<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

<p>The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South&nbsp / Africa / a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute&nbsp / carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline&nbsp / frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot&reg / and Multiplex AS-PCR genotyping assays, and&nbsp / also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the&nbsp / University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the&nbsp / Cape Metropolitan area. Two SNaPshot&reg / Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for&nbsp / the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific &ndash / PCR (MAS-PCR) genotyping&nbsp / system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was&nbsp / conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan&nbsp / large numbers of samples for novel genetic variations.&nbsp / </p>

Magister Scientiae - MSc / The aim of this study was to investigate the genetic diversity of the SLC22A1 gene and to deduce its possible pharmacogenetic implications within the Cape Coloured population of South Africa; a uniquely admixed population of immigrant Europeans, Asians and the indigenous populations. Recent studies have reported an abundance of polymorphic variants within this solute carrier transporter gene encoding for the organic cation transporter 1, as well as evidence linking these variants to an effect on metformin uptake. This study included establishing baseline frequency distribution of previously reported alleles for 20 SNP variants within the SLC22A1 gene, as well as the development of SNaPshot® and Multiplex AS-PCR genotyping assays, and also exploring the possibility of using High-resolution melt (HRM) analysis as a costeffective alternative for SNP genotyping. Ethics clearance was obtained from the Ethics Committee of the University of the Western Cape. Biological samples in the form of buccal (oral) swabs were collected from 132 unrelated voluntary donors from the Cape Coloured population residing in the Cape Metropolitan area. Two SNaPshot® Multiplex Systems were specifically designed for the study,successfully optimized and used for genotyping. Hundred genetic profiles were then generated for a total of 20 SNP variants on SLC22A1 gene, using this primer extension-based genotyping method that enables multiplexing up 10 SNPs. Population genetics data obtained for the investigated SNPs were analysed using various statistical analysis software. Important population genetic parameters were calculated, and possible pharmacogenetics implications were then discussed. Among others, allelic and genotypic frequencies, as well as linkage disequilibrium were determined and compared with world populations. Minor deviation from Hardy- Weinberg equilibrium was observed in the Cape Coloured population. No significantLinkage Disequilibrium between the investigated SNPs was observed in this population. A Multiplex allele specific – PCR (MAS-PCR) genotyping system was successfully designed and optimized for the genotyping of 10 SNPs from the SLC22A1. This system, also developed specifically for this study, was made of 2 multiplexes each covering 5 SNPs. It is an inexpensive genotyping assay that allows for efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. A pilot study was conducted to explore the possibility of using High-resolution melt (HRM) analysis as a cost-effective alternative for SNP genotyping. In addition to genotyping, HRM analysis can be used to scan large numbers of samples for novel genetic variations. / South Africa

