Le virus de la panachure jaune (RYMV) est une contrainte majeure pour la riziculture en Afrique. Deux gènes contrôlant des résistances récessives ont précédemment été décrits : RYMV1, qui code pour eIF(iso)4G1, un facteur d’initiation de la traduction et RYMV2, qui code pour CPR5-1, un probable composant du pore nucléaire impliqué dans la régulation des mécanismes de défense. Cependant, la capacité du virus à contourner ces résistances justifie la caractérisation de sources de résistance originales, présentes dans les espèces de riz africain O. glaberrima et O. barthii. Trois approches complémentaires ont été mises en œuvre afin d’identifier les facteurs génétiques contrôlant ces résistances.Une approche de cartographie génétique dans des populations bi-parentales a permis l’identification du gène RYMV3, contrôlant la résistance de l’accession Tog5307 et sans doute également de l’accession Tog5672. Il s’agit de la première résistance dominante identifiée dans le pathosystème riz/RYMV. RYMV3 a été cartographié dans un intervalle de 15 kb où deux gènes sont annotés, dont un gène NB-LRR. Des comparaisons de séquences entre accessions résistantes et accessions sensibles suggèrent que le polymorphisme responsable de la résistance est une mutation ponctuelle dans le domaine LRR du gène NB-LRR.Les deux autres approches ont reposé sur l’exploitation de données de séquençage Illumina de 163 accessions O. glaberrima et 84 accessions O. barthii. Les accessions O. glaberrima ont été phénotypées à la fois pour la résistance élevée et pour la résistance partielle au RYMV, et une partie des accessions O. barthii a été évaluée pour la résistance élevée. L’analyse de la variabilité allélique aux trois gènes majeurs de résistance a permis l’identification d’un probable nouvel allèle de résistance à RYMV1 et de six à RYMV2. Ces allèles sont actuellement en cours de validation. D’autre part, une approche de génétique d’association réalisée sur 125 accessions O. glaberrima a mis en évidence deux QTL de résistance partielle sur les chromosomes 6 et 11, dont l’un colocalise, en première approche, avec le gène RYMV3.Ce travail a ainsi permis l’identification d’un gène majeur, de deux QTL et de nouveaux allèles de résistance qui contribuent à une meilleure compréhension des interactions riz/RYMV et sont utilisables en sélection pour améliorer la durabilité des variétés résistantes. / The Rice yellow mottle virus (RYMV) is a major constraint for rice production in Africa. Two genes controlling recessive resistances have been previously described : RYMV1 coding for eIF(iso)4G1, a translation initiation factor, and RYMV2, coding for CPR5-1, a probable component of the nuclear pore complex involved in the regulation of defense mechanisms. However, the virus' ability to overcome these resistances highlight the need to characterize new sources of resistance in the African rice species O. glaberrima and O. barthii. Three complementary approaches were carried out in order to identify the genetic factors controlling these resistances.A genetic mapping strategy in bi-parental populations led to the identification of the RYMV3 gene, controlling resistance in the Tog5307 accession and probably also in the Tog5672 accession. It is the first dominant resistance identified in the rice/RYMV pathosystem. RYMV3 mapped in a 15 kb interval in which two genes annotated occur, including one NB-LRR gene.The two other strategies used were based on the utilization of Illumina sequencing data of 163 O. glaberrima accessions and 84 O. barthii accessions. O. glaberrima accessions were phenotyped for both high and partial resistance to RYMV, and the high resistance of a portion of the O. barthii accessions was assessed. Analysis of allelic variability at the previously identified genes led to the identification of a probable new resistance allele at RYMV1 and of six others at RYMV2. These alleles are currently undergoing validation. Furthermore, a genome wide association study was carried out on 125 O. glaberrima accessions, revealing two partial resistance QTLs on chromosomes 6 and 11, including one colocalized with RYMV3.This work has thus allowed the identification of one major resistance gene, of two QTLs and of new resistance alleles, contributing to a better understanding of rice/RYMV interactions and creating new prospects for the breeding for resistant varieties.

L'évolution climatique a été marquée ces dernières décennies par des changements importants, notamment une augmentation de température et une variation de la pluviométrie. Une des conséquences du changement climatique est son impact actuel et futur sur l'agriculture et la sécurité alimentaire. En région sahélienne, la sécurité alimentaire repose essentiellement sur quelques céréales, parmi lesquelles le mil occupe une place fondamentale. Un des traits qui permettent l'adaptation au climat de cette espèce est la date de floraison, dont la variation permet d'accomplir le cycle de vie de la plante dans des saisons pluvieuses plus ou moins courtes. Les bases génétiques de ce caractère adaptatif sont encore peu connues. Nous avons développé une méthodologie de cartographie d'association phénotype-génotype, afin d'identifier des gènes impliqués dans la variation de ce caractère. Cette étude a permis d'identifier deux gènes candidats, PHYC et MADS11, associés à la date de floraison et à la variation morphologique chez le mil. Nous avons validé ces associations par des analyses QTLs. Pour PHYC, nous avons aussi étudié le pattern de déséquilibre de liaison sur une zone d'environ 80 kb autour du gène, et développé une approche de Markov Chain Monte Carlo (MCMC) pour comparer les gènes identifiés dans la région. Cette analyse suggère que les polymorphismes à l'intérieur de PHYC sont les meilleurs candidats expliquant l'effet phénotypique observée dans cette région du génome. La seconde partie de ce projet a été consacrée à l'examen méthodologique de la cartographie d'association pour l'étude des interactions entre le génotype et l'environnement. Les résultats de cette thèse ont été discutés notamment dans la perspective de gestion de l'impact du changement climatique sur le mil, céréale majeure des zones semi-arides / In last decades, climate changes led to temperature increase and rainfall variation across the globe. One of the key consequences of these changes is their impact on agriculture and food security. In sahelian countries, food security relies on a few cereal crops, among which pearl millet plays a crucial role for population food supply. Sahel region is facing the impact of rainfall variability and drought since the 1970s. Flowering time variation is one of the main adaptations that allow pearl millet cultivation in drier and shorter rainy seasons. The genetic bases of this complex trait are still understudied. We developed an association mapping framework for the analysis of genotype-phenotype relationship in pearl millet. We successfully identified two genes associated with flowering time variation in pearl millet (PHYC and MADS11). We confirmed these associations using QTL studies. For PHYC, we also examined the pattern of linkage disequilibri um on a chromosomal region extending to 80 kb around the gene, and we developed a Markov Chain Monte Carlo approach (MCMC) to compare six genes identified in this region. Our results suggest that, among the polymorphisms observed in this region, polymorphisms in PHYC are the best candidate for a direct causative role. The second part of this project addressed methodological examination of association mapping framework to deal with genotype by environment interactions. The results of this work were discussed with regard to the challenge of pearl millet crop adaptation to climate change.

