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Sets of polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high- and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 × PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest.

The domestication history of rubber tree (Hevea brasiliensis) is very recent and was characterized by a severe bottle-neck when introduced from South America to Asia more than a century ago. This event might have greatly influenced the structure of genome and gene diversity of the species, and reflects, among other traits, in the capacity to cope with a major foliar disease. South American Leaf Blight (SALB) is a fungal disease caused by the Ascomycota Microcyclus ulei that is until now limited to the South American continent where it hampers the expansion of rubber cultivation. There is no known evidence of any source of tolerance or resistance to this disease among Asiatic rubber cultivars. Conversely, many resistance mechanisms were found among Brazilian or Peruvian accessions. Their characterization was primarily undertaken by QTL analysis within progenies of four resistant genotypes crossed with susceptible Asiatic clones. These studies allowed to differentiating various genetic loci responsible for resistance mechanisms. A more in depth analysis was undertaken for two of these resistant cultivars (MDF 180 and FX3899) by sequencing and annotation of cDNA libraries obtained after artificial inoculation by the fungus. The identification of numerous candidate genes differentially regulated during the infection process in both cultivars suggested a general scheme for major mechanisms implied in reaction to an infestation by M. ulei. Another study on NGS transcriptomic data showed also that few genes are shared at interspecific level within the genus Hevea, in response to SALB inoculation.

Corynespora cassiicola, causing agent of the Corynespora Leaf Fall (CLF) of rubber tree, is an Ascomycete fungus able to infect a large range of plants, mostly in tropical areas or green houses. Over the past 20 years, the CLF disease has gained increasing impo1tance in all Asian and African rubber producing countries but has not been declared so far in South America. lt causes massive sporadic leaf falls, thus impacting the yield of rubber production. Susceptibility of the various rubber clones to the disease varies depending on the geographical location, suggesting an important diversity of the natural inoculum. The biology of the fungus is complex since it has been described as a necrotrophic pathogen as well as an endophyte or a saphophyte. In this presentation, I will give a broad description of the research program that we are developing for the characterization of fungal effectors involved in the H. brasiliensis/C. cassiicola interaction. Our objective is to describe the diversity of the C. cassiicola species, trying to connect the genetic races with specific effectors potentially involved in virulence or host specialization of the isolates. In this program, we have first used biochemical and molecular approaches, which lead to the characterization of the cassiicolin toxin, a small secreted protein (SSP) putatively involved in the pathogenicity of C. cassiicola (isolate CCP) (Barthe et al., 2007; Breton, Sanier, & d'Auzac, 2000; de Lamotte et al., 2007; Deon et al., 2012a). A phylogenetic analysis based on C. cassiicola isolates of various geographical origins and host plants identified at least 8 phylogenetic clades (Deon et al., 2014). In addition, a classification based on the cassiicolin gene diversity was proposed (toxin classes). We found a rather good coherence between the toxin classes and the phylogenetic clades. However, 53% of the isolates did not carry any casssicolin gene, although some of them are virulent, suggesting the existence of effectors other than cassiicolin. This analysis is currently being extended to new isolates recently collected in West Africa (preliminary results will be presented). We are also conducting a genomic/transcriptomic approach with the objective to identify all putative effectors involved in the interaction with rubber tree. We have obtained the first reference sequence of the C. cassiicola genome, in frame of the DOE-JGI "1000 Fungal Genome" program. The assembled genome is around 45 Mb and contains about 17000 genes. We are now conducting comparative genomic analysis. The analysis pipeline is presented below. Jnterspecific comparative genomics with 45 fungal genomes, mostly pathogens from the Dothideomycetes (Schoch et al. , 2009), aims to describe the potential pathogenesis effectors arsenal, including the secondary metabolites, SSPs, lipases, proteases and CAZymes. lntraspecific comparative genomics among 37 C. cassiicola isolates of our collection, covering the known diversity, will help defining new and complete effector patterns that will be analyzed comparatively togenome-wide phylogenetic profiles. Finally, a transcriptomic analysis (RNAseq) of fungal genes differentially expressed after inoculation of detached rubber tree leaves (sensitive clone PB260) with a spore suspension (isolate CCP) is being conducted, in order to identify functional and pertinent effector candidates. The best candidates will be validated by functional genetics (through the knock-down of effector encoding genes). This study will provide knowledge and molecular tools for the characterization of C. cassiicola populations in epidemiological studies. This knowledge will help adapting the breeding strategies, taking into account the diversity of the pathogen, for the selection of more tolerant rubber tree clones.

