This master thesis deals with typing of Klebsiella pneumoniae isolates. The first part of the thesis introduces molecular typing methods. Then bacterial genomes and Klebsiella pneumoniae are characterized. Following part describes data validation, assembly of genomes and proposed algorithm for finding genes with high variability. In last part obtained results are presented and validated on other genomes of Klebsiella pneumoniae.

This thesis deals with automatic bacteria genotyping by rep-PCR method. Its theoretical part presents various methods of DNA typing, basic information on electrophoresis and modern electrophoretic approaches, including their problems, misleading data distortion. In order to automate typing, there has been introduced a program for phylogenetic sample classification from rep-PCR, also applicable for data from chip capillary electrophoresis. The program consists of three main parts: digitization, bandmatching and clustering apparatus to bacterial type classification. The result of the algorithm is a phylogenetic tree, which indicates the cluster of sapmles according to bacterial type. The program has a graphical user interface for possible use in the Children's Hospital. Finally, the program is tested with data from the Children´s Hospital.

MSc (Microbiology) / Department of Microbiology

Wheat is the most widely grown crop in the world and as such, is an essential source of energy and nutrition. The challenges that breeders presently face is to increase production to feed the rising population of the world, while also accounting for climate change, pollution, water and environmental stresses. As genetic uniformity of modern cultivars has increased vulnerability to pests and diseases, the wild relatives of wheat offer a rich source of genetic diversity and stress tolerance traits, that can be harnessed and transferred in to modern wheat. In this study, we used array-based genotyping to explore genetic diversity in 385 domesticated and non-domesticated lines of wheat and their wild relatives. Genetic characterization using the wheat 90K array, and subsequent filtering and validation mapped 9,570 single nucleotide polymorphic markers onto the wheat reference genome. Phylogenetic analyses illustrated four major clades, clearly separating the wild species from the domesticated, and the ancient Triticum turgidum species from modern T. turgidum cultivars. Using this diverse germplasm, a genome-wide association study (GWAS) was performed for leaf rust, the most widespread rust disease of wheat. Identification of novel sources of resistance is necessary to maintain disease resistance and stay ahead in the plant-pathogen evolutionary arms race. GWAS was conducted using eight statistical models for infection types against six leaf rust isolates and leaf rust severity rated in field trials for 3-4 years at 2-3 locations in Canada. Functional annotation of genes containing significant quantitative trait nucleotides (QTNs) identified 96 disease-related nucleotide associated with leaf rust resistance. A total of 21 QTNs were in haplotype blocks or within flanking markers of at least 16 known leaf rust (Lr) resistance genes. The remaining significant QTNs were considered loci that putatively harbor new Lr resistance genes. Future efforts to validate these loci will help understand their role in disease resistance and promote their utility for marker-assisted selection in pre-breeding.

Weed management options in agronomic crop production have been severely limited by widespread populations of weeds resistant to herbicides, including waterhemp [Amaranthus tuberculatus (Moq.) Sauer (syn. rudis)] resistant to foliar applications of herbicides that inhibit protoporphyrinogen oxidase (PPO; EC 1.3.3.4) activity (PPO-R). Herbicides within this site of action (WSSA site of action #14) remain efficacious when soil-applied to PPO-R waterhemp populations. Therefore, the continued use of these herbicides for soil-residual control of PPO-R waterhemp, especially in soybean production, is paramount with limited postemergence herbicides that remain effective. An improved understanding of the selection for PPO-R waterhemp would provide information to help minimize future loss of residual PPO-inhibiting herbicide activity. Five studies, consisting of 14 experiments, were conducted to improve our understanding of the selection for herbicide-resistant individuals. Soil-residual herbicides have been suspected to select for herbicide-resistant individuals; however, this phenomenon has never been observed experimentally in field conditions. This dissertation provides direct evidence from greenhouse and field experiments that significant selection pressure can occur from soil-residual herbicides; however, this selection for resistance could be delayed when using full commercial herbicide rates and effective herbicides from multiple sites of action. Also, the frequency of heterozygous individuals (RS) and PPO-inhibiting herbicide efficacy on RS individuals is a factor in the selection for herbicide resistance; however, current information on the these topics is limited. To provide additional information on RS individuals, a large-scale genotypic and phenotypic screen of multiple PPO-R waterhemp populations was conducted. It was determined that RS individuals were less frequent than expected and PPO-inhibiting herbicide efficacy on RS individuals was population-dependent. Finally, the hormetic effects of soil-residual herbicides have been paradoxically implicated as a means of both mitigating and exacerbating the selection for herbicide resistant biotypes; however, limited information was available on the hormetic effects of soil-residual PPO inhibitors. Greenhouse and growth chamber experiments were conducted to improve understanding of hormesis and soil-residual PPO-inhibiting herbicides. Experiments indicated that PPO-inhibiting herbicides may exert a limited hormetic effect on waterhemp germination below doses that cause a phytotoxic effect of the emerging seedling, indicating this effect may exacerbate the issue of selection for PPO-R waterhemp. Overall, data presented in this dissertation provides important information on the under-studied interaction between PPO-inhibiting herbicides and PPO-R waterhemp to safeguard the sustained efficacy of herbicides within this site of action.

