The cocoa genome hub, an integrated platform to access the Criollo genome V2

The first draft genome of the species, from the Belizian Criollo B97-61/B2 cultivar, was published in 2011. Although a useful resource, some improvements were possible, including to identify misassemblies, to reduce the number of scaffolds and gaps, and to anchor un-anchored sequences to the 10 chromosomes. In 2017, we used a combination Next Generation Sequencing data to produce the version 2 of the assembly. We corrected misassembled regions and reduced the number of scaffolds from 4,792 in assembly V1 to 554 in V2 with a N50 increased from 0.47 Mb in V1 to 6.5 Mb in V2. A total of 96.7% of the assembly was anchored to the 10 chromosomes compared to 66.8% in the previous version. Unknown sites (Ns) were reduced from 10.8% to 5.7%. In addition, we updated the functional annotations and performed a new RefSeq structural annotation based on RNAseq evidence. In that context and to support post-genomics efforts, we developed the Cocoa Genome Hub (http://cocoagenome- hub.southgreen.fr/), an integrated web-based database providing centralized access to T. cacao genome and analysis tools to facilitate basic, translational and applied research in cocoa. We provide access to the complete criollo genome sequence V2 along with gene structure, gene product information, metabolism, gene families, transcriptomics (ESTs, RNA-Seq), genetic markers and genetic maps. The hub relies on generic software (e.g. GMOD tools) for easy querying, visualizing and downloading research data. It includes a Genome Browser enhanced by a Community Annotation System, enabling the improvement of automatic gene annotation through an annotation editor. (Résumé d'auteur

Ethylene-regulated genes in Hevea brasiliensis: effect of ethylene and wounding in young budded plants of three clones with contrasting metabolisms : [Draft]

Ethephon, an ethylene generator, stimulates both latex flow and regeneration. Given ethylene triggers the expression of numerous genes in latex cells, a transcriptomics approach was taken in order to understand the molecular mechanisms underlying latex production controlled by this hormone. Two cDNA libraries were constructed using Suppression Subtractive Hybridization (SSH) technology from 4-year-old trees of clone PB 260 in their immature period without stimulation or stimulated with 2.5% ethephon stimulated. Among 1158 sequenced clones, 158 unique transcripts were identified. Putative functions were assigned by sequence analysis using BLASTX, which showed a large number of genes related to transcription and protein synthesis, unknown functions or defence proteins. A high density filter was completed with genes involved in latex metabolism such as rubber biosynthesis and ROS-scavenging protein. Macro-array analysis revealed a general differential expression between clones with a contrasting metabolism. A large proportion of genes was up-regulated for the active metabolic clone PB 260, and by contrast, a down-regulation was observed for lower metabolisms such as PB 217 or RRIM 600. Discrimination of the response to ethylene for these clones was significant for 35 genes, and 5 of them might discriminate between the responses of the 3 clones. The differential gene expression by Real Time PCR upon ethylene stimulation was confirmed for some of these candidate genes. These genes could be used as markers of expression under stress in a marker-assisted selection programme. (Résumé d'auteur

Identification of Theobroma cacao genes differentially expressed during Phytophthora infection

Pod rot, caused by different species belonging to the genus Phytophthora, is the main cause of harvest losses worldwide for cocoa production. Between 15% to 80% of losses could be observed according to the Phytophthora species, P. megakarya being the most aggressive. Varieties¿ improvement with a sustainable resistance has been identified as a priority of research programs of producer countries with about 14 millions of workers getting their income from the cocoa cultivation. Cocoa resistance to Phytophthora is quantitative and polygenic. The objectives of this project is to improve our knowledge of molecular mechanisms involved in the partial cocoa resistance in order to develop efficient tools of breeding to increase the resistance level of cocoa trees. This work aims to develop functional genomic approaches to identify candidate genes involved in this partial resistance. Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to cocoa/Phytophthora interactions. More than 15,000 ESTs were sequenced (in the frame of a GENOSCOPE project) and used for these studies. ESTs were analyzed by Blastn and/or Blastx search against NCBI data base. A little part of the clones had no homology with sequences and/or function already describe. The other part had significant matches to known genes. Among them, sequence homologies were found with pathogenesis related function knowledge, as PR protein (PR-1, glucanase, chitinase...), kinases, receptors (LRR), and transduction factors. Gene expression was conducted on leaf tissues of a progeny created in Papoua Nouvelle Guinea derived from a cross implied forastero and haut amazonien. Two resistant individuals with the better allele combination and two susceptible individuals with the less allele combination were kept and used for this work. Nylon cDNA macro arrays were used to assess the differentially expressed genes of the resistant and the susceptible cocoa clones infected by Phytophthora megakarya.. Libraries were screened with the inoculated-subtracted probes and non-inoculated reverse-subtracted probes, to reduce the candidate clones. We developed a novel set of macro arrays and obtained expression profiles during the several steps of phytophthora infection kinetic. Several genes differentially regulated between resistant/susceptible individuals revealed in this study are already known as integrated in signal transduction or in plant defence responses of other species. (Texte intégral

