Contribution of subgenomes to the transcriptome and their intertwined regulation in the allopolyploid Coffea arabica L. grown in contrasted temperatures : W190

Polyploidy occurs throughout the evolutionary history of many plants, giving rise to novel phenotypes and leading to ecological diversification and colonization of new niches. Coffea arabica a recent allopolyploid between two low divergent diploid species, Coffea eugenioides and Coffea canephora, can be grown in regions with marked variations in thermal amplitude while the parental species are less adapted to temperature variations. To assess the contribution of subgenomes to the C. arabica transcriptome and its variation in relation to the adaptation to variable culture conditions, the transcriptome of leaves of C. arabica plants grown in two contrasted thermal regimes were examined using RNA sequencing (RNA-seq) approach. Then the relative homoeologous gene expression was compared to the relative expression between the modern-day diploid progenitor species. Whatever the growing conditions, 65% of the studied genes showed equivalent level of homoeologous gene expression, and for a large majority (92%), the relative homoeologous gene expression between both growing conditions varied less than 10 %. In C. arabica growing conditions do not disrupt the relative homoeologous gene expression and the overall gene expression appears to be regulated by intertwined mechanisms. Unlike others allopolyploids analyzed previously, C. arabica showed no preferentially expressed subgenome illustrating the interest of considering the age of the allopolyploidization event and the evolutionary divergence of progenitor species when studying allopolyploidy. (Résumé d'auteur

The Coffee Microarray Project: a new tool to discover candidate genes correlated to quality traits

Coffee is a product of mass consumption, with worldwide consumption estimated at 2.2 billion cups per day. The annual turnover generated is approximately 25 billion Euros and so coffee is the third biggest source of international trade, after oil and cereals. Coffee trees have not, however, resulted in any significant seedling industry involvement, which is in sharp contrast with their economic importance. However, biotechnology tools used for improving other species with economic impact are gradually adapted to coffee trees and used for guiding and improving coffee trees performances. Over the past few years, Coffee research programs also include Functional Genomics studies for the discovery of the genes and biosynthesis pathways involved in characteristics of agricultural, industrial or qualitative interest. The Coffee Microarray Project is based on scientific collaboration between NESLTE and CIRAD/IRD granted by ANR (National Research Agency) via GENOPLANTE. PUCE CAFE project has two main objectives 1) Create the first Coffee 16 K oligo microarray and 2) validate and use this new tool to analyze gene expression patterns during coffee grain maturation in Coffea arabica and Coffea canephora (robusta). (Texte intégral

Organisation, sequence diversity and evolution of a disease-resistance gene cluster in coffee tree (Coffea l.)

Pathogen resistance (R) genes of the NBS-LRR class (for nucleotide binding site and leucine-rich repeat) are found in all plant species and confer resistance to a diverse spectrum of pathogens. Although the ability of plant species to survive over evolutionary time might depend on their ability to maintain and generate useful levels of diversity at resistance loci, little is known about the mechanisms that drive NBS-LRR gene evolution. Various genetic mechanisms, including point mutations, recombination, unequal crossing over, and gene conversion has been proposed to account for the evolution of R-genes. Furthermore polyploidy (whole genome duplication) that gives rise to organisms with multiple (3x, 4x...) sets of chromosomes, is a major evolutionary process in plants. It is now well established that most, if not all, wild and cultivated plants including those with small genomes (rice, Arabidopsis, grapevine) have undergone at least one cycle of whole-genome duplication during the course of their evolution. Coffea arabica is a young allopolyploid species and represents a good model to study mechanisms of gene evolution after polyploidization. By combining physical mapping using a bacterial artificial chromosome (BAC) library of C. arabica and sequencing of two BACs, organization of a cluster of disease resistance genes was established on the constitutive genomes of C. arabica. The relationships (orthology versus paralogy) of the different members of the resistance gene family identified on the two homeologous chromosomes was found out and sequence comparison between the two homeologous regions was carried out. In addition, the complete sequences of identified disease resistance genes were determined in different diploid coffea species representing the different biogeographical groups of coffee tree species. Evolutionary relationships and mechanisms were assessed based on DNA sequence analysis. Consequences in terms of speciation and evolution within the genus Coffea and coffee genetic resources preservation will be presented and discussed. (Texte intégral

Sequence organisation and conservation at homeologous regions ln the recent allotetraploid coffee (Coffea arabica L.)

