Dissertations / Theses: 'SNPs genotyping'

1

Simmons, Stacy. "Genotyping for Response to Physical Training." Wright State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wright1565880927061102.

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2

Hammond, Naomi Rachel. "Improved approaches to multiplexed PCR and to the genotyping of SNPs by mass spectrometry." Thesis, University of Southampton, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.438509.

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3

Chacon, Cortes Diego Fernando. "Study of miRNA polymorphisms and their potential association with breast cancer risk in Australian Caucasian populations." Thesis, Queensland University of Technology, 2015. https://eprints.qut.edu.au/89768/15/89768%28thesis%29.pdf.

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This project established a large and well characterised prospective breast cancer DNA biobank and used this biobank to conduct genetic studies in breast cancer. The thesis presented the results of these high-throughput genotyping studies in two separate Australian Caucasian case-control populations and identified association between three novel genetic variants in microRNA genes and breast cancer risk.

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4

Merchant-Patel, Shreema. "Development of rapid and highly resolving combinatorial genotyping schemes for Campylobacter jejuni and Campylobacter coli." Thesis, Queensland University of Technology, 2009. https://eprints.qut.edu.au/33194/1/Shreema_Merchant-Patel_Thesis.pdf.

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Campylobacter jejuni followed by Campylobacter coli contribute substantially to the economic and public health burden attributed to food-borne infections in Australia. Genotypic characterisation of isolates has provided new insights into the epidemiology and pathogenesis of C. jejuni and C. coli. However, currently available methods are not conducive to large scale epidemiological investigations that are necessary to elucidate the global epidemiology of these common food-borne pathogens. This research aims to develop high resolution C. jejuni and C. coli genotyping schemes that are convenient for high throughput applications. Real-time PCR and High Resolution Melt (HRM) analysis are fundamental to the genotyping schemes developed in this study and enable rapid, cost effective, interrogation of a range of different polymorphic sites within the Campylobacter genome. While the sources and routes of transmission of campylobacters are unclear, handling and consumption of poultry meat is frequently associated with human campylobacteriosis in Australia. Therefore, chicken derived C. jejuni and C. coli isolates were used to develop and verify the methods described in this study. The first aim of this study describes the application of MLST-SNP (Multi Locus Sequence Typing Single Nucleotide Polymorphisms) + binary typing to 87 chicken C. jejuni isolates using real-time PCR analysis. These typing schemes were developed previously by our research group using isolates from campylobacteriosis patients. This present study showed that SNP + binary typing alone or in combination are effective at detecting epidemiological linkage between chicken derived Campylobacter isolates and enable data comparisons with other MLST based investigations. SNP + binary types obtained from chicken isolates in this study were compared with a previously SNP + binary and MLST typed set of human isolates. Common genotypes between the two collections of isolates were identified and ST-524 represented a clone that could be worth monitoring in the chicken meat industry. In contrast, ST-48, mainly associated with bovine hosts, was abundant in the human isolates. This genotype was, however, absent in the chicken isolates, indicating the role of non-poultry sources in causing human Campylobacter infections. This demonstrates the potential application of SNP + binary typing for epidemiological investigations and source tracing. While MLST SNPs and binary genes comprise the more stable backbone of the Campylobacter genome and are indicative of long term epidemiological linkage of the isolates, the development of a High Resolution Melt (HRM) based curve analysis method to interrogate the hypervariable Campylobacter flagellin encoding gene (flaA) is described in Aim 2 of this study. The flaA gene product appears to be an important pathogenicity determinant of campylobacters and is therefore a popular target for genotyping, especially for short term epidemiological studies such as outbreak investigations. HRM curve analysis based flaA interrogation is a single-step closed-tube method that provides portable data that can be easily shared and accessed. Critical to the development of flaA HRM was the use of flaA specific primers that did not amplify the flaB gene. HRM curve analysis flaA interrogation was successful at discriminating the 47 sequence variants identified within the 87 C. jejuni and 15 C. coli isolates and correlated to the epidemiological background of the isolates. In the combinatorial format, the resolving power of flaA was additive to that of SNP + binary typing and CRISPR (Clustered regularly spaced short Palindromic repeats) HRM and fits the PHRANA (Progressive hierarchical resolving assays using nucleic acids) approach for genotyping. The use of statistical methods to analyse the HRM data enhanced sophistication of the method. Therefore, flaA HRM is a rapid and cost effective alternative to gel- or sequence-based flaA typing schemes. Aim 3 of this study describes the development of a novel bioinformatics driven method to interrogate Campylobacter MLST gene fragments using HRM, and is called ‘SNP Nucleated Minim MLST’ or ‘Minim typing’. The method involves HRM interrogation of MLST fragments that encompass highly informative “Nucleating SNPS” to ensure high resolution. Selection of fragments potentially suited to HRM analysis was conducted in silico using i) “Minimum SNPs” and ii) the new ’HRMtype’ software packages. Species specific sets of six “Nucleating SNPs” and six HRM fragments were identified for both C. jejuni and C. coli to ensure high typeability and resolution relevant to the MLST database. ‘Minim typing’ was tested empirically by typing 15 C. jejuni and five C. coli isolates. The association of clonal complexes (CC) to each isolate by ‘Minim typing’ and SNP + binary typing were used to compare the two MLST interrogation schemes. The CCs linked with each C. jejuni isolate were consistent for both methods. Thus, ‘Minim typing’ is an efficient and cost effective method to interrogate MLST genes. However, it is not expected to be independent, or meet the resolution of, sequence based MLST gene interrogation. ‘Minim typing’ in combination with flaA HRM is envisaged to comprise a highly resolving combinatorial typing scheme developed around the HRM platform and is amenable to automation and multiplexing. The genotyping techniques described in this thesis involve the combinatorial interrogation of differentially evolving genetic markers on the unified real-time PCR and HRM platform. They provide high resolution and are simple, cost effective and ideally suited to rapid and high throughput genotyping for these common food-borne pathogens.

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5

Söderholm, Simon. "The Complex Genetics of Multiple Sclerosis : A preliminary study of MS-associated SNPs prior to a larger genotyping project." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-129423.

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Biomedical research have been revolutionized by recent technological advances, both in the fields of molecular biology and computer science, turning the biomolecular and genetic research into “big data science”. One of the main objectives have been to improve our understanding of complex human diseases. Among those diseases, multiple sclerosis (MS) is considered as one of the most common. MS is a chronic autoimmune disease that cause inflammation and damage to the central nervous system. In this study, a set of bioinformatics analyses have been conducted on SNP data, as an initial step to gain more information prior to an upcoming genotyping project. The results showed extensive regulatory properties for the 761 selected SNPs, which is consistent with current scientific knowledge, and also identified another 332 SNPs in linkage to these. However, during the study some issues have also been identified, which need to be addressed going forward.

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6

Carvalho, Thaysa Buss. "Avaliação de SNPs (Single Nucleotide Polymorphisms) nas diferentes formas clínicas da doença de Chagas." Universidade Estadual Paulista (UNESP), 2018. http://hdl.handle.net/11449/152931.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A doença de Chagas (DC), causada pelo protozoário Trypanosoma cruzi (T. cruzi), ainda é considerada como um problema de saúde pública em muitos países da América Latina. De acordo com a Organização Mundial da Saúde, estima-se que entre seis a sete milhões de pessoas no mundo estejam infectadas. Indivíduos na fase crônica da doença podem ser classificados como assintomáticos ou sintomáticos (estes, desenvolvendo as formas clínicas cardíaca, digestiva ou mista). Os assintomáticos correspondem a 70% dos indivíduos nessa fase e, embora apresentem sorologia positiva para anticorpos anti T-cruzi, não desenvolvem manifestações clínicas da doença. O motivo pelo qual alguns pacientes permanecem assintomáticos, e outros desenvolvem sintomas severos, ainda é desconhecido. Fatores genéticos do hospedeiro são bastante relevantes e podem explicar a heterogeneidade encontrada em pacientes que vivem com a doença em áreas endêmicas. Diante disso, o presente trabalho teve como objetivo avaliar SNPs (Single Nucleotide Polymorphisms) no gene TNF-α (rs1800629) e ACAT-1 (rs1044925) em indivíduos com DC crônica e verificar se os mesmos estão relacionados com a susceptibilidade para manifestação de formas clínicas sintomáticas com uso da técnica PCR-RFLP. Foram genotipadas 124 amostras para o gene TNF-α e 135 para o gene ACAT-1. Foi observada associação significativa da presença do alelo A do gene TNF- α em indivíduos sintomáticos em relação aos assintomáticos (p = 0,045). Também houve associação significativa entre o alelo G (p = 0,008) e o genótipo GG (p = 0,001) do gene TNF-α e os genótipos AA (p = 0,047) e AC (p = 0,016) do gene ACAT-1 nos indivíduos assintomáticos em relação aos sintomáticos. Nossos resultados sugerem que a presença do alelo A do gene TNF-α possa estar relacionada com a presença de manifestações clínicas sintomáticas na fase crônica da doença e o alelo G, bem como, genótipo GG possam estar associados com ausência de sintomas clínicos em indivíduos nessa fase. A respeito do SNP do gene ACAT-1, nossos dados sugerem efeito protetor dos genótipos AA e AC segundo apresentação de sintomas da doença na fase crônica, o que representa dado inédito em chagásicos.
Chagas disease (CD), caused by the protozoan Trypanosoma cruzi (T. cruzi), is still considered a public health problem in many Latin America countries. According to the World Health Organization, it is estimated that between six and seven million people worldwide are infected. Disease’s chronic phase individuals may be classified as asymptomatic or symptomatic (these, developing as clinical cardiac, digestive or mixed forms). Asymptomatic individuals account for 70% of the patients at this stage and, although they have positive serology for anti-T-cruzi antibodies, they do not develop it’s clinical manifestations. The reason why some patients remain asymptomatic, and others develop severe symptoms, is still unknown. Host’s genetic factors are quite relevant and may explain the heterogeneity found in patients living with the disease in endemic areas. The objective of this study was to evaluate SNPs in the TNF-α (rs1800629) and ACAT-1 (rs1044925) genes in individuals with chronic CD and to verify if the polymorphisms are related to the susceptibility to manifestation of symptomatic clinical forms using the PCR-RFLP technique. Were genotyped 124 samples for the TNF-α gene and 135 for the ACAT-1 gene. Significant association for the presence of the A allele of the TNF-α gene was observed for symptomatic individuals in relation to the asymptomatic ones (p = 0.045). There was also a significant association between the G allele (p = 0.008) and the GG genotype (p = 0.001) of the TNF-α gene and the AA (p = 0.047) and AC (p = 0.016) genotypes of the ACAT-1 gene for asymptomatic patients. Our results suggests that the presence of the TNF-α gene A allele may be related to the presence of symptomatic clinical manifestations in the chronic phase of the disease and the G allele as well as the GG genotype may be associated with absence of clinical symptoms in individuals at this stage. Regarding the ACAT-1 gene SNP, our data suggests a protective effect of AA and AC genotypes according to the to the presentation of chronic disease symptoms, which is an unprecedented finding in chagasic patients.
CAPES: 1578310

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7

Stephens, Alex J. "The development of rapid genotyping methods for methicillin-resistant Staphylococcus aureus." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/20172/1/Alexander_Stephens_Thesis.pdf.

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Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that is endemic in hospitals all over the world. It has more recently emerged as a serious threat to the general public in the form of community-acquired MRSA. MRSA has been implicated in a wide variety of diseases, ranging from skin infections and food poisoning to more severe and potentially fatal conditions, including; endocarditis, septicaemia and necrotising pneumonia. Treatment of MRSA disease is complicated and can be unsuccessful due to the bacterium's remarkable ability to develop antibiotic resistance. The considerable economic and public health burden imposed by MRSA has fuelled attempts by researchers to understand the evolution of virulent and antibiotic resistant strains and thereby improve epidemiological management strategies. Central to MRSA transmission management strategies is the implementation of active surveillance programs, via which unique genetic fingerprints, or genotypes, of each strain can be identified. Despite numerous advances in MRSA genotyping methodology, there remains a need for a rapid, reproducible, cost-effective method that is capable of producing a high level of genotype discrimination, whilst being suitable for high throughput use. Consequently, the fundamental aim of this thesis was to develop a novel MRSA genotyping strategy incorporating these benefits. This thesis explored the possibility that the development of more efficient genotyping strategies could be achieved through careful identification, and then simple interrogation, of multiple, unlinked DNA loci that exhibit progressively increasing mutation rates. The baseline component of the MRSA genotyping strategy described in this thesis is the allele-specific real-time PCR interrogation of slowly evolving core single nucleotide polymorphisms (SNPs). The genotyping SNP set was identified previously from the Multi-locus sequence typing (MLST) sequence database using an in-house software package named Minimum SNPs. As discussed in Chapter Three, the genotyping utility of the SNP set was validated on 107 diverse Australian MRSA isolates, which were largely clustered into groups of related strains as defined by MLST. To increase the resolution of the SNP genotyping method, a selection of binary virulence genes and antimicrobial resistance plasmids were tested that were successful at sub typing the SNP groups. A comprehensive MRSA genotyping strategy requires characterisation of the clonal background as well as interrogation of the hypervariable Staphylococcal Cassette Chromosome mec (SCCmec) that carries the β-lactam resistance gene, mecA. SCCmec genotyping defines the MRSA lineages; however, current SCCmec genotyping methods have struggled to handle the increasing number of SCCmec elements resulting from a recent explosion of comparative genomic analyses. Chapter Four of this thesis collates the known SCCmec binary marker diversity and demonstrates the ability of Minimum SNPs to identify systematically a minimal set of binary markers capable of generating maximum genotyping resolution. A number of binary targets were identified that indeed permit high resolution genotyping of the SCCmec element. Furthermore, the SCCmec genotyping targets are amenable for combinatorial use with the MLST genotyping SNPs and therefore are suitable as the second component of the MRSA genotyping strategy. To increase genotyping resolution of the slowly evolving MLST SNPs and the SCCmec binary markers, the analysis of a hypervariable repeat region was required. Sequence analysis of the Staphylococcal protein A (spa) repeat region has been conducted frequently with great success. Chapter Five describes the characterisation of the tandem repeats in the spa gene using real-time PCR and high resolution melting (HRM) analysis. Since the melting rate and precise point of dissociation of double stranded DNA is dependent on the size and sequence of the PCR amplicon, the HRM method was used successfully to identify 20 of 22 spa sequence types, without the need for DNA sequencing. The accumulation of comparative genomic information has allowed the systematic identification of key MRSA genomic polymorphisms to genotype MRSA efficiently. If implemented in its entirety, the strategy described in this thesis would produce efficient and deep-rooted genotypes. For example, an unknown MRSA isolate would be positioned within the MLST defined population structure, categorised based on its SCCmec lineage, then subtyped based on the polymorphic spa repeat region. Overall, by combining the genotyping methods described here, an integrated and novel MRSA genotyping strategy results that is efficacious for both long and short term investigations. Furthermore, an additional benefit is that each component can be performed easily and cost-effectively on a standard real-time PCR platform.