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Hevea brasiliensis est la seule source de caoutchouc naturel, agro-matériau utilisé principalement (70%) dans l'industrie du pneumatique. En Asie et en Afrique, l'hévéa est sensible à une maladie foliaire due au champignon Corynespora cassiicola. Ce champignon sécrète une toxine protéique, la cassiicoline, dont six isoformes différentes ont été identifiées1. L'isoforme Cas1, issue d'une souche très agressive originaire des Philippines (CCP), jouerait un rôle important en tant qu'effecteur de la maladie2-5. Dans un objectif de sélection précoce, une méthode d'évaluation au laboratoire de la sensibilité des clones à cette maladie a été mise au point, par application sur folioles détachées de la toxine Cas1 purifiée ou de filtrats de culture de différentes souches du champignon. La toxicité induite est quantifiée après 48 heures par des mesures portant d'une part sur le pourcentage de fuite d'électrolyte R (conductimétrie), et d'autre part sur la surface de nécrose S. On a étudié la réponse de 20 clones d'hévéa d'une part à quatre concentrations de la toxine (0, 1, 5 et 10 ng/?l), et d'autre part à 12 filtrats de culture de souches différentes (dont la souche CCP) et à un témoin CZ (milieu de culture Czapeck). Les mesures de R sont apparues beaucoup plus sensibles que celles de S. L'intensité des symptômes augmente avec la concentration de la toxine, sans effet d'interaction entre clones et concentrations (hors témoin 0). Les profils de sensibilité des clones en réponse à la toxine Cas1 purifiée ou au filtrat CCP sont globalement les mêmes, renforçant l'idée que la toxine Cas1 serait le déterminant majeur de la toxicité du filtrat CCP. Un effet d'interaction entre clones et filtrats de culture a été mis en évidence, notamment entre les 4 filtrats les plus agressifs : CCP, CSRi5, CIND3 et CTHA3, avec des sensibilités différentes des clones selon les filtrats. Les classements de sensibilité des clones selon ce test ne rendent cependant compte que partiellement des comportements en champ. Il est donc nécessaire de poursuivre cette recherche, notamment par la caractérisation d'autres effecteurs que Cas1.

La maladie CLF (" Corynespora Leaf Fall ") provoquée par le champignon ascomycète Corynespora cassiicola (classe des Dothideomycetes1) est devenue depuis une cinquantaine d'année la principale pathologie foliaire de l'hévéa (Hevea brasiliensis) en Asie et en Afrique. L'hévéa est l'unique source commerciale de caoutchouc naturel dans le monde, avec 92 % de la production mondiale provenant d'Asie et 5 % d'Afrique. C. cassiicola représente donc un handicap majeur pour la filière caoutchouc. Une petite glycoprotéine secrétée par C. cassiicola, nommée cassiicoline, a été identifiée comme étant un effecteur de maladie important pour l'hévéa. Une étude de la structure et de la diversité de cette toxine a été entreprise, afin d'avoir une meilleure compréhension du pouvoir pathogène de C. cassiicola. Nous décrirons dans un premier temps la purification et la caractérisation moléculaire de la cassiicoline à partir d'une souche pathogène provenant des Philippines2,3,4. Ensuite, nous présenterons une étude de la diversité du gène de cassiicoline au sein d'une collection de souches d'origines géographiques et d'hôtes variés4,5. Cette étude a révélé l'existence d'au moins six isoformes de cassiicoline, permettant de grouper les souches en classes toxiniques. Une analyse phylogénétique de la collection des souches de C. cassiicola sur la base de 4 locus combinés a permis de mettre en évidence une importante diversité au sein de l'espèce C. cassiicola, en relative concordance avec les classes toxiniques. Les souches porteuses de l'isoforme de cassiicoline Cas1 semblent être les plus agressives chez l'hévéa. Cependant, certaines souches dépourvues de gène de cassiicoline présentent néanmoins un niveau de virulence faible à moyen chez l'hévéa, ce qui suggère l'existence d'autres effecteurs que la cassiicoline. Un test de la sensibilité des cultivars d'hévéa à C. cassiicola basé sur la sensibilité aux toxines secrétées par le champignon (en particulier Cas1) est en cours de validation. Il permettra le phénotypage de deux descendances pour la sélection d'individus tolérants. L'analyse comparative du déterminisme de la tolérance de l'hévéa aux toxines purifiées d'une part et au champignon d'autre part permettra de mieux comprendre le rôle de ces toxines dans la maladie CLF chez l'hévéa.