Coffee is one of the world's largest traded commodities produced in more than 60 countries. Coffee species belong to the Rubiaceae family and commercial coffee production relies mainly on two closely related species: Coffea arabica and C. canephora, which account respectively for 65 and 35% of the coffee production. All coffee species are diploid (2n=2x=22) and generally self-incompatible, except for C. arabica which is the only tetraploid (2n=4x=44) and self-fertile. Molecular phylogenies have indicated that C. arabica is a recent allotetraploid (CaEa genome) formed by hybridisation between two related diploid species: C. canephora (C genome) and C. eugenioides (E genome) or ecotypes related to those diploid species. In spite of the close relationship between the two constitutive sub-genomes, C. arabica displays diploid-like meiotic behaviour with bivalent formation. In order to estimate the sub-genome divergence at fine scale, a sequence comparison was performed between orthologous regions from both Ca and Ea sub-genomes of C. arabica. In particular two homeologous BAC clones, named clones 45 and 52, and sharing around 180 Kb, were analyzed for gene composition, sequence divergence and for presence/absence of repetitive elements. The analyzed regions include 17 putatively functional genes. All predicted genes were strictly conserved in the same order and orientation on both subgenomes. On more than 31 Kb of coding sequence, around 1% of substitution rate was observed, corresponding to 1.5% of amino acid substitutions. In intronic regions the substitution rate increased to around 2%. Some repetitive elements were shared by the two sub-genomes, indicating that their insertion predated the divergence between C. canephora and C. eugenioides. Based on the comparison of the predicted coding sequences of both clones with the publicly available C. canephora ESTs, the clones 45 and 52 were attributed to the Ca and Ea sub-genomes, respectively. Our data point out the high sequence and structure conservation between the Ca and Ea subgenomes of C. arabica. This result is consistent with the evolutionary history of Arabica progenitors, where diploid C- and E-genome species involved in the allopolyploidization event derived from the same ancestor approximately 150,000-350,000 yr BP. Moreover, limited changes in genome structure of the analysed regions appear to be associated with polyploidy in C. arabica. Although partial, this study provides us with the first view of Arabica genome organisation and will be very useful when defining a whole genome sequencing strategy. (Texte intégral

Gene expression divergences between the allopolyploid Coffea arabica and its diploids relatives appear environment-dependant

Polyploidy is widespread among many major crops. In coffee, the main cultivated species, Coffea arabica, is an allotetraploid containing two diploid subgenomes which originated from two different diploid species, C. canephora and C. eugenioides. Here we showed that the gene expression changes between the natural but recent coffee allopolyploid species in its two diploid relatives is environment-specific. Using spotted 70-mer oligo-gene microarrays targeting 15522 unigenes, leaf gene expression patterns from plants growing in two temperature conditions were compared between the two parental species and C. arabica. At the lowest temperature, we observed a massive dominance and transgressive expression in C. arabica when compared to its two relatives since 47 to 49 % of unigenes were differentially expressed with the proportions of up- or down-regulation approximately equal (23-24%). Surprisingly at the warmest temperature, we observed a strong disequilibrium. The divergence between C. arabica and C. eugenioides was rather identical to that observed at the lowest temperature since we observed over 40% of the unigenes differentially expressed, but on the other hand the divergence between C. arabica and C. canephora were only 9%. These data show that numerous genes in C. arabica are non-additively expressed and that divergences in gene expression pattern between allo and diploid genomes are function of the environment conditions. These results reinforce the hypothesis of a better functional plasticity of the allopolyploids in comparison to their related diploids species and consequently the evolutionary advantage of this genome architecture. (Texte intégral

Combining ability of doubled haploids in Coffea canephora P.

Afin d'étudier leur aptitude à la combinaison, cinquante-cinq haploïde-doublées (HD) de #Coffea canephora ont été croisés avec soit des génotypes hétérozygotes soit des génotypes HD. Trois essais agronomiques ont été établis. Une vigueur hybride marquée est observée pour tous les caractères analysés dont la production. Des différences importantes sont mises en évidence entre HD produits à partir d'un même clone reflétant le haut niveau d'hétérozygotie des clones. L'analyse d'un plan factoriel de croisement indique que la variance génétique est essentiellement due aux effets additifs pour la production ainsi que pour la hauteur des arbres et la forme des feuilles. L'aptitude générale à la combinaison apparaît aussi prédominante pour le diamètre au collet et la résistance à la rouille bien que des effets d'interaction soient détectés. Des combinaisons hybrides ont une productivité semblable à celle de variétés clones témoins. Les implications de ces résultats pour l'amélioration génétique de #Coffea canephora sont discutées. Particulièrement, le développement de variété hybride F1 est envisagé. (Résumé d'auteur

Sources of resistance to root-knot nematodes in coffee : characterisation, genetic bases and perspectives