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8

Stephens, Alex J. "The development of rapid genotyping methods for methicillin-resistant Staphylococcus aureus." Queensland University of Technology, 2008. http://eprints.qut.edu.au/20172/.

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Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that is endemic in hospitals all over the world. It has more recently emerged as a serious threat to the general public in the form of community-acquired MRSA. MRSA has been implicated in a wide variety of diseases, ranging from skin infections and food poisoning to more severe and potentially fatal conditions, including; endocarditis, septicaemia and necrotising pneumonia. Treatment of MRSA disease is complicated and can be unsuccessful due to the bacterium's remarkable ability to develop antibiotic resistance. The considerable economic and public health burden imposed by MRSA has fuelled attempts by researchers to understand the evolution of virulent and antibiotic resistant strains and thereby improve epidemiological management strategies. Central to MRSA transmission management strategies is the implementation of active surveillance programs, via which unique genetic fingerprints, or genotypes, of each strain can be identified. Despite numerous advances in MRSA genotyping methodology, there remains a need for a rapid, reproducible, cost-effective method that is capable of producing a high level of genotype discrimination, whilst being suitable for high throughput use. Consequently, the fundamental aim of this thesis was to develop a novel MRSA genotyping strategy incorporating these benefits. This thesis explored the possibility that the development of more efficient genotyping strategies could be achieved through careful identification, and then simple interrogation, of multiple, unlinked DNA loci that exhibit progressively increasing mutation rates. The baseline component of the MRSA genotyping strategy described in this thesis is the allele-specific real-time PCR interrogation of slowly evolving core single nucleotide polymorphisms (SNPs). The genotyping SNP set was identified previously from the Multi-locus sequence typing (MLST) sequence database using an in-house software package named Minimum SNPs. As discussed in Chapter Three, the genotyping utility of the SNP set was validated on 107 diverse Australian MRSA isolates, which were largely clustered into groups of related strains as defined by MLST. To increase the resolution of the SNP genotyping method, a selection of binary virulence genes and antimicrobial resistance plasmids were tested that were successful at sub typing the SNP groups. A comprehensive MRSA genotyping strategy requires characterisation of the clonal background as well as interrogation of the hypervariable Staphylococcal Cassette Chromosome mec (SCCmec) that carries the β-lactam resistance gene, mecA. SCCmec genotyping defines the MRSA lineages; however, current SCCmec genotyping methods have struggled to handle the increasing number of SCCmec elements resulting from a recent explosion of comparative genomic analyses. Chapter Four of this thesis collates the known SCCmec binary marker diversity and demonstrates the ability of Minimum SNPs to identify systematically a minimal set of binary markers capable of generating maximum genotyping resolution. A number of binary targets were identified that indeed permit high resolution genotyping of the SCCmec element. Furthermore, the SCCmec genotyping targets are amenable for combinatorial use with the MLST genotyping SNPs and therefore are suitable as the second component of the MRSA genotyping strategy. To increase genotyping resolution of the slowly evolving MLST SNPs and the SCCmec binary markers, the analysis of a hypervariable repeat region was required. Sequence analysis of the Staphylococcal protein A (spa) repeat region has been conducted frequently with great success. Chapter Five describes the characterisation of the tandem repeats in the spa gene using real-time PCR and high resolution melting (HRM) analysis. Since the melting rate and precise point of dissociation of double stranded DNA is dependent on the size and sequence of the PCR amplicon, the HRM method was used successfully to identify 20 of 22 spa sequence types, without the need for DNA sequencing. The accumulation of comparative genomic information has allowed the systematic identification of key MRSA genomic polymorphisms to genotype MRSA efficiently. If implemented in its entirety, the strategy described in this thesis would produce efficient and deep-rooted genotypes. For example, an unknown MRSA isolate would be positioned within the MLST defined population structure, categorised based on its SCCmec lineage, then subtyped based on the polymorphic spa repeat region. Overall, by combining the genotyping methods described here, an integrated and novel MRSA genotyping strategy results that is efficacious for both long and short term investigations. Furthermore, an additional benefit is that each component can be performed easily and cost-effectively on a standard real-time PCR platform.

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9

Montes, Vergara Donicer Eduardo [UNESP]. "Prospecção de assinaturas de seleção em regiões de QTL associadas com características reprodutivas em novilhas Nelore." Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/137897.

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Características reprodutivas, como a ocorrência de prenhez precoce, são mais importantes economicamente ao comparar-se com as características de crescimento. Desta forma, o aumento da taxa de fertilidade e emprego de animais geneticamente superiores é determinante no progresso da produtividade nas fazendas comerciais de produção de carne bovina. A seleção modifica as frequências alélicas de uma população ao transmitir as variantes gênicas mais interessantes. Considerando o desequilíbrio de ligação, alguns locos adjacentes às mutações favoráveis são transmitidos ao longo das gerações. Estes são conhecidos como assinaturas de seleção e podem ser identificados com o uso de “chips” de SNP e metodologias estatísticas adequadas. Com o objetivo de identificar assinaturas de seleção recentes em QTL previamente mapeados para características reprodutivas de fêmeas bovinas ligadas à precocidade sexual, foram genotipadas 2.035 fêmeas da raça Nelore (Bos taurus indicus) com o chip “Illumina BovineHD BeadChip”. Posteriormente foi inferida a fase de ligação dos SNPs e a reconstrução dos haplótipos. A detecção de assinaturas de seleção foi realizada por meio da aplicação da metodologia “Relative Extended Haplotype Homozygosity” (REHH). A identificação de genes que contribuem para a importância da característica nestas regiões foi feita com a ferramenta Map Viewer do “National Center for Biotechnology Information”- NCBI e GBrowse carregada com o genoma bovino versão UMD 3.1. Foram detectadas 2.756 regiões núcleo, com tamanho médio 27,6 ± 29,1 Kb, abrangendo 70,1 Mb dos 25 cromossomos estudados. Dos SNPs utilizados, 17.312 participaram da formação das regiões núcleo, com o mínimo de 10 no BTA27 e o máximo de 20 SNPs nos cromossomos 1, 3-7, 9-15,18-21, e 23-24. Foram identificadas 40 assinaturas de seleção recentes com diferentes níveis de significância e 56 genes A maioria dos genes localizados nas regiões de assinaturas de seleção tem relação com os processos biológicos de metabolismo mitocondrial, desenvolvimento pós-embrionário, regulação da taxa de ovulação e fertilidade, resposta imune, metabolismo de triglicerídeo, proliferação celular e neurônios receptores olfativos. A investigação de mecanismos regulatórios da expressão dos genes associados aos processos biológicos descritos pode oferecer conhecimentos sobre os mecanismos moleculares que afetam a característica ocorrências de prenhez precoce, na raça Nelore.
Some reproductive traits such as early pregnancy are more profitable than those related to growth. Increasing fertility rate and using genetically superior animals are crucial in productivity of meat commercial farms. Artificial selection modifies allele frequencies of a cattle population by transmitting the most significant gene variants. Considering linkage disequilibrium, some loci adjacent to favorable mutations are transmitted across generations. Known as signatures of selection, such locations can be identified by the SNP chips, and appropriate statistical methods. To determine recent selection signature in quantitative trait loci (QTL) previously mapped for reproductive cow features linked to sexual precocity, 2,035 Nelore (Bos taurus indicus) females were genotyped by Illumina Bovine chip. After, inferring the connection phase of SNPs allowed haplotype reconstruction. Selection signatures were detected by Relative Extended Haplotype Homozygosity (REHH) method. Genes supposedly important were recognized by Map Viewer from the National Center for Biotechnology Information (NCBI), and also through a loaded GBrowse with bovine genome UMD, version 3.1. A total of 2,756 core regions were detected, with an average size of 27.6 ± 29.1 Kb, covering 70.1 Mb of 25 chromosomes. 17,312 SNPs are involved in the formation of core regions with at least 10 on BTA27, and a maximum of 20 SNPs on 1, 3-7, 9-15, 18-21, and 23-24 chromosomes. We identify 40 possible recent selection signatures, with different levels of significance, and 56 positional candidate genes. Most of genes located in selection signature regions are related to biological processes of mitochondrial metabolism, post-embryonic development, ovulation rate regulation and fertility, immune response, triglyceride metabolism, cell proliferation, and olfactory receptor neurons. The investigation of regulatory mechanisms of gene expression associated with biological processes described can provide knowledge on the molecular mechanisms affecting characteristic of early pregnancy occurrences in Nellore.

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Price, Erin Peta. "Development of novel combinatorial methods for genotyping the common foodborne pathogen Campylobacter jejuni." Thesis, Queensland University of Technology, 2007. https://eprints.qut.edu.au/16601/1/Erin_Peta_Price_Thesis.pdf.

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Campylobacter jejuni is the commonest cause of bacterial foodborne gastroenteritis in industrialised countries. Despite its significance, it remains unclear how C. jejuni is disseminated in the environment, whether particular strains are more pathogenic than others, and by what routes this bacterium is transmitted to humans. One major factor hampering this knowledge is the lack of a standardised method for fingerprinting C. jejuni. Therefore, the overall aim of this project was to develop systematic and novel genotyping methods for C. jejuni. Chapter Three describes the use of single nucleotide polymorphisms (SNPs) derived from the multilocus sequence typing (MLST) database of C. jejuni and the closely related Campylobacter coli for genotyping these pathogens. The MLST database contains DNA sequence data for over 4000 strains, making it the largest comparative database available for these organisms. Using the in-house software package "Minimum SNPs", seven SNPs were identified from the C. jejuni/C. coli MLST database that gave a Simpson's Index of Diversity (D), or resolving power, of 0.98. An allele-specific real-time PCR method was developed and tested on 154 Australian C. jejuni and C. coli isolates. The major advantage of the seven SNPs over MLST is that they are cheaper, faster and simpler to interrogate than the sequence-based MLST method. When the SNP profiles were combined with sequencing of the rapidly evolving flaA short variable region (flaA SVR) locus, the genotype distributions were comparable to those obtained by MLST-flaA SVR. Recent technological advances have facilitated the characterisation of entire bacterial genomes using comparative genome hybridisation (CGH) microarrays. Chapter Four of this thesis explores the large volume of CGH data generated for C. jejuni and eight binary genes (genes present in some strains but absent in others) were identified that provided complete discrimination of 20 epidemiologically unrelated strains of C. jejuni. Real-time PCR assays were developed for the eight binary genes and tested on the Australian isolates. The results from this study showed that the SNP-binary assay provided a sufficient replacement for the more laborious MLST-flaA SVR sequencing method. The clustered regularly interspaced short palindromic repeat (CRISPR) region is comprised of tandem repeats, with one half of the repeat region highly conserved and the other half highly diverse in sequence. Recent advances in real-time PCR enabled the interrogation of these repeat regions in C. jejuni using high-resolution melt differentiation of PCR products. It was found that the CRISPR loci discriminated epidemiologically distinct isolates that were indistinguishable by the other typing methods (Chapter Five). Importantly, the combinatorial SNP-binary-CRISPR assay provided resolution comparable to the current 'gold standard' genotyping methodology, pulsed-field gel electrophoresis. Chapter Six describes a novel third module of "Minimum SNPs", 'Not-N', to identify genetic targets diagnostic for strain populations of interest from the remaining population. The applicability of Not-N was tested using bacterial and viral sequence databases. Due to the weakly clonal population structure of C. jejuni and C. coli, Not-N was inefficient at identifying small numbers of SNPs for the major MLST clonal complexes. In contrast, Not-N completely discriminated the 13 major subtypes of hepatitis C virus using 15 SNPs, and identified binary gene targets superior to those previously found for phylogenetic clades of C. jejuni, Yersinia enterocolitica and Clostridium difficile, demonstrating the utility of this additional module of "Minimum SNPs". Taken together, the presented work demonstrates the potentially far-reaching applications of novel and systematic genotyping assays to characterise bacterial pathogens with high accuracy and discriminatory power. This project has exploited known genetic diversity of C. jejuni to develop highly targeted assays that are akin to the resolution of the current 'gold standard' typing methods. By targeting differentially evolving genetic markers, an epidemiologically relevant, high-resolution fingerprint of the isolate in question can be determined at a fraction of the time, effort and cost of current genotyping procedures. The outcomes from this study will pave the way for improved diagnostics for many clinically significant pathogens as the concept of hierarchal combinatorial genotyping gains momentum amongst infectious disease specialists and public health-related agencies.

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11

Price, Erin Peta. "Development of novel combinatorial methods for genotyping the common foodborne pathogen Campylobacter jejuni." Queensland University of Technology, 2007. http://eprints.qut.edu.au/16601/.

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Campylobacter jejuni is the commonest cause of bacterial foodborne gastroenteritis in industrialised countries. Despite its significance, it remains unclear how C. jejuni is disseminated in the environment, whether particular strains are more pathogenic than others, and by what routes this bacterium is transmitted to humans. One major factor hampering this knowledge is the lack of a standardised method for fingerprinting C. jejuni. Therefore, the overall aim of this project was to develop systematic and novel genotyping methods for C. jejuni. Chapter Three describes the use of single nucleotide polymorphisms (SNPs) derived from the multilocus sequence typing (MLST) database of C. jejuni and the closely related Campylobacter coli for genotyping these pathogens. The MLST database contains DNA sequence data for over 4000 strains, making it the largest comparative database available for these organisms. Using the in-house software package "Minimum SNPs", seven SNPs were identified from the C. jejuni/C. coli MLST database that gave a Simpson's Index of Diversity (D), or resolving power, of 0.98. An allele-specific real-time PCR method was developed and tested on 154 Australian C. jejuni and C. coli isolates. The major advantage of the seven SNPs over MLST is that they are cheaper, faster and simpler to interrogate than the sequence-based MLST method. When the SNP profiles were combined with sequencing of the rapidly evolving flaA short variable region (flaA SVR) locus, the genotype distributions were comparable to those obtained by MLST-flaA SVR. Recent technological advances have facilitated the characterisation of entire bacterial genomes using comparative genome hybridisation (CGH) microarrays. Chapter Four of this thesis explores the large volume of CGH data generated for C. jejuni and eight binary genes (genes present in some strains but absent in others) were identified that provided complete discrimination of 20 epidemiologically unrelated strains of C. jejuni. Real-time PCR assays were developed for the eight binary genes and tested on the Australian isolates. The results from this study showed that the SNP-binary assay provided a sufficient replacement for the more laborious MLST-flaA SVR sequencing method. The clustered regularly interspaced short palindromic repeat (CRISPR) region is comprised of tandem repeats, with one half of the repeat region highly conserved and the other half highly diverse in sequence. Recent advances in real-time PCR enabled the interrogation of these repeat regions in C. jejuni using high-resolution melt differentiation of PCR products. It was found that the CRISPR loci discriminated epidemiologically distinct isolates that were indistinguishable by the other typing methods (Chapter Five). Importantly, the combinatorial SNP-binary-CRISPR assay provided resolution comparable to the current 'gold standard' genotyping methodology, pulsed-field gel electrophoresis. Chapter Six describes a novel third module of "Minimum SNPs", 'Not-N', to identify genetic targets diagnostic for strain populations of interest from the remaining population. The applicability of Not-N was tested using bacterial and viral sequence databases. Due to the weakly clonal population structure of C. jejuni and C. coli, Not-N was inefficient at identifying small numbers of SNPs for the major MLST clonal complexes. In contrast, Not-N completely discriminated the 13 major subtypes of hepatitis C virus using 15 SNPs, and identified binary gene targets superior to those previously found for phylogenetic clades of C. jejuni, Yersinia enterocolitica and Clostridium difficile, demonstrating the utility of this additional module of "Minimum SNPs". Taken together, the presented work demonstrates the potentially far-reaching applications of novel and systematic genotyping assays to characterise bacterial pathogens with high accuracy and discriminatory power. This project has exploited known genetic diversity of C. jejuni to develop highly targeted assays that are akin to the resolution of the current 'gold standard' typing methods. By targeting differentially evolving genetic markers, an epidemiologically relevant, high-resolution fingerprint of the isolate in question can be determined at a fraction of the time, effort and cost of current genotyping procedures. The outcomes from this study will pave the way for improved diagnostics for many clinically significant pathogens as the concept of hierarchal combinatorial genotyping gains momentum amongst infectious disease specialists and public health-related agencies.

