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

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations.

Selection of Reference Genes for Expression Study in Pulp and Seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum.

Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality-selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality.

Isolation and characterization of twelve polymorphic microsatellite Loci for the cocoa mirid bug Sahlbergella singularis.

Mirids are the primary pests affecting cocoa production in Africa, but no genetic studies have been conducted on these insects. Here we report the isolation and characterization of 12 polymorphic microsatellite loci for Sahlbergella singularis. A microsatellite-enriched genomic DNA library was developed and screened to identify marker loci. Twelve polymorphic loci were identified by screening 28 individuals collected from one presumed population in cocoa plantations in Southern Cameroon. The number of alleles ranged from 5 to 25, whereas the observed and the expected heterozygosities ranged from 0.179 to 0.786 and from 0.671 to 0.946, respectively. Tests showed significant deviations from HW equilibrium for four loci, but no linkage disequilibrium was detected at any of the loci. No cross-species amplification was observed in two other mirid pests in Africa.

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.

BACKGROUND: Theobroma 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. RESULTS: Fifty-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. CONCLUSION: This 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.

Identification and sequencing of ESTs from the halophyte grass Aeluropus littoralis.

Aeluropus littoralis (Gouan) Parl. is a C4 perennial halophyte monocotyledonous plant belonging to the same family as wheat. Growing as weed in dry salty areas or marshes, it is salt-secreting, rhizomatous and is used as forage. It is diploid (2n=2X=14) and has a relative small genome of around 342 Mb. A. littoralis is highly salt-tolerant since this plant has the ability to secrete salt. Thus, A. littoralis has the potential to become an important genetic resource for biotechnological strategies to improve salt and drought tolerance in economically important crops such as wheat. We have constructed SSH (Suppression Subtractive Hybridization) cDNA libraries from root (RSD45) and leaf (LSD45) tissues of 45 days old plants grown in the presence of 300 mM NaCl. We have also constructed full-length cDNA library from 15 days old salt stressed (300 mM NaCl) roots (RSTL15). Sequencing revealed 25 and 42 independent transcripts from the RSD45 and LSD45 cDNA libraries respectively, in both cases this was less than 25% of the clones sequenced. In contrast, 425 (60%) of the clones from the RSTL15 library revealed independent transcripts. After comparison with protein databases using BlastX, 335 (68%) ESTs (Expressed Sequence Tag) were classified into putative known functions and unclassified proteins, 59 (12%) have homology only to unidentified homologous sequences. A total of 98 (20%) of the ESTs have no homologies to known sequences in the protein databases which can be considered as novel.

Genomic DNA methylation of juvenile and mature Acacia mangium micropropagated in vitro with reference to leaf morphology as a phase change marker.

Genomic DNA methylation was analyzed in Acacia mangium Willd. microshoots micropropagated in vitro from juvenile and mature explants, and in relation to leaf morphology of the microshoots, which is considered a phase change indicator. Based on high performance liquid chromatography (HPLC) analyses, we found more DNA methylation in microshoots exhibiting juvenile leaf morphology (22.4%) than in microshoots of the mature phyllode morphological type (20.7%), irrespective of the age of the source material. Overall, the degree of DNA methylation in A. mangium microshoots was consistent with values reported for other angiosperms. Complementary investigations based on methylation sensitive amplification polymorphism (MSAP) techniques established that, of 1204 fragments revealed by the different primer pairs used, 49 (i.e., 4.08%) were derived from C(5m)CGG methylated sites. Three of these C(5m)CGG sites were exclusive to the juvenile plant material, and three sites were exclusive to the mature source. No fragments were associated specifically with leaf morphology, rather than with plant age. Thus, although the two age classes could not be distinguished based on a quantitative HPLC measure of DNA methylation, qualitative differences existed, as demonstrated by the six age-specific markers identified by MSAP. The reliability of the MSAP data was confirmed on a larger sample of juvenile plant material, which suggested that the total of six methylation markers detected is probably an underestimation of the age-related differences in DNA methylation that may exist between juvenile and mature plant materials.