Les nématodes à galles (Meloidogyne spp.) sont des pathogènes cosmopolites extrêmement polyphages. L’emploi de la majorité des nématicides chimiques étant désormais interdit, la meilleure alternative repose sur l’utilisation de gènes majeurs de résistance (gènes R). Cependant, il existe un risque de contournement de ceux-ci et les ressources génétiques en termes de gènes R sont limitées. Une gestion qui permette de pérenniser l’utilisation de ces ressources d’intérêt agronomique est donc primordiale. Me1 et Me3 sont deux gènes R à large spectre du piment actuellement utilisés dans les programmes de sélection. La confrontation vis-à-vis de M. incognita de différents génotypes possédant l’un de ces deux gènes a montré que (1) le fond génétique de la plante joue un rôle clé sur l’efficacité de ces gènes R, (2) il n’existe pas d’effet du dosage d’allèles pour ces gènes R. Suite à ces résultats, une recherche de loci à caractère quantitatif (QTLs) a été réalisée afin d’identifier et de localiser des facteurs de résistance partielle susceptibles d’expliquer les différences observées entre les différents fonds génétiques. L’étude de tels facteurs vis-à-vis de M. incognita, M. arenaria et M. javanica, les trois principales espèces de nématodes à galles, a mis en évidence quatre nouveaux QTLs. Tous sont regroupés sur le chromosome P1 du piment, sauf un efficace vis-à-vis de M. javanica situé sur le chromosome P9. C’est la première fois que des facteurs de résistance aux nématodes à galles sont localisés sur le chromosome P1. Ce travail ouvre de nouvelles perspectives quant à la création de nouvelles variétés avec un potentiel accru en termes de résistance aux Meloidogyne. / Root-knot nematodes (RKNs), Meloidogyne spp., are extremely polyphagous plant parasites worldwide. Since the use of most chemical nematicides is being prohibited, genetic resistance is an efficient alternative way to protect crops against these pests. However, nematode populations proved able to breakdown plant resistance, and genetic resources in terms of resistance genes (R-genes) are limited. Sustainable management of these valuable resources is thus a key point of R-gene durability. In pepper, Me1 and Me3 are two dominant major R-genes, currently used in breeding programs to control M. arenaria, M. incognita and M. javanica, the three main RKN species. Challenging these two genes in different genetic backgrounds against M. incognita demonstrated that (1) the efficiency of the R-genes in reducing the reproductive potential of RKNs is strongly affected by the plant genetic background, (2) the allelic status of the R-genes has no effect on nematode reproduction. According to these first results, a QTL analysis was performed to identify and to localize partial resistance factors against RKNs which could explain the differences observed between the genetic backgrounds. Focusing on M. incognita, M. arenaria and M. javanica, four new major QTLs were localized. They are all regrouped on pepper chromosome P1 except one QTL efficient against M. javanica, which was located on pepper chromosome P9. The cluster on chromosome P1, regrouping most of the newly discovered resistance factors, is described for the first time with respect to RKN resistance. As a conclusion, this work should contribute to the breeding of new pepper varieties with a high level of resistance against RKNs.

Melampyrum nemorosum agg. is very complicated group of hemiparasitic plants. According to the traditional concept, 15 species is distinguished. Recent molecular analyses show a need of critical taxonomic revision of group and a potential importance of ancient hybridization. Analysis of 3 regions of cpDNA (trnTUGU-trnLUAA, psbA-trnHGUG, rpl32-trnLUAG) and 2 regions of nuclear DNA (Agt1 and At103) reveals well supported lineage with limited geographical distribution. Morphology and genome size of genetically supported lineages were compared.

Melampyrum nemorosum agg. is very complicated group of hemiparasitic plants. According to the traditional concept, 15 species is distinguished. Recent molecular analyses show a need of critical taxonomic revision of group and a potential importance of ancient hybridization. Analysis of 3 regions of cpDNA (trnTUGU-trnLUAA, psbA-trnHGUG, rpl32-trnLUAG) and 2 regions of nuclear DNA (Agt1 and At103) reveals well supported lineage with limited geographical distribution. Morphology and genome size of genetically supported lineages were compared.

