A genetic linkage map was created from 146 cacao trees (Theobroma cacao), using an F2 population produced by selfing an F1 progeny of the cross Sca6 and ICS1. Simple sequence repeat (SSR) markers (170) were used principally for this map, with 12 candidate genes [eight resistance gene homologues (RGH) and four stress related WRKY genes], for a total of 182 markers. Joinmap software was used to create the map, and 10 linkage groups were clearly obtained, corresponding to the 10 known chromosomes of cacao. Our map encompassed 671.9 cM, approximately 100 cM less than most previously reported cacao maps, and 213.5 cM less than the one reported high-density map. Approximately 27% of the markers showed significant segregation distortion, mapping together in six genomic areas, four of which also showed distortion in other cacao maps. Two quantitative trait loci (QTL) for resistance to witches' broom disease were found, one producing a major effect and one a minor effect, both showing important dominance effects. One QTL for trunk diameter was found at a point 10.2 cM away from the stronger resistance gene. One RGH flanked the minor QTL for witches' broom resistance, implying possible association. QTLs mapped in F2 populations produce estimates of additive and dominance effects, not obtainable in F1 crosses. As dominance was clearly shown in the QTL found in this study, this population merits further study for evaluation of dominance effects for other traits. This F2 cacao population constitutes a useful link for genomic studies between cacao and cotton, its only widely grown agronomic relative.

-Cacao was introduced in Bahia in

Resistance gene homologue (RGH) sequences have been developed into useful genetic markers for marker-assisted selection (MAS) of disease resistant Theobroma cacao. A plasmid library of amplified fragments was created from seven different cultivars of cacao. Over 600 cloned recombinant amplicons were evaluated. From these, 74 unique RGHs were identified that could be placed into 11 categories based on sequence analysis. Primers specific to each category were designed. The primers specific for a single RGH category amplified fragments of equal length from the seven different cultivars used to create the library. However, these fragments exhibited single-strand conformational polymorphism (SSCP), which allowed us to map six of the RGH categories in an F(2) population of T. cacao. RGHs 1, 4 and 5 were in the same linkage group, with RGH 4 and 5 separated by less than 4 cM. As SSCP can be efficiently performed on our automated sequencer, we have developed a convenient and rapid high throughput assay for RGH alleles.

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.

Molecular markers (RAPD, AFLP and microsatellites) were used to generate a linkage map and to identify QTLs associated to witches' broom (Crinipellis perniciosa) resistance in cacao (Theobroma cacao), using 82 individuals of an F 2 population derived from the clones ICS-1 (susceptible) and Scavina-6 (resistant). Fifteen evaluations of the number of brooms have been carried out in six years (1997)(1998)(1999)(2000)(2001)(2002). In order to increase the precision and accuracy in the measures of resistance, each F 2 plant was cloned in three replications in a randomized block design with singletree plots and evaluated over 2 years. Three hundred and forty-two markers were obtained, being 33 microsatellites, 77 AFLPs and 232 RAPDs. The distribution of the number of brooms in the F 2 population was skewed to resistance, suggesting the involvement of major genes controlling resistance and the repeatability estimated for resistance was 44%. A strong putative QTL was detected as being related to witches' broom resistance. Associated to this QTL, the microsatellite mTcCIR35 explained 35.5% of the phenotypic variation in resistance. This marker is being used for marker-assisted selection in Scavina-6 progenies, including those selected in private plantations, as an auxiliary tool to the phenotypic selection.

Microsatellite markers of Crinipellis perniciosa , with three and four repeats, were developed from sequence database and evaluated for their usefulness in detecting genetic polymorphism. Thirty-three primers produced unambiguous amplification products of 28 microsatellite-containing loci and 14 microsatellite-like polymorphic loci, with two to seven alleles at each locus. Three loci were useful to distinguish isolates from different biotypes and isolates from different countries. Amplification of the markers in the closely related fungi Moniliophthora roreri indicates that their usefulness in population's studies may go beyond the present study of the C. perniciosa and may have applications in population genetics of M. roreri .

Sexual compatibility limits the production of cacao plantations, being an important selection criterion in breeding programs. However, the current method for characterizing compatibility, based on the frequency of flower setting after controlled pollination, is time consuming, requiring a long time to identify self-compatible individuals. The identification of molecular markers in genomic regions can be an alternative to allow early selection of self-compatible plants. The present study aimed to identify SNP markers associated with sexual compatibility in cacao, by utilizing genome-wide association (GWAS) mapping. A population of 295 individuals mostly from third-generation breeding populations, but also founder clones, was used. This population was phenotypically characterized by hand pollinating 8199 flowers and evaluating the flower retention 15 days after pollination. In addition, leaf samples of each individual were collected and DNA extracted for genotyping by sequencing, generating 5301 SNP markers after cleaning. Genome-wide association mapping analysis was performed using Synbreed, GCTA, and TASSEL softwares. Significant markers associated to incompatibility, likely in strong linkage disequilibrium, were found within a region of 196 kb, in the proximal end of chromosome 4, suggesting the existence of a major gene in that region. However, this result should be validated in a larger population, considering that only 295 trees were used here. When the SNP effects were treated as random in the estimation process, many other regions in the genome appears to be involved with sexual incompatibility in cacao. Candidate genes were found not only in the proximal end of chromosome 4 but also spread in several other regions of the genome.

Cocoa butter is an important raw material for the chocolate, pharmaceutical, and cosmetic industries. The butter content and quality in cocoa beans are genetically controlled characteristics, and affect its commercial value and industrial applicability. In the present work, an F 2 population derived from the cross between the ICS-1 and Scavina-6 cocoa clones was used for molecular mapping. A linkage map was constructed based on amplified fragment length polymorphism, random amplified polymorphic DNA, and simple sequence repeat markers, resulting in a total of 273 markers, distributed in 14 linkage groups (LGs). Phenotyping of butter content was performed after ether extraction and butter hardness was determined by sweeping differential calorimetry. One quantitative trait locus (QTL) associated to butter content was mapped at linkage group 9 (LG9) and two QTLs for butter hardness were identified at linkage groups 9 and 7 (LG9 and LG7). The two QTLs mapped at the LG9 explained 51.0% and 28.8% of the phenotypic variation for butter content and hardness, respectively. These QTLs were concentrated in the same map region, suggesting a close genetic linkage or pleiotropic effect. The QTLs identified may be useful in further marker-assisted selection breeding programs aimed at cocoa butter quality improvement.