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Santos, Renato Oliveira dos. "Avaliação da importância dos genes PTPRM e IL1B na epilepsia de lopo temporal mesial com atrofia hipocampal através da genotipagem de SNPS." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/308726.

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Orientadores: Cláudia Vianna Maurer-Morelli, Íscia Lopes-Cendes
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: As epilepsias formam um grupo de doenças neurológicas crônicas caracterizadas por crises epilépticas, que podem ser definidas como um distúrbio intermitente do sistema nervoso causado por descarga elétrica anormal, súbita e sincronizada dos neurônios cerebrais. A epilepsia de lobo temporal mesial (ELTM) é o tipo de epilepsia mais frequente, representando aproximadamente 30% dos casos em adultos e tem como manifestação típica, a crise parcial complexa. Este tipo de acometimento é frequentemente refratário ao tratamento medicamentoso e os principais sintomas são gerados, predominantemente, pelo acometimento das estruturas mediais do lobo temporal. De fato, a relação entre ELTM e esclerose mesial temporal (EMT) vem de longa data, no entanto os mecanismos responsáveis por este tipo de achado ainda não estão bem esclarecidos. Recentemente, alguns genes ligados a processos inflamatórios, como a Interleucina-1 beta (IL1B) têm sido implicados na ELTM em humanos e em modelos animais. Outro fato importante é que foi identificada uma expressão diferencial para o gene PTPRM em tecido hipocampal ressecado cirurgicamente das ELTM refratárias apontando o gene PTPRM como um gene candidato para a epilepsia. A partir dessas observações foi investigada a associação dos genes IL1B e PTPRM com a ELTM associada à EMT. Foram selecionados 179 pacientes do HC-UNICAMP e 24 do HC da USP de Ribeirão Preto com diagnóstico estabelecido de ELTM com EH e 204 indivíduos saudáveis, sem histórico de epilepsia, como grupo controle. O estudo empregou a técnica SNPlex para a genotipagem de 119 SNPs no gene PTPRM e 7 SNPs no gene IL1B. Além desses, um SNP adicional para o gene IL1B foi genotipado por PCR e digestão enzimática. Através da análise dos dados foram encontrados 20 SNPs do gene PTPRM e um SNP do gene IL1B em associação com a ELTM com EMT . Ambos os genes possuem funções prévias estabelecidas e que sugerem sua participação no mecanismo fisiopatológico da doença. Embora maiores progressos tenham sido feitos com a caracterização de genes envolvidos em formas raras e monogênicas de epilepsia, as formas comuns e que mostram um padrão complexo ainda permanecem como um desafio para a identificação de genes. Neste aspecto, este estudo contribui com novos conhecimentos ao indicar os genes IL1B e PTPRM como genes de predisposição associados com a ELTM com sinais de EMT
Abstract: Epilepsies are a group of chronic neurological disease characterized by seizures; an intermittent disorder of the nervous system caused by an abnormal and synchronized electrical discharge of the neurons. Mesial temporal lobe epilepsy (MTLE) is the most common form of epilepsy, representing approximately 30% of cases in adults and has the complex partial seizure as a typical manifestation. MTLE is frequently related with medically refractory seizures and the main symptoms are predominantly generated by medial temporal lobe structures. In fact, the relationship between MTLE and mesial temporal sclerosis (MTS) is well established, however the mechanisms responsible for this finding are poorly understood. Recently, genes linked to inflammatory processes, such as IL1B has been involved with MTLE in humans and in animal models. In addition, we have identified a differential expression in human hippocampi that were surgically extracted from refractory MTLE for the PTPRM gene. Since we had these observations, we investigated the association of the IL1B and PTPRM genes and MTLE associated with HS. One hundred seventy nine patients were selected from HC-UNICAMP and 24 patients from HC-USP Ribeirão Preto, diagnosed with MTLE with mangnetic resonance imaging (MRI) signs of MTS and 204 healthy individuals with no history of epilepsy, to compose the control group. To this study we employed the SNPlex system to genotype 119 SNPs in PTPRM gene and seven SNPs in the IL1B gene. Besides, one additional SNP in the IL1B gene was genotyped by PCR and enzymatic digestion. Twenty SNPs in the PTPRM gene and one SNP in the IL1B gene were found in association with the MTLE with MRI signs of MTS. Our association study shows that there is a relationship between IL1B as well PTPRM genes and MTLE. Although much progress has been made in the characterization of genes for the monogenic and rare forms of epilepsy the common epilepsy syndromes, usually showing complex inheritance remain a major challenge for gene identification. In this way, our study hopes to shed some light into this area
Mestrado
Ciencias Biomedicas
Mestre em Ciências Médicas

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Jardim, Priscila Magalhães da Veiga. "Mapeamento genético de marcadores SNPs (Single Nucleotide Polymorphisms) em cana-de-açúcar (Saccharum spp.)." Universidade Federal de Goiás, 2018. http://repositorio.bc.ufg.br/tede/handle/tede/9009.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Sugarcane is an important culture quite relevant to the Brazilian economy. The production is growing as well as a cultivated area is increasing every year. The genome of this culture is still deficient due to complications such as high ploidy and the big genome that presents. Among the different genetic characterization studies, the development of genetic maps is important for providing information about the genome structure of a species. In addition, it can help develop the techniques of interpretation and use of genetic information. They provide more understanding of how genetic information is organized in the genome of sugarcane supplying a lack in the basic element in this culture. The maps constructed for sugarcane, so far, not shown saturated. This work was obtained a map for sugarcane using SNP markers based on genotyping-by-sequencing technology in next-generation sequencing using the target sequencing strategy (RAPiD-Seq). For obtaining the map, 103 clones RB97327 and RB72454 were used. Probes were designed based on sequence similarity using the sorghum genome as a reference. The construction of the binding groups, considering as a binding criterion of a recombination fraction equal 0.20; allowed the identification of 249 binding groups for the biparental population with 1: 1 segregation. A total of 20555 polymorphic were scored in the analysis. The sum of the average sizes of homeologia groups identified, using the sorghum genome as a reference, was 3964.68 cM for the female parent and 3797.05 cM for the male parent.
A cana-de-açúcar é uma cultura de importância bastante relevante para a economia brasileira. A produção de cana-de-açúcar no Brasil é crescente assim como a área cultivada vem aumentando a cada ano. A compreensão do genoma da cana-de-açúcar ainda é deficiente devido a complicações como alta ploidia e o grande genoma que a cultura apresenta. Dentre os diferentes estudos de caracterização genética, o desenvolvimento de mapas genéticos é importante por fornecer informações acerca da estrutura do genoma de uma espécie. Além disso, pode auxiliar no desenvolvimento das técnicas de interpretação e uso da informação genética. Eles possibilitam a compreensão mais abrangente da organização da informação genética no genoma da cana-de-açúcar suprindo uma carência do estudo básico sobre essa cultura. Os mapas construídos para cana-de-açúcar, até agora, não se mostraram completos. Neste trabalho foi obtido um mapa de ligação para cana-de-açúcar utilizando marcadores SNPs baseados na tecnologia de genotipagem por sequenciamento de nova geração utilizando a estratégia de target sequencing (RAPiD-Seq). Para a obtenção do mapa foram utilizados 103 genótipos obtidos do cruzamento entre os clones RB97327 e RB72454. Foram desenhadas sondas baseadas em sequenciamento de semelhança utilizando o genoma de sorgo como referência. A construção dos grupos de ligação, considerando-se como critério de ligação uma fração de recombinação de 0,20; permitiu a identificação de 249 grupos de ligação para a população biparental com segregação 1:1. Foram consideradas 20555 marcas polimórficas nas análises de construção do mapa de ligação. A soma dos tamanhos médios dos grupos de homeologia identificados, utilizando-se o genoma de sorgo como referência, foi de 3964,68 cM para o genitor feminino e de 3797,05 cM para o genitor masculino.

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Pantalião, Gabriel Feresin. "Mapeamento associativo para produtividade em arroz sob déficit hídrico." Universidade Federal de Goiás, 2013. http://repositorio.bc.ufg.br/tede/handle/tede/4431.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Drought is an environmental factor which narrows crop production, such as upland rice (Oryza sativa L.). The knowledge of aspects related to drought stress, and plant response to it, may furnish plant breeding programs essential data for the development of tolerant cultivars, and hence with higher yields under such conditions. Association mapping has been a successful approach to elucidate the genetic basis of economically important traits in plants, and afterward in the implementation of marker assisted selection (MAS). Next-generation sequencing (NGS) technologies have been applied in a variety of contexts, including SNP identification and development. Among methodologies for marker discovery and high-throughput genotyping, GBS (Genotyping by Sequencing) points out by its low cost and speed at which samples can be analyzed. The aim of this work was to identify, by GBS, the polymorphism from SNP markers within 283 upland accessions from Embrapa Rice Core Collection (ERiCC) and associate them to yield under drought stress. After filtering the raw data of predetermined stringent parameters, 285.379 SNP were identified in the 12 rice chromosomes. For the association mapping, molecular and phenotypic data were combined for the identification of SNP associated to drought, aiming the subsequent development of a marker set for MAS besides the identification of genes for genetic engineering. The analysis identified 48 SNP associated with the evaluated traits, 13 associated to drought susceptibility index (DSI) and 35 to yield under drought stress. Among the 48 SNP, 35 was anchored in 31 rice genes. Seven genes, out of the 31, possessed SNP associated to DSI, and the other 24 genes to yield under drought stress. These genes may be evaluated to be effectively employed for MAS. If the overexpression of such genes provides an enhanced drought tolerance, they may be used in the development of tolerant rice cultivars.
A seca é um fator ambiental que limita a produção das culturas, como a do arroz de terras altas (Oryza sativa L.). O conhecimento de fatores envolvidos na tolerância à deficiência hídrica e das respostas das plantas a esse estresse podem fornecer subsídios aos programas de melhoramento para o desenvolvimento de cultivares tolerantes, e, consequentemente, com uma maior produtividade sob essas condições. O mapeamento associativo, ou análise de associação, tem sido aplicado com sucesso em plantas, sendo utilizado primeiramente na identificação de genes associados a caracteres de importância econômica, e posteriormente, na implementação de seleção assistida por marcadores (SAM). Tecnologias de sequenciamento de nova geração (NGS) têm sido recentemente utilizadas em projetos de sequenciamento e resequenciamento para identificar, validar e avaliar um grande número de SNPs, os quais podem ser utilizados em estudos de mapeamento associativo. Dentre os métodos desenvolvidos para a descoberta de marcadores moleculares e genotipagem de alto desempenho, destaca-se pela rapidez e baixo custo a genotipagem por sequenciamento (GBS). Esse trabalho objetivou detectar, via GBS, o polimorfismo de marcadores SNPs em 283 acessos de arroz de terras altas componentes da CNAE (Coleção Nuclear de Arroz da Embrapa) e associá-los à produtividade sob déficit hídrico. Após a filtragem dos dados brutos de acordo com parâmetros de estringência pré-definidos, foram contabilizados 285.379 SNPs distribuídos ao longo dos 12 cromossomos do arroz. As informações moleculares foram integradas aos dados fenotípicos derivados do experimento de avaliação de produtividade e Índice de Suscetibilidade à Seca (ISS), conduzido no ano de 2010 em Porangatu (GO) em ambiente com e sem deficiência hídrica, para possibilitar a análise de mapeamento associativo, e com isso, detectar marcadores SNPs relacionados à tolerância à seca e oportunizar o desenvolvimento de um conjunto de marcadores úteis para a seleção assistida para esse caráter, assim como genes para estudos de engenharia genética do arroz. Através da análise de associação, foram detectados 48 SNPs relacionados com os caracteres avaliados, dentre os quais 13 foram relacionados ao ISS e 35 à produtividade em condição de déficit hídrico. Dentre os 48 SNPs, foram identificados 35 SNPs ancorados em 31 genes de arroz. Dentre os genes identificados, sete deles continham SNPs associados ao ISS, enquanto que os restantes 24 genes continham SNPs associados à produtividade dos acessos em ambiente com deficiência hídrica. Esses genes podem ser avaliados para serem efetivamente utilizados na seleção assistida por marcadores. Adicionalmente, esses genes podem ser superexpressos para avaliar sua capacidade de aumentar a tolerância à seca, e em caso positivo, gerar cultivares comerciais de arroz geneticamente modificadas mais tolerantes a esse estresse.

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Rathnayake, Irani Udeshika. "Molecular characterisation of environmental enterococci derived from water samples and assessment of associated public health hazards." Thesis, Queensland University of Technology, 2012. https://eprints.qut.edu.au/52631/1/Irani_Rathnayake_Thesis.pdf.