The South American Leaf Blight (SALB), due to the Ascomycota Microcyclus ulei, threatens the world production of natural rubber, based on the cultivation of nearly only one species: the tropical tree Hevea brasiliensis. In connection with a breeding program, conducted in Brazil by Michelin and Cirad and aiming to create resistant cultivars of rubber tree, the genetic determinism of three sources of natural resistance to SALB has been analyzed. We developed a classical approach of QTL mapping on a segregating population issued from a crosses implying a quantitative and durable resistant in MDF180 genotype (Le Guen et al., 2008), a quantitative and by-passed resistant in Fx3899 genotype, and a qualitative resistance in Fx2784 genotype. Disease resistance of the progenies was assessed under natural infestation in field trials, in Brazil and French Guyana, or under controlled inoculations with isolated strains of Microcyclus ulei. Genetic mapping revealed unexpected genetic determinisms underlying the observed resistances: whereas a complex genetic determinism implying major resistance genes and several minor QTLs can be bypassed by the pathogen (Le Guen et al. 2007), a durable partial resistance appeared to be governed by only 2 major resistance loci (Le Guen et al. 2011). To initiate a comprehensive approach of these resistances, eleven cDNA libraries were built, sequenced and annotated from MDF180, Fx3899, PB314 (susceptible) and PB260 (susceptible) genotypes (Garcia et al., 2011). We sequenced 20,493 expressed sequence tags (EST), developed array expression analysis and identified of 212 candidate genes differentially regulated in MDF180 and 30 genes in Fx3899 during the infection process. The integrated analysis of gene expression and systems biology allowed designing a general scheme of major mechanisms associated with durable resistance of MDF180 genotype and susceptibility of PB314 genotype to South American leaf blight. Concomitantly, transcriptomic NGS data give us the possibility to analyze large data set of inoculated and non- inoculated leaves of 3 Hevea species, H. brasiliensis, H. pauciflora and H. bentamiana. Comparison of the genes differentially expressed (inoculated vs non-inoculated) indicated few common genes involved in the response to SALB between the genera of Hevea. These results, in accordance with genetic mapping, constitute a favorable context for developing a strategy of pyramiding of the resistance. Work supported by ANR, Michelin, FAPESB, CNPq. (Texte intégral)

The South American Leaf Blight (SALB), due to the Ascomycota Microcyclus ulei, threatens the world production of natural rubber, based on the cultivation of nearly only one species: the tropical tree Hevea brasiliensis. In connection with a breeding program, conducted in Brazil by Michelin and Cirad and aiming to create resistant cultivars of rubber tree, we analysed the genetic determinism of 3 sources of natural resistance to SALB. We developed a classical approach of QTL mapping on 5 segregating populations issued from crosses implying 3 resistant Hevea accessions, showing contrasting type of resistance: partial or complete, bypassed or sustainable. Disease resistance of the progenies was assessed under natural infestation in field trials, in Brazil and French Guyana, or under controlled inoculations with isolated strains of Microcyclus ulei. Genetic mapping revealed unexpected genetic determinisms underlying the observed resistances: whereas a complex genetic determinism implying major resistance genes and several minor QTLs can be bypassed by the pathogen (Le Guen et al. 2007), a durable partial resistance appeared to be governed by only 2 major resistance loci (Le Guen et al. 2011). These original results constitute a favorable context for a marker?aided selection of cultivars combining high latex yield and SALB tolerance.

Aims Hevea brasiliensis cultivation is nearly the only source of production of natural rubber (latex), an irreplaceable strategic biopolymer for various industrial sectors. Worldwide production is threatened by the South American Leaf Blight (SALB) due to the fungus Microcyclus ulei (Ascomycota). CIRAD and Michelin collaborate, since 1992, on a program (CMB, Cirad-Michelin-Brésil) aiming at the creation of new varieties (grafted clones), combining high latex yield and tolerance to SALB. The ultimate objective is both to allow rubber farming development in the American inter-tropical zone endemically affected by the disease, and to prevent the risk of accidental introduction of the pathogen in the current Asian and African producing regions. The aim of the GENESALB project was to reinforce genetic mapping and candidate genes identification in order to speed up the characterization of genetics factors governing natural resistance to SALB and, ultimately, to set up a marker-aided selection program. Our strategy was based on the analysis of 6 mapping populations issued from 4 diversified tolerant progenitors, evaluated for SALB resistance in 2 locations (Brazil and/or French Guiana) in order to assess genetic diversity of resistance factors (major genes/loci and QTLs). Results Our results show that SALB resistance is mainly governed by major resistance genes/loci and each of the cultivar analyzed by genetic mapping led to the identification of a specific major resistance gene. These original results constitute a favorable context for a marker-aided selection of cultivars combining high yield and high level of SALB tolerance. In addition, it illustrates the richness of wild Amazonian genetic resources and their usefulness for disease resistance improvement in rubber tree. The project produced molecular resources for expression studies and breeding: cloning and sequencing of 6958 expressed genes associated with the disease, characterization of 489 candidate genes and set up of 125 additional genetic markers. The project strengthens the leadership of Cirad and Michelin on this topic, and helped generating new partnership in Brazil. Perspectives Genetic mapping allowed identification of resistance factors, but also of SALB resistant individuals with a potentially improved latex yield. Indeed, all the mapping populations we analyzed correspond to crosses between a high yielding, susceptible progenitor by a low yielding but tolerant one. Available genotyping data allow marker aided selection of the individuals, with the resistance alleles at the resistance loci. that have to be, next years, more precisely evaluated in larger scale trials for other traits of interest such as latex yield and growth vigor. Other segregating populations issued from different tolerant progenitors are available in Brazil and QTL mapping of this material might reveal additional SALB resistance loci. One of these populations shows a complete inheritance of SALB tolerance, with most of the progenies showing a high level of resistance. We plan to apply genetic mapping of this population, which allows performing QTL detection of yield or growth without phytosanitary treatments of the trees against Microcyclus. Development of genetic markers in EST-SSH will be continued. Around 350 ESTs from the SSH libraries, and containing a SSR motif, remain to be tested for EST-SSR polymorphism and mapping. Development of SNP markers will be necessary in order to map a greater number of the candidate genes we identified.

