability to germinate. To establish more durable resistance additional genetic variation needs to be identified that reduces the <i>S. hermonthica </i>parasitism in sorghum, but also reduces the parasitic weed seed bank by promoting suicidal germination. To that end, the PP37 multi-parent advanced generation inter-cross (MAGIC) population was developed, originally as a recurrent selection population that was developed to recombine sorghum accessions with different putative resistance mechanisms to <i>S. hermonthica. </i>Whole genome sequences were developed for approximately 1,006 individuals of the PP37 MAGIC population. The population was phenotyped for <i>S. hermonthica </i>resistance during the 2016 and 2017 growing season in Northwestern Ethiopia. There was significant spatial variation in the <i>S. hermonthica </i>natural infestations that were partially attenuated for with artificial inoculation. The data was used to conduct a genome-wide association study that detected several subthreshold peaks, including the previously mapped <i>LGS1. </i>The highly quantitative nature of <i>S. hermonthica </i>resistance confounded with the complex spatial variation in the parasite infestations across a given location make it difficult to detect highly heritable variation across years and environments. </p> <p> In addition to <i>S. hermonthica </i>resistance, the plant architecture of the PP37 MAGIC was also assessed at a location in Northwestern Ethiopia that is free of the parasite, as it significantly reduces plant height. To asses plant architecture the total plant height, the height of the panicle base, flag leaf height, and pre-flag leaf height were collected using a relatively high-throughput barcoded measurement system. Sorghum head exertion and panicle length were derived from this data. The actual measures of plant architecture and the derived traits were used to conduct a genome-wide association study. The high heritability of this trait demonstrated the statistical power of the PP37 mapping population. Highly significant peaks were detected that resolved the <i>dwarf3</i> locus and an uncharacterized qHT7.1 that had only been previously resolved using a recombinant inbred line population. Furthermore, a novel significant locus was associated with exertion on chromosome 1. The random mating that was utilized to develop the PP37 MAGIC has broken the population structure that when present can hinder our ability associate regions of the genome to a given phenotype. As a result, novel candidate gene lists have been developed as an outcome of this research that refined the potential genes that need to be explored to validate qHT7.1 and the novel association on chromosome 1. </p> <p>This research demonstrated the power of MAGIC populations in determining the genomic regions that influence complex phenotypes, that facilitates future work in sorghum genetic improvement through plant breeding. This research however also demonstrates a large international research effort. The nuisances and lessons learned while conducting this international research project are also discussed to help facilitate and guide similar research projects in the future. The broader impacts of this research on the society at large are also discussed, to highlight the unique potential broader impacts of international research in the plant sciences. The broader impacts of this research include germplasm development and extensive human capacity building in plant breeding genetics for developing country students and aspiring scientists. Overall this research attempts to serve as a model for highlighting the interdisciplinary nature and complexity of conducting international plant science research, while also making significant strides in improving our understanding the genetic architecture of quantitative traits of agronomic importance in sorghum.</p><br><p></p>

Genome-wide association (GWA) studies have been successfully applied in detection of susceptibility loci for complex diseases, but most of the identified variants have a large to moderate effect, and explain only a limited proportion of the heritability of the diseases. It is believed that the majority of the latent risk alleles have very small risk effects that are difficult to be identified and GWA study may have inadequate power in dealing with those small effect variants. Researchers will often collect other phenotypic information in addition to disease status to maximize the output from the study. Some of the phenotypes can be on the pathway to the disease, i.e., intermediate phenotype. Statistical methods based on both the disease status and intermediate phenotype should be more powerful than a case-control study as it incorporates more information. Meta-analysis has been used in genetic association analysis for many years to combine information from multiple populations, but never been used in a single population GWA study. In this study, simulations were conducted and the results show that when an intermediate phenotype is available, the meta-analysis incorporating the disease status and intermediate phenotype information from a single population has more power than a case-control study only in GWA study of complex diseases, especially for identification of those loci that have a very small effect. And compared with Fisher's method, the modified inverse variance weighted meta-analysis method is more robust as it is more powerful and has a lower type I error rate at the same time, which provides a potent approach in detecting the susceptibility loci associated with complex diseases, especially for those latent loci whose effect are very small. In the meta-analysis of lung cancer with smoking data, the results replicate the signal in \emph{CHRNA3} and \emph{CHRNA5} genes on chromosome 15q25. Some new signals in \emph{CYP2F1} on chromosome 19, \emph{SUMF1} on chromosome 3, and \emph{ARHGAP10} on chromosome 4 are also detected. And the \emph{CYP2F1} gene, close to the already known cigarette-induced lung cancer gene \emph{CYP2A6}, is highly possible another cytochrome P450 (CYP) gene that is related to the smoking-involved lung cancer. The meta-analysis of rheumatoid arthritis with anti-cyclic citrullinated peptide (anti-CCP) data identified new signals on 9q24 and 16q12. There are evidences these two regions are involved in other autoimmune diseases and different autoimmune/inflammatory diseases may share same genetic susceptibility loci. Both the theoretical and empirical studies show that the modified variance weighted meta-analysis method is a robust method and is a potent approach in detecting the susceptibility loci associated with complex diseases when an intermediate phenotype is available.