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Enterococci are versatile Gram-positive bacteria that can survive under extreme conditions. Most enterococci are non-virulent and found in the gastrointestinal tract of humans and animals. Other strains are opportunistic pathogens that contribute to a large number of nosocomial infections globally. Epidemiological studies demonstrated a direct relationship between the density of enterococci in surface waters and the risk of swimmer-associated gastroenteritis. The distribution of infectious enterococcal strains from the hospital environment or other sources to environmental water bodies through sewage discharge or other means, could increase the prevalence of these strains in the human population. Environmental water quality studies may benefit from focusing on a subset of Enterococcus spp. that are consistently associated with sources of faecal pollution such as domestic sewage, rather than testing for the entire genus. E. faecalis and E. faecium are potentially good focal species for such studies, as they have been consistently identified as the dominant Enterococcus spp. in human faeces and sewage. On the other hand enterococcal infections are predominantly caused by E. faecalis and E. faecium. The characterisation of E. faecalis and E. faecium is important in studying their population structures, particularly in environmental samples. In developing and implementing rapid, robust molecular genotyping techniques, it is possible to more accurately establish the relationship between human and environmental enterococci. Of particular importance, is to determine the distribution of high risk enterococcal clonal complexes, such as E. faecium clonal complex 17 and E. faecalis clonal complexes 2 and 9 in recreational waters. These clonal complexes are recognized as particularly pathogenic enterococcal genotypes that cause severe disease in humans globally. The Pimpama-Coomera watershed is located in South East Queensland, Australia and was investigated in this study mainly because it is used intensively for agriculture and recreational purposes and has a strong anthropogenic impact. The primary aim of this study was to develop novel, universally applicable, robust, rapid and cost effective genotyping methods which are likely to yield more definitive results for the routine monitoring of E. faecalis and E. faecium, particularly in environmental water sources. To fullfill this aim, new genotyping methods were developed based on the interrogation of highly informative single nucleotide polymorphisms (SNPs) located in housekeeping genes of both E. faecalis and E. faecium. SNP genotyping was successfully applied in field investigations of the Coomera watershed, South-East Queensland, Australia. E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles respectively. This study showed the high longitudinal diversity of E. faecalis and E. faecium over a period of two years, and both human-related and human-specific SNP profiles were identified. Furthermore, 4.25% of E. faecium strains isolated from water was found to correspond to the important clonal complex-17 (CC17). Strains that belong to CC17 cause the majority of hospital outbreaks and clinical infections globally. Of the six sampling sites of the Coomera River, Paradise Point had the highest number of human-related and human-specific E. faecalis and E. faecium SNP profiles. The secondary aim of this study was to determine the antibiotic-resistance profiles and virulence traits associated with environmental E. faecalis and E. faecium isolates compared to human pathogenic E. faecalis and E. faecium isolates. This was performed to predict the potential health risks associated with coming into contact with these strains in the Coomera watershed. In general, clinical isolates were found to be more resistant to all the antibiotics tested compared to water isolates and they harbored more virulence traits. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). However, tetracycline, gentamicin, ciprofloxacin and ampicillin resistance was observed in water isolates. The virulence gene esp was the most prevalent virulence determinant observed in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium), and this gene has been described as a human-specific marker used for microbial source tracking (MST). The presence of esp in water isolates (16.36% of E. faecalis and 19.14% of E. faecium) could be indicative of human faecal contamination in these waterways. Finally, in order to compare overall gene expression between environmental and clinical strains of E. faecalis, a comparative gene hybridization study was performed. The results of this investigation clearly demonstrated the up-regulation of genes associated with pathogenicity in E. faecalis isolated from water. The expression study was performed at physiological temperatures relative to ambient temperatures. The up-regulation of virulence genes demonstrates that environmental strains of E. faecalis can pose an increased health risk which can lead to serious disease, particularly if these strains belong to the virulent CC17 group. The genotyping techniques developed in this study not only provide a rapid, robust and highly discriminatory tool to characterize E. faecalis and E. faecium, but also enables the efficient identification of virulent enterococci that are distributed in environmental water sources.

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Cabral, Cléia Santos. "Análise genética da resistência a Fusarium oxysporum f. sp. lactucae raça 1 em alface : aplicação de marcadores do tipo RGA e de SNPs derivados de genotyping-by-sequencing." reponame:Repositório Institucional da UnB, 2016. http://repositorio.unb.br/handle/10482/22462.

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Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, Programa de Pós-Graduação em Fitopatologia, 2016.
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A alface (Lactuca sativa L.) é uma das hortaliças mais importantes no Brasil e no mundo. A murcha de fusário (causada por distintas raças do fungo Fusarium oxysporum f. sp. lactucae – FOLAC) é uma das principais doenças de da alface em regiões tropicais e subtropicais. Devido à dificuldade de implementação de estratégias eficazes de controle químico e cultural, o uso de cultivares com resistência genética é o método mais prático de manejo da doença. Fontes estáveis de resistência à raça FOLAC 1 foram encontradas, mas a base genética dessa característica ainda se encontra mal caracterizada. A integração da seleção assistida por marcadores (SAM) em programas de melhoramento convencional irá acelerar o desenvolvimento e lançamento de cultivares de alface com resistência genética a esse patógeno. O presente estudo teve como objetivos: elucidar os fatores genéticos associados à resistência a murcha de fusário (Capítulo 2) e idenficar marcadores moleculares potencialmente ligados aos fatores de resistência a isolados de FOLAC raça 1 identificados na cultivar ‘Vanda’ (Capítulo 3). O estudo da herança genética da resistência a FOLAC raça 1 foi conduzido com populações segregantes (F2 e F3) obtidas do cruzamento entre um parental suscetível ‘Gizele’ e ‘Vanda’. Populações foram inoculadas com uma suspensão de esporos do patógeno (3 x 106 microconídios/mL) por meio de corte e imersão de raízes e avaliadas quanto à resistência por meio de escala de notas. O DNA genômico das plantas resistentes e suscetíveis foi extraído e usado como molde em diferentes sistemas de marcadores (RAPD, SCAR, DR analogs, SRR e CAPS), visando identificar polimorfismos ligados a essa característica. Com relação à resistência a FOLAC raça 1, os estudos indicaram um controle genético relativamente simples na cultivar ‘Vanda’, com os resultados de segregação indicando um locus monogênico com uma provável combinação de efeitos de dosagem e penetrância incompleta. No entanto, os diferentes sistemas de marcadores moleculares utilizados nessa primeira etapa do trabalho não permitiram encontrar polimorfismos com estreita ligação com o(s) fator(es) de resistência. Desta forma, uma nova etapa do trabalho foi conduzida visando identificar por meio do método genotyping-by-sequencing (GBS) marcadores moleculares ligados a resistência (Capítulo 4). O GBS foi explorado na identificação de SNPs (single nucleotide polymorphisms) empregando DNA extraído dos dois parentais e de um conjunto de 82 indivíduos da população F2 derivados do cruzamento entre ‘Vanda’ x ‘Gizele’. Cada indivíduo foi genotipado utilizando o Illumina Hiseq 3000, que produziu 4,5 milhões de reads por amostra. Um total de 10.017 SNPs foi identificado entre os parentais. Estes SNPs foram avaliados para ligação com o fenótipo de resistência nos 82 indivíduos F2. Os dados genotípicos foram condensados em 1.484 scaffolds ou supercontigs. Um subconjunto de 417 scaffolds contendo polimorfismos foi selecionado para a construção de um mapa de ligação após filtragem baseada em missing data (< 20%), teste de qui-quadrado (3:1 p>0,05) e número de SNPs por scaffold. O mapa foi composto por 17 grupos de ligação, com um comprimento total de 1.132,984 cM. A análise de QTL (quantitative trait loci) para resistência a FOLAC raça 1 foi realizada no mapa de ligação com os conjuntos de dados de severidade da doença obtido nas populações F2 e F3. Dois QTLs de efeito maior foram identificados no cromossomo 9, explicando 30 a 40% da variação fenotípica observada na população F3 e F2, respectivamente. Um QTL de efeito menor foi detectado no cromossomo 4, explicando 0,06% da variação fenotípica na população F3. Desta forma, o presente estudo estabelece a porção mediana do cromossomo 9 como sendo a localização física do principal locus de resistência para FOLAC raça 1 presente na cultivar ‘Vanda’. Portanto, os marcadores moleculares localizados na proximidade desta região genômica são candidatos para o desenvolvimento de ferramentas de SAM em programas de melhoramento genético visando incorporar essa característica em linhagens elite de alface.
Lettuce (Lactuca sativa L.) is one of the most important leafy vegetable crops in Brazil and worldwide. Fusarium wilt (caused by distinct races of the fungus Fusarium oxysporum f. sp. lactucae – FOLAC) is one of the main soil-borne diseases of lettuce in tropical and subtropical regions. Due to the complexity of implementing effective cultural and chemical control, the most practical disease management strategy has been the use of cultivars with genetic resistance. Stable sources of resistance to FOLAC race 1 have been found, but the genetic basis of this trait is yet poorly characterized. The integration of marker assisted selection (MAS) in conventional breeding programs would be an important contribution to accelerate the development and release of lettuce cultivars with genetic resistance to this pathogen. In this context, the present study aimed to elucidate the inheritance of resistance to FOLAC race 1 detected in the cultivar Vanda (Chapter II) and to search for molecular markers linked to FOLAC race 1 resistance factor(s) (Chapter III). Inheritance studies were carried out using segregating populations (F2 and F3 families) derived from the cross between a susceptible cultivar (Gizele) and a FOLAC race 1 resistant cultivar (Vanda). The parental lines and the segregating populations were inoculated via root dipping technique with a conidial suspension adjusted to 3 x 106 microconidia/mL. Reaction to one FOLAC race 1 isolate was evaluated using a disease rating scale ranging from 1 (= no symptoms) to 5 (= dead plant). The studies indicated a simple genetic control of FOLAC race resistance in cultivar Vanda, with segregation results indicating a single gene locus with a likely combination of dosage effects and incomplete penetrance. Genomic DNA of resistant and susceptible plants was extracted and used as a template in PCR assays with different marker systems (RAPD, SCAR, DR Analogs, SSRs and CAPS) aiming to identify polymorphisms linked to the resistant reaction. However, none of the evaluated molecular techniques were able to identify markers in close linkage with the resistance factor(s). Therefore, a new phase of the present study was conducted with the objective of searching for markers linked to resistance through the employment of the genotyping-by-sequencing (GBS) strategy (Chapter IV). The GBS strategy was employed using DNA extracted from the parental lines and 82 phenotyped F2 individuals derived from the same cross between Gizele x Vanda. Each individual was genotyped using the Illumina Hiseq 3000. A total of 4.5 million reads was obtained per sample with 10,017 SNPs being identified among the parental lines as well as among the 82 F2 individuals. Genotypic data were condensed into 1484 scaffolds. Four hundred seventeen (417) scaffolds containing polymorphisms were selected for the construction of the linkage map after filtering based on missing data (< 20%), chi-square test (3: 1 p> 0.05) and number of SNPs per scaffold. The final map consisted of 17 linking groups with a total length of 1,132.984 cM. The QTL analysis for resistance to FOLAC race 1 was performed on the linkage map based upon the disease severity datasets of F2 population and F3 families. Two major effect QTLs were identified on chromosome 9, explaining 30 to 40% of the phenotypic variation observed in the population F3 and F2, respectively. One QTL of minor effect was also identified on chromosome 4, explaining 0.06% of the phenotypic variation in the F3 population. This study establishes the central region of chromosome 9 as the physical location of a major resistance locus to FOLAC race 1 found in Vanda. Molecular markers in close proximity to this genomic region are candidates for the development of MAS tools for breeding programs aiming to incorporate this trait into elite lettuce lines.

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MESQUITA, Adriano Queiroz de. "Associação entre polimorfismos de nucleotídeo único (SNPs) no gene codificador do Toll-like receptor 4 (TLR4) e contagem celular somática." Universidade Federal de Goiás, 2010. http://repositorio.bc.ufg.br/tede/handle/tde/831.

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A mastite tem sido considerada, mundialmente, a doença de maior impacto nos rebanhos leiteiros, devido à elevada prevalência e aos prejuízos econômicos que determina. As desordens decorrentes da mastite por agente etiológico de origem bacteriana são complexas, dependentes do microrganismo envolvido, e desencadeiam inúmeros processos de reconhecimento. As estruturas moleculares dos microrganismos são conhecidas como padrões moleculares associados aos patógenos (PAMPS) e os receptores nas células do hospedeiro como receptores de reconhecimento de padrões (PRR). O presente trabalho foi desenvolvido com o objetivo de identificar a presença de polimorfismos de nucleotídeo único no gene codificador do TLR4 em vacas leiteiras da raça holandesa em uma propriedade leiteira em Goiás, avaliando a relação dos alelos identificados, com a ocorrência de mastite subclínica e contagem celular somática. Foram coletadas 150 amostras de leite individual de vacas para identificação de microrganismos, contagem celular somática e composição centesimal, e 150 amostras de sangue para genotipagem em uma propriedade rural do Estado de Goiás. A discriminação alélica foi realizada por meio da técnica de PCR em tempo real, baseada em 4 SNPs de referência no gene codificador do TLR4 depositados no NCBI (rs8193046, rs8193047, rs8193060 e rs29017188). Os resultados obtidos revelam maior frequência de microrganismos Gram negativos na propriedade de estudo (52,47%) e que, animais identificados com os genótipos AACCCC, GGTCGG e GACCGC são os mais indicados para seleção assistida por marcadores moleculares.
Mastitis has been considered, worldwide, the disease of greatest impact in dairy herds because of the high prevalence and the economic losses that determines. The disorders caused by mastitis causative agent of bacterial origin are complex, depending on the microrganism involved, and trigger numerous processes of recognition. The molecular structures of microrganisms are known as Pathogen- Associated Molecular Patterns (PAMPs) and the receptors on host cells as pattern recognition receptors (PRR). This study was developed with the aim of identifying the presence of single nucleotide polymorphisms in TLR4 in Holstein dairy cows on a dairy farm in Goiás, evaluating the relationship between identified alleles, occurrence of subclinical mastitis and somatic cell count. 150 milk samples from individual cows were collected for identification of microrganisms, somatic cell count and composition, and 150 blood samples for genotyping on a farm in the State of Goiás. The allelic discrimination was performed by Real-time PCR, based on four reference SNPs in TLR4 gene from NCBI (rs8193046, rs8193047, rs8193060 and rs29017188). The results showed higher frequency of Gram negative microrganisms (52.47%) and that animals with the genotypes AACCCC, GGTCGG GACCGC are best suited for marker-assisted selection.

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Rayner, Rachael E. "Investigating the population structure of Queensland invasive Streptococcus pneumoniae isolates in children: Using a modified multi-locus variable number of tandem repeat analysis and a novel minimum SNPs capsular typing method." Thesis, Queensland University of Technology, 2015. https://eprints.qut.edu.au/87096/1/Rachael_Rayner_Thesis.pdf.

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This research investigated the use of DNA fingerprinting to characterise the bacteria Streptococcus pneumoniae or pneumococcus, and hence gain insight into the development of new vaccines or antibiotics. Different bacterial DNA fingerprinting methods were studied, and a novel method was developed and validated, which characterises different cell coatings that pneumococci produce. This method was used to study the epidemiology of pneumococci in Queensland before and after the introduction of the current pneumococcal vaccine. This study demonstrated that pneumococcal disease is highly prevalent in children under four years, that the bacteria can `switch' its cell coating to evade the vaccine, and that some DNA fingerprinting methods are more discriminatory than others. This has an impact on understanding which strains are more prone to cause invasive disease. Evidence of the excellent research findings have been published in high impact internationally refereed journals.

