With an increasing amount of whole genome sequence data becoming available on a daily basis we have an opportunity to study the interactions and dynamics of different organisms on a whole genome level. In the past, reports of horizontal gene transfer have focused mainly on the identification of single genes that show distorted phylogenetic profiles to that of the organism it was isolated from. This study firstly did whole genome comparisons between the rice nuclear and plastid genomes to determine the level and dynamics gene transfer and insertion of the chloroplast ad mitochondrial genomes into that of the nuclear genome of rice. Secondly, it looked to identify sequence similarities between the rice genome and microbial genomes by performing whole genome comparisons between the rice genome and that of several microbial genomes. These sequences were analyzed further to identify possible instances of horizontal transfer of DNA from microbes to the rice genome. Using this approach, this study reports several fragments in the rice genome with significant sequence similarity to that of microbial DNA fragments. This study also provides evidence supporting horizontal transfer of several of these fragments. This study provides valuable information regarding intra- as well as inter-genome DNA transfer dynamics. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted

Comparisons of flowering plant genomes reveal multiple rounds of ancient polyploidy characterized by large intragenomic syntenic blocks. Three such whole-genome duplication (WGD) events, designated as rho (rho), sigma (sigma), and tau (tau), have been identified in the genomes of cereal grasses. Precise dating of these WGD events is necessary to investigate how they have influenced diversification rates, evolutionary innovations, and genomic characteristics such as the GC profile of protein-coding sequences. The timing of these events has remained uncertain due to the paucity of monocot genome sequence data outside the grass family (Poaceae). Phylogenomic analysis of protein-coding genes from sequenced genomes and transcriptome assemblies from 35 species, including representatives of all families within the Poales, has resolved the timing of rho and sigma relative to speciation events and placed tau prior to divergence of Asparagales and the commelinids but after divergence with eudicots. Examination of gene family phylogenies indicates that rho occurred just prior to the diversification of Poaceae and sigma occurred before early diversification of Poales lineages but after the Poales-commelinid split. Additional lineage-specific WGD events were identified on the basis of the transcriptome data. Gene families exhibiting high GC content are underrepresented among those with duplicate genes that persisted following these genome duplications. However, genome duplications had little overall influence on lineage-specific changes in the GC content of coding genes. Improved resolution of the timing of WGD events in monocot history provides evidence for the influence of polyploidization on functional evolution and species diversification.

Whole genome doubling (WGD) occurs early in tumorigenesis and generates genetically unstable tetraploid cells that fuel tumor development. Cells that undergo WGD (WGD+) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can subsequently be exploited therapeutically, is unclear. Using sequencing data from ~10,000 primary human cancer samples and essentiality data from ~600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD+ cells are more dependent on spindle assembly checkpoint signaling, DNA replication factors, and proteasome function than WGD– cells. We also identify KIF18A, which encodes for a mitotic kinesin, as being specifically required for the viability of WGD+ cells. While loss of KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGD– cells, its loss induces dramatic mitotic errors in WGD+ cells, ultimately impairing cell viability. Collectively, our results reveal new strategies to specifically target WGD+ cancer cells while sparing the normal, non-transformed WGD– cells that comprise human tissue.

No description available.

Yes / Pantoea stewartii is known to be the causative agent of Stewart's wilt, which usually affects sweet corn (Zea mays) with the corn flea beetle as the transmission vector. In this work, we present the whole-genome sequence of Pantoea stewartii strain M009, isolated from a Malaysian tropical rainforest waterfall. / University of Malaya via High Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01 no. A-000001- 50001 and UM C/625/1/HIR/MOHE/CHAN/14/1 no. H-50001-A000027)

Like many angiosperms, Brassica rapa underwent several rounds of whole genome duplication during its evolutionary history. Brassica rapa is particularly valuable for studying genome evolution because it also experienced whole genome triplication shortly after it diverged from the common ancestor it shares with Arabidopsis thaliana about 17-20 million years ago. While many B. rapa genes appear resistant to paralog retention, close to 50% of B. rapa genes have retained multiple, paralogous loci for millions of years and appear to be multi-copy tolerant. Based on previous studies, gene function may contribute to the selective pressure driving certain genes back to singleton status. It is suspected that other factors, such as gene expression patterns, also play a role in determining the fate of genes following whole genome triplication. Published RNA-seq data was used to determine if gene expression patterns influence the retention of extra gene copies. It is hypothesized that retention of genes in duplicate and triplicate is more likely if those genes are expressed in a tissue-specific manner, as opposed to being expressed globally across all tissues. This study shows that genes expressed specifically in flowers and roots in B. rapa are more resistant to fractionation than globally expressed genes following whole genome triplication. In particular, there appears to have been selection on genes expressed specifically in flower tissues to retain higher copy numbers and for all three copies to exhibit the same flower-specific expression pattern. Future research to determine if these observations in Brassica rapa are consistent with other angiosperms that have undergone recent whole genome duplication would confirm that retention of flower-specific-expressed genes is a general feature in plant genome evolution and not specific to B. rapa.