The resistance of the MDF180 Hevea brasiliensis cultivar to South American leaf blight (SALB) caused by the fungus Microcyclus ulei has been studied under both controlled conditions of inoculation and natural infestation in the Brazilian state of Bahia. Inoculated isolates on this cultivar in most cases developed sporulating lesions and produced conidiospores, but at a moderate level, characteristic of a race non-specific, partial resistance. The teleomorph of M. ulei has never been reported on this cultivar, either in controlled conditions or in natural infestation, whereas it is commonly observed on other susceptible and partially resistant cultivars. This type of resistance has seldom been described for other pathosystems. MDF180 resistance now enduring for more than 30 years in areas very conducive to the disease, it can therefore be described as a durable resistance. To initiate a comprehensive approach of the resistance, five cDNA libraries were built and analyzed comparing up-regulated genes of MDF180 and PB314 (a susceptible genotype) from 6 h post-infection (hpi) to 58 days post-infection (dpi). We sequenced 8,027 expressed sequence tags (EST). After control of their quality, trimming and assembling, we obtained 1,165 singlets and 458 contigs. Based on sequence similarity 49% of the sequences could be assigned to functional categories, whereas 21% corresponded to already described sequences but with unknown function and 30% did not show any significant similarity with other sequences. Macroarray expression analysis allowed the identification of 311 genes differentially regulated during the infection process of both genotypes. Focusing on candidate genes associated with stress and defense, twelve genes were up-regulated in young lesions of MDF180 (e.g. NBS-LRR, cf5, Peroxydase, Pathogenesis-Related-Protein, cystatin) while seven genes appeared up-regulated in older lesions of PB314 (e.g. SOD, LRR, chitinase). These results provided a first contribution to the understanding of molecular mechanisms of the MDF180 resistance to South American leaf blight and provide information for the further exploration of signalization pathways involved in this durable resistance.

In Cirad and the research unit " AGAP ", the two groups EGV (Marc Seguin et al.) and BURST (Pascal Montoro et al.) work on rubber genetics and molecular physiology respectively. Whereas BURST group is related with the IRRDB biotechnology group, EGV is more closely associated with the IRRDB breeding group. The main rubber research activities of EGV group are the development of molecular genetic markers for application to rubber breeding including clonal identification, genetic diversity analysis, and QTL-mapping. QTL-mapping was notably applied to the analysis of genetic determinism of SALB resistance sources in Wickham x Amazonian families (CMB = Cirad-Michelin-Brazil project), and extended to a Wickham x Wickham family for the study of growth, latex production and molar mass distribution of rubber chains in native rubber. In the CMB project, SSH banks were created for the generation of ESTs and candidate gene identification, from which a new series of EST-SSR markers were produced. In Brazil, the CMB project also develops a conventional programme of creation and selection of clones, either productive and tolerant to SALB, or adapted to supoptimal areas. Hevea x Corynespora interactions are studied with view to develop resistance breeding. In partnership with IFC (French Rubber Institute), a collaborative network of Large Scale Clonal Trials (LSCT) was developed for characterizing rubber clones, including the IRCA clones issued from a CNRA-Cirad cooperation in Côte d'Ivoire, thus allowing the up-dating of clonal recommendations in Africa.