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Maia, Mayara dos Santos. "Estudo da associação de SNPs dos genes do mecanismo de reparo por excisão de nucleotídeo em carcinoma basocelular no Estado da Paraíba." Universidade Federal da Paraíba, 2017. http://tede.biblioteca.ufpb.br:8080/handle/tede/9441.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Basal Cell Carcinoma (BCC) is a frequent neoplasm in humans and its main etiological factor is exposure to solar radiation. Although genetic and epigenetic changes can activate proto-oncogenes, inactivate tumor suppressor genes and repair mechanism genes, the cell has several mechanisms that contribute to the maintenance of genomic stability. Mutations in repair genes can lead to tumor progression and loss of genome integrity leading to the onset of cancer. Nucleotide excision repair (NER) is an important mechanism primarily used to repair injuries caused by UV. The objective of this study was to evaluate single nucleotide polymorphisms (SNP) of XPA and XPC genes and the risk of developing BCC. One hundred samples of paraffined tissue from patients from the State of Paraíba with histopathological diagnosis of BCC were analyzed for each polymorphism. The results were obtained by a newly developed genotyping method, the Dideoxy Unique Allele Specific - PCR, a method that presents high sensitivity and low cost. Graphpad Prism 6.01 software was used for the statistical analysis and application of Chi-square and Fisher's exact test. The SNP rs535425175 of the XPC gene showed a significant association with the BCC in the analyzed samples (X2 = 14.51 and P <0.005). Whereas the SNPs rs745769173 of the XPA gene and rs761106780 of the XPC gene are in the Hardy-Weinberg equilibrium, not showing any association with the neoplasia. The results suggest that the SNP rs535425175 of the XPC gene may be considered a risk factor associated with the development of BCC.
O Carcinoma Basocelular (CBC) é uma neoplasia frequente em seres humanos e seu principal fator etiológico é a exposição à radiação solar. Embora alterações genéticas e epigenéticas possam ativar proto-oncogenes, inativar genes supressores de tumor e genes do mecanismo de reparo, a célula apresenta vários mecanismos que contribuem para a manutenção da estabilidade genômica. Mutações em genes de reparo podem levar a progressão tumoral e à perda da integridade do genoma levando ao surgimento do câncer. O reparo por excisão de nucleotídeo (NER) é um importante mecanismo utilizado principalmente para reparar lesões causadas por UV. O objetivo deste trabalho foi avaliar polimorfismos de nucleotídeo único (SNP) dos genes XPA e XPC e o risco de desenvolver CBC. Foram analisadas 100 amostras de tecido parafinado de pacientes do Estado da Paraíba com diagnóstico histopatológico de CBC para cada polimorfismo. Os resultados foram obtidos por um método de genotipagem recentemente desenvolvido, o Didesoxi Único Alelo Específico – PCR, método que apresenta alta sensibilidade e de baixo custo. O software Graphpad Prism 6.01 foi utilizado para as análises estatísticas e aplicação de teste Qui-quadrado e Exato de Fisher. O SNP rs535425175 do gene XPC apresentou associação significativa com o CBC nas amostras analisadas (X2=14,51 e P<0,005). Enquanto que os SNP rs745769173 do gene XPA e rs761106780 do gene XPC estão no equilíbrio de Hardy-Weinberg, não apresentando associação com a neoplasia. Os resultados sugerem que o SNP rs535425175 do gene XPC pode ser considerado um fator de risco associado ao desenvolvimento de CBC. Palavras-chaves: Carcinoma Basocelular, Família XP, Reparo por excisão de nucleotídeo, Polimorfismo de nucleotídeo único, Genotipagem. VII

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20

Du, Plessis Jana. "Medium-throughput SNP genotyping and linkage mapping in Haliotis midae." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/71701.

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Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Haliotis midae (locally also known as perlemoen) is the largest of five endemic species found along the coast of South Africa. It is the only species with commercial value contributing to the exploitation of these animals. Due to declines of natural stocks, farming practices were established during the early 1990s in order to supply the international demand. To facilitate efficient breeding methods and ensure the sustainability of these commercial populations, genetic management, which can be accomplished with the use of molecular markers such as single nucleotide polymorphisms (SNPs), is necessary. Single nucleotide polymorphisms have become the markers of choice in various applications in aquaculture genetics due to their abundance in genomes, reduction in developmental costs and increased throughput of genotyping assays. Identification of SNPs in non-model species such as H. midae can be achieved by in silico approaches. In silico methods are suitable for de novo SNP identification and are both cost- and time-efficient. It is based on the analysis of multiple alignments where mismatches may be reported as candidate SNPs. Various medium-throughput genotyping methods are available to confirm putative SNPs, but the ideal method depends on factors such as cost, accuracy and multiplexing capacity. Although SNP markers can have various applications within the aquaculture environment the focus for this current study was saturating the linkage map of H. midae with additional markers. This would assist in the identification of quantitative trait loci associated with economically important traits, which in turn could ultimately be employed for marker-assisted selection and improved molecular breeding programs. In order to identify in silico SNPs, sequenced transcriptome data from a previous study was used and subjected to a series of criteria: minor allele frequency 10%, minimum coverage 80, 60 bp flanking regions. Selected loci were genotyped using a 192-plex assay with the Illumina GoldenGate genotyping assay with the VeraCode technology on the BeadXpress platform, in individuals from six mapping families. A conversion rate of 69.35% and global success rate of 76.34% was achieved. Polymorphic loci were subjected to linkage analysis using JoinMap® v.4.1 to create sex-average and sex-specific maps and to saturate the current linkage map for H. midae. Along with previously developed markers, 54% of the newly developed SNPs could be successfully incorporated into the linkage map of H. midae. A total of 18 linkage groups were observed with an average marker spacing of 6.9 cM and genome coverage of 79.1%. Bioinformatic analyses and setting stringent criteria to identify SNPs from sequenced transcriptomic data proved to be an efficient way for SNP discovery in the current study. Genotyping of the identified loci with the GoldenGate genotyping assay demonstrated a high success rate; providing a genotyping assay adequate for species with little genomic information. The linkage map created in this study illustrated the utility of SNP markers in conjunction with microsatellite markers for linkage map construction and the adequate marker spacing obtained provides a step closer to quantitative trait loci mapping in this species.
AFRIKAANSE OPSOMMING: Haliotis midae (plaaslik ook bekend as perlemoen) is die grootste van vyf inheemse spesies wat langs die kus van Suid-Afrika aangetref word. Dit is die enigste spesie van kommersiële waarde wat bydraend is tot die uitbuiting van hierdie diere. As gevolg van die afname in hierdie natuurlike hulpbron het boerdery praktyke gedurende die vroeë 1990's ontstaan om in die internasionale aanvraag te voorsien. Ten einde doeltreffende teelmetodes te beoefen en die volhoubaarheid van hierdie kommersiële populasies te verseker is genetiese bestuur, wat bewerkstellig kan word deur die gebruik van molekulêre merkers soos enkel nukleotied polimorfismes (ENPs), baie belangrik. Enkel nukleotied polimorfismes is gewilde merkers in verskeie toepassings in akwakultuur genetika as gevolg van hul oorvloed in genome, verlaagde ontwikkelingskoste en verhoogde deurset van ENP-genotiperingstoetse. Identifisering van ENPs in nie-model spesies soos H. midae kan uitgevoer word deur in siliko benaderings te gebruik wat geskik is vir de novo ENP identifisering en ook tyd- en koste-effektief is. Dit word gebaseer op die analise van veelvuldige inlynstellings waar nukleotiedes wat nie ooreenstem nie as kandidaat ENPs gerapporteer kan word. Om kandidaat ENPs te bevestig, kan verskeie medium-deurset genotiperingsmetodes uitgevoer word, maar die ideale metode word bepaal deur faktore soos koste, akkuraatheid en multipleks kapasiteit. Alhoewel ENP merkers in verskeie toepassing binne die akwakultuur omgewing gebruik kan word was die fokus van die huidige studie om die koppelingskaart van H. midae te versadig. Dit sal bydrae tot die identifisering van kwantitatiewe eienskap lokusse wat gekoppel kan word aan ekonomies belangrike eienskappe wat dan op die beurt weer vir merkerbemiddelde seleksie gebruik kan word en uiteindelik ten opsigte van die verbetering van molekulêre teelprogramme aangewend kan word. Ten einde in siliko ENPs te identifiseer is transkriptoomdata van 'n vorige studie gebruik en onderwerp aan 'n reeks kriteria: geringste alleelfrekwensie 10%, minimum dekking 80, 60 bp gebiede weerskante van polimorfisme. Geïdentifiseerde lokus-genotipering is met behulp van 'n 192-pleks toets uitgevoer met die Illumina GoldenGate genotiperingstoets met die VeraCode tegnologie op die BeadXpress-platform, in individue afkomsitg vanaf ses karteringsfamilies. 'n Omskakelingskoers van 69.35% en 'n algehele sukseskoers van 76.34% is bereik. Polimorfiese lokusse is onderwerp aan koppelings-analise met behulp van JoinMap® v.4.1 om geslags-gemiddelde en geslags-spesifieke kaarte te skep asook om die kaart wat beskikbaar is vir H. midae te versadig. Saam met voorheen ontwikkelde merkers is 54% van die nuut ontwikkelde ENPs suksesvol opgeneem in die kaart van H. midae. 'n Totaal van 18 koppelingsgroepe is verkry met 'n gemiddelde merker-spasiëring van 6.9 cM en 'n genoomdekking van 79.1%. Die gebruik van bioinformatiese analises en streng kriteria om ENPs vanaf transkriptoomdata te identifiseer blyk doeltreffend te wees in hierdie studie. Genotipering van die geïdentifiseerde lokusse met die GoldenGate genotiperingstoets dui op 'n hoë suksessyfer en verskaf 'n voldoende genotiperingstoets aan spesies met min genomiese inligting. Die koppelingskaart in hierdie studie het geïllustreer dat die ENP merkers suksesvol saam met mikrosatelliet merkers gebruik kan word vir koppelingskaart konstruksie en dat die voldoende merker-spasiëring verkry 'n stap nader aan kwantitatiewe eienskap lokus kartering in hierdie spesie bied.

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21

Gibson, Spencer James. "Application of SNP genotyping technologies to breed authentication and pork quality markers." Thesis, University of Nottingham, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491007.

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The main hypothesis for the study presented was that current genotyping technologies could be us used to genotype DNA from various meat sources. The second hypothesis was that augmenting of 3 SNPs in the actin gene (McAnulty, 2002) with three non-sense SNP mutations in the calpastatin gene (Ciobanu et al, 2004) would improve the level of discrimination seen between pig breeds.

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Halpern, Micah. "DEVELOPMENT AND FORENSIC APPLICATION OF DYE PROBE FLUORESCENCE RESONANCE ENERGY TRANSFER FOR IMPROVED DETECTION OF CHANGES IN DN." Master's thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2871.

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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.
M.S.
Department of Chemistry
Sciences
Forensic Science MS

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23

Scholz, S. W. "The promise of genome-wide SNP genotyping : from population genetics to disease gene identification." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/19892/.

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Advances in single nucleotide polymorphism (SNP) genotyping technologies have revolutionised our ability to scrutinise the human genome. My PhD research focuses on using these new technologies to catalogue genetic variability in a collection of diverse populations from around the globe, and to determine the role of genetic variants in neurological diseases. First, I present and discuss the analysis of genome‐wide SNP data in individuals from ethnically and geographically diverse human populations uncovering the diversity of genotype, haplotype and copy number variation in these populations. Second, I will describe an autozygosity mapping approach in three Brazilian dystoniaparkinsonism families which lead to the identification of a novel disease‐segregating mutation in the gene PRKRA. Third, I will report on a large genome‐wide association study in Parkinson’s disease, uncovering genetic variability at the SNCA and MAPT loci that are strongly associated with risk for developing disease. Forth, I provide compelling evidence that genetic variants at the SNCA locus are also significantly associated with risk for developing multiple system atrophy. This finding represents the first reproducible risk gene for this devastating disorder, and causally links this condition to the more common neurodegenerative disorder Parkinson’s disease. Finally, I present the results of a comprehensive mutational screening study investigating the frequency and spectrum of sequence and copy number mutations in the parkinsonism genes PRKN and PINK in individuals with early-onset Parkinson’s disease, in multiple system atrophy patients and in normal controls. In summary, the data presented in this thesis emphasise the critical role that genetic variability plays in the pathogenesis of neurological disorders.

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Silva, Junior Orzenil Bonfim da. "Development and applications of high-throughput SNP genotyping technologies in non-model plant genomes." Universidade Cat??lica de Bras??lia, 2017. https://bdtd.ucb.br:8443/jspui/handle/tede/2273.

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In the last twenty-five years, we have witnessed the wide adoption of DNA markers for the study of genetic variation in many organisms. A DNA marker must have two or more identifiable allelic DNA sequences to be useful. It usually does not have a biological effect, but instead functions as a traceable landmark in the genome, found in a specific location, and transmitted by the standard laws of inheritance from one generation to the next. Its application goes beyond genetic mapping and includes the analysis of genetic diversity, marker-trait association studies, marker assisted selection and, more recently, with the advent of wholegenome sequencing, whole-genome association and genomic selection. Among the several types of DNA sequence polymorphisms that can be used as DNA marker, Single Nucleotide Polymorphisms (SNPs) are the most powerful for large-scale variation analysis. There are vast numbers of SNPs in every genome and they can be typed by methods that have been proven easy to automate. Detection of alternative alleles is rapid and effortless because it is based on well-known polymerase chain reaction and DNA oligomer hybridization assays. Various strategies have been devised to discriminate alleles at a SNP, including fixed DNA arrays technologies, solution hybridization techniques and many sequencing-based genotyping. In our study, we have developed high-throughput DNA marker systems for non-model, highly heterozygous, diploid tree species. We took advantage of the combined power of Next Generation Sequencing (NGS) technologies, well-established highly automated methods of SNP typing and bioinformatics algorithms to perform genome-wide DNA variation analysis. We used whole genome resequencing of pooled individuals to develop a high-density 60K SNP chip for Eucalyptus species (EucHIP60k) providing a 96% genome-wide coverage with 1 SNP/12???20 kbp, and 47,069 SNPs at ??? 10 kb from 30,444 of the 33,917 genes in the Eucalyptus genome. We then used high-density SNP data and whole-genome pooled resequencing to examine the landscape of population recombination (??) and theta (??), assess the extent of linkage disequilibrium (r2) and build the highest density linkage maps for Eucalyptus to date. Chromosome-wide ancestral recombination graphs allowed us to date the split of Eucalytpus grandis (1.7???4.8 million yr. ago) and identify a scenario for the recent demographic history of the species. In a final set of studies, we built the first genome assembly for a Neotropical forest tree, the Pink Ip?? (Handroanthus impetiginosus), a highly-valued keystone timber species. Genome sequence was screened for the development of a targeted-capture sequencing system for SNP genotyping consisting of nearly 24,000 probe sequences. This genotyping system showed flexibility as it allowed the identification of SNPs across different populations of the species in moderate sample sizes. The good genome coverage, consistent Ts/Tv ratio estimated across samples and fair technical reproducibility between replicates, in terms of recall and precision of the SNP calling and accuracy on genotypes, indicate that this genotyping platform can be confidently used to estimate population genetics parameters and carry out population genomics investigations at the genome-wide scale
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25

Mangano, E. "Genomic profiling of chromosomal instability in renal carcinoma primary cultures and cell lines by SNP array technology." Doctoral thesis, Università degli Studi di Milano, 2009. http://hdl.handle.net/2434/61979.