Strangles is considered the main respiratory disease in horses. The etiologic agent is the bacterium Streptococcus equi subsp. equi (S. equi), responsible for approximately 30% of horse diseases worldwide notifications. The clinical signs of strangles are fever, nasal secretion and lymph node enlargement. The last one occurs due the incomplete phagocytosis of S. equi by defense cells because bacterial hyaluronic acid capsule and M protein (SeM). The epidemiology, the risk factors and the strangles control are poorly understood. The 5' end of the seM gene sequence has been used for isolate differentiation by characterization of the alleles. Therefore, this thesis aimed to study the prevalence of Streptococcus equi subsp. equi (S. equi) in healthy horses, the alleles frequency and the risk factors involved on equine adenitis. One thousand and ten nasal swabs were obtained from healthy horses from 341 farms. Twenty four horses were positive for S. equi, confirmed by PCR and DNA sequencing. The prevalence of S. equi per equine was 2.37%, and 20 farms were positive (5.86%). Risk factor analysis showed by confirming and quantifying statistically that: the number of agglomeration events that horses participate (RR: 1.6), the situation with food container shared (RR: 3.74) and the positive diagnosis for adenitis (RR: 3.20) are significant risk factors for Strangles. These results provide important epidemiological contribution to the equine industry and can give support for the disease control. In addition, the 5 'end of the gene seM was amplified by PCR and sequenced and allele characterized. It was found the same allele (SEM-61) in all the samples. These results support the hypothesis of natural selection of alleles apparently more suited to survive, persist and perpetuate in the population studied. / A adenite equina é uma doença infecto-contagiosa que acomete o trato respiratório superior, sendo uma das principais doenças respiratórias de equinos. O agente etiológico dessa enfermidade é o Streptococcus equi subespécie equi (S. equi), responsável por aproximadamente 30% das notificações em todo o mundo. Os principais sinais clínicos da adenite são febre, secreção nasal e enfartamento de linfonodos, que ocorre pela dificuldade de fagocitose do S. equi por células de defesa devido a presença da cápsula de ácido hialurônico e proteína M. O entendimento sobre a epidemiologia, a análise de fatores de risco para adenite equina e o controle dessa enfermidade ainda são limitados. Estudos moleculares demonstram diferenças na extremidade 5 da sequência do gene (seM) codificador da proteína M de S. equi. Esta região do gene já foi utilizada na diferenciação de isolados por meio da caracterização de diferentes alelos. Por tudo isso, essa tese objetivou obter resultados de prevalência, e também análise de fatores de risco para adenite equina através de um desenho experimental para coleta de suabes nasais. Foram obtidos 1.010 suabes nasais de equinos sadios em 341 fazendas, de onde foram identificados 24 equinos positivos para S. equi em isolamento, que posteriormente foram confirmados por PCR e sequenciamento de DNA. A prevalência estimada por equino foi de 2.37%, e 20 fazendas foram consideradas positivas (5.86%). Na análise de fatores de risco, foi comprovado e quantificado estatisticamente que: número de eventos de aglomeração que os equinos participam (RR:1.06), o ato de compartilhar recipiente de alimento (RR:3.74) e ter tido diagnóstico positivo para adenite (RR:3.20) são fatores de risco relevantes para adenite equina. Estes resultados oferecem contribuições epidemiológicas importantes para a indústria de equinos e pode apoiar o controle da doença. Em paralelo, a região 5 terminal do gene seM das 24 amostras positivas foi amplificada por PCR e sequenciada para caracterização de alelos, sendo identificado o mesmo alelo (seM-61) em todas as amostras. Esses resultados evidenciam a hipótese de seleção natural de alelos aparentemente mais adaptados a sobreviver, persistir e se perpetuar na população estudada.

NADPH-P450 oxidoreductase (POR) is a membrane bound flavoprotein that donates electrons to a wide spectrum of heme-containing proteins, among which are several steroidogenic and many xenobiotics-metabolizing enzymes. Given the important role of POR protein in drug metabolism and pharmacogenomics, there is a particular need to understand the contributions of POR genetic variants to these processes. Mutations in POR gene cause a disorder called POR deficiency, which manifests with a wide phenotypic spectrum ranging from disordered steroidogenesis to skeletal malformation, namely, Antley-Bixler syndrome (ABS). The aim of the present work was to investigate the POR gene in patients suspected to have POR deficiency syndrome from Czech Republic and to perform genotyping in Czech and Jewish control populations. We analyzed 644 alleles in unrelated individuals from the general Czech population and 1128 alleles in Jewish population, where 330 alleles were of Askhenazi and 798 of Sephardic Jews. We have also studied the impact of selected new genetic variants on POR activity and identified fourteen amino acid variations, two of which we have studied in detail to establish their influence on POR activity. Using the available human POR three-dimensional structure, we then modelled the newly identified variants...

Magister Scientiae - MSc / The objective of this study was to develop and validate a six Non-CODIS miniSTR genotyping system and to determine its suitability for forensic casework in South Africa. In Non-CODIS miniSTR genotyping systems, smaller PCR products are amplified and the primers are positioned as close as possible to the repeat region. For this reason, these systems can be valuable in a variety of scenarios including complex paternity cases, missing persons work, and mass fatality disasters. / South Africa

Magister Scientiae - MSc / The aim of the present study was to evaluate the power of discrimination and genetic (diversity) parameters in the Y chromosome extended haploytpe markers in populations of Tanzania for forensic and populations studies. Eleven Y chromosome extended haplotype markers were selected for this study, these includes Minimal haplotypes markers i.e. DYS19, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS389I/II and two additional markers DYS438 and DYS439. Six populations of Tanzania were investigated under this study. These populations were selected based on the language family categories; Niger Congo (Kuria and Sukuma), Nilo Saharan (Luo and Maasai) and Afro Asiatic (Iraqw and Alagwa). / South Africa

A dissertação apresenta os resultados obtidos com o desenvolvimento de um programa de computação abrangente em interface gráfica para ambiente Windows visando a estimativa de parâmetros de interesse genético-populacional (freqüências alélicas, respectivos erros-padrão e intervalos de confiança a 95%) e o teste de hipóteses genéticas (equilíbrio de Hardy-Weinberg e análise de estruturação hierárquica populacional), por meio de métodos tradicionais e por meio de testes exatos obtidos com procedimentos de simulação (bootstrap e jackknife). / The dissertation presents the results obtained with the development of a comprehensive computation program (software), running on the Windows (MS) graphic interface, with the aim of: (a) estimating parameters of population genetic interest (such as allelic frequencies and their corresponding standard errors and 95% confidence intervals); and (b) performing the testing of genetic hypotheses (Hardy-Weinberg population ratios and analysis of population hierarchical structure) by means of traditional methods as well as through exact tests obtained with computer simulation procedures (bootstrap and jackknife methods).

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.