La maladie de la Sharka est due à un virus de quarantaine, le Plum Pox Virus (PPV), infectant les arbres fruitiers du genre Prunus. Il est nécessaire de trouver des moyens de lutte, telle que la sélection de plantes résistantes. Or chez ces espèces, les sources de résistance sont à l’heure actuelle en nombre limité, voire inexistantes. Il a été montré, au laboratoire, que ce virus est capable d’infecter Arabidopsis thaliana et qu’il existe chez cette espèce une grande diversité de réponse à l’infection. En effet nous avons pu observer que les accessions St-0 et JEA avais un comportement résistant, alors que l'accession Cvi-1 été partiellement résistante. Deux méthodes d’inoculation ont été comparées: une inoculation mécanique à partir de feuilles de Nicotiana benthamiana inoculées avec pICPPVnkGFP et une inoculation par agro-infection à partir d’une souche Agrobacterium tumesfasciens contenant l’isolat viral pBINPPVnkGFP. L'emploi de ces deux méthodes d'inoculation nous a permis de mettre en évidence une variabilité de la réponse au PPV en fonction de la méthode utilisée. En conséquence, cette étude visait donc à identifier le ou les facteur(s) de la plante hôte impliqué(s) dans l'infection virale. L'agro-infection de populations recombinantes (F2 et RIL), de lignées multi-parentales ainsi que l'emploi de la génétique d'association a mis en évidence chez St-0 ainsi que dans plusieurs accession distinct (sept) un locus majeur sur le groupe de liaison 3, appelé sha3. Il apparait indispensable dans le mouvement longue distance du PPV. De plus l'utilisation de la génétique d'association a permis d'initier la cartographie fine de sha3 et de réduire considérablement le nombre de gènes candidats. Un criblage de mutants a été initié afin de déterminer le ou les gènes candidats contrôlant le phénotype Sha3. Après inoculation mécanique, l’analyse d'une population recombinante a mis en évidence la présence d’un locus majeur, distinct de sha3 et positionné au milieu du bras long du groupe de liaison 1. Ce locus co-localise avec rpv1, locus identifié précédemment dans la descendance Cvi x Ler (Sicard, Loudet et al. 2008). Ce même locus a été également confirmé à la fois dans une population multi-parentale et par une approche de génétique d'association. Un gène candidat est actuellement en cours de validation au laboratoire. Une étude visant à décomposer le mécanisme de résistance porté par l’accession JEA a été mise en place. Dans ce cas, il apparait que la propagation du virus est inhibée dans les feuilles de la rosette mais pas dans les tissus floraux. Ainsi, la résistance/sensibilité au PPV chez JEA est fortement conditionnée par les stades physiologiques de la plante hôte. Des travaux complémentaires seront indispensables afin de décrire plus finement ce mécanisme de résistance très particulier. Au terme de cette thèse, nous nous attendons à ce que l’identification de ces nouveaux gènes de résistance chez Arabidopsis permette, après transfert, d’accroître la diversité des sources de résistance à la Sharka chez les arbres fruitiers. / The Plum Pox Virus (PPV) infects Prunus species (stone fruit) and is the causal agent of the Sharka disease. This disease is vastly devastating for fruit and plant productivity and quality. Its cost reaches 10 billions of euros over the last 30 years. Breeding programs have been carried out with the aim to implement resistant cultivars but the number of sources of resistance in Prunus species is rather limited. It has been shown in the laboratory that this virus is able to infect Arabidopsis thaliana with a wide range of response to infection. Indeed, we observed that accessions St-0 and JEA had a resistant behavior, while accession Cvi-1 was partially resistant. Two inoculation methods were compared: mechanical inoculation from Nicotiana benthamiana leaves inoculated with pICPPVnkGFP and agro-inoculation infection from an Agrobacterium strain containing the viral isolate tumesfasciens pBINPPVnkGFP. The use of these two methods of inoculation allows us to highlight variability in the response to PPV depending on the method used. This study aims to identify the factor (s) of the host (s) involved in viral infection. Agro-infection of recombinant populations (F2 and RIL), multi-parental lines and the use of genetic association demonstrate in St-0 and several distinct accessions (seven) a major locus on linkage group 3, called sha3. It appears essential in the long-distance movement of PPV. Use of association genetics helped initiate the fine mapping of sha3 and significantly reduce the number of candidate genes. Screening of mutants was initiated to determine the gene controlling the phenotype Sha3. After mechanical inoculation, the analysis of a recombinant population revealed the presence of a major locus positioned in the middle of the long arm of linkage group 1. This locus co-localizes with rpv1, previously identified in Cvi x Ler offspring (Sicard, Loudet et al. 2008). The same locus was also confirmed with a multi-parental population and by a genetic association approach. A candidate gene is currently being validated in the laboratory. The study of the resistance mechanism carried by the accession JEA was initiated. In this case, it appears that the spread of the virus is inhibited in basal leaves but not in floral stem. The resistance / susceptibility to PPV in JEA appear to be strongly influenced by the physiological stages of the host plant. Further work will be necessary to describe more precisely this resistance mechanism very special. At the end of this thesis, we expect that the identification of these new resistance genes in Arabidopsis allows, after transfer, to increase the diversity of sources of resistance to plum pox virus in fruit trees.

