Michel Boccara

A meta-QTL analysis of disease resistance traits of Theobroma cacao L.

Theobroma cacao, is a tropical understorey tree that is a major economic resource to several tropical countries. However, the crop is under increased threat from several diseases that are responsible for 30% loss of harvest globally. Although QTL data related to the genetic determinism of disease resistance exist in cocoa, QTL mapping experiments are heterogeneous, thus making comparative QTL mapping essential for marker assisted selection (MAS). Sixteen QTL experiments were analysed, and the 76 QTLs detected were projected on a progressively established consensus map. Several hot spots, with QTLs related to different Phytophthora species and other diseases, were observed. The likely number of “real” QTLs was estimated by using a meta-analysis implemented in BioMercator software. There was a twofold reduction in average confidence interval observed when compared to the confidence interval of individual QTLs. This alternative approach confirms the existence of several sources of resistance to different diseases of cocoa which could be cumulated in new varieties to increase the sustainability of cocoa resistance using MAS strategies.

Discovery and mapping of a new expressed sequence tag-single nucleotide polymorphism and simple sequence repeat panel for large-scale genetic studies and breeding of Theobroma cacao L.

Theobroma cacao is an economically important tree of several tropical countries. Its genetic improvement is essential to provide protection against major diseases and improve chocolate quality. We discovered and mapped new expressed sequence tag-single nucleotide polymorphism (EST-SNP) and simple sequence repeat (SSR) markers and constructed a high-density genetic map. By screening 149 650 ESTs, 5246 SNPs were detected in silico, of which 1536 corresponded to genes with a putative function, while 851 had a clear polymorphic pattern across a collection of genetic resources. In addition, 409 new SSR markers were detected on the Criollo genome. Lastly, 681 new EST-SNPs and 163 new SSRs were added to the pre-existing 418 co-dominant markers to construct a large consensus genetic map. This high-density map and the set of new genetic markers identified in this study are a milestone in cocoa genomics and for marker-assisted breeding. The data are available at http://tropgenedb.cirad.fr.

Increasing Accuracy and Throughput in Large-Scale Microsatellite Fingerprinting of Cacao Field Germplasm Collections

In this study we report on increasing the rate and accuracy of microsatellite fingerprinting of accessions in Theobroma cacao L. field germplasm collections with a medium-throughput capillary sequencing system. We examined the use of a reduced number of microsatellite loci to decrease the time and materials required for fingerprinting and determined the best available microsatellite loci for accurately separating accessions. A subset of nine informative loci that could separate sixty accessions into the same genetic groupings as a complete set of 37 loci was found. Stringent probability of identity values (i.e. chance of unique accession) was highly influenced (r = −0.996; P < 0.001) by the number of alleles utilised in the fingerprinting set but the composition of the primer set was more important when choosing discriminatory loci. DNA pooling to reduce the number of samples was also investigated. There was a relatively high level of mixture within plots (59% of 54 plots examined) of the field genebank, which opposed the use of a pooling strategy to fingerprint the multiple trees of an accession plot in the collection.

Molecular characterization of an earliest cacao (Theobroma cacao L.) collection from Upper Amazon using microsatellite DNA markers

Cacao (Theobroma cacao L.) is indigenous to the Amazon region of South America. The river basins in the Upper Amazon harbor a large number of diverse cacao populations. Since the 1930s, several numbers of populations have been collected from the present-day Peruvian Amazon and maintained as ex situ germplasm repositories in various countries, with the largest held in the International Cacao Genebank in Trinidad. The lack of information on population structure and pedigree relationship and the incorrect labeling of accessions are major concerns for efficient conservation and use of cacao germplasm. In the present study, we assessed the individual identity, sibship, and population structure in cacao populations collected from the present-day Loreto Region, Peru in the 1930–1940s. Using a capillary electrophoresis genotyping system, we analyzed the simple sequence repeat variation of 612 cacao accessions collected from the Marañon, Nanay, and Ucayali river systems. A total of 180 cases of mislabeling were identified using a Bayesian clustering method for admixture detection. Using maximum likelihood-based methods, we reconstructed 78 full-sib families nested in 48 half-sib families, indicating that the pods collected in the 1930s were from 48 mother trees, maximum. Likelihood simulation also identified eight probable parents that are responsible for 117 pairs of mother–offspring relationships in this collection. Principal coordinate analysis (PCoA) and the Bayesian clustering method cohesively demonstrated a pronounced structure of genetic diversity, stratified by the river systems of the Peruvian Amazon. Our results also show that, in spite of the high level of allelic diversity in this collection, it was composed of a large number of related family members collected from a relatively small area, including a couple of sites in the Ucayali and Nanay rivers, as well as the lower Marañon river near Iquitos. The vast majority of the Peruvian Amazon, especially the upper Marañon River and its tributaries, have not been sampled by collecting expeditions. The improved understanding of the individual identities, genealogical relationships, and geographical origin of cacao germplasm in this collection will contribute to more efficient conservation and utilization of these germplasm. Additionally, this study also provides more baseline information to help guide future collecting expeditions in the Peruvian Amazon.

Insight into the Wild Origin, Migration and Domestication History of the Fine Flavour Nacional Theobroma cacao L. Variety from Ecuador

Ecuador’s economic history has been closely linked to Theobroma cacao L cultivation, and specifically to the native fine flavour Nacional cocoa variety. The original Nacional cocoa trees are presently in danger of extinction due to foreign germplasm introductions. In a previous work, a few non-introgressed Nacional types were identified as potential founders of the modern Ecuadorian cocoa population, but so far their origin could not be formally identified. In order to determine the putative centre of origin of Nacional and trace its domestication history, we used 80 simple sequence repeat (SSR) markers to analyse the relationships between these potential Nacional founders and 169 wild and cultivated cocoa accessions from South and Central America. The highest genetic similarity was observed between the Nacional pool and some wild genotypes from the southern Amazonian region of Ecuador, sampled along the Yacuambi, Nangaritza and Zamora rivers in Zamora Chinchipe province. This result was confirmed by a parentage analysis. Based on our results and on data about pre-Columbian civilization and Spanish colonization history of Ecuador, we determined, for the first time, the possible centre of origin and migration events of the Nacional variety from the Amazonian area until its arrival in the coastal provinces. As large unexplored forest areas still exist in the southern part of the Ecuadorian Amazonian region, our findings could provide clues as to where precious new genetic resources could be collected, and subsequently used to improve the flavour and disease resistance of modern Ecuadorian cocoa varieties.

Elucidation of genetic identity and population structure of cacao germplasm within an international cacao genebank

Theobroma cacao L., or cacao, is the source of cocoa products used in the making of chocolate. These tropical trees are conserved in living genebanks. The International Cocoa Genebank, Trinidad is one of the largest ex situ collections in the public domain. Mislabelling is a critical problem and the correction of this problem is vital to improve the accuracy and efficiency of genebank management. Using microsatellite DNA markers, we assessed the level of mislabelling in a group of Refractario cacao that originated from Ecuador and determined their population memberships through Bayesian clustering analysis. The microsatellite data revealed a synonymous rate of 7.5% and an error rate of 39.4% in this germplasm subset. The analysis of the population structure grouped the Refractario accessions into four subclusters, indicating intra-population heterogeneity in this germplasm group. Based on the results, we recommend that when the assignment test is used for cacao genotype identification, it should (a) use duplicated samples as internal checks, (b) choose suitable reference accessions, including a known homogeneous group and (c) employ subclustering checks to obtain reliable results. The information framework generated is discussed in relation to the management of the collection, population enhancement and future research of the collection.