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Génomique Comparative Et Fonctionnelle De Familles De Gènes Liés Au Métabolisme Secondaire De La Vigne (Vitis Vinifera) Et De Ses Proches Parents Gautier Arista

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Génomique Comparative Et Fonctionnelle De Familles De Gènes Liés Au Métabolisme Secondaire De La Vigne (Vitis Vinifera) Et De Ses Proches Parents Gautier Arista Génomique comparative et fonctionnelle de familles de gènes liés au métabolisme secondaire de la vigne (Vitis vinifera) et de ses proches parents Gautier Arista To cite this version: Gautier Arista. Génomique comparative et fonctionnelle de familles de gènes liés au métabolisme secondaire de la vigne (Vitis vinifera) et de ses proches parents. Génomique, Transcriptomique et Protéomique [q-bio.GN]. Université de Strasbourg, 2017. Français. NNT : 2017STRAJ010. tel- 01726947 HAL Id: tel-01726947 https://tel.archives-ouvertes.fr/tel-01726947 Submitted on 8 Mar 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Présentée à l’Université de Strasbourg Pour l’obtention du titre de Docteur d’Université Discipline : Aspects moléculaires et cellulaires de la biologie Gautier ARISTA Génomique comparative et fonctionnelle de familles de gènes liés au métabolisme secondaire de la vigne (Vitis vinifera) et de ses proches parents. Soutenue le 31 janvier 2017 devant le Jury : Rapporteurs : P. FAIVRE-RAMPANT, Chargée de recherche INRA, Evry E. PAUX, Directeur de recherche INRA, Clermont-Ferrand Examinateurs : H. SCHALLER, Directeur de recherche CNRS, Strasbourg V. GEFFROY, Directrice de recherche INRA, Paris-Saclay Encadrants : P. HUGUENEY, Directeur de recherche INRA, Colmar C. RUSTENHOLZ, Maître de conférences Université de Strasbourg Financée par : Résumé La vigne (Vitis vinifera) possède un métabolisme secondaire particulièrement riche donnant naissance à une large palette de molécules dont certaines sont impliquées dans les défenses contre les pathogènes et d'autres dans la grande diversité d’arômes qui fait la renommée des vins. L’analyse de la séquence de référence du génome de la vigne a permis de mettre en évidence une remarquable expansion de certaines familles de gènes liés au métabolisme secondaire par rapport aux autres plantes. Dans ce travail, j'ai étudié les familles gènes codant pour les cytochromes P450, dont certains sont impliqués dans la production d’arômes, les gènes codant pour les stilbènes synthases (STS), les endo-β-1,3-glucanases et les gènes de résistance de type NBS impliqués dans les défenses de la vigne. Ma thèse vise à proposer des hypothèses expliquant l’organisation structurale de ces familles de gènes et ainsi à mieux comprendre pourquoi certaines familles présentent une amplification dans le génome de la vigne. Des approches bioinformatiques ont été utilisées afin d’étudier ces différentes familles de gènes. Les gènes cytochromes P450 et gènes R de type NBS ont tout d'abord été annotés de manière manuelle dans le génome de référence de la vigne. L’expression des gènes endo-β-1,3- glucanases, STS et cytochromes P450 a été analysée en utilisant une approche transcriptomique à grande échelle. Pour ce faire, un outil a été développé durant cette thèse pour estimer le niveau d’expression des gènes à partir de données RNA-Seq disponibles dans les banques de données publiques. Parallèlement, des données de reséquençage d’ADN de 56 cépages et espèces de vigne ont été analysées, afin de déterminer les variations structurales de type CNV au sein des familles de gènes à domaine NBS et de gènes STS. Ces différents travaux ont permis de montrer que l’amplification des familles de gènes étudiées n’est pas spécifique du génome de référence mais est retrouvée dans l'ensemble du genre Vitis, mais également de mettre en évidence des variations structurales au sein des différents génomes étudiés. L'analyse de la famille STS a montré que ces gènes sont organisés en blocs de duplication, et que les gènes plus conservés sont aussi les plus exprimés. Nous avons également montré que les gènes à domaine NBS sont organisés en cluster, dont certains sont particulièrement soumis à variation. Ces travaux contribuent à une meilleure connaissance de facteurs de défense efficaces et durables ainsi que des gènes impliqués dans la synthèse d’arômes dans la vigne. Ces connaissances pourront bénéficier aux programmes de création variétale mis en œuvre à l’INRA de Colmar. Mots clés : vigne, familles de gènes, génomique comparative, transcriptomique, CNV Abstract Grapevine (Vitis vinifera) has a particularly rich secondary metabolism, giving rise to a wide range of molecules, some of which are involved in defences against pathogens and others in the great diversity of aromas that make wines famous. Analysis of grapevine reference genome has shown a remarkable expansion of certain families of genes linked to secondary metabolism in comparison with the other plants. In this work, I have