A caracterização e o entendimento das variações genômicas e morfológicas, bem como a estrutura genética de variedades locais armazenadas em bancos de germoplasma é importante para sua efetiva utilização em programas de melhoramento visando tolerância a estresses. Neste trabalho um conjunto de 192 variedades oriundas de diferentes regiões geoclimáticas do Japão foram testadas quanto à suas características morfológicas e produtivas, utilizando ensaios de campo e metodogias de fenotipagem de alto desempenho. A fenotipagem por meio da metodologia de camadas de herbicida (Aminotriazol+Diuron+2,4D, 100mg/plant) alocado a 30 centímetros de profundidade foi possível detectar variação entre as variedades para comprimento de raiz e velocidade de emissão de raiz sendo possível a distinção de variedades com sistema radicular profundo e sistema radicular superficial baseando-se na sua pontuação no ensaio de herbicida, destacando-se 20 genótipos como possíveis doadores de genes para comprimento, densidade e velocidade de emissão de raízes. Ensaios a campo foram conduzidos em 4 localidades expondo as variedades as mais distintas condições climáticas, buscando analisar a diversidade fenotípica para caracteres agromorfológicos. Os dados fenotípicos obtidos pelos marcadores morfológicos geraram um total de 15 grupos de acessos quando utilizados os 13 caracteres avaliados. A média do índice de sensibilidade a seca foi de 0,99 havendo materiais tolerantes com índices próximos a 0,6 e materiais sensíveis com índice próximos a 1,12. Os 384 marcadores SNP detectaram um total de 73728 alelos indicando alta porcentagem de A (40,8%) e G (34,6%) comparado com C (15,6%) e T (3,6%). Quanto aos heterozigotos, a maior porcentagem foi observada de A/G (0,54%) e a menor porcentagem de A/T (0,04%), sendo a maior parte dos heterozigotos observados nos cromossomos 3 e 8 comparado com outros cromossomos. As análises caracterizaram os acessos japoneses como 98,4% pertencentes à subespécie Japônica. Para associação entre marcadores e fenótipos, foi utilizada a abordagem de modelo linear misto (MLM), o qual incorpora informações de estrutura populacional e parentesco. Os resultados obtidos deverão ser investigados futuramente a fim de confirmar as associações em diferentes populações. O aumento do estresse hídrico teve efeito significativo no desempenho dos acessos, e a interação genótipo e estresse hídrico foi significativa para rendimento final e tamanho de panícula. Entre os acessos estudados foi observada variação genética para as características relacionadas com a tolerância a estresse hídrico e encontraram-se acessos com reduções no rendimento devido ao déficit hídrico comparáveis com o das testemunhas, embora com tamanho de panícula menor, inclusive em condições ótimas. Dos 384 marcadores utilizados, 10 foram responsáveis por associações significativas com o índice de sensibilidade a seca, com base nos diferentes métodos de correção para múltiplos testes. Estas associações foram selecionadas para verificar o efeito alélico sobre o genótipo observado, gerando informações preliminares para a aplicação futura de seleção assistida por marcadores (SAM). O tamanho dos blocos de ligação foram estimados em ~100 kb (r < 0,05) e ~75kb (r < 0,1). / Germplasm characterization and the knowledge of its diversity and population structure are important to effective utilization of genetic resources in breeding programs specially drought breeding program. In this work 192 landraces from all over Japan were evaluate for their morphological and productive characteristics, using field trials and high throughput screening methods. The screening using herbicide barrier approach at 30 cm depth was able to detect genetic diversity between the landraces for root length and clearly distinguish between deep root landraces and shallow root landraces. With this approach was possible to select 20 landraces with deep root system as possible donor of drought tolerance genes. Aiming characterize the landraces to agromorphological characteristics field trials were carried in 4 different locations exposing the landraces for a diversity environment effect. Using 13 traits phenotypically data generate a total of 15 groups during cluster analysis. The average result for drought sensitive index was 0.99, however this results presents a huge variability having landraces with scores about 0.6 to landraces with score up to 1.12. Drought effect was huge and statistically significant affecting directly yield and panicle size. The landraces presented genetic variability for drought tolerance and some landraces presenting yield and panicle size reductions due to drought comparable with drought-tolerant controls were detected. A total of 73728 alleles were detected by the SNP markers, indicated a high percentage of A (40,8%) and G (34,6%) alleles compared to C(15,6%) and T (3.6%). Heterozygocity of A/G was highest (0,54%) and lowest in A/T (0.04%). Of 3 chromosomes of rice, chromosome 8 produced highest percentage of heterozygocity compared to other chromosomes. Accessions were classified as 98.4% belonging to japônica subspecies. Association between markers and phenotypes was performed using a mixed linear approach (MLM), which incorporates information regarding population structure and kinship. Among the 384 markers used, 10 were responsible for significant associations with drought sensibility index, based on different criteria to correct for multiple tests.These associations were selected to determine the allelic effects over the traits, in order to generate preliminary data for marker assisted selections (MAS). Estimated size of haplotype blocks were ~100 kb (r <0.05) and ~75 kb (r <0.1). Future studies should confirm marker trait associations here found using different populations.

