Genetic and environmental links between traits of cocoa beans and pods clarify the phenotyping processes to be implemented

The average weight of cocoa beans is not generally taken into account during breeding processes, although it is a trait of interest. Several studies indicate that the weight of the beans has a high heritability in Theobroma cacao. However, the values obtained from different countries for the same clone often vary. In this study, we analyzed the effect of different factors on the weight of the beans. Apart from the clone effect, three main factors had an impact: i) the number of beans per pod: a good filling of the pod with beans tended to limit the weight of the beans, ii) the position of the beans in the pod: beans in the apical part of the pod were significantly lighter than the others and iii) the longer the duration of the fructification cycle the heavier the beans were (positive genetics correlation). These results lead us to propose protocols aimed at normalizing the phenotypic values of the genetic material. To obtain a reliable estimate of the bean weight, the following is proposed: either to use beans obtained from manual pollination to saturate the pods with beans, or to systematically use the number beans in the pods as a covariable

Combined genetic and genomic approaches to characterize a durable Hevea resistance to South American leaf blight : S01T07

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

Evaluation de la résistance à Phytophthora palmivora et P. Capsici des 186 clones de la "core collection" du groupe génétique "Guiana"

La pourriture brune des cabosses, causée par des Straménopyles du genre Phytophthora, provoque d'importantes pertes de production dans toutes les zones cacaoyères. Pour réduire l'impact de ces bio-agresseurs, la lutte génétique utilisant des variétés résistantes est privilégiée; dans ce but, les sélectionneurs recherchent des sources de résistance, particulièrement chez les cacaoyers spontanés. Des prospections ont ainsi été réalisées dans les populations naturelles de cacaoyers du sud-est de la Guyane française entre 1985 et 1995 et ont permis de collecter un abondant matériel végétal appartenant à un groupe génétique particulier, le groupe "Guiana". Une "core collection" a ensuite été établie ex situ par le Cirad à Sinnamary (Guyane). A la suite de nombreux travaux ponctuels ayant montré l'intérêt potentiel de ce groupe génétique comme nouvelle source de résistance aux Phytophthora (megakarya et palmtvora), nous présentons ici les résultats d'une étude globale d'évaluation de la résistance des 186 clones de la "core collection" du groupe "Guiana" vis-à-vis de deux souches guyanaises des espèces P. palmivora (souche GY 27) et P. capsici (souche Reg 2-6). Cette étude, menée en Guyane de 2008 à 2011, utilisant une méthodologie performante (dix séries de tests sur disques de feuilles et un test statistique adapté à la nature ordinale des données élémentaires), confirme que le groupe génétique "Guiana" constitue bien une importante source de résistance à P. palmivora. Cinquante neuf clones se montrent aussi résistants que le témoin de résistance Scavina 6, tandis que certains (9) sont statistiquement plus résistants. Concernant P. capsici, la souche locale Reg 2-6, prélevée vers Régina dans les reliques des vieilles plantations du XVIIIème siècle, se révèle particulièrement virulente: le témoin Scavina 6, pourtant référence internationale en matière de résistance aux Phytophthora, n'y est pas résistant, tout comme les autres témoins de résistance de l'étude, et seuls quelques clones "Guiana" le sont. Néanmoins, vis-à-vis de cette souche, des dizaines de clones "Guiana" se révèlent statistiquement plus résistants que Scavina 6, montrant ainsi l'intérêt du groupe contre des souches "normalement" virulentes de P. capsici . En complément, une vingtaine de clones parmi les plus résistan à P. palmivora (et parfois aussi à P. capsici) ont été transférés au Cirad à Montpellier (France) où 15 ont pu être testés vis-à-vis de deux souches agressives de P. megakarya, NS269, du Cameroun, et NGR20, du Nigeria et en présence de 4 témoins, dont Scavina 6. Les tests montrent que 9 clones sur les 15 ont un niveau de résistance supérieur à Scavina 6, confirmant des résultats antérieurs sur la valeur du groupe génétique Guiana comme source de résistance à P. megakarya. Ainsi, parmi les clones du groupe Guiana plus résistants que Scavina 6, en particulier à P. palmivora et à P. megakarya, certains, présentant par aillems d'autres qualités notables, pourraient être intégrés dans de nombreux programmes d'amélioration génétique du cacaoyer. (Résumé d'auteur

Genetic and genomic diversity response of rubber tree to a major fungal disease

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. (Résumé d'auteur

GENESALB (2007-2010): GENEtic analysis of resistance to South American Leaf Blight (SALB) in rubber tree (Hevea spp.)

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. (Résumé d'auteur

Obtaining isolates of Microcyclus ulei, a fungus pathogenic to rubber trees, from ascospores

Microcyclus ulei, a fungus causing South American leaf blight of rubber tree, can only be isolated from asexual spores, which remain on a lesion just for a few days. This is a serious problem when wishing to study the diversity of the fungus on wild trees, which bear stromatic lesions almost exclusively on old leaves. The proposed technique can be used to isolate the fungus easily from sexual spores occurring in these stromata, even on leaves that are several months old.(Résumé d'auteur