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The work described in this PhD thesis aimed to characterize 9 clear cell renal carcinoma primary cultures (RCCpc) and their parental tumor tissues and 5 commercial RCC cell lines, using the Affymetrix GeneChip® SNP array technology (50K and 250K platforms). We performed a genome-wide analysis of copy number alterations (CNAs) and LOH events, together with allele dosage, using CNAG (v3.0) and Affymetrix GTC (v2.0) software. RCCpc and parental tumor tissues were assessed comparing each culture and parental tissue to its corresponding blood sample, while cell lines were analyzed using 48 HapMap CEU samples as normal controls. Also, a comparison was performed between these samples and the typical RCC genomic signature, in order to assess if primary cultures and cell lines were a good in vitro model to study this pathology. The results here obtained indicated that our RCCpc were a reliable model to study RCC pathology, much better than RCC cell lines. Also, comparing RCCpc and parental tumor tissues, we demonstrated that RCCpc provided greater cell homogeneity and an enrichment in tumor cells with respect to heterogeneous bioptic tissues, thus facilitating the characterization of CNAs and the identification of novel genetic elements potentially involved in RCC etiology and useful in clinical applications.

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26

Blaauw, Sonja. "SNP screening and validation in Haliotis midae." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/19976.

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Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Haliotis midae (commonly referred to as perlemoen) is the only one of five endemic species in South Africa that is commercially valued both locally and internationally. Unfortunately, natural perlemoen populations have become a dwindling resource due to commercial exploitation, poaching and the influx of natural threats, such as the West Coast rock lobster, Jasus lalandii. To preserve the natural diversity and sustainability of natural populations as well as commercial stocks, genetic management and improvement of perlemoen is critical. Genetic management requires the utilisation of molecular markers, which aid in the construction of linkage maps and the identification of quantitative trait loci (QTL) associated with economically significant traits. This will allow improvement of commercial stock management in terms of broodstock selection as well as provide valuable insight into natural population dynamics. Single Nucleotide Polymorphisms (SNPs) were selected as the marker of choice due to their successful employment as molecular markers and their wide distribution and abundance within the genomes of various marine species. This study focuses on the characterisation of novel SNPs from transcript sequences generated by Next Generation Sequencing technology. Approximately 40% of the transcripts facilitated the isolation of 105 putative markers, indicating a SNP frequency of ~1% within the H. midae genome. A subset of 24 markers, in addition to 24 previously developed markers, was characterised using the Illumina GoldenGate genotyping assay with the VeraCode technology, a medium to high-throughput genotyping technology. This is the first reported medium- to highthroughput characterisation of SNPs in H. midae. The selected markers were used to determine the efficiency and overall success rate of the GoldenGate platform. Marker characterisation was completed in both natural and commercial populations to determine the utility of these markers for genetic diversity and population structure inference. An 85% genotyping success rate was achieved with the platform. Statistical analysis indicated that the markers developed in this study are suitable for applications including population genetic structure inference, genetic diversity estimation and possibly other downstream applications such as linkage mapping. These markers are considered to be invaluable for future work regarding the genetic management and conservation of H. midae.
AFRIKAANSE OPSOMMING: Haliotis midae (ook bekend as perlemoen) is die enigste van vyf inheemse spesies in Suid-Afrika wat noemenswaardige kommersiële waarde toon plaaslik sowel as internasionaal. Ongelukkig het kommersiële uitbuiting, wildstropery en natuurlike bedreiging (bv. die Weskus kreef Jasus lalandii), wilde perlemoen populasies noemenswaardig verminder. Dus, om natuurlike diversiteit en die voortbestaan van beide wilde en kommersiële populasies te beskerm, is genetiese bestuur en verbetering absoluut noodsaaklik. Genetiese bestuur vereis die gebruik van molekulêre merkers as ’n hulpmiddel in die opstellingvan koppelingskaarte, en die identifisering van die relevante kwantitatiewe eienskap loki (QTL) tipies geassosieer met ekonomies belangrike eienskappe. Die laasgenoemde beoog om kommersiële voorraad bestuur te verbeter, kragtens deur broeidier seleksie sowel as om insig te verskaf m.b.t. wilde bevolking dinamika. Enkel Nukleotied Polimorfismes (SNPs) is gekies as die toepaslike merker vanweë die omvattende toepaslikheid van hierdie merkers binne die genome van verskeie mariene spesies. Hierdie studie fokus op die karakterisering van nuwe SNPs vanuit transkript volgordes ontwikkel deur middel van Volgende Generasie Volgordebepaling (“Next Generation Sequencing”). ’n Beraamde 40% van transkripte het gelei tot die ontwikkeling van 105 potensiëlemerkers, aanduidend van ’n SNP frekwensie van ~1% binne die H. midae genoom. ’n Sub-versameling van 24 merkers, tesame met 24 bestaande merkers, is gekarakteriseer deur die Illumina GoldenGate genotiperings toets met die VeraCode tegnologie, ’n medium tot hoë deurvloei genotiperingstegnologie. Hierdie is die eerste berig van medium tot hoë deurvloei karakterisering van SNPs in H. midae. Die geselekteerde merkers is gebruik om die doeltreffendheid van die GoldenGate platform te bepaal. Merker karakterisering is uitgevoer in beide wilde en kommersiële bevolkings om die effektiewe bruikbaarheid van hierdie merkers m.b.t. genetiese diversiteit, en bevolking struktuur bepaling, te ondersoek. Die platform het ’n 85% genotiperingsukses syfer getoon. Statistiese analise dui daarop dat merkers ontwikkel tydens hierdie studie toepaslik is vir bevolking genetiese struktuur bepaling, genetiese diversiteitberaming en moontlik ook genetiese koppelingskartering. Hierdie merkers word bestempel as onmisbaar vir toekomstige navorsing in genetiese bestuur en bewaring van H. midae.

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27

Melo, Caroline Oliveira de Araújo. "Avaliação de polimorfismos nos genes de reparo e detoxificação e a associação com o dano no DNA em etilistas." Universidade Federal de Goiás, 2017. http://repositorio.bc.ufg.br/tede/handle/tede/7656.

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
Alcohol abuse is related to approximately 3.3 million annual deaths worldwide and is considered the first risk factor for the global burden of disease in countries of the Americas. Long-term abuse of alcohol induces numerous molecular and biochemical changes in alcohol-related tissues. It is believed that the toxic effects of alcohol are mediated by DNA damage by several mechanisms, such as by the induction of oxidative damage, DNA adducts, crosslinks and DNA strand breaks. In this sense, the aim of this study was to verify the frequency of polymorphisms in the repair and detoxification genes and the association with the DNA damage in alcoholics. The analysis of the obtained results showed a greater genotoxic damage in the alcoholics, when carrying out the Comet Assay. A point of variation was found in the GSTP1 gene and another point of variation in the XRCC1 gene. However, no statistically significant differences were observed between GSTM1 and GSTT1 genotypes and genetic damage. In this context, it can be concluded that the Comet Assay is a very effective and inexpensive method for the analysis of genotoxic damage.
O consumo abusivo do álcool está relacionado a aproximadamente 3,3 milhões de mortes anuais em todo o mundo, sendo considerado o primeiro fator de risco para a carga global de doenças em países das Américas. O abuso da ingestão de álcool em longo prazo induz inúmeras alterações moleculares e bioquímicas nos tecidos relacionadas ao álcool. Acredita-se que os efeitos tóxicos do álcool sejam mediados por danos ao DNA por vários mecanismos, como pela indução dos danos oxidativos, pelos adutos de DNA, pelos crosslinks e pelas quebras de fitas de DNA. Nesse sentido, o objetivo do presente trabalho foi de verificar a frequência de polimorfismos nos genes de reparo e detoxificação e a associação com o dano no DNA em etilistas. A análise dos resultados obtidos mostrou um maior dano genotóxico nos etilistas, ao realizar o Ensaio Cometa. Foi encontrado um ponto de variação no gene GSTP1 e um ponto de variação no gene XRCC1. No entanto, não foram observadas diferenças estatisticamente significativas entre os genótipos GSTM1 e GSTT1 e os danos genéticos. Nesse contexto, pode-se concluir que o Ensaio Cometa é um método bastante eficaz e barato para a análise de danos genotóxicos.

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28

Dahlgren, Andreas. "Analysis of Complex Genetic Traits in Population Cohorts using High-throughput Genotyping Technology." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8291.

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29

Cai, Jin. "Meta-analysis of QTL for Fusarium head blight resistance in Chinese wheat landraces using genotyping by sequencing." Diss., Kansas State University, 2016. http://hdl.handle.net/2097/32166.

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Doctor of Philosophy
Department of Agronomy
Guihua Bai
Guorong Zhang
Fusarium head blight (FHB) is a devastating fungal disease in wheat, reducing not only grain yield but also quality. The pathogen produces the mycotoxin deoxynivalenol (DON) that induces severe toxicological problems in human and animals. Using host resistance has been the most efficient way to control the disease. To identify quantitative trait loci (QTLs) for FHB resistance in Chinese landrace Haiyanzhong (HYZ), a recombinant inbred lines (RILs) population derived from a cross between HYZ and Wheaton was developed. The RILs were evaluated for percentage of symptomatic spikelets (PSS) in three greenhouse experiments, and genotyped using simple sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) developed from genotyping-by-sequencing (GBS). Eight QTLs were identified for type II (PSS) resistance on chromosomes 5A, 6B, 7D, 2B (2), 3B, 4B, and 4D, with 5A as the major QTL. Ten SNPs closely linked to 5A, 6B, and 2B QTLs were successfully converted to Kompetitave allelic specific PCR (KASP) assays. To identify common QTLs across different populations, we constructed high-density GBS-SNP maps in an additional four RIL populations derived from the Chinese landraces, Wangshuibai (WSB), Baishanyuehuang (BSYH), Huangfangzhu (HFZ), and Huangchandou (HCD) and conducted meta-analysis of the QTLs for FHB resistance using a consensus map developed from the five populations. We identified six MQTLs on chromosomes 3BS (2), 3A, 3D, 2D, and 4D and 23 tightly linked GBS-SNPs to the MQTLs. These GBS-SNPs were successfully converted to KASPs. The KASPs linked to MQTLs can be used for pyramiding these QTL in breeding programs. To quickly reduce FHB damage in U.S. hard winter wheat (HWW), we transferred Fhb1, a major QTL with stable effects on FHB resistance, from Ning7840 into three adapted HWW cultivars Overland, Jagger, and Overley, by marker-assisted backcross (MAB), and assessed the effect of Fhb1 on FHB resistance in these different backgrounds. The results showed that Fhb1 can significantly lower FHB severity, Fusarium-damaged kernel (FDK), and DON accumulation in the all the three HWW backgrounds. Some of the selected lines showed high levels of FHB resistance, but agronomically similar traits as recurrent parents, can be used as resistant parents to improve HWW FHB resistance.

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30

BARDINI, MICHELA. "Infant ALL with MLL-AF4 is sustained by t(4;11) as the sole genetic abnormality, and it is initiated in mice by phenotypically and functionally distinct leukemic stem cell subsets." Doctoral thesis, Università Vita-Salute San Raffaele Milano, 2010. http://hdl.handle.net/10281/44105.