A estrutura populacional e o desequilíbrio de ligação são dois processos fundamentais para estudos evolutivos e de mapeamento associativo. Tradicionalmente, ambos têm sido investigados por meio de métodos clássicos comumente utilizados. Tais métodos certamente forneceram grandes avanços no entendimento dos processos evolutivos das espécies. No entanto, em geral, nenhum deles utiliza uma visão genealógica de forma a considerar eventos genéticos ocorridos no passado, dificultando a compreensão dos padrões de variação observados no presente. Uma abordagem que possibilita a investigação retrospectiva com base no atual polimorfismo observado é a teoria da coalescência. Assim, o objetivo deste trabalho foi analisar, com base na teoria da coalescência, a estrutura populacional e o desequilíbrio de ligação de um painel mundial de acessos de sorgo (Sorghum bicolor). Para tanto, análises de mutação, migração com fluxo gênico e recombinação foram realizadas para cinco regiões genômicas relacionadas à altura de plantas e maturidade (Dw1, Dw2, Dw4, Ma1 e Ma3) e sete populações previamente selecionadas. Em geral, elevado fluxo gênico médio (&Mu; = m/&mu; = 41,78 &minus; 52,07) foi observado entre as populações considerando cada região genômica e todas elas simultaneamente. Os padrões sugeriram intenso intercâmbio de acessos e história evolutiva específica para cada região genômica, mostrando a importância da análise individual dos locos. A quantidade média de migrantes por geração (&Mu;) não foi simétrica entre pares recíprocos de populações, de acordo com a análise individual e simultânea das regiões. Isso sugere que a forma pela qual as populações se relacionaram e continuam interagindo evolutivamente não é igual, mostrando que os métodos clássicos utilizados para investigar estrutura populacional podem ser insatisfatórios. Baixas taxas médias de recombinação (&rho;L = 2Ner = 0,030 &minus; 0,246) foram observadas utilizando o modelo de recombinação constante ao longo da região. Baixas e altas taxas médias de recombinação (&rho;r = 2Ner = 0,060 &minus; 3,395) foram estimadas utilizando o modelo de recombinação variável ao longo da região. Os métodos tradicional (r2) e via coalescência (E[r2 rhomap]) utilizados para a estimação do desequilíbrio de ligação mostraram resultados próximos para algumas regiões genômicas e populações. No entanto, o r2 sugeriu padrões descontínuos de desequilíbrio em várias ocasiões, dificultando o entendimento e a caracterização de possíveis blocos de associação. O método via coalescência (E[r2 rhomap]) forneceu resultados que pareceram ter sido mais consistentes, podendo ser uma estratégia eventualmente importante para um refinamento dos padrões não-aleatórios de associação. Os resultados aqui encontrados sugerem que o mapeamento genético a partir de um único pool gênico pode ser insuficiente para detectar associações causais importantes para características quantitativas em sorgo. / Population structure and linkage disequilibrium are two fundamental processes for evolution and association mapping studies. Traditionally, both have been investigated using classical methods that are commonly used. These methods certainly provided important advances for the understanding of the evolution processes of the species. However, in general, none of them uses a genealogical view to consider genetic events occurred in the past, making difficult the understanding of the variation patterns observed in the present. An approach that enables the retrospective investigation based on the actual observed polymorphism is the coalescent theory. Here, we used the coalescent theory to analyze the population structure and linkage disequilibrium of a worldwide sorghum (Sorghum bicolor) accession panel. To reach this purpose, analyses of mutation, migration with gene flow and recombination were performed to five genomic regions related to plant height and maturity (Dw1, Dw2, Dw4, Ma1 e Ma3) and seven previously selected populations. In general, high average gene flow (&Mu; = m/&mu; = 41,78 &minus; 52,07) was observed between populations considering each genomic region and all the regions simultaneously. The patterns suggested a high exchance of accessions between populations and a specific evolutionary history for each genomic region, showing that the individual analysis of each locus was important. The average number of migrants per generation (&Mu;) was not symmetric between reciprocal pairs of populations, according to the specific and simultaneous analyses of the regions. This result suggests that the historical and recent evolutionary relations between populations are not equal, showing that the classical methods to investigate population structure may be unsatisfactory. Low average recombination rates (&rho;L = 2Ner = 0,030 &minus; 0,246) were observed using a constant recombination model along the region. Low and high average recombination rates (&rho;r = 2Ner = 0,060 &minus; 3,395) were estimated using a variable recombination model along the region. Both traditional (r2) and coalescent (E[r2 rhomap]) methods for the estimation of linkage disequilibrium showed similar results for some genomic regions and populations. However, r2 suggested discontinuous patterns of linkage disequilibrium in several cases, making difficult the understanding and definition of the association blocks. The coalescent method (E[r2 rhomap]) provided results that seemed to be more consistent and could be an eventually important strategy to refine the non-random association patterns. The results detected here suggest that the genetic mapping from a unique gene pool may be insufficient to detect important causal associations for quantitative traits in sorghum.

