Olivier Sounigo

Towards the understanding of the cocoa transcriptome: Production and analysis of an exhaustive dataset of ESTs of Theobroma cacao L. generated from various tissues and under various conditions

BackgroundTheobroma cacao L., is a tree originated from the tropical rainforest of South America. It is one of the major cash crops for many tropical countries. T. cacao is mainly produced on smallholdings, providing resources for 14 million farmers. Disease resistance and T. cacao quality improvement are two important challenges for all actors of cocoa and chocolate production. T. cacao is seriously affected by pests and fungal diseases, responsible for more than 40% yield losses and quality improvement, nutritional and organoleptic, is also important for consumers. An international collaboration was formed to develop an EST genomic resource database for cacao.ResultsFifty-six cDNA libraries were constructed from different organs, different genotypes and different environmental conditions. A total of 149,650 valid EST sequences were generated corresponding to 48,594 unigenes, 12,692 contigs and 35,902 singletons. A total of 29,849 unigenes shared significant homology with public sequences from other species.Gene Ontology (GO) annotation was applied to distribute the ESTs among the main GO categories.A specific information system (ESTtik) was constructed to process, store and manage this EST collection allowing the user to query a database.To check the representativeness of our EST collection, we looked for the genes known to be involved in two different metabolic pathways extensively studied in other plant species and important for T. cacao qualities: the flavonoid and the terpene pathways. Most of the enzymes described in other crops for these two metabolic pathways were found in our EST collection.A large collection of new genetic markers was provided by this ESTs collection.ConclusionThis EST collection displays a good representation of the T. cacao transcriptome, suitable for analysis of biochemical pathways based on oligonucleotide microarrays derived from these ESTs. It will provide numerous genetic markers that will allow the construction of a high density gene map of T. cacao. This EST collection represents a unique and important molecular resource for T. cacao study and improvement, facilitating the discovery of candidate genes for important T. cacao trait variation.

Genetic diversity and structure of farm and GenBank accessions of cacao (Theobroma cacao L.) in Cameroon revealed by microsatellite markers

The genetic diversity of 400 accessions collected in cacao farms, 95 GenBank, and 31 reference accessions was analyzed using the 12 microsatellite markers. The GenBank and reference accessions were subdivided into 12 accession groups (AG) that belong to the traditional cacao genetic groups (GG) Lower Amazon Forastero (LA), Upper Amazon Forastero (UA), Trinitario, and Criollo (Cr). The 12-microsatellite loci revealed a total of 125 alleles, 113 of which were present in the farm accession group (FA). The within and between group variation for all AGs accounted respectively for 81% and 19% of the total molecular variation. The average Fis for the FA was 0.15 suggesting a moderate level of inbreeding. Significant differences for the level of gene diversity were found between the farm (0.50), GenBank (0.42 to 0.62), and reference (0.10 to 0.60) AGs. Genetic differentiation among AGs was variable with Fst values varying between 0.14 and 0.57 for the different AGs. Analysis using a Bayesian model-based method showed the existence of a high level of admixture for the farm accessions group. The LA genes were most represented in the FA (54%), followed by UA (33%) and Cr (7%). The genes of LA were also the most represented in the GenBank (48%), followed by UA (24%) and Cr (14%). Only 14% and 6% of the genes of the GenBank and farm accessions, respectively, could not be attributed to any of the reference GGs. The results suggest the predominating presence of LA genes in the Cameroon farm accessions and a high level of admixture, with apparent presence of genes of more than three GGs in most accessions. The traditional Trinitario types appear to have almost disappeared from farmers fields. The admixture must be the result of hybridization and recombination of these genes from the different GGs in seed gardens and in farmers’ fields. The use of selected farm accessions will depend on the GG that it belongs to and also on their level of heterozygosity. Further implications of the results for breeding and for introduction of new germplasm into the Cameroon GenBank are discussed.

Methods of analysis for studying cocoa (Theobroma cacao L.) susceptibility to mirids

Two species of mirids, Distantiella theobromae (Dist.) and Sahlbergella singularis Hagl., cause substantial damage to cocoa plantations in West Africa. Cocoa susceptibility to mirid attack was observed in the Côte d’Ivoire in a trial involving progenies obtained from crosses according to a factorial mating design, and in a trial involving clones. As spatial distribution of mirid damage was not uniform within the plots, a method was developed for experimental design of completely randomized single-tree elementary plots, based on analysis of covariance and taking into account the spatial within-plot effect of the damage caused. This method resulted into a more precise estimation of genetic effects and heritabilities adjusting for mirid susceptibility heterogeneity in the field.

Parentage analysis and outcrossing patterns in cacao (Theobroma cacao L.) farms in Cameroon.

The present study investigates the parentage of farm accessions in Cameroon using data from 12 microsatellite loci. Bayesian analysis suggests that 25.5% of the 400 farm accessions studied is still closely related to the traditional Amelonado variety called ‘German Cocoa’ by the farmers. Another 46.3% of the farm accessions were found to be direct descendants (20.8% first-generation (F1) hybrids and 25.5% selfed genotypes) from 24 parental clones used in biclonal seed gardens (BSGs) established in the 1970s in southern and western Cameroon. Furthermore, 28.3% of farm accessions appeared to descent from uncontrolled pollination events in cacao farms, which could be related to a common practice of cacao growers to use seeds collected in their own farm for new plantings. All farm accessions descending from BSG could be individually related through parentage analysis to the 24 progenitors of the BSG. Only 25% of progenies distributed from BSG corresponded to F1 hybrids combinations originally planned to be released. Significant biparental inbreeding estimates were observed for all ‘traditional’ farms and for most ‘F1 hybrids’ farms due to presence of a high proportion of selfed accessions. Biparental inbreeding occurs when plants receive pollen from genetically related neighbors. High levels of outcrossing observed in ‘mixed’ farms might be explained by the admixture of traditional varieties and BSG progenies. The implications of our finding for management of seed gardens and for further breeding using farm accessions in Cameroon are discussed.

Deciphering the Theobroma cacao self-incompatibility system: from genomics to diagnostic markers for self-compatibility

Two different mechanisms controlling the Theobroma cacao self-incompatibility system were identified at two separate loci together with candidate genes differentially expressed between self-compatible/incompatible reactions, and diagnostic markers predicting self-compatible varieties were produced.