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.
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.
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.
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.
Aiming to evaluate an indirect screening approach for Ceratocystis wilt resistance, the leaf disc (LD) method, two assays were conducted to assess its efficiency in discriminating resistant/susceptible genotypes. The first one used five and the second 16 cacao clones. For each assay, 40 discs of 2month-old leaves were cut, wounded at the midrib, laid down in wet sterilized foam and inoculated (20 μL-3 × 10 4 CFU/ mL suspension of Ceratocystis cacaofunesta spores). The number of perithecia formed per leaf disc was assessed 4 days after inoculation (DAI). To assess the correlation between the LD method and field resistance, 16 branches of the same 16 clones were randomly selected and inoculated (30 μL/ 3 × 10 4 CFU/mL). The lesion length, width and area were measured 60 DAI. In both assays it was possible to distinguish resistant from susceptible clones. The clonal heritabilities were high (h 2 clo ≥ 77.19) for the assays in the lab and in the field, the highest estimate being obtained for perithecia number (h 2 clo = 95.82). The correlation of perithecia number with lesion width was positive and highly significant (r = 0.65, pvalue = 0.009). Therefore, high genetic gains on field resistance can be achieved by indirectly selecting through the LD method.
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