Common bean is a major dietary component in several countries, but its productivity is negatively affected by abiotic stresses. Dissecting candidate genes involved in abiotic stress tolerance is a paramount step toward the improvement of common bean performance under such constraints. Thereby, this thesis presents a systematic analysis of the DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) gene subfamily, which encompasses genes that regulate several processes during stress responses, but with limited information for common bean. First, a series of in silico analyses with sequences retrieved from the P. vulgaris genome on Phytozome supported the categorization of 54 putative PvDREB genes distributed within six phylogenetic subgroups (A-1 to A-6), along the 11 chromosomes. Second, we cloned four novel PvDREB genes and determined their inducibility-factors, including the dehydration-, salinity- and cold-inducible genes PvDREB1F and PvDREB5A, and the dehydration- and cold-inducible genes PvDREB2A and PvDREB6B. Afterwards, nucleotide polymorphisms were searched through Sanger sequencing along those genes, revealing a high number of single nucleotide polymorphisms within PvDREB6B by the comparison of Mesoamerican and Andean genotypes. The nomenclature of PvDREB6B is discussed in details. Furthermore, we used the BARCBean6K_3 SNP platform to identify and genotype the closest SNP to each one of the 54 PvDREB genes. We selected PvDREB6B for a broader study encompassing a collection of wild common bean accessions of Mesoamerican origin. The population structure of the wild beans was accessed using sequence polymorphisms of PvDREB6B. The genetic clusters were partially associated with variation in latitude, altitude, precipitation and temperature throughout the areas such beans are distributed. With an emphasis on drought stress, an adapted tube-screening method in greenhouse conditions enabled the phenotyping of several drought-related traits in the wild collection. Interestingly, our data revealed a correlation between root depth, plant height and biomass and the environmental data of the location of the accessions. Correlation was also observed between the population structure determined through PvDREB6B and the environmental data. An association study combining data from the SNP array and DREB polymorphisms enabled the detection of SNP associated with drought-related traits through a compressed mixed linear model (CMLM) analysis. This thesis highlighted important features of DREB genes in common bean, revealing candidates for further strategies aimed at improvement of abiotic stress tolerance, with emphasis on drought tolerance / O feijoeiro é um componente essencial na dieta em diversos países, no entanto, sua produção é afetada negativamente por estresses abióticos. O estudo de genes candidatos envolvidos na adaptação aos estresses é uma etapa fundamental para o melhoramento da performance do feijoeiro sob tais estresses. Desse modo, esta tese apresenta uma análise sistemática da subfamília de genes DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB), que reúne genes envolvidos em diversos processos em resposta a estresses, mas pouco estudados no feijoeiro. Primeiramente, uma série de análises in silico com sequências de feijoeiro obtidas da plataforma Phytozome possibilitaram a categorização de 54 genes PvDREB putativos, distribuídos em seis subgrupos (A-1 até A-6) nos 11 cromossomos da espécie. Posteriormente, quatro novos genes PvDREB foram clonados e seus padrões de inducibilidade foram determinados. PvDREB1F e PvDREB5A foram induzidos por desidratação, baixa temperatura e salinidade, enquanto PvDREB2A e PvDREB6B foram predominantemente induzidos por desidratação e baixa temperatura. Polimorfismos de nucleotídeos foram buscados através de sequenciamento por método derivado de Sanger, revelando elevado número de SNP no gene PvDREB6B. A nomenclatura desse gene foi discutida detalhadamente ao longo da tese. A plataforma de marcadores SNP BARCBean6K_3 foi acessada para identificar o SNP mais próximo de cada um dos 54 PvDREB. O gene PvDREB6B foi selecionado para um estudo mais amplo, envolvendo uma coleção de acessos selvagens de origem Mesoamericana. A estrutura populacional destes genótipos foi analisada a partir de polimorfismos na sequência de PvDREB6B. Os grupos genéticos apresentaram associação parcial com variação da latitude, altitude, precipitação e temperatura das áreas em que os acessos naturalmente ocorrem. Com ênfase no estudo do déficit hídrico, uma plataforma de fenotipagem destes acessos em casa de vegetação, utilizando um sistema de tubos plásticos, foi elaborada para a análise de diversos parâmetros relacionados ao estresse por déficit hídrico. Os dados revelaram correlação entre profundidade de raízes, altura das plantas e a biomassa e as variáveis ambientais de cada local. A correlação também foi detectada entre a estrutura populacional estudada por PvDREB6B e os dados ambientais. Finalmente, um estudo de associação genética foi realizado entre os SNP da plataforma e ligados a DREB e os parâmetros fenotípicos, permitindo a identificação de marcadores SNP associados a caracteres específicos, usando um modelo linear misto (CMLM). Esta tese apresentou importantes aspectos sobre os genes DREB em feijoeiro, revelando candidatos para seu uso em estratégias de melhoramento para tolerância a estresses abióticos, com ênfase em déficit hídrico