Towards a bioinformatics platform for the Musa research community : [Abstract W076]

Current experiments in genomics produce a large amount of data that needs to be organized into databases and broadly accessible. Like other species, the Musa genomics community would benefit from centralized and innovative ways to study its genome. Over the past years, genetic and genomic data (e.g. BAC, EST, Markers) have been generated and stored in databases. Several pipelines of analyses were implemented for gene, transposable element, and expression data analyses, and for comparative genomics such as ortholog predictions via a phylogenomic approach (GreenPhyl). Web tools have been developed or implemented to facilitate access to data, such as genetic makers (TropGeneDB), genetic maps (CMap), a physical Map (GBrowse), and Expressed sequence Tags (ESTtik) gene/TE predictions, and to allow online manual genome annotation (GnpAnnot). The number of tools may continue to grow, in particular with the near release of the Musa genome sequence, and the increase of Next-Generation Sequencing (NGS) facilities. The GMGC website (http://www.musagenomics.org) is a place where data can be shared, and where databases and tools can be listed in an homogeneous way to serve the Musa genomics community. It is intended to provide the researchers interested in Musa with a common set of resources in order to work more efficiently and effectively. (Texte intégral

Identification de gènes de Theobroma cacao exprimés de façon différentielle pendant une infection par Phytophthora

La pourriture brune des cabosses, provoquée par plusieurs espèces de Phytophthora, est présente dans toutes les zones de production cacaoyère et constitue l'une des principales causes de perte de récolte de T. cacao. Les traitements chimiques contre cette maladie sont possibles mais ils sont contraignants, coûteux, polluants et ne s'intègrent pas dans un processus durable de gestion des plantations de cacao. La résistance du cacaoyer à Phytophthora est quantitative et polygénique. L'objectif de ce travail est de faire progresser notre connaissance des mécanismes moléculaires intervenant dans la résistance partielle du cacaoyer, de façon à développer des outils efficaces de sélection pour augmenter le degré de résistance des cacaoyers. Ces travaux visent à développer des approches génomiques fonctionnelles pour identifier les gènes candidats impliqués dans cette résistance. Des ressources de séquences exprimées ont été accumulées au cours des dernières années sur les interactions T. cacao/P. palmivora. Parmi celles-ci, des séquences correspondant aux gènes impliquées dans les réponses de stress biotique ont été sélectionnées et utilisées pour démarrer des études de génomique fonctionnelle. Une puce à ADN en lien avec les interactions cacaoyer/Phytophthora a été construite à partir d'une collection unigène de 3200 séquences de la plante, 50 séquences de Phytophthora sp. (base de données NCIS) et 20 séquences génériques pour la normalisation des puces. Des gènes exprimés de façon différentielle ont été observés dans le génotype résistant Scavina6 avant et après des inoculations de P. palmivora par des tests artificiels effectués sur les feuilles. Des échantillons des feuilles infectées ont été collectés 0, 1, 4, 8, 24, 48, 72 et 96 heures après l'inoculation. Des copies des gènes exprimées à chacun de ces moments ont été étiquetés et des hybridations de puce ADN ont été effectuées sur la plateforme Biopuces de Toulouse (http://biopuces.genotoul.fr/). Après les analyses des images, les données ont été analysées par le logiciel BioPlot/Biodust accessible sur la plateforme. Près de 250 gènes surexprimés entre 3 et 20 fois dans les feuilles inoculées ont été identifiés. Parmi eux, 80 ont été spécifiquement induits 8 heures après l'inoculation tandis que 50 ont été induits 48 h après. Les autres gènes étaient répartis à différents horaires après l'inoculation. L'expression des gènes fortement induits ou associés avec les régions des QTL de résistance (communes à plusieurs génotypes et identifiées sur les chromosomes 1, 4 et 9) ont été confirmées par une Rt-PCR quantitative. (Texte intégral