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Ogni giorno, circa mille miliardi di nuove cellule vengono prodotte dal midollo osseo e messe in circolazione nel sangue periferico per far fronte alle necessità del nostro organismo. Tutte le cellule che compongono il nostro sangue, derivano da una stessa cellula-madre, chiamata cellula staminale ematopoietica, un precursore primordiale e immaturo che ha la capacità di espandersi e la potenzilità di differenzare a eritrociti, linfociti, leucociti, piastrine. Il processo di differenziamento che va dalla cellula staminale alle cellule mature circolanti è detto emopoiesi. La leucemia è un tipo di tumore circolante che può avere origine da una cellula qualunque del sangue, a un qualsiasi stadio differenziativo, la quale inizia a proliferare all’impazzata, sfuggendo ad ogni controllo, e prende il sopravvento sulle altre cellule sane. Il risultato è l’espansione aberrante di un singolo tipo cellulare, a discapito degli altri compartimenti, che causa una disfunzione del sistema emopoietico. La leucemia non può essere considerata un’ unica malattia, poichè ne esistono molti diversi tipi, a seconda del tipo cellulare interessato, e del tipo di mutazione genica che la caratteizza. Nel nostro laboratorio, presso il Centro di Ricerca M. Tettamanti dell’Opedale S. Gerardo di Monza, ci occupiamo delle leucemie del bambino. In particolare, nel mio periodo di dottorato, mi sono occupata di studiare una particolare forma di leucemia linfoblastica acuta, detta infant LLA, ad insorgenza nel primo anno di vita, che presenta il riarrangiamento del gene MLL. Questa leucemia rappresenta la forma prevalente in età neonatale, ed è purtoppo una malattia molto aggressiva con prognosi infausta che in Italia colpisce circa 10-12 nuovi nati all’anno. In particolare, i casi che presentano la traslocazione t(4;11) con espressione del prodotto di fusione MLL-AF4 rappresentano il sottogruppo genetico a incidenza maggiore in età neonatale. Per moltissimi aspetti, sia dal punto di vista clinico che biologico, la LLA infant rappresenta una malattia peculiare, diversa dalle altre forme di leucemia dell’età pediatrica e adulta. In questi quattro anni di dottorato mi sono occupata nello specifico di due progetti. Nel primo, in collaborazione con il Prof. Jacobsen in Svezia, ci siamo proposti di identificare, isolare e caratterizzare dal punto di vista biologico e funzionale la ‘cellula staminale leucemica’ della LLA infant con riarrangiamento del gene MLL. Nel secondo studio, pubblicato su ‘Leukemia’ abbiamo voluto valutare se il solo riarrangiamento del gene MLL fosse di per se sufficiente per dare origine alla malattia, oppure se alterazioni genetiche ulteriori, secondarie al riarrangiamento di MLL che costituisce il primo evento, fossero necessarie per la manifestazione clinica della leucemia. La composizione cellulare di ogni singolo tumore umano è altamente eterogenea, e non tutte le cellule che lo compongono sono capaci di sostenerne la crescita. Per spiegare queste osservazioni è stata postulata l’esistenza di un sottogruppo ristretto di cellule, dette staminali tumorali (CSC), o staminali leucemiche (LSC) nel caso si tratti di leucemia, che hanno la potenzialità di auto-rigenerarsi e di riprodurre l’intera massa tumorale. Secondo il modello della ‘cellula staminale tumorale’, in tutti i tumori solo un ristretto gruppo di cellule è capace di generare l’intera massa tumorale e sostenerne la crescita all’infinito. Poter bersagliare in modo specifico questa cellula significa eradicare definitivamente la malattia, che altrimenti inevitabilmente si rigenererebbe. Questa ipotesi è in contrasto con il precedente modello stocastico secondo cui ogni cellula che compone il tumore è potenzialmente capace di sostenerne la crescita: tuttavia la eterogenicità tumorale, e la sua bassa tumorigenicità sono dovute alla scarsa probalilità per una cellula di iniziare il ciclo cellulare e proliferare. Tuttavia recenti studi mettono in discussione l’ipotesi stessa dell’esistenza di una CSC, ed attualmente l’opinione scientifica è divisa riguardo al fatto se il modello gerarchico della CSC, piuttosto che il classico modello stocastico, siano più adatti per spiagere il meccanismo biologico alla base dell’insorgenza dei tumori umani. Infatti, benchè il concetto di CSC sia intuitivamente immediato, la sua effettiva identificazione e caratterizzazione si è rivelata inaspettatamente complicata, e ad oggi, non esiste una definizione universale di CSC. Il termine ‘leucemia’ comprende in realtà tantissimi tipi differenti di tumore, e tale eterogeneità si riflette sulla LSC. La specifica alterazione genetica, la cellula di origine (cioè la cellula inizialmente colpita dal primo evento trasformante da cui il tumore ha avuto luogo), le diverse metodologie in vivo applicate, l’influenza del microambiente midollare e l’alta variabilità fenotipica tra diversi pazienti affetti dalla medesima forma di leucemia, hanno portato inevitabilmente alla generazione di risultati apparentementente contrastanti tra i vari gruppi di ricerca che indipendentemente si sono riproposti di identificare la LSC. Il modello sperimentale d’elezione per identificare la LSC è il trapianto in vivo di blasti leucemici in topi umanizzati immunocompromessi, capaci di accettare cellule umane senza rigettarle e permetterne l’attecchimento. Nel periodo all’estero trascorso nel laboratorio del Porf. Jacobsen a Lund, mi sono occupata di mettere a punto il modello di trapianto in vivo di cellule leucemiche da pazienti affetti da infant LLA in topi unamizzati. Previa irradiazione subletale del topo ricevente, che ha il duplice scopo di eliminare le cellule circolanti murine mature e creare spazio per le cellule umane trapiantate, i blasti leucemici vengono iniettati nel topo per via sistemica attraverso la vena della coda.. Se l’attecchimento è avvenuto, già dopo poche settimane, i topi mostreranno alti livelli di ricostituzione nel midollo e nei compartimenti extramidollari (come milza, fegato e sangue periferico), con conseguente manifestazione dei sintomi clinici della malattia (quali splenomegalia, pelo arruffato, letargia, anoressia, perdita di peso e decesso). L’attecchimento viene valutato eseguendo periodiche biopsie midollari (dopo 6, 9, 12 o più settimane dal trapianto) dalla sede femorale, in anestesia generale, e successivamente la presenza di cellule leucemiche umane è rilevata nell’aspirato midollare tramite analisi successive. Nel primo lavoro inviato a ‘Cancer Cell’ abbiamo osservato che nella LLA infant MLL-AF4 positiva la LSC risiede nella frazione CD19+, costituita da linfociti B a diversi stadi maturativi. Infatti, la frazione CD19- è composta sia da cellule staminali normali residue (non leucemiche) che danno luogo a una ricostituzione normale in vivo, che da cellule leucemiche (MLL riarrangiate), tuttavia prive di potenziale leucemogenico, poiché incapaci di ripopolare il midollo di un topo irradiato e dare origine alla leucemia. Inoltre abbiamo dimostrato che all’interno della frazione CD19+, esistono molteplici sottopolazioni LSC con diverso potenziale leucemogenico e diverse caratteristiche biologiche e funzionali in vivo (es. penetranza, latenza di insorgenza della malattia e fenotipo della leucemia risultante). In conclusione, i nostri risultati dimostrano che, a differenza del modello convenzionale, esistono molteplici sottopopolazioni LSC con caratteristiche biologiche e funzionali distinte, che possono essere distinte tra loro e dalla restante popolazione leucemica (non tumorigenica) sulla base dell’espressione di determinati antigeni di superficie. Nel secondo studio pubblicato su ‘Leukemia’, ci siamo chiesti se la sola presenza della traslocazione del gene MLL fosse di per se sufficiente per la manifestazione clinica della malattia, o se mutazioni accessorie cooperanti fossero necessarie. Per fare ciò abbiamo analizzato a livello genomico un gruppo consistente di pazienti affetti da LLA infant MLL-AF4 positiva, tramite tecniche sofisticate di screening su tutto il genoma ad alta risoluzione, e abbiamo osservato che, contrariamente ai pazienti affetti da altri tipi di LLA pediatrica ad insorgenza dopo l’anno, nella LLA infant MLL-AF4 positiva non si rilevano mutazioni aggiuntive. Questi risultati dimostrano che il solo riarrangiamento di MLL è necessario e sufficiente per la manifestazione, in tempi brevi, della malattia conclamata, a differenza di altre forme di leucemia del bambino e dell’adulto, in cui mutazioni genetiche aggiuntive sono indispensabili. Inoltre tali evidenze ribadiscono ulteriormente che la LLA infant con riarangiamento di MLL è una malattia nica dal punto di vista biologco, diversa dagli altri tipi di leucemia, e che che il meccanismo di leucemogenesi può essere distinto. Considerando l’alta mortalità, l’alto rischio di ricaduta, la prognosi infausta della LLA infant MLL positiva e l’urgente necessità di avere a disposizione nuove ed efficaci terapie antitumorali, questi studi sono di rilevante importanza, in quanto non solo aiutano a comprendere meglio i meccanismi di insorgenza della LLA infant e fare luce sulla biologia e l’aggressività di questa malattia, ma rappresentano anche il punto di partenza per identificare nuove strategie terapeutiche al fine bersagliare la LSC ed eradicare la malattia in modo definitivo.

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31

Watts, Annabelle Monica. "High resolution melt analysis : a novel method for studying the genetic relatedness of Pseudomonas aeruginosa isolates from clinical and environmental sources." Thesis, Queensland University of Technology, 2013. https://eprints.qut.edu.au/63094/2/Annabelle_Watts_Thesis.pdf.

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A novel method was developed for studying the genetic relatedness of Pseudomonas aeruginosa isolates from clinical and environmental sources. This bacterium is ubiquitous in the natural environment and is an important pathogen known to infect Cystic Fibrosis (CF) patients. The transmission route of strains has not yet been defined; current theories include acquisition from an environmental source or through patient-to-patient spread. A highly discriminatory, bioinformatics based, DNA typing method was developed to investigate the relatedness of clinical and environmental isolates. This study found a similarity between the environmental and several CF clonal strains and also highlighted occurrence of environmental P. aeruginosa strains in CF infections.

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32

Küpper, Anita, Harish K. Manmathan, Darci Giacomini, Eric L. Patterson, William B. McCloskey, and Todd A. Gaines. "Population Genetic Structure in Glyphosate-Resistant and -Susceptible Palmer Amaranth (Amaranthus palmeri) Populations Using Genotyping-by-sequencing (GBS)." FRONTIERS MEDIA SA, 2018. http://hdl.handle.net/10150/627054.

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Palmer amaranth (Amaranthus palmeri) is a major weed in United States cotton and soybean production systems. Originally native to the Southwest, the species has spread throughout the country. In 2004 a population of A. palmeri was identified with resistance to glyphosate, a herbicide heavily relied on in modern no-tillage and transgenic glyphosate-resistant (GR) crop systems. This project aims to determine the degree of genetic relatedness among eight different populations of GR and glyphosate-susceptible (GS) A. palmeri from various geographic regions in the United States by analyzing patterns of phylogeography and diversity to ascertain whether resistance evolved independently or spread from outside to an Arizona locality (AZ-R). Shikimic acid accumulation and EPSPS genomic copy assays confirmed resistance or susceptibility. With a set of 1,351 single nucleotide polymorphisms (SNPs), discovered by genotyping-by-sequencing (GBS), UPGMA phylogenetic analysis, principal component analysis, Bayesian model-based clustering, and pairwise comparisons of genetic distances were conducted. A GR population from Tennessee and two GS populations from Georgia and Arizona were identified as genetically distinct while the remaining GS populations from Kansas, Arizona, and Nebraska clustered together with two GR populations from Arizona and Georgia. Within the latter group, AZ-R was most closely related to the GS populations from Kansas and Arizona followed by the GR population from Georgia. GR populations from Georgia and Tennessee were genetically distinct from each other. No isolation by distance was detected and A. palmeri was revealed to be a species with high genetic diversity. The data suggest the following two possible scenarios: either glyphosate resistance was introduced to the Arizona locality from the east, or resistance evolved independently in Arizona. Glyphosate resistance in the Georgia and Tennessee localities most likely evolved separately. Thus, modern farmers need to continue to diversify weed management practices and prevent seed dispersal to mitigate herbicide resistance evolution in A. palmeri.

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Costello, Mary-Ellen Clare. "Single nucleotide polymorphism (SNP) - genotyping of Community Acquired Methicillin-Resistant Staphylococcus aureus, including the subtyping of PVL toxin producers using Real-Time PCR." Thesis, Queensland University of Technology, 2010. https://eprints.qut.edu.au/37662/1/Mary-Ellen_Costello_Thesis.pdf.

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Staphylococcus aureus is a common pathogen that causes a variety of infections including soft tissue infections, impetigo, septicemia toxic shock and scalded skin syndrome. Traditionally, Methicillin-Resistant Staphylococcus aureus (MRSA) was considered a Hospital-Acquired (HA) infection. It is now recognised that the frequency of infections with MRSA is increasing in the community, and that these infections are not originating from hospital environments. A 2007 report by the Centers for Disease Control and Prevention (CDC) stated that Staphylococcus aureus is the most important cause of serious and fatal infections in the USA. Community-Acquired MRSA (CA-MRSA) are genetically diverse and distinct, meaning they are able to be identified and tracked by way of genotyping. Genotyping of MRSA using Single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring MRSA, specifically ST93 (Queensland Clone) dissemination in the community. It has been shown that a large proportion of CA-MRSA infections in Queensland and New South Wales are caused by ST93. The rationale for this project was that SNP analysis of MLST genes is a rapid and cost-effective method for genotyping and monitoring MRSA dissemination in the community. In this study, 16 different sequence types (ST) were identified with 41% of isolates identified as ST93 making it the predominate clone. Males and Females were infected equally with an average patient age of 45yrs. Phenotypically, all of the ST93 had an identical antimicrobial resistance pattern. They were resistant to the β-lactams – Penicillin, Flu(di)cloxacillin and Cephalothin but sensitive to all other antibiotics tested. Virulence factors play an important role in allowing S. aureus to cause disease by way of colonising, replication and damage to the host. One virulence factor of particular interest is the toxin Panton-Valentine leukocidin (PVL), which is composed of two separate proteins encoded by two adjacent genes. PVL positive CA-MRSA are shown to cause recurrent, chronic or severe skin and soft tissue infections. As a result, it is important that PVL positive CA-MRSA is genotyped and tracked. Especially now that CA-MRSA infections are more prevalent than HA-MRSA infections and are now deemed endemic in Australia. 98% of all isolates in this study tested positive for the PVL toxin gene. This study showed that PVL is present in many different community based ST, not just ST93, which were all PVL positive. With this toxin becoming entrenched in CA-MRSA, genotyping would provide more accurate data and a way of tracking the dissemination. PVL gene can be sub-typed using an allele-specific Real-Time PCR (RT-PCR) followed by High resolution meltanalysis. This allows the identification of PVL subtypes within the CA-MRSA population and allow the tracking of these clones in the community.

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Lau, Ka-po, and 劉嘉寶. "Multiplex ARMS PCR for SNP genotyping and its association with HbF expression and other clinical phenotypes in beta-thalassaemia patientsin Hong Kong." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44659726.

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Maushammer, Maria [Verfasser], and Jörn [Akademischer Betreuer] Bennewitz. "Analyse komplexer Merkmale beim Schwein mittels SNP-Chip Genotypen, Darmmikrobiota- und Genexpressionsdaten / Maria Maushammer ; Betreuer: Jörn Bennewitz." Hohenheim : Kommunikations-, Informations- und Medienzentrum der Universität Hohenheim, 2018. http://d-nb.info/1153464020/34.

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36

Ahlford, Annika. "Applications of Four-Colour Fluorescent Primer Extension Technology for SNP Analysis and Discovery." Doctoral thesis, Uppsala universitet, Molekylär medicin, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-129221.

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Studies on genetic variation can reveal effects on traits and disease, both in humans and in model organisms. Good technology for the analysis of DNA sequence variations is critical. Currently the development towards assays for large-scale and parallel DNA sequencing and genotyping is progressing rapidly. Single base primer extension (SBE) is a robust reaction principle based on four-colour fluorescent terminating nucleotides to interrogate all four DNA nucleotides in a single reaction. In this thesis, SBE methods were applied to the analysis and discovery of single nucleotide polymorphism (SNP) in the model organism Drosophila melanogaster and in humans. The tag-array minisequencing system in a microarray format is convenient for intermediate sized genotyping projects. The system is scalable and flexible to adapt to specialized and novel applications. In Study I of the thesis a tool was established to automate quality control of clustered genotype data. By calculating “Silhouette scores”, the SNP genotype assignment can be evaluated by a single numeric measure. Silhouette scores were then applied in Study I to compare the performance of four DNA polymerases and in Study III to evaluate freeze-dried reagents in the tag-array minisequencing system. The characteristics of the tag-array minisequencing system makes it suitable for inexpensive genome-wide gene mapping in the fruit fly. In Study II a high-resolution SNP map, and 293 genotyping assays, were established across the X, 2nd and 3rd chromosomes to distinguish commonly used Drosophila strains. A database of the SNP markers and a program for automatic allele calling and identification of map positions of mutants was also developed. The utility of the system was demonstrated by rapid mapping of 14 genes that disrupt embryonic muscle patterning. In Study III the tag-array minisequencing system was adapted to a lab-on-a-chip format for diagnostic testing for mutations in the TP53 gene. Freeze-drying was evaluated for storing reagents, including thermo-sensitive enzymes, on the microchip to reduce the complexity of the integrated test. Correct genotyping results were obtained using freeze-dried reagents in each reaction step of the genotyping protocol, both in test tubes and in single polymer test chambers. The results showed the potential of the approach to be implemented in fully integrated systems. The four-colour chemistry of SBE has been developed further to allow massively parallel sequencing (MPS) of short DNA fragments as in the Genome Analyzer system (Solexa/Illumina). In Study IV MPS was used to compare Nimblegen arrays and the SureSelect solution-based system for targeted enrichment of 56 continuous human candidate-gene regions totalling 3.1 Mb in size. Both methods detected known SNPs and discovered novel SNPs in the target regions, demonstrating the feasibility for complexity reduction of sequencing libraries by hybridization methods.

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Sigurdsson, Snaevar. "Large-Scale Genotyping for Analysis of the Type I Interferon System in Autoimmune Diseases." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6792.

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CASELLA, LAURA. "SNP ANALYSIS FOR DROUGHT-RELATED CANDIDATE GENES IN A GERMPLASM COLLECTION AND A TILLING POPULATION OF ITALIAN RICE." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/203361.