3-Methylindole and 4-methylphenol are cytotoxic and malodorant compounds derived from tryptophan and tyrosine, respectively. Each is present in swine waste lagoons and contributes to malodorous emissions from agricultural facilities. Clostridium scatologenes ATCC 25775 produces both compounds and serves as a model organism to study their metabolism and function. Through the repeated assembly and annotation of the Clostridium scatologenes genome, we propose a novel pathway for tryptophan degradation and 3-methylindole production by this organism. The genome of Clostridium scatologenes was sequenced, and re-assembled into contigs. Key elements of the tryptophan and shikimate pathways were identified. Contigs containing these elements were extracted from assemblies and matched to the reference genome of Clostridium carboxidivorans. Sequence for both pathways was then extended and defined using these joined sequence fragments. This sequence could serve as a starting point for the isolation of genes related to 3-methylindole synthesis using biochemical and enzyme analysis

Despite the convergence of rapid technological advances in genomics and the maturing field of ecoimmunology, our understanding of the genes that regulate immunity in wild populations is still nascent. Previous work to assess immune function has relied upon relatively crude measures of immunocompetence. However, with next-generation RNA-sequencing, it is now possible to create a profile of gene expression in response to an immune challenge. In this study, captive zebra finch (Taeniopygia guttata; adult males) were challenged with bacterial lipopolysaccharide (2 mg/Kg BW; dissolved in 0.9% saline) or vehicle (0.9% saline) to stimulate the immune system. Two hours after injection, birds were euthanized and hypothalami, spleen, and red blood cells (RBCs) were collected. Taking advantage of the fully sequenced genome of zebra finch, total RNA was isolated, sequenced, and partially annotated in these tissue/cells. The data show 628 significantly upregulated transcripts in the hypothalamus, as well as 439 and 121 in the spleen and RBCs, respectively, relative to controls. Also, 134 transcripts in the hypothalamus, 517 in the spleen, and 61 in the RBCs were significantly downregulated. More specifically, a number of immunity-related transcripts (e.g., IL-1β, RSAD2, SOCS3) were upregulated among tissues/cells. Additionally, transcripts involved in metabolic processes (APOD, LRAT, RBP4) were downregulated, suggesting a potential trade-off in expression of genes that regulate immunity and metabolism. Unlike mammals, birds have nucleated RBCs, and these results suggest a novel transcriptomic response of RBCs to immune challenge. Lastly, molecular biomarkers could be developed to rapidly screen bird populations by simple blood sampling in the field.

Chloroplast genomes of land plants show conservation of structure and gene arrangement. The spinach chloroplast genome is comprised of a covalently closed. circular DNA molecule of 150 kilobases and is typical of these plants. Approximately 20% of the proteins found in the spinach chloroplast are encoded by the chloroplast genome and translated on chloroplast ribosomes. The remainder are encoded on chromosomes in the nucleus, translated on cytoplasmic ribosomes and transported into the chloroplast. Spinach chloroplast DNA was isolated from crude 2 chloroplast preparations by a new method. Chloroplasts were lysed with alkaline sodium dodecyl sulfate, contaminating macromolecules precipitated with acidified potassium acetate and plastid DNA was purified by phenol:chloroform extraction and ethanol:ammonium acetate precipitation. The yield was approximately 50 ug chloroplast DNA per 100 grams leaf material. The DNA consisted of 10% circular molecules and 90% linear molecules. The chloroplast DNA was digested with restriction enzyme PstI and the fragments were cloned into the plasmid vector pUC9. Several recombinant plasmids were isolated and the chloroplast DNA inserts identified. The recombinant plasmid pRD105 containing the PstI #5 fragment was subjected to further investigation. The ClaI restriction sites of the PstI #5 fragment were mapped and the insert was subcloned into the plasmid vector pGEM4, which bears bacteriophage SP6 and T7 RNA polymerase promoter sequences.