Discovering, screening, and associating changes in DNA sequence are important to a broad range of disciplines and play a central role in Forensic Science. The typical types of changes include sequence variations [single nucleotide polymorphisms (SNP)] and length variations [short tandem repeats (STR)]. The steps for forensic DNA sample processing are similar for both types of changes but diverge at the point of detection. A number of approaches are being explored for SNP genotyping while STR analysis primarily consists of size-based analysis by capillary electrophoresis. Limitations exist for all current detection methods that pose significant impacts to forensic analysis. Bi-allelic SNPs result in three possible genotypes with a minimal amount of information generated per marker. Limitations for SNP analysis are due to the inability to amplify a suitable number of SNP markers from low DNA content samples to provide an appropriate level of discrimination. Multi-allelic STR markers are currently the marker of choice for forensic typing but a variety of experimental artifacts are possible that consist of either biology or technology related causes. Molecular genotyping methods developed across other disciplines have potential to alleviate some of these shortcomings but no current approach is capable of genotyping both SNP and STR loci with a single chemistry. The need for a more effective, efficient, and generalized approach led to development of a unique method called Dye Probe Fluorescence Resonance Energy Transfer (dpFRET) and determination of its suitability for forensic analysis. The development phase of the research consisted of synthetic testing to establish proof of concept for the chemistry followed by polymerase chain reaction (PCR) based assays to demonstrate real world applications. Following successful development, the boundaries and limitations for the technology were established (sensitivity, allelic dropout, mixed samples) and efforts were made to improve the approach. In the process, parallel testing for other fields including molecular pathology and conservation biology were incorporated to explore potential widespread application of this new approach. The overall goal of this project was to develop and explore the limitations for a unique approach to genotyping both SNPs and STRs. A majority of the work involved development of the method itself with the ultimate objective of application for forensic science. The focus of this project was to address and alleviate some of the shortcomings of current approaches that result in potential limitations for forensic analysis. It is expected that future applications of this technology might impact a wide range of disciplines to aid in discovery, screening and association of changes in DNA sequence.

Biological evidence may contain any number of cells in any proportion. Extreme low-template DNA samples are often very difficult to interpret due to complex signal or peaks which may be indistinguishable from baseline noise. Current solutions focus on increasing the amount of amplicon detected by adjusting PCR cycle number or capillary electrophoresis injection parameters. Consensus profiling is an additional option. However, the aforementioned solutions are often not helpful for extreme low-template samples due to the high occurrence of allelic drop-out. Additionally, PCR is a destructive technique that causes one amplification to completely exhaust this type of sample, making further typing and analysis impossible. Therefore, a technique that allows for the re-generation of a DNA template in order to amplify it multiple times would be an extremely useful tool. This study outlines the development of a method that allows for the recursive amplification of a DNA sample. Amplification was performed using biotinylated primers for an STR locus and the resulting product was cleaned using streptavidin-coated magnetic beads to sequester the amplicons. Subsequent centrifugal filtration was used to remove the remaining PCR components, thus isolating the original genomic DNA. Re-amplification was then successfully performed at a different STR locus. Though successful, multiple run-throughs of the method indicated retention of signal from the original amplification as well as significant genomic DNA loss during the process. This study outlines experiments seeking to characterize the cause(s) of these imperfections in order to effectively direct method optimization. A computer generated dynamic model was also created and used to simulate the recursive amplification process to assist in development. When optimized, it is expected that recursive amplification can significantly reduce the difficulties associated with low-template DNA analysis and eradicate the concept of an ‘exhaustive’ DNA sample.

The DNA technology applied to forensic science has improved significantly in recent years. As a result of these advancements, DNA profiles can be generated from a low-template amount of DNA. This advancement, however, can lead to more complex mixtures due to the instrumentation picking up trace amounts of DNA. This leads to the need for these more challenging profiles to be interpreted. Due to the lack of standardization in DNA profile interpretation, one complex DNA profile is likely to draw several different conclusions from DNA analysts when assessing the number of contributors (NoC). Probabilistic genotyping software (PGS) is a possible solution to the problems of complex DNA profile interpretations. DNA-View® Mixture Solution™, developed by Dr. Charles Brenner, is a continuous modelling PGS that considers genotypes, peak height, stutter, dropout, and other artifacts that result from stochastic effects in the interpretation of DNA profiles. Mixture Solution has the potential to minimize some of the uncertainty inherent in DNA analysis of profiles having multiple contributors. In this project, DNA mixture analysis with Mixture Solution was applied to two and three-person mixtures having ratios ranging from 1:1 to 8:1 and 1:1:1 to 8:1:8. Two scenarios with several hypotheses were tested regarding each contributor as if they were the person of interest (POI) in a real case. Mixture Solution assigns the most favorable hypothesis for and against the POI and calculates an LR representing the comparison of those two hypotheses. In the final reports, trends previously observed in two-person mixture ratios were also observed in three-person mixtures. The main factor driving low LR assignment in three-person mixtures is low template DNA. Low peak heights and dropout are the factors driving low LR assignment. The factors that make manual DNA profile interpretation challenging can also challenge PGS. However, the robustness of Mixture Solution was demonstrated throughout the project with complex three-person mixtures.

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