L’amélioration de la qualité du fruit de tomate dépend largement de la variation génétique. A la suite de la domestication et de la sélection moderne, la diversité moléculaire chez la tomate a été profondément réduite, limitant les possibilités d’amélioration. De nouveaux marqueurs moléculaires révélant les polymorphismes nucléotidiques (SNP) constituent des outils précieux pour saturer les cartes génétiques et identifier des QTL (quantitative trait loci) et des associations chez une espèce peu polymorphe comme la tomate. Les objectifs de cette étude étaient de caractériser la diversité génétique de la tomate au niveau moléculaire et de tenter d’identifier des QTL, des gènes et des protéines responsables de la variation de caractères de qualité du fruit. Pour cela, trois études indépendantes ont conduit à (1) la découverte de nouveaux marqueurs SNP, (2) leur utilisation en génétique d’association et (3) l’analyse de la diversité du protéome en relation avec des caractères physiologiques du fruit.Dans la première étude, nous avons comparé deux plateformes de reséquençage pour reséquencer des zones ciblées couvrant environ 0.2% du génome de deux accessions contrastées. Plus de 3000 SNP sont été identifiés. Nous avons ensuite validé 64 SNPs en développant des marqueurs CAPS. Nous avons ainsi montré l’intérêt des techniques de reséquençage pour la découverte de SNP chez la tomate et produit des marqueurs simples qui peuvent être utiles pour caractériser de nouvelles ressources.Nous avons ensuite développé un ensemble de 192 SNPs et génotypé une collection de 188 accessions comportant des accessions cultivées, des types “cerise” et des formes sauvages apparentées et recherché des associations avec 10 caractères de qualité du fruit. Une quarantaine d’associations a été détectée dans des régions où des QTL avaient été préalablement identifiés. D’autres associations ont été identifiées dans de nouvelles régions. Nous avons ainsi confirmé le potentiel de la génétique d’association pour la découverte de QTL chez la tomate. Finalement une approche combinant l’analyse du protéome, du métabolome et de traits phénotypiques a été mise en oeuvre pour étudier la variabilité naturelle de la qualité du fruit de huit lignées contrastées et de quatre de leurs hybrides, à deux stades de développement (expansion cellulaire et orange-rouge). Nous avons identifié 424 spots protéiques variables en combinant électrophorèse bidimensionnelle et nano LC MS/MS et construit une carte de référence du protéome de fruit de tomate. En parallèle, nous avons mesuré la variation de teneurs en 34 métabolites, les activités de 26 enzymes et cinq caractères phénotypiques. La variabilité génétique et les modes d’hérédité ont été décrits. L’intégration des données a été réalisée par construction de réseaux de corrélations et régression sPLS. Plusieurs associations ont été détectées intra et inter niveau d’expression, permettant une meilleure compréhension de la variation de la qualité des fruits de tomate / Fruit quality in tomato is highly dependent on genetic variation. Following domestication and modernbreeding, molecular diversity of tomato has been strongly reduced, limiting the possibility to improvetraits of interest. New molecular markers such as single nucleotide polymorphisms (SNP) constituteprecious tools to saturate tomato genetic maps and identify quantitative trait loci (QTL) andassociations in a poorly polymorphic species like tomato. The objectives of this study were tocharacterize tomato genetic diversity at the molecular levels and to try to identify QTLs, genes andproteins responsible for fruit quality traits in tomato. For this purpose, three independent studies wereconducted leading to the discovery of new SNP markers, their use for association study and finally theanalysis of proteome diversity in relation to physiological phenotypes. We first used two nextgenrationsequencing platforms (GA2 Illumina and 454 Roche) to re-sequence targeted sequencescovering about 0.2% of the tomato genome from two contrasted accessions. More than 3000 SNPswere identified between the two accessions. We then validated 64 SNPs by developing CAPS markers.We thus showed the value of NGS for the discovery of SNPs in tomato and we produced low costCAPS markers which could be used to characterize other tomato collections. A SNPlexTM arraycarrying 192 SNPs was then developed and used to genotype a broad collection of 188 accessionsincluding cultivated, cherry type and wild tomato species and to associate these polymorphisms to tenfruit quality traits using association mapping approach. A total of 40 associations were detected andco-localized with previously mapped QTLs. Some other associations were identified in new regions.We showed the potential of using association genetics in tomato. Finally, a new analytical approachcombining proteome, metabolome and phenotypic profiling were applied to study natural geneticvariation of fruit quality traits in eight diverse accessions and their four corresponding F1s at cellexpansion and orange-red stages. We identified 424 variable spots by combining 2-DE and nano LCMS/MS and built the first comprehensive proteome reference map of the tomato fruit pericarp at twodevelopmental stages from the 12 genotypes. In parallel, we measured the variation of 34 metabolites,26 enzyme activities and five phenotypic traits. A large range of variability and several inheritancemodes were described in the four groups of traits. Data integration was achieved through sPLS andcorrelation networks. Many significant associations were detected within level and between levels ofexpression. This systems biology approach provides better understanding of networks of elements(proteins, enzymes, metabolites and phenotypic traits) in tomato fruits

