2013 Annual Report
1a.Objectives (from AD-416):
1. Identify, map, and characterize host-plant resistance genes for priority cacao diseases and insect pests, and develop genetic markers for those genes.
1.A. For frosty pod (FP) caused by Moniliophthora roreri, sequence RNA from a time course of resistant or susceptible pods inoculated with frosty pod. Identify differentially expressed genes and correlate to QTLs for resistance or susceptibility to frosty pod.
1.B. For black pod (BP) caused by Phytophthora spp., identify QTLs for host-plant resistance from several populations. For QTLs stable over populations, identify candidate genes by bioinformatic analyses. Measure expression of candidate genes in inoculated tissues by real-time qPCR.
1.C. For witches' broom (WB) caused by Moniliophthora perniciosa, identify QTLs for host-plant resistance from several populations. For QTLs stable over populations, identify candidate genes by bioinformatic approaches. Measure expression of candidate genes in inoculated tissues by real-time qPCR.
1.D. For cacao swollen shoot virus (CSSV), improve the PCR assay for both DNA and RNA forms of virus, and determine sensitivity of assay through a standard curve and digital PCR.
2. Conduct expanded national and international field trials and phenotypic evaluations of cacao germplasm and breeding populations.
2.A. Establish new cacao field trials in Puerto Rico and Hawaii for evaluating newly developed cultivars from the breeding program of INIAP and CATIE. 2.B*. Continue to conduct existing field trials in Ecuador, Costa Rica, Brazil, Ghana, Côte d’Ivoire, Cameroon, Nigeria, Papua New Guinea and Indonesia. Continue with the phenotypic evaluation of resistance to FP, BP, WB, Ceratocystis, CSSV, VSD, Mirids and CPB.
3: Apply association mapping and genomic selection approaches to genomic and phenotypic data to accelerate the breeding of cacao having improved productivity, host-plant resistance to biotic stresses, tolerance to environmental extremes and superior product quality.
3.A. Perform association mapping procedures to isolate new QTLs from cacao germplasm collections in Puerto Rico and Trinidad.
3.B. Apply different genome-wide selection (GWS) models to predict genomic breeding values (GBV) in full-sib family crosses of cacao planted in Ecuador and Costa Rica.
4. With ARS cooperators develop and maintain an expanded database of genetic, genomic, and phenotypic data for cacao.
1b.Approach (from AD-416):
Single Nucleotide Polymorphism (SNP)s, both as single base pair substitutions and single base pair insertions/deletions (indels) are the most common sequence
differences found between alleles. Methods have been developed for high-throughput detection of SNPs, but these methods require a priori knowledge of the SNP being assayed or sequence information surrounding the SNP. As more cacao EST sequence data become available, we can use it to screen for SNPs. In addition, SNP markers are completely portable and can be added into the growing international database (CocoaGenDB). Microsatellite allele calls are platform-dependent and there has been no way to efficiently share data among the research groups currently genotyping cacao.
Once SNPs have been identified, they can be employed in a genetic assay that does not require electrophoresis or a dedicated molecular genetics facility staffed with highly trained technicians. Because breeding trials are established in cacao producing countries where such molecular genetics facilities do not exist, it is imperative to develop high throughput assays that can be performed and analyzed in the field to be able to use the molecular data for Marker Assisted Selection (MAS). Continue with existing field trials in Costa Rica, Ecuador, Brazil, and Ghana assisted by MAS to validate putative resistance to FP, BP, WB, Ceratocystis, CSSV and VSD. Establish new field trials in Cameroon, Ghana, Ivory Coast and Nigeria. Preventative breeding for Frosty Pod (FP) and Witches Broom (WB) in West Africa and South Asia. Using the markers flanking the Quantitative Trait Loci (QTL) for WB resistance on LG 1 and LG 9, selection of seedlings can be made from within families with ‘SCA6’ or ‘SCA12’ as a parent, that contain the genes conferring resistance to WB.
Saturated maps of the ‘UF273’ x ‘Pound 7’ and ‘TSH1188’ x ‘CCN51’ were constructed using the 6000 SNP chip data. QTL analysis is currently in progress and the cacao genome sequence is currently being used for the identification of gene models involved in disease resistance.
Collection of phenotypic data in segregating populations at all field locations (Ecuador, Costa Rica, Brazil, Ghana, Cameroon, Nigeria, Cote d'Ivoire, and PNG) is currently in progress. Leaf samples for SNP fingerprinting have been collected in Ecuador and Costa Rica. In addition, the evaluation of disease resistant plants selected in our collaborative breeding effort in Ecuador and Costa Rica is also underway.
The Genomics-Assisted Breeding program continues to move forward. Gathering of phenotypic and molecular data continued for all the breeding projects. As of this time, forty six new selections developed in Ecuador with resistance to WB and FP were moved to the quarantine facility in Miami. Eight clones have been recently released from quarantine; these selections will be distributed to breeding programs in West Africa, East Asia and Central and South America for use as parents and testing as clones. Two fully saturated linkage maps were produced using the SNP markers and these were used to complement the genome assembly and to refine the QTL areas for BP, FP, and WB resistance.