International audience; ARCAD is an initiative supported by Agropolis Fondation and the Region Languedoc Roussillon (France). ARCAD aims at setting up a new open multi-function (conservation, research and training) platform devoted to the assessment and better use of plant agrobiodiversity in Mediterranean and tropical regions. The programme's scientific agenda will prioritize the study of history and patterns of crop domestication and adaptation as well as the analysis of key parameters underpinning adaptation and diversity structure, at various time scales, through studies of evolutionary genomics, population genetics and social sciences. The research will focus on Population comparative genomics, Crop adaptation to climate change and Cereal crops in Africa. These activities will be complemented with technological and methodological components for the conservation (DNA bank, cryopreservation) and analysis (bioinformatics, linkage disequilibrium) of crop diversity. A major objective of the programme is also to set up a demand-oriented capacity building platform, based upon the educational facilities offered by universities in Montpellier and the development of specific training modules. The ARCAD programme is jointly developed by CIRAD, INRA, IRD, Montpellier SupAgro and University of Montpellier 2, in partnership with numerous South and international institutions. As an open platform, ARCAD will continuously seek the involvement of interested partners.

Germplasm is the substrate used by plant breeders to create novel plant gene combinations and select crop varieties more suited to the needs of diverse agricultural systems. Crop germplasm diversity has been shaped by domestication, a complex anthropogenic process caused by a multitude of human populations with specific habits and needs. Genome analysis tools now provide access to thousands of polymorphisms, thus considerably enriching our approaches to the evaluation of diversity. This involves describing global diversity patterns, monitoring variations along the genome and investigating local sequence variation in regions of interest. The past few years have seen major progress in unravelling the drivers of crop evolution, including foundations, introgression, admixture, mutations, translocations, in a background of drift and selection. Results on rice, sorghum, banana, cacao, rubber tree, groundnut, among others, provide a range of situations which highlight targeted approaches to access the diversity conserved in ex situ germplasm collections and help build strategies for further germplasm collections. Soon it will be possible to determine and compare the whole sequence of hundreds of accessions. We therefore advocate identification for each important species of a common core set of reference materials to help R.E.A.D. (Represent existing diversity - Enter the whole collection - Assess phenotypic variation - Dissect trait-gene associations) germplasm through concerted efforts within the research community. This is being implemented by the Generation Challenge Programme (www.generationcp.org) of the CGIAR for the major food crops. The process of crop domestication is being analysed for a range of tropical crops by the Agropolis Resource Center for Crop Conservation, Adaptation and Diversity (www.arcad-project.fr) through a multidisciplinary approach associating ethnological studies to genomic surveys in search for selection signatures. This will help focus on specific germplasm compartments that enable high resolution genetic analyses in support for breeding as well as deploy renewed efforts for under-utilised species.

La maladie sud-américaine des feuilles (ou SALB) de l'hévéa, provoquée par Microcyclus ulei, touche aujourd'hui uniquement l'Amérique du Sud. Outre les dégâts causés sur les plantations sud-américaines, Microcyclus ulei représente une réelle menace pour l'hévéaculture asiatique. Une meilleure connaissance de la diversité des populations, du système de reproduction et de la dispersion de ce champignon phytopathogène profiteraient à la lutte contre le SALB. Cela pourrait également permettre d'envisager les stratégies à adopter en cas d'introduction de l'agent pathogène dans une région exempte de maladie. C'est pourquoi nous avons entrepris l'étude de la diversité et de la structure génétique de populations sud-américaines de Microcyclus ulei. Un nouveau jeu de marqueurs microsatellites a été développé afin de compléter l'existant. Trente-quatre marqueurs ont été testés parmi lesquels 17 ont été sélectionnés pour réaliser des études de populations. Au total, 124 individus provenant de quatre populations sud-américaines (Brésil, Equateur, Guatemala et Guyane Française) ont été génotypés. Des diversités génique et génotypique importantes ont été observées dans l'ensemble des populations. L'importance de la diversité génotypique et l'absence de déséquilibre de liaison entre les marqueurs montrent que les recombinaisons sont fréquentes et suggère que Microcyclus ulei est hétérothallique. La structuration génétique entre les différentes populations est extrêmement forte (Fst = 0.70) et indique l'absence de flux de gènes réguliers à l'échelle continentale couplée à de forts effets de fondation. Nous avons également constaté que la diversité des populations diminuait lorsque l'on s'éloignait de la zone d'origine supposée de l'agent pathogène. Une étude de génétique du paysage à l'échelle régionale sera réalisée autour de cette zone d'origine afin de comprendre l'importance, dans l'épidémie de SALB, du compartiment forêt relativement aux plantations industrielles. (Texte intégral)