BackgroundTheobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders.ResultsWe describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation.ConclusionsWe report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits.

Traditional fermentations of the local Ecuadorian cocoa type Nacional, with its fine flavor, are carried out in boxes and on platforms for a short time. A multiphasic approach, encompassing culture-dependent and -independent microbiological analyses of fermenting cocoa pulp-bean samples, metabolite target analyses of both cocoa pulp and beans, and sensory analysis of chocolates produced from the respective fermented dry beans, was applied for the investigation of the influence of these fermentation practices on the yeast and bacterial species diversity and community dynamics during cocoa bean fermentation. A wide microbial species diversity was found during the first 3 days of all fermentations carried out. The prevailing ethanol-producing yeast species were Pichia kudriavzevii and Pichia manshurica, followed by Saccharomyces cerevisiae. Leuconostoc pseudomesenteroides (glucose and fructose fermenting), Fructobacillus tropaeoli-like (fructose fermenting), and Lactobacillus fermentum (citrate converting, mannitol producing) represented the main lactic acid bacterial species in the fermentations studied, resulting in intensive heterolactate metabolism of the pulp substrates. Tatumella saanichensis and Tatumella punctata were among the members of the family Enterobacteriaceae present during the initial phase of the cocoa bean fermentations and could be responsible for the production of gluconic acid in some cases. Also, a potential new yeast species was isolated, namely, Candida sorbosivorans-like. Acetic acid bacteria, whose main representative was Acetobacter pasteurianus, generally appeared later during fermentation and oxidized ethanol to acetic acid. However, acetic acid bacteria were not always present during the main course of the platform fermentations. All of the data taken together indicated that short box and platform fermentation methods caused incomplete fermentation, which had a serious impact on the quality of the fermented dry cocoa beans.

Cacao (Theobroma cacao L.) is an important cash crop in tropical growing regions of the world and particularly for small cacao farmers. Over the past two decades, ‘CCN 51’ has become one of the most planted cultivars in Ecuador, mainly as a result of its high productivity and disease resistance. Intermixing of Nacional fine flavor Ecuadorian beans with beans of ‘CCN 51’ has become common practice, reducing overall bean quality and decreasing value. The primary goals of this study were to determine the genetic identity, structure, and allelic richness of ‘CCN 51’, its maternal origin and to compare ‘CCN 51’s’ agronomic characteristics against a composite group of Nacional cultivars. To investigate the complex genetic background of this cultivar, 70 simple sequence repeat loci were used. The high heterozygosity observed (56 of 70 loci) for ‘CCN 51’ is not characteristic of traditional Nacional cultivars. Comparison of agronomic characteristics between ‘CCN 51’ and several Nacional cultivars indicates significant differences in cacao dry bean weight, yield potential, production efficiency, percent healthy pods, and witches' broom [Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora] disease incidence. Additionally, physical, chemical, and organoleptic characteristics suggest that ‘CCN 51’ is different from those of Nacional lineage. Based on population structure analysis, the predominant ancestries for ‘CCN 51’ are Iquitos (45.4%), Criollo (22.2%), and Amelonado (21.5%) genetic groups. A lesser proportion of its genome was accounted for by genetic groups Contamana (3.9%), Purús (2.5%), Marañon (2.1%), and Nacional (1.1%) admixtures. Results of phylogenetic analyses using the unweighted pair group method with arithmetic mean yielding high bootstrap values strongly support the relatedness of ‘CCN 51’ with Iquitos, Criollo, and Amelonado genetic groups. Moreover, seven mitochondrial simple sequence repeat loci revealed that ‘CCN 51’ maternally inherited the ‘IMC 67’ cytotype. ‘CCN 51’ constitutes a valuable cacao genetic resource that is currently used not only in its country of origin, but also in many other national breeding and selection programs worldwide.

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.

Cacao (Theobroma cacao) is a globally important crop, and its yield is severely restricted by disease. Two of the most damaging diseases, witches’ broom disease (WBD) and frosty pod rot disease (FPRD), are caused by a pair of related fungi: Moniliophthora perniciosa and Moniliophthora roreri, respectively. Resistant cultivars are the most effective long-term strategy to address Moniliophthora diseases, but efficiently generating resistant and productive new cultivars will require robust methods for screening germplasm before field testing. Marker-assisted selection (MAS) and genomic selection (GS) provide two potential avenues for predicting the performance of new genotypes, potentially increasing the selection gain per unit time. To test the effectiveness of these two approaches, we performed a genome-wide association study (GWAS) and GS on three related populations of cacao in Ecuador genotyped with a 15K single nucleotide polymorphism (SNP) microarray for three measures of WBD infection (vegetative broom, cushion broom, and chirimoya pod), one of FPRD (monilia pod) and two productivity traits (total fresh weight of pods and % healthy pods produced). GWAS yielded several SNPs associated with disease resistance in each population, but none were significantly correlated with the same trait in other populations. Genomic selection, using one population as a training set to estimate the phenotypes of the remaining two (composed of different families), varied among traits, from a mean prediction accuracy of 0.46 (vegetative broom) to 0.15 (monilia pod), and varied between training populations. Simulations demonstrated that selecting seedlings using GWAS markers alone generates no improvement over selecting at random, but that GS improves the selection process significantly. Our results suggest that the GWAS markers discovered here are not sufficiently predictive across diverse germplasm to be useful for MAS, but that using all markers in a GS framework holds substantial promise in accelerating disease-resistance in cacao.