BACKGROUND: Black rot is a destructive bacterial disease causing large yield and quality losses in Brassica oleracea. To detect quantitative trait loci (QTL) for black rot resistance, we performed whole-genome resequencing of two cabbage parental lines and genome-wide SNP identification using the recently published B. oleracea genome sequences as reference. RESULTS: Approximately 11.5 Gb of sequencing data was produced from each parental line. Reference genome-guided mapping and SNP calling revealed 674,521 SNPs between the two cabbage lines, with an average of one SNP per 662.5 bp. Among 167 dCAPS markers derived from candidate SNPs, 117 (70.1%) were validated as bona fide SNPs showing polymorphism between the parental lines. We then improved the resolution of a previous genetic map by adding 103 markers including 87 SNP-based dCAPS markers. The new map composed of 368 markers and covers 1467.3 cM with an average interval of 3.88 cM between adjacent markers. We evaluated black rot resistance in the mapping population in three independent inoculation tests using F₂:₃ progenies and identified one major QTL and three minor QTLs. CONCLUSION: We report successful utilization of whole-genome resequencing for large-scale SNP identification and development of molecular markers for genetic map construction. In addition, we identified novel QTLs for black rot resistance. The high-density genetic map will promote QTL analysis for other important agricultural traits and marker-assisted breeding of B. oleracea.

Compared to microarray-based genotyping, next-generation whole genome-sequencing (WGS) studies have the strength to provide greater information for the identification of rare variants, which likely account for a significant portion of missing heritability of common human diseases. In WGS, family-based studies are important because they are likely enriched for rare disease variants that segregate with the disease in relatives. We propose a multilevel model to detect disease variants using family-based WGS data with longitudinal measures. This model incorporates the correlation structure from family pedigrees and that from repeated measures. The iterative generalized least squares (IGLS) algorithm was applied to estimation of parameters and test of associations. The model was applied to the data of Genetic Analysis Workshop 18 and compared with existing linear mixed effect (LME) models. The multilevel model shows higher power at practical p-value levels and a better type I error control than LME model. Both multilevel and LME models, which utilize the longitudinal repeated information, have higher power than the method that only utilize data collected at one time point.

Next-generation sequencing technologies are revolutionising our ability to characterise and investigate infectious diseases. Utilising the power of high throughput sequencing, this study reports, the development of a sensitive, non-PCR based, unbiased amplification method, which allows the rapid and accurate sequencing of multiple microbial pathogens directly from clinical samples. The method employs Φ29 DNA polymerase, a highly efficient enzyme able to produce strand displacement during the polymerisation process with high fidelity. Problems with DNA secondary structure were overcome and the method optimised to produce sufficient DNA to sequence from a single bacterial cell in two hours. Evidence was also found that the enzyme requires at least six bases of single stranded DNA to initiate replication, and is not capable of amplification from nicks. Φ29 multiple displacement amplification was shown to be suitable for a range of GC contents and bacterial cell wall types as well as for viral pathogens. The method was shown to be able to provide relative quantification of mixed cells, and a method for quantification of viruses using a known standard was developed. To complement the novel molecular biology workflow, a data analysis pipeline was developed to allow pathogen identification and characterisation without prior knowledge of input. The use of de novo assemblies for annotation was shown to be equivalent to the use of polished reference genomes. Single cell Φ29 MDA samples had better assembly and annotation than non-amplification controls, a novel finding which, when combined with the very long DNA fragments produced, has interesting implications for a variety of analytical procedures. A sampling process was developed to allow isolation and amplification of pathogens directly from clinical samples, with good concordance shown between this method and traditional testing. The process was tested on a variety of modelled and real clinical samples showing good application to sterile site infections, particularly bacteraemia models. Within these samples multiple bacterial, viral and parasitic pathogens were identified, showing good application across multiple infection types. Emerging pathogens were identified including Onchocerca volvulus within a CSF sample, and Sneathia sanguinegens within an STI sample. Use of Φ29 MDA allows rapid and accurate amplification of whole pathogen genomes. When this is coupled with the sample processing developed here it is possible to detect the presence of pathogens in sterile sites with a sensitivity of a single genome copy.