Understanding complex traits is a fundamental challenge in plant genetics and a prerequisite for molecular breeding. Tools for trait dissection are markers, maps, and quantitative trait locus (QTL) analysis. Marker-assisted selection (MAS) is an application that integrates these tools. In this thesis research, a new sequence-based marker was evaluated, maps were constructed and used, and QTLs were detected using two types of populations. Marker-assisted selection was used to develop a novel class of barley. Restriction-site Associated DNA (RAD), a sequence based-marker technology, allows for simultaneous high-density single nucleotide polymorphism (SNP) discovery and genotyping. We assessed the value of RAD markers for linkage map construction using the Oregon Wolfe Barley (OWB) mapping population. We compared a RAD-based map to a map generated using Illumina GoldenGate Assay (EST-based SNPs). The RAD markers generated a high quality map with complete genome coverage. We then used the RAD map to locate QTL for agronomic fitness traits. A paper describing this research was published (Chutimanitsakun et al., 2011). Marker-assisted selection was used to rapidly develop fall-sown barley germplasm for human food uses. The target traits were high grain β-glucan, vernalization sensitivity (VS) and low temperature tolerance (LTT). The target loci were WX and VRN-H2. Marker-assisted selection was effective in fixing target alleles at both loci and waxy starch led to increase in grain β-glucan. Unexpected segregation at VRN-H1 and VRN-H3, revealed by genome-wide association mapping (GW-AM), led to unanticipated phenotypic variation in VS and LTT. We found that GW-AM is an efficient and powerful method for identifying the genome coordinates of genes determining target traits. Precise information is obtained with perfect markers; additional research may be needed when multiple alleles are segregating at target loci and significant associations are with markers in linkage disequilibrium (LD) with the target loci. A paper describing this research will be submitted for publication. / Graduation date: 2012

The genome can be considered the blueprint for an organism. Composed of DNA, it harbours all organism-specific instructions for the synthesis of all structural components and their associated functions. The role of carriers of actual molecular structure and functions was believed to be exclusively assumed by proteins encoded in particular segments of the genome, the genes. In the process of converting the information stored genes into functional proteins, RNA – a third major molecule class – was discovered early on to act a messenger by copying the genomic information and relaying it to the protein-synthesizing machinery. Furthermore, RNA molecules were identified to assist in the assembly of amino acids into native proteins. For a long time, these - rather passive - roles were thought to be the sole purpose of RNA. However, in recent years, new discoveries have led to a radical revision of this view. First, RNA molecules with catalytic functions - thought to be the exclusive domain of proteins - were discovered. Then, scientists realized that much more of the genomic sequence is transcribed into RNA molecules than there are proteins in cells begging the question what the function of all these molecules are. Furthermore, very short and altogether new types of RNA molecules seemingly playing a critical role in orchestrating cellular processes were discovered. Thus, RNA has become a central research topic in molecular biology, even to the extent that some researcher dub cells as “RNA machines”. This thesis aims to contribute towards our understanding of RNA-related phenomena by applying Bioinformatics means. First, we performed a genome-wide screen to identify sites at which the chemical composition of DNA (the genotype) critically influences phenotypic traits (the phenotype) of the model plant Arabidopsis thaliana. Whole genome hybridisation arrays were used and an informatics strategy developed, to identify polymorphic sites from hybridisation to genomic DNA. Following this approach, not only were genotype-phenotype associations discovered across the entire Arabidopsis genome, but also regions not currently known to encode proteins, thus representing candidate sites for novel RNA functional molecules. By statistically associating them with phenotypic traits, clues as to their particular functions were obtained. Furthermore, these candidate regions were subjected to a novel RNA-function classification prediction method developed as part of this thesis. While determining the chemical structure (the sequence) of candidate RNA molecules is relatively straightforward, the elucidation of its structure-function relationship is much more challenging. Towards this end, we devised and implemented a novel algorithmic approach to predict the structural and, thereby, functional class of RNA molecules. In this algorithm, the concept of treating RNA molecule structures as graphs was introduced. We demonstrate that this abstraction of the actual structure leads to meaningful results that may greatly assist in the characterization of novel RNA molecules. Furthermore, by using graph-theoretic properties as descriptors of structure, we indentified particular structural features of RNA molecules that may determine their function, thus providing new insights into the structure-function relationships of RNA. The method (termed Grapple) has been made available to the scientific community as a web-based service. RNA has taken centre stage in molecular biology research and novel discoveries can be expected to further solidify the central role of RNA in the origin and support of life on earth. As illustrated by this thesis, Bioinformatics methods will continue to play an essential role in these discoveries. / Das Genom eines Organismus enthält alle Informationen für die Synthese aller strukturellen Komponenten und deren jeweiligen Funktionen. Lange Zeit wurde angenommen, dass Proteine, die auf definierten Abschnitten auf dem Genom – den Genen – kodiert werden, die alleinigen Träger der molekularen - und vor allem katalytischen - Funktionen sind. Im Prozess der Umsetzung der genetischen Information von Genen in die Funktion von Proteinen wurden RNA Moleküle als weitere zentrale Molekülklasse identifiziert. Sie fungieren dabei als Botenmoleküle (mRNA) und unterstützen als Trägermoleküle (in Form von tRNA) die Zusammenfügung der einzelnen Aminosäurebausteine zu nativen Proteine. Diese eher passiven Funktionen wurden lange als die einzigen Funktionen von RNA Molekülen angenommen. Jedoch führten neue Entdeckungen zu einer radikalen Neubewertung der Rolle von RNA. So wurden RNA-Moleküle mit katalytischen Eigenschaften entdeckt, sogenannte Ribozyme. Weiterhin wurde festgestellt, dass über proteinkodierende Abschnitte hinaus, weit mehr genomische Sequenzbereiche abgelesen und in RNA Moleküle transkribiert werden