Olivier Fouet

The genome of Theobroma cacao

We sequenced and assembled the draft genome of Theobroma cacao, an economically important tropical-fruit tree crop that is the source of chocolate. This assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of these genes anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example, flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions.

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.

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.

Tracing the native ancestors of the modern Theobroma cacao L. population in Ecuador.

The native Theobroma cacao L. population from Ecuador, known as Nacional, is famous for its fine cocoa flavour. From the beginning of the twentieth century, however, it has been subjected to genetic erosion due principally to successive introductions of foreign germplasm whose hybrid descendants gradually replaced the native plantations, implying a decrease in cocoa quality. We attempted to trace this native cacao within a wide pool of modern Ecuadorian cacao population. Three hundred and twenty-two cacao accessions collected from different geographical areas along the pacific coast of Ecuador and maintained in two living collections were analysed using 40 simple-sequence repeat markers. Most of Ecuadorian cacao accessions displayed a high diversity and heterozygosity level. A factorial analysis of correspondence (FAC) showed a continuous variation among them, with a few ones, grouped at an extreme side of the FAC cloud, showing higher levels of homozygosity and lower introgression level by foreign cacaos. A paternity analysis revealed that these highly homozygous individuals are the most probable ancestors of the modern Nacional hybrid pool. These particular accessions studied could represent the native Nacional cacao present in Ecuador before the foreign introductions. Their identification will help to conserve valuable genetic material and to improve cocoa quality in new cacao varieties.

A genomewide admixture mapping study for yield factors and morphological traits in a cultivated cocoa (Theobroma cacao L.) population

The selection of productive varieties of modern Criollo cocoa, showing fine aromatic qualities in their beans, is of major interest for some producing countries, such as Venezuela. Cultivated populations of Modern Criollo or Trinitario varieties may be suitable for admixture mapping analysis, as large blocks of alleles derived from two identified divergent ancestors, recently admixed, are still preserved, after a few generations of recombination, similar to experimental mapping progenies. Two hundred and fifty-seven individuals from a cultivated population of Modern Criollo were selected and analysed with 92 microsatellite markers distributed along the genome. This population exhibited a wide range of variability for yield factors and morphological features. Population structure analysis identified two main subgroups corresponding to the admixture from the two ancestors Criollo and Forastero. Several significant associations between markers and phenotypic data (yield factors and morphological traits) were identified by a least squares general linear model (GLM) taking into account the population structure and the percentage of admixture of each individual. Results were compared with classical QTL analyses previously reported for other cacao populations. Most markers associated to quantitative traits were very close to QTLs detected formerly for the same traits. Associations were also identified between markers and several qualitative traits including the red pigmentation observed in different organs, mainly associated to common markers in linkage group 4.

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.

Evolutionary forces affecting synonymous variations in plant genomes

Base composition is highly variable among and within plant genomes, especially at third codon positions, ranging from GC-poor and homogeneous species to GC-rich and highly heterogeneous ones (particularly Monocots). Consequently, synonymous codon usage is biased in most species, even when base composition is relatively homogeneous. The causes of these variations are still under debate, with three main forces being possibly involved: mutational bias, selection and GC-biased gene conversion (gBGC). So far, both selection and gBGC have been detected in some species but how their relative strength varies among and within species remains unclear. Population genetics approaches allow to jointly estimating the intensity of selection, gBGC and mutational bias. We extended a recently developed method and applied it to a large population genomic dataset based on transcriptome sequencing of 11 angiosperm species spread across the phylogeny. We found that at synonymous positions, base composition is far from mutation-drift equilibrium in most genomes and that gBGC is a widespread and stronger process than selection. gBGC could strongly contribute to base composition variation among plant species, implying that it should be taken into account in plant genome analyses, especially for GC-rich ones.

The cacao Criollo genome v2.0: An improved version of the genome for genetic and functional genomic studies

BackgroundTheobroma cacao L., native to the Amazonian basin of South America, is an economically important fruit tree crop for tropical countries as a source of chocolate. The first draft genome of the species, from a Criollo cultivar, was published in 2011. Although a useful resource, some improvements are possible, including identifying misassemblies, reducing the number of scaffolds and gaps, and anchoring un-anchored sequences to the 10 chromosomes.MethodsWe used a NGS-based approach to significantly improve the assembly of the Belizian Criollo B97-61/B2 genome. We combined four Illumina large insert size mate paired libraries with 52x of Pacific Biosciences long reads to correct misassembled regions and reduced the number of scaffolds. We then used genotyping by sequencing (GBS) methods to increase the proportion of the assembly anchored to chromosomes.ResultsThe scaffold number decreased from 4,792 in assembly V1 to 554 in V2 while the scaffold N50 size has increased from 0.47 Mb in V1 to 6.5 Mb in V2. A total of 96.7% of the assembly was anchored to the 10 chromosomes compared to 66.8% in the previous version. Unknown sites (Ns) were reduced from 10.8% to 5.7%. In addition, we updated the functional annotations and performed a new RefSeq structural annotation based on RNAseq evidence. ConclusionTheobroma cacao Criollo genome version 2 will be a valuable resource for the investigation of complex traits at the genomic level and for future comparative genomics and genetics studies in cacao tree. New functional tools and annotations are available on the Cocoa Genome Hub (http://cocoa-genome-hub.southgreen.fr).

A newly identified locus controls complete resistance to Microcyclus ulei in the Fx2784 rubber clone

Using cultivars which are genetically resistant to South American leaf blight (SALB) caused by the fungus Microcyclus ulei is the only way to plant rubber trees in disease-affected areas. Numerous field observations led to the hypothesis that the resistance of the cultivar Fx2784 to SALB is likely to be monogenic. In this study, we investigated this hypothesis by examining the distribution of the trait in a cross between the resistant cultivar and a susceptible one. The individuals resulting from this cross were planted in field trials in French Guiana and Brazil. The resistance of all the trees was assessed by field observations. Bulk segregant analysis (BSA) using microsatellite markers was performed in French Guiana to determine which markers were genetically linked to resistance, and the results were validated by field observations in Brazil. In both locations, a 1:1 segregation of the resistance trait was observed, thus reinforcing the monogenic hypothesis. BSA showed tight linkage between resistance and the microsatellite markers located in linkage group 2 in the Hevea genome and enabled to pinpoint the resistance locus. The location was confirmed by observations on the trees planted in Brazil. This result should facilitate the use of Fx2784 resistance in future breeding programs for SALB resistance. This is the third major locus conferring resistance to SALB identified in rubber tree (Hevea spp.). These three loci are genetically independent, a favorable situation for genetic improvement of SALB resistance.