Citrus es el género de la subfamilia Aurantioideae de mayor importancia económica. Su origen es la región sureste de Asia, en un área que incluye China, India y la península de Indochina y los archipiélagos de los alrededores. Aunque se han realizado múltiples estudios, la taxonomía del género Citrus aun no está bien definida, debido al alto nivel de diversidad morfológica encontrado en este grupo, la compatibilidad sexual entre sus especies y la apomixis de muchos genotipos. En la presente tesis doctoral se ha estudiado una amplia diversidad del género Citrus, especies relacionadas y otros taxones de la subfamilia Aurantioideae, para poder aclarar su organización y filogenia mediante el empleo de diferentes tipos de marcadores moleculares y métodos de genotipado. Más concretamente, el germoplasma de mandarino juega un papel muy importante en la mejora de variedades y patrones, pero su organización genética no está bien definida. Por lo tanto, se ha realizado un análisis en profundidad de su diversidad y organización genética. El desarrollo de marcadores moleculares de Inserción-Deleción (indel), por primera vez en cítricos, ha permitido demostrar su utilidad para estudios de diversidad y filogenia en el género Citrus. En combinación con los marcadores de tipo microsatélite (SSR), se ha cuantificado la contribución de los tres principales taxones de cítricos (C. reticulata, C. maxima and C. medica) a los genomas de las especies secundarias y cultivares modernos. También se ha definido su estructura genética a partir de los datos obtenidos en la secuenciación de 27 fragmentos de genes nucleares relacionados con la biosíntesis de compuestos que determinan la calidad de los cítricos y genes relacionados con la respuesta de la planta a estreses abióticos. El análisis de la filogenia nuclear ha permitido determinar la relación existente entre la especie C. reticulata y Fortunella, que se diferencian claramente del grupo formado por las otras dos principales especies de cítricos (C. maxima y C. medica). Este resultado está en concordancia con el origen geográfico de las especies estudiadas. A partir de este estudio, se han desarrollado marcadores moleculares de tipo SNP con un alto valor filogenético, que han sido transferidos a géneros relacionados de los cítricos. Estos marcadores han dado un resultado muy positivo en el género Citrus y serán de gran utilidad para el establecimiento de la huella genética del germoplasma en un nivel de diversidad más amplio. Se ha estudiado la organización genética dentro del germoplasma mandarino (198 genotipos de tipo mandarino pertenecientes a dos colecciones, INRA-CIRAD e IVIA), así como la introgresión de otros genomas mediante el uso de 50 y 24 marcadores de tipo SSR y indel, respectivamente, además de cuatro marcadores InDel mitocondrial (ADNmt). Se ha observado que muchos genotipos, que se creía que eran mandarinos puros, presentan introgresión de otros genomas ancestrales. Dentro del germoplasma de mandarino, se han identificado a nivel nuclear cinco grupos parentales, a partir de los cuales se originaron muchos genotipos, dando lugar a estructuras hibridas complejas. Se ha observado incluso, genotipos con un origen maternal no mandarino, determinado por los marcadores de ADNmt. La presente tesis doctoral ha aportado nueva información sobre las relaciones filogenéticas entre las especies del género Citrus, géneros cercanos, así como de las especies secundarias. Además, se han desarrollado nuevos marcadores moleculares que se complementan entre sí. Se ha establecido una nueva organización genética del germoplasma mandarino y se han caracterizado adecuadamente las dos colecciones de cítricos en estudio. Por lo tanto, todas estas contribuciones, ayudarán a los programas de mejora para la obtención de nuevas variedades de cítricos de alta calidad y permitirán optimizar la conservación y uso de los recursos genéticos existentes, así como su caracterización genética y fenotípica. / García Lor, A. (2013). Organización de la diversidad genética de los cítricos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31518 / TESIS

Leber’s hereditary optic neuropathy (LHON) is a genetic disease that causes the patients to become blind, first in one eye and then the other, around the ages of 10-75 years. The disease is caused by mutations in the mitochondrial DNA, which disturbs the respiratory chain leading to the deterioration of the retinal ganglion cells. This study’s aim is to optimize a multiplex amplification-refractory mutation system PCR for detection of three primary mutations causing LHON. This was done through a series of PCRs, including PCR aimed at the ß-globin gene, conventional simplex PCR and a simplex ARMS-PCR aimed at the three primary mutations causing LHON. This study was, however, terminated prematurely due the Covid-19 outbreak and the optimization of the ARMS-PCR could therefore not be done. This study’s aim was adapted to the new circumstances to instead provide guidance on how to perform the optimization using the results from the PCRs that were done before the termination. The results found that for the ARMS-PCR 2 mM of magnesium would suffice as a start point overall and the need to solve the problems with the two 14484 plasmids was evident. The ARMS-PCR is one of many methods that can be used to the detect single nucleotide polymorphism, but its availability and robustness makes this a method worth optimizing. To continue with the optimization of the ARMS-PCR several factors would have to be tested, including annealing temperature, primer concentrations and magnesium concentration.

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.

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.