als angenommen. Darüber hinaus wurden sehr kleine und neuartige RNA Moleküle identifiziert, die entscheidend bei der Koordinierung der Genexpression beteiligt sind. Diese Entdeckungen rückten RNA als Molekülklasse in den Mittelpunkt moderner molekularbiologischen Forschung und führten zu einer Neubewertung ihrer funktionellen Rolle. Die vorliegende Promotionsarbeit versucht mit Hilfe bioinformatorischer Methoden einen Beitrag zum Verständnis RNA-bezogener Phänomene zu leisten. Zunächst wurde eine genomweite Suche nach Abschnitten im Genom der Modellpflanze Arabidopsis thaliana vorgenommen, deren veränderte chemische Struktur (dem Genotyp) die Ausprägung ausgewählter Merkmale (dem Phänotyp) entscheidend beeinflusst. Dabei wurden sogenannte Ganz-Genom Hybridisierungschips eingesetzt und eine bioinformatische Strategie entwickelt, Veränderungen der chemischen Struktur (Polymorphismen) anhand der veränderten Bindung von genomischer DNA aus verschiedenen Arabidopsis Kultivaren an definierte Proben auf dem Chip zu detektieren. In dieser Suche wurden nicht nur systematisch Genotyp-Phänotyp Assoziationen entdeckt, sondern dabei auch Bereiche identifiziert, die bisher nicht als proteinkodierende Abschnitte annotiert sind, aber dennoch die Ausprägung eines konkreten Merkmals zu bestimmen scheinen. Diese Bereiche wurden desweiteren auf mögliche neue RNA Moleküle untersucht, die in diesen Abschnitten kodiert sein könnten. Hierbei wurde ein neuer Algorithmus eingesetzt, der ebenfalls als Teil der vorliegenden Arbeit entwickelt wurde. Während es zum Standardrepertoire der Molekularbiologen gehört, die chemische Struktur (die Sequenz) eines RNA Moleküls zu bestimmen, ist die Aufklärung sowohl der Struktur als auch der konkreten Funktion des Moleküls weitaus schwieriger. Zu diesem Zweck wurde in dieser Arbeit ein neuer algorithmischer Ansatz entwickelt, der mittels Computermethoden eine Zuordnung von RNA Molekülen zu bestimmten Funktionsklassen gestattet. Hierbei wurde das Konzept der Beschreibung von RNA-Sekundärstrukturen als Graphen genutzt. Es konnte gezeigt werden, dass diese Abstraktion von der konkreten Struktur zu nützlichen Aussagen zur Funktion führt. Des weiteren konnte demonstriert werden, dass graphen-theoretisch abgeleitete Merkmale von RNA-Molekülen einen neuen Zugang zum Verständnis der Struktur-Funktionsbeziehungen ermöglichen. Die entwickelte Methode (Grapple) wurde als web-basierte Anwendung der wissenschaftlichen Welt zur Verfügung gestellt. RNA hat sich als ein zentraler Forschungsgegenstand der Molekularbiologie etabliert und neue Entdeckungen können erwartet werden, die die zentrale Rolle von RNA bei der Entstehung und Aufrechterhaltung des Lebens auf der Erde weiter untermauern. Bioinformatische Methoden werden dabei weiterhin eine essentielle Rolle spielen.

Le riz est l'une des céréales les plus importantes au monde. Au Vietnam, le riz est également considéré comme un produit agronomique clé pour l'exportation. Cependant, le stress dû à la sécheresse menace la production de riz de plus en plus fréquemment et sur de plus longues périodes. Les racines coronaires constituent une partie importante du système racinaire du riz et jouent un rôle crucial dans le maintien du rendement en cas de sécheresse. Le nombre de racines coronaires affecte la biomasse de racines et détermine la capacité de la plante à extraire les ressources du sol. Un QTL de NRC, qNCR11, localisé sur le chromosome 11, avait été détecté dans une étude d’association précédente utilisant un panel de riz vietnamiens. Dans notre étude, son effet sur le NCR, léger, a été validé par cartographie de QTL en utilisant une population biparentale . Afin de déterminer les gènes sous-jacents à qNCR11 et régissant l'initiation et le développement des racines coronaires, une étude de séquençage du génome entier et une étude d'expression ont été réalisées. Deux gènes candidats, NCR2 (NBS-LRR) et NCR3 (OsbHLH014) ont été identifiés. NCR2 portait un SNP non synonyme dans son ORF, provoquant un codon stop prématuré corrélé avec un NCR élevé; NCR3 était moins exprimé dans les bases de la tige des plantes à haplotype NCR élevé que chez les plantes à haplotype NCR faible. Des mutations dans ces gènes ont été obtenues à l'aide du système CRISPR / Cas9 et le phénotypage des lignées obtenues est en cours. Le QTL qNCR11 à effet mineur pourrait être utile aux sélectionneurs pour produire des variétés de riz avec un NCR augmenté ou diminué pour les différents écosystèmes-cibles, afin de favoriser l'extraction de l'eau dans des conditions de sécheresse. / Rice is one of the most important cereals worldwide. In Vietnam, rice is also known as a key agronomic product for exportation. However, drought stresses threaten rice production with an increasing frequency and for longer periods. Crown roots are a major component of rice root system and play a crucial role in maintaining yield under drought. The number of crown roots (NCR) impacts on root biomass and determines the ability of a plant to acquire soil resources. qNCR11, a QTL for NCR located on chromosome 11, was detected in a previous genome-wide association study using a Vietnamese rice panel. qNCR11 was validated to have a slight effect on NCR by QTL mapping using a biparental population in this study. To determine the genes underlying qNCR11 and governing crown root initiation and development, whole genome sequencing and expression study were performed. Two candidate genes, NCR2 (NBS-LRR) and NCR3 (OsbHLH014) were identified. NCR2 carried a non-synonymous SNP inside its ORF, causing a premature stop-codon that correlates with the high NCR trait; NCR3 was less expressed in stem bases of the high NCR haplotype plants relative to the low NCR haplotype plants. Mutations in these genes were obtained using the CRISPR/Cas9 system and the phenotyping of the obtained lines is on-going. The minor-effect qNCR11 could be useful for breeders to generate rice varieties with increased or decreased NCR for different target agro-systems, in order to enhance water extraction under drought stress.