By introducing coffee in Latin America, this was exposed to a range of root-knot nematodes (RKNs) with some of them proved to be highly pathogens such as the three main species for this crop: Meloidogyne paranaensis, M. incognita and M. exigua. Genetic control, using resistant cultivars, is the mainstay of a low cost integrated RKNs management aiming avoidance of highly-toxic nematicides. The main Coffea arabica cultivars traditionally cultivated are highly susceptible to RKNs. Sources of resistance to RKNs were prospected in wild accessions of C. arabica and C. canephora. Resistance to M. paranaensis, M. incognita and M. exigua and even to some other minor species like M. arabicida has been identified in C. canephora germplasm. In the case of wild C. arabica accessions, resistance to M. paranaensis, M. arabicida and M. incognita have been found. On the other hand, none resistance to M. exigua has been found in this species. Resistance phenotypes on coffee have been partially characterized for M. exigua and M. incognita infestations. However more detailed cellular and molecular characterization of resistance mechanisms to RKNs in coffee roots must be carried out in order to indentify the timing and pathways of the plant responses. Depending on the RKN species and coffee genotypes, different resistance traits have been observed at different infestation steps from penetration restraint to hypersensitive like reactions. These cell death processes were observed during migration of infective J2 in the roots toward the plant vascular system and also in and/or close to giant-cell-formed feeding sites leading to none or weak reproduction of the nematode. Knowledge on inheritance of resistances to RKNs is essential for breeding programs. Among known resistance to RKNs in coffee, only genetic determinism of the resistance to M. exigua has been described with the identification of one dominant gene: Mex-1 whose mapping is in progress looking for nearby markers among a large F2 population segregating for resistance to this nematode. Precise and reliable phenotyping is a key step for genetic studies on plant resistances. For further studies on inheritance of resistance to other RKNs such as to M. paranaensis, as well as for mapping the regions carrying the involved R genes, we developed a phenotyping platform that allows a nematode-reproduction-rate based on screening of large number of plant F2 populations in a fast, reliable and reproducible way. On an applied point of view, we aim at the development of RKN R gene marker that will permit a high-throughput assisted selection also more reliable than the phenotypic selection. The next step of research on coffee RKN R genes will be the functional validation of the identified gene sequences. These studies will be shortly initiated in the case of Mex-1 R gene. (Texte intégral

Construction of a molecular linkage map in coffee

Une carte de liaison génétique du caféier (#Coffea canephora$) totalisant 1402 cM a été établie sur la base d'une population de plantes haploïde-doublées. Des marqueurs RFLP comme des marqueurs utilisant la PCR (RAPD) ont permis la construction de 15 groupes de liaison. Des clones de DNA génomiques de caféier ainsi que des DNAc ont été utilisés. Au total, 47 RFLP et 100 RAPD sont positionnés sur la carte de liaison. Un niveau relativement faible de polymorphisme est observé. 81 % des marqueurs RAPD et 85 % des marqueurs RFLP présentent une ségrégation significativement non différente (P < 0.01) du ration 1:1. La disponibilité d'une carte de liaison moléculaire du caféier offre de nombreuses perspectives tant pour des études génétiques que pour l'amélioration des espèces de caféiers qui constituent une culture de grande importance économique. (Résumé d'auteur

Synténie et génome évolution chez le caféier allotétraploïde (Coffea arabica) : étude de la région SH3

Coffee leaf rust caused by the obligate parasitic fungus Hemileia vastatrix is an economically important disease and a major limiting factor for arabica coffee (Coffea arabica) production. While the rust resistance genes identified in C. arabica, a recent allotetraploid species (CaEa), have not provided durable resistance, resistance genes from diploid related coffee species such as C. liberica (i.e. SH3 gene) and C. canephora have provided long-lived protection under field conditions. Positional cloning of the SH3 gene has been therefore undertaken in order to enhance opportunities for genomics-enabled breeding and to gain molecular insight into rust durable resistance. Hence, we explored the possibility to utilize the exponentially increasing sequence information from model plants such as Arabidopsis and Tomato. By combining a search of Arabidopsis sequences homologous to coffee BAC-end sequences belonging to the related SH3 BAC contig and use of orthologous sequence markers, we demonstrated microsynteny between coffee and Arabidopsis duplicated counterparts. The complex duplication history of Arabidopsis did not prevent the use of Arabidopsis as model for coffee species. Furthermore, an extended colinearity between the coffee and tomato genomes was revealed for the chromosome arm carrying the SH3 locus using comparative genetic molecular mapping. This finding highlights the possibility to share genomic and genetic information among these two related crop plants. Finally, the complete sequences of homeologous coffee BAC clones (i.e. belonging to the two different genomes) from the SH3 region were determined. Preliminary analysis showed a low sequence divergence and a highly conserved colinearity between the corresponding segments of the Ca and Ea genomes. (Texte intégral