analysed gene families coding for cytochromes P450, some of them being involved in the production of aromas, genes coding for stilbene synthases (STS), endo-β-1,3-glucanases and NBS type resistance genes involved in grapevine defences. My thesis intends to propose hypothesis to explain the structural organisation of these families and therefore better understand why some of these families are amplified in the grapevine genome. Bioinformatic approaches have been used to study these different genes families. The cytochromes P450 and R genes of NBS type were manually annotated to improve the knowledge of these families of genes. The expression of endo-β-1,3-glucanases, STS and cytochromes P450 genes has been quantified using a large-scale transcriptomic approach. To this purpose, a tool has been developed during this thesis to estimate the level of genes expression from RNA- Seq data available in public databases. In the meantime, DNA resequencing data from 56 cultivars and grapevine species have been analysed to identify structural variations of CNV types within the genes with a NBS domain and the STS genes. These works showed that the amplification of the gene families of interest was not specific to the reference genome but occurred at the scale of the Vitis genus, but also to highlighted structural variations in different genomes. Regarding the STS genes, blocks of duplication and more conserved and expressed genes were identified. For the genes with NBS domain, a clustered organisation has been highlighted with some clusters varying more than others in the studied genotypes. These works contribute to a better knowledge of gene families for efficient and durable defence against pathogens and optimal aromas synthesis in grapevine. This knowledge will benefit to breeding programs currently in progress at INRA Colmar. Key words: grapevine, family of genes, comparative genomics, transcriptomics, CNV "Research is what I'm doing when I don't know what I'm doing" Wernher von Braun Remerciements Je tiens tout d’abord à remercier Claire Parage qui, grâce à sa thèse, a permis de poser les bases de ce projet. Sans ses résultats, ce travail n’aurait jamais vu le jour. Je voudrais remercier l’INRA et la région Alsace qui ont financé cette thèse. Mais aussi, Camille Rustenholz et Philippe Hugueney qui ont décidé de me recruter après un entretien via webcam interposée, sans savoir si je portais un pantalon pour compléter le haut de mon costume et malgré la féroce concurrence d’une gagnante d’un concours de Miss pour le poste. J’aimerais remercier Patricia Faivre-Rampant, Valérie Geffroy, Etienne Paux, Hubert Schaller, Camille Rustenholz, et Philippe Hugueney d’avoir accepté de faire partie de mon jury de thèse. Mais aussi, Raquel Tavares et Hadi Quesneville pour avoir participé à mon comité de thèse. Je tiens tout particulièrement à remercier Camille Rustenholz qui, tout au long de ce projet, a fait preuve d’un encadrement exceptionnel. Merci pour tous ces moments, bons et moins bons, pour ces échanges et ces discussions plus ou moins enflammées, pour tes conseils, astuces et pistes pour me permettre d’avancer. Je ressors grandi de cette expérience et j’espère que toi aussi. Merci pour cette complicité et bonne entente dont, au final, peu de doctorants peuvent se vanter. Je ne peux qu’envier les ‘‘petits poussins’’ qui restent sous ton aile (Guillaume et maintenant Sophie et Amandine) mais aussi les prochains avec qui tu partageras ton savoir, comme tu l’as fait avec moi, et qui réaliseront à quel point tu es formidable. Merci à Philippe Hugueney, d’une part pour l’accueil au sein de l’équipe ‘‘Métabolisme Secondaire de la Vigne’’ et, d’autre part, pour l’encadrement exemplaire, les discussions plus que fructueuses et le point de vue plus extérieur qui parfois manquait pour faire avancer ce projet. Merci aussi d’avoir instauré un groupe de tarot sur l’heure de midi, je ne compte plus les rois coupés, les petits volés et le nombre de rois que tu as mis dans tes écarts… Le tarot a permis de renforcer la cohésion et la bonne entente dans l’équipe et, bien que cela reste un plaisir, nous parvenons à rester raisonnable et à ne pas en abuser. Bref, merci à vous deux pour l’aide inestimable apportée tout au long de la thèse et particulièrement sur les derniers moments qui se font toujours dans le rush malgré les précautions prises. Merci à toute l’équipe MSV pour l’accueil et la bonne ambiance au quotidien ainsi que les différents gâteaux et pâtisseries apparaissant aux pauses café. Tout particulièrement à Anne pour m’avoir permis de retourner en Belgique à plusieurs occasions. Merci à Guillaume Barnabé pour sa collaboration dans ce travail mais aussi pour l’aide qu’il a apportée au fil du temps permettant la résolution de bugs (plus ou moins faciles à trouver, vive l’informatique), la découverte et l’utilisation de nouveaux outils et les échanges d’idées.
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