Legionella bacteria, the causative agent of Legionaries’ disease and Pontiac fever, are ubiquitous in fresh-water environments including man-made water systems. Incidence of legionellosis is increasing in the United States resulting in thousands of cases every year. Infection via aerosols generated by showers, faucets, cooling towers, spas, fountains, and other water fixtures has been identified as the primary source of transmission. Legionella bacteria pose a significant public health threat, particularly in health care and long term care settings as Legionella can readily colonize the plumbing systems and infect the vulnerable patient population. One species, Legionella pneumophila (Lp), is responsible for over 90% of the known cases of Legionnaires’ disease. The importance of genetic diversity of Lp and non-pneumophila strains in human disease remains an area of ongoing research. Little is known in regard to the phylogenetic diversity of environmental strains, particularly strains that colonize facilities with high risk populations such as hospitals. Whole-genome sequencing (WGS) analysis, is an emerging tool used to support epidemiological investigation of cases of legionellosis and can be used to describe and establish phylogenetic relationships between environmental strains and clinical cases. The advantage of this method is the ability to differentiate bacteria down to the level of single nucleotide polymorphisms (SNPs). However, it was unknown whether current WGS methods accurately represent the potential SNP diversity among Lp isolates from the same environmental sample. It is unclear as to why certain strains tend be associated with clinical cases more than others, but certain genes referred to as virulence factors may be related to the relative pathogenicity of Legionella strains. Further investigation into virulence factors and antibiotic resistance factors could be used in future risk assessment of environmental Legionella. Additionally, Legionella have the potential for high genetic diversity due to recombination events, and gene transfer can occur between distinct Legionella species and strains. There is a lack of research on the potential sharing of virulence factor genes between Legionella strains typically associated with disease and those considered to be non-virulent. The goal of the work presented in this thesis is to describe the diversity of phylogenetic relationships between Lp isolates found in hospital premise plumbing systems, to estimate the genetic diversity among Lp found in the same environmental sample, and to identify virulence and antibiotic resistance genes shared between Legionella strains. A better understanding of the genetic diversity of environmental Lp could inform future surveillance and outbreak investigations by demonstrating the need to collect samples from multiple sites within a facility, and identifying shared virulence and antibiotic resistance genes between Legionella species and strains could apprise future risk assessment. WGS was utilized to describe the phylogenetic relationships of 81 Lp isolates from five hospitals. Individual hospitals were found to have distinct strains of Lp. For some strains, highly conserved subpopulations were collected from the same room over time, whereas other strains did not cluster by room. Using prospectively collected isolates from two hospitals, the mean number of SNP differences among isolates from the same environmental sample was found to differ between hospitals (0.4 versus 7.5). The presence of virulence factors and antibiotic resistance genes in Legionella species and strains was described. An analysis of 10 virulence factor genes revealed that Lp likely did not share these genes with Legionella anisa, a species generally considered to be non-virulent. Within Lp strains there was no clear difference between the Lp strains considered to be more virulent and those considered to be less virulent. A few antibiotic resistance genes were also identified. Following an in vitro assay, only the identified genes associated with macrolide resistance, LpeA and LpeB, were found to impact a quantifiable measure of antimicrobial resistance. The results of these studies emphasize the importance of understanding the context of an individual facility in Legionella related studies. Importantly, the observations or trends of one facility should not necessarily be applied to another. Legionella genetic diversity was highly conserved in some facilities, whereas in others there was greater diversity as measured by SNP differences. Within sample SNP differences was also variable between hospitals. The virulence findings gave a clear indication of the limited virulence capacity of L. anisa. These findings could explain the limited potential of L. anisa to cause disease in humans. However, a lack of difference among Lp strains may be cause to reassess the potential risk of these other strains especially in diagnostic practices. Finally, some strains of Lp have genes that may contribute to resistance to the leading antibiotic treatments for Legionnaires’ disease. Overall, this research further demonstrates the power of WGS as multiple questions can be addressed using this methodology.