2010 Annual Report
1a.Objectives (from AD-416)
The development and implementation of an international Marker Assisted Selection (MAS) program for cacao is the major objective of this project. This objective involves a combination of hypothesis-driven and non-hypothesis driven research and includes the training of scientists from cacao producing countries in plant breeding, genetics, and the use of molecular markers in a MAS program.
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.
Marker development expanded greatly with 300,000 potential SNP markers identified from the genome sequencing project. From these we have developed an Illumina Infinium custom SNP chip tailored for genotyping of several mapping populations, that consists of 6,000 SNPs (~4500 loci). Testing and validation of the SNP chip is underway prior to the genotyping of 1100 individuals in the three large mapping populations.
Progress continued in the breeding effort with new field trials planted in West Africa and Ecuador. Phenotypic evaluation at all field locations (Ecuador, Costa Rica, Brazil, Ghana, Cameroon, Nigeria, Cote d'Ivoire, and PNG) continued. Foreign collaborating scientists from Brazil (1) and Malaysia (2) were trained in molecular marker technology in the Miami lab this year.
Significant progress has been made towards sequencing the entire genome of cacao. Using the additional funds provided by Mars a complete physical map has been integrated with our recombination map. Ten pseudo molecules, representing the 10 chromosomes of cacao, have been created. BAC ends for every 20Kb of the minimum tiling path, approximately 36,000 BACs (72,000 ends), have been sequenced. Several paired end libraries (3kb and 8kb, as well as the ends of ~55,000 fosmids) have been sequenced to assist with the genome assembly. Three genome assemblies have been partially completed and a complete genome sequence is expected to be completed this calendar year. The website (http://www.cacaogenomedb.org) has been expanded with a breeders ‘tool kit’ and contains all the information for the cacao genome sequencing project.
The cacao genome sequencing project has moved ahead with surprising speed. Full integration of the physical map and recombination map has been accomplished and 10 pseudo-molecules that represent the 10 chromosomes of cacao constructed. Approximately 10x and 50x coverage of the cacao genome has been accomplished using 454 and Illumina sequencing technologies respectively. This information has been assembled and is being integrated with the physical and recombination maps. To assist with the assembly, new paired end libraries have been developed (3 kb, 8 kb) along with end sequences from 55,000 fosmids. Paired end sequences have been generated from all these libraries and used to assist with whole genome assembly. Three draft genome assemblies have been completed and these are currently being compared and integrated to complete the genome assembly.
The MAS breeding program continues to move forward. Phenotypic and molecular data continued to be collected for all the breeding projects. Six new varieties were released in Central and South America, two from CATIE in Costa Rica and four from INIAP in Ecuador. All these new varieties are high yielding disease resistant clones. Over 300K new SNP markers were identified from the cacao genome sequencing project and a subset of these, 6K, have been verified and are appropriate for use in a SNP chip. This SNP chip is being designed by Illumina and will allow us to fully saturate current linkage maps and to saturate new maps being developed at the Mars Center for Cocoa Science farm in Brazil.