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Rice is the most important food crop in the world, representing the main source of caloric intake for more than one third of the world’s population. Although a great part of the global rice production comes from the developing countries such as China, India, Indonesia and Bangladesh, northern Italy plays a relevant role in terms of rice production providing about 50% of the total European paddy rice production. Due to the nature of the area devoted to rice growth, characterized by large availability of water and an efficient water distribution net, rice in Italy developed in the last two centuries as a water demanding crop, completing its growth cycle under submersion. However in the last decades also these regions experienced a reduction in water availability, with consequences on production and quality. The development of new varieties able to cope with water scarcity is therefore becoming of utmost importance for a sustainable rice cultivation in Italy. This study aims at identifying new alleles with added value for the improvement of drought resistance in Italian rice. The EMS-induced genetic variability in drought-related candidate genes was then explored in a TILLING population developed in the Italian rice variety Volano. The Volano TILLING platform was validated through the screening of three relevant target genes. A mutation density of 1/374 kb was estimated, proving the effectiveness of this approach for targeted rice crop improvement of Italian germplasm. The collection, currently consisting of 1860 mutant lines that are being enlarged with new mutagenized lines, represents an interesting source of variation exploitable in terms of response to drought stress and directly of use for targeted breeding programs. The mutant lines identified, affecting genes shown to be involved in plant drought escape and avoidance strategies, not only are relevant for Volano breeding programs, but represent a powerful genetic material in view of breeding for drought improvement in Italian rice. The second part of the work aimed at understanding the genetic determinants of root system architecture in the Italian rice germplasm, considering the profound implications of root development on the ability of the plant to cope with water deficits. The first Genome-Wide Association study on root traits was then performed on a germplasm collection including local accessions representing the genetic diversity of rice cultivated in Italy and a set of foreign varieties from temperate areas adapted to Italian climatic conditions. Whole genome genotyping was performed at Cornell University using the GBS (Genotyping-by-Sequencing) approach, a novel NGS strategy previously applied with success to maize and barley that uses libraries based on reducing genome complexity by methylation-sensitive restriction enzymes. In parallel, a thorough phenotypic screening for root morphological features was performed under controlled greenhouse conditions using a novel root phenotyping method that combines an optimized plant growing system (plastic cylindrical baskets coupled with PVC pipes) with an efficient imaging analysis (WinRHIZO image analysis software). The results of genome-wide association analyses performed on a first set of root phenotypic traits were very encouraging. All the detected significant associations were co-localizing with root QTLs previously identified in bi-parental mapping populations. Moreover, four of the detected regions co-localized with drought-avoidance QTLs, strongly supporting the hypothesis of their possible involvement in plant ability to cope with water scarcity. This work provides an initial study paving the way towards improvement of Italian rice varieties in terms of drought resistance.

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Geduld, Tasneem. "Reconstruction of major male and female lineages of the Strand Muslim community." Thesis, University of the Western Cape, 2010. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_7976_1299130289.

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Initially, a pilot study was carried out in order to reconstruct the major paternal and maternal lineages of the Muslim population living in the Cape metropolitan area. The Study has shown the ability of molecular genetic tools to give insight into the origins and history of local communities. The study was also used as a point of reference for the Strand Muslim Community project. Genetic variations of the Y-chromosome and mitochondrial DNA for the pilot study were analyzed using the RFLP technique. The SNaPshot mini-sequencing technique was used to genotype single nucleotide polymorphisms (SNP) on the Y-chromosome and mitochondrial DNA in 115 males from the Strand Muslim community.

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Silva, Fabiane de Lima. "Estratégias de imputação de genótipos de marcadores SNP para estudos de associação genômica em animais da raça Nelore." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/11/11139/tde-17122013-121145/.

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Temperamento em bovinos é definido como a reação dos animais em resposta ao contato com o ser humano, geralmente atribuído ao medo ocasionado no manejo. Animais agitados são mais difíceis de manejar nas fazendas. Estudos na literatura mostram que características de temperamento influenciam o desempenho produtivo dos rebanhos, devido à sua correlação com outras características dentre elas, ganho de peso diário, taxa de prenhez, qualidade e rendimento de carcaça. Todavia, estudos visando o melhor entendimento dos mecanismos biológicos, e da arquitetura genética destas características são escassos. Com a evolução das tecnologias de genotipagem em alta densidade com marcadores de polimorfismo único (SNP), observou-se um aumento de pesquisas nas áreas de seleção genômica (GS) e associação genômica (GWAS). Entretanto, devido ao alto custo que a genotipagem em larga escala apresenta, torna-se inviável a genotipagem para todos os animais candidatos à seleção. Porém, existe a possibilidade de utilizar painéis de baixa densidade de SNP e inferir estes genótipos desconhecidos para painéis de alta densidade. Neste contexto foram desenvolvidos dois estudos. No primeiro, o objetivo foi identificar regiões cromossômicas associadas com características de temperamento em animais da raça Nelore. As características avaliadas foram: velocidade de saída (VS) e mediana do escore composto (EC_mediana), em que 599 e 575 animais foram utilizados, respectivamente. Todos os animais foram genotipados com Illumina BovineHD BeadChip (800K), e para o GWAS dois modelos estatísticos foram aplicados. O primeiro modelo utilizado foi genômico de única etapa (ssGBLUP), em que os efeitos dos SNP são derivados da predição dos valores genéticos genômicos dos animais. O segundo modelo foi linear misto, similar ao modelo anterior, porém várias análises, SNP por SNP (regressão simples) foram realizadas. Nos dois modelos foram incluídos os efeitos de grupo de contemporâneo como fixo, idade do animal como covariável e efeito poligênico do animal e ambiente permanente como aleatórios. Os componentes de variância foram estimados pelo método de máxima verossimilhança restrita. Os coeficientes de herdabilidade apresentaram baixa magnitude com estimativas de 0,02 e 0,05 para VS e EC_mediana, respectivamente. Diferentes regiões cromossômicas foram associados com as características estudadas nesta população, de acordo com os modelos utilizados, contribuindo para o entendimento da arquitetura genética dessas características. No segundo, o objetivo foi avaliar a acurácia de imputação também nesta população, usando dois painéis de baixa densidade (3K e 6K) e um de média densidade (50K) imputados para o painel de alta densidade (800K). Os animais genotipados com 800K foram divididos em população de validação e população de referência. Na população de validação, os animais tiveram seus genótipos mascarados para os chips Illumina 3K, 6K e 50K. A imputação de 3K, 6K e 50K para 800K foi realizada utilizando os softwares de imputação fastPHASE e Beagle. O software fastPHASE apresentou maiores valores de acurácia de imputação em comparação ao Beagle. O painéis 6K e 50K apresentaram maiores valores de acurácia.
Temperament in cattle is generally defined as the reaction of an animal in response to contact with human, usually attributed to the fear. Animals agitated are harder to manage in farms. Studies in the literature reported that temperamental traits have been found to influence the productive performance of herds, due to its correlation with other traits such as carcass quality, daily gains, pregnancy rate and feed efficiency. However, almost nothing is known regarding the genetic landscape controlling temperamental animal variation in cattle. With the advances in genotyping technologies for high density genotyping platforms to markers of single nucleotide polymorphisms (SNP), there was an increase in research in the areas of genomic selection (GS) and genome-wide association studies (GWAS). However, genotyping many animals with a high-density marker panel can be expensive and economically unfeasible. An alternative in this context is to use a lower density marker panel on a larger number of animals, and impute the missing genotypes. In this context two studies were developed. In the first study, the objective was to identify chromosomal regions associated with temperament traits in Nellore cattle. The temperamental traits evaluated in this study were: exit velocity (VS) and the median score temperament (EC_mediana) assessed in 599 and 575 animals, respectively. All animals were genotyped with Illumina BeadChip BovineHD (800K), and two GWAS statistical models were applied. The first model used was genomic single step (ssGBLUP) in which the effects of SNP are derived from the genomic prediction values of the animals. The second was linear mixed model, similar to the previous model, but a series of models, one for each SNP (single regression), were performed. In both models were included the effects of contemporary group as fixed, the animal\'s age as a covariate effect and polygenic animal and permanent environment as random effect. Variance components were estimated by restricted maximum likelihood. The heritability coefficients showed low magnitude estimative of 0.02 and 0.05 for VS and EC_mediana, respectively. Different chromosomal regions were associated with the traits studied in this population, according with the models used, and moreover to contribute in the understanding of the genetic architecture of these traits. In the second study, the objective was to evaluate the accuracy of imputation in a same population, using two panels of low-density (3K and 6K) and one medium-density (50K) panel markers imputed up to higher density of 800K. The animals genotyped with 800K markers panel were split into a reference population and validation population. The validation population the animals had markers masked to Illumina chip 3K, 6K e 50K. Imputation from 3K, 6K and 50K up to 770K markers was performed using fastPHASE and Beagle. The software fastPHASE had higher imputation accuracy compared to Beagle. The 6K and 50K panels showed higher accuracy.

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Smith, Scott Matthew. "Application of Genome Reduction, Next Generation Sequencing, and KASPar Genotyping in Development, Characterization, and Linkage Mapping of Single Nucleotide Polymorphisms in the Grain Amaranths and Quinoa." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/3548.

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The grain amaranths (Amaranthus sp.) and quinoa (Chenopodium quinoa Willd.) are important seed crops in South America. These crops have gained international attention in recent years for their nutritional quality and tolerance to abiotic stress. We report the identification and development of functional single nucleotide polymorphism (SNP) assays for both amaranth and quinoa. SNPs were identified using a genome reduction protocol and next generation sequencing. SNP assays are based on KASPar genotyping chemistry and were detected using the Fluidigm dynamic array platform. A diversity screen consisting of 41 amaranth accessions showed that the minor allele frequency (MAF) of the amaranth markers ranged from 0.05 to 0.5 with an average MAF of 0.27. A diversity screen of 113 quinoa accessions showed that the MAF of the quinoa markers ranged from 0.02 to 0.5 with an average MAF of 0.28. Linkage mapping in amaranth produced a linkage map consisting of 16 linkage groups, presumably corresponding to each of the 16 amaranth haploid chromosomes. This map spans 1288 cM with an average marker density of 3.1 cM per marker. Linkage mapping in quinoa resulted in a linkage map consisting of 29 linkage groups with 20 large linkage groups, spanning 1,404 cM with a marker density of 3.1 cM per SNP marker. The SNPs identified here represent important genomic tools needed for genetic dissection of agronomically important characteristics and advanced genetic analysis of agronomic traits in amaranth and quinoa. We also describe in detail the scalable and cost effective SNP genotyping method used in this research. This method is based on KBioscience's competitive allele specific PCR amplification of target sequences and endpoint fluorescence genotyping (KASPar) using a FRET capable plate reader or Fluidigm's dynamic array high throughput platform.

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Rönn, Ann-Charlotte. "Analysis of Nucleotide Variations in Non-human Primates." Doctoral thesis, Uppsala University, Molecular Medicine, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7904.

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Many of our closest relatives, the primates, are endangered and could be extinct in a near future. To increase the knowledge of non-human primate genomes, and at the same time acquire information on our own genomic evolution, studies using high-throughput technologies are applied, which raises the demand for large amounts of high quality DNA.

In study I and II, we evaluated the multiple displacement amplification (MDA) technique, a whole genome amplification method, on a wide range of DNA sources, such as blood, hair and semen, by comparing MDA products to genomic DNA as templates for several commonly used genotyping methods. In general, the genotyping success rate from the MDA products was in concordance with the genomic DNA. The quality of sequences of the mitochondrial control region obtained from MDA products from blood and non-invasively collected semen samples was maintained. However, the readable sequence length was shorter for MDA products.

Few studies have focused on the genetic variation in the nuclear genes of non-human primates. In study III, we discovered 23 new single nucleotide polymorphisms (SNPs) in the Y-chromosome of the chimpanzee. We designed a tag-microarray minisequencing assay for genotyping the SNPs together with 19 SNPs from the literature and 45 SNPs in the mitochondrial DNA. Using the microarray, we were able to analyze the population structure of wild-living chimpanzees.

In study IV, we established 111 diagnostic nucleotide positions for primate genera determination. We used sequence alignments of the nuclear epsilon globin gene and apolipoprotein B gene to identify positions for determination on the infraorder and Catarrhini subfamily level, respectively, and sequence alignments of the mitochondrial 12S rRNA (MT-RNR1) to identify positions to distinguish between genera. We designed a microarray assay for immobilized minisequencing primers for genotyping these positions to aid in the forensic determination of an unknown sample.

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Eldon, Desiree Rochelle. "Population Genetic Structure of Bromus tectorum in the American Desert Southwest." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/4273.

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Following its introduction to North America in the late nineteenth century, Bromus tectorum L., an inbreeding invasive winter annual grass, has become dominant on millions of hectares of sagebrush steppe habitat throughout Intermountain Western North America. It appears that within the last 30-40 years, B. tectorum has expanded its range southward into the Mojave Desert and also into more climatically extreme salt desert environments. Previous research using microsatellite markers and experimental studies has suggested that lineages found in desert habitats are genetically distinct from those found in the sagebrush-steppe habitat and possess suites of traits that pre-adapt them to these environments. To provide additional support for our hypothesis that desert habitat-specific haplotypes dominate and are widely distributed across warm and salt desert habitats, we genotyped approximately 20 individuals from each of 39 B. tectorum populations from these habitats and adjacent sagebrush steppe habitats using 71 single nucleotide polymorphic (SNP) markers. Our data clearly demonstrate that populations throughout the Mojave Desert region, as well as in salt desert habitats further north, are dominated by a small number of closely related SNP haplotypes that belong to the desert clade. In contrast, populations from adjacent environments are largely dominated by haplotypes of the common clade, which is widely distributed throughout the North American sagebrush steppe. Populations across all habitats were usually dominated by 1-2 SNP haplotypes. This suggests that inbreeding B. tectorum lineages can often maintain their genetic integrity. It also explains the strong association between marker fingerprints and suites of adaptive traits in this species.

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Ortega, Maria Andrea. "Identification of Molecular Markers Associated with the Rps8 locus in Soybean and Evaluation of Microsporogenesis in Rps8/rps8 Heterozygous Lines." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1259772038.

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45

Bayerl, Helmut [Verfasser], Ralph [Akademischer Betreuer] Kühn, Jürgen [Gutachter] Geist, Roman [Gutachter] Gula, and Ralph [Gutachter] Kühn. "From low-throughput SSR genotyping to high-throughput SNP analyses of natural populations: validation of their application focused on non-invasive samples / Helmut Bayerl ; Gutachter: Jürgen Geist, Roman Gula, Ralph Kühn ; Betreuer: Ralph Kühn." München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/1201482356/34.

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46

Benazir, Katarina Marquez. "Molecular Marker Applications in Oat (Avena Sativa L.) Breeding and Germplasm Diagnostics." Thèse, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31148.

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The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.

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Hultin, Emilie. "Genetic Sequence Analysis by Microarray Technology." Doctoral thesis, Stockholm : School of Biotechnology, Royal Institute of Technology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4330.

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48

O'Meara, Deirdre. "Molecular Tools for Nucleic Acid Analysis." Doctoral thesis, Stockholm : Tekniska högsk, 2001. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3220.

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Graf, Justin T. "Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/25913/1/Justin_Graf_Thesis.pdf.

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This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

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50

Graf, Justin T. "Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation." Queensland University of Technology, 2008. http://eprints.qut.edu.au/25913/.

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Abstract:

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

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