2012 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.
The complete assembled and annotated genome of the cacao cultivar 'Matina 1-6' Ver 0.9 was released on September 15, 2010 and the updated version Ver 1.1 has been completed and is undergoing preparation for release.
A 6000 SNP chip was developed and only 5,388 SNPs provided genetic information. Three mapping populations consisting of over 1000 individuals were analyzed using these markers and new saturated recombination maps were produced. These saturated recombination maps from ‘UF273’ x ‘P7’, ‘TSH1188’ x ‘CCN51’ and ‘KA2-101’ x ‘K82’ were used to complete the genome assembly. Quantitative trait loci (QTLs) have been re-maped and refined using these saturated maps. In addition using the cacao genome sequences, the identification of gene models involved in disease resistance is currently in progress.
Progress continued in the breeding effort, with phenotypic evaluation at all field locations (Ecuador, Costa Rica, Brazil, Ghana, Cameroon, Nigeria, Cote d'Ivoire, and PNG). A foreign collaborating scientist from IITA in Nigeria were trained in molecular marker technology in the Miami lab this year and ARS scientist and collaborators traveled to Ghana and Cote d'Ivoire.
The MAS breeding program continues to move forward. Collection of phenotypic and molecular data continued for all the breeding projects. Fourteen new selections developed in Ecuador with resistance to Witches Broom (WB) and Frosty Pod (FP) were moved to the quarantine facility in Miami. After release 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. The complete genome of the clone 'Matina 1-6' was assembled, annotated and released in a user friendly database. A 6,000 SNP (single nucleotide polymorphism) Illumina chip was designed and used on over 1000 genotypes. Three fully saturated linkage maps were produced using the SNP and simple sequence repeat markers and these were used to complement the genome assembly and to refine the quantitative trait loci areas for FP, WB and VSD resistance.
Saski, C.A., Feltus, F.A., Staton, M.E., Blackmon, B.P., Ficklin, S.P., Kuhn, D.N., Schnell Ii, R.J., Shapiro, H., Motamayor, J.C. 2011. A genetically anchored physical framework for Theobroma cacao cv. Matina 1-6. Biomed Central (BMC) Genomics. 12:413. DOI: 10.1186/1471-2164-12-413.
Haiminen, N., Kuhn, D.N., Parida, L., Rigoutsos, I. 2011. Evaluation of Methods for de novo Genome assembly from High-throughput Sequencing Reads Reveals Dependencies that Affect the Quality of the Results. PLoS One. 6(9): e24182.
Kuhn, D.N., Livingstone, D., Main, D., Zheng, P., Saski, C., Feltus, F.A., Mockaitis, K., Farmer, A., May, G., Schnell Ii, R.J., Motamayor, J.C. 2011. Identification and mapping of conserved ortholog set(COS) II sequences of cacao and their conversion to SNP markers for marker-assisted selection in Theobroma cocoa and comparative genomics studies. Tree Genetics and Genomes. 8:97–11.
Boza, E.J., Irish, B.M., Meerow, A.W., Rodriguez, O.A., Ventura-Lopez, M., Gomez, J.A., Moore, J.M., Zhang, D., Motamayor, J.C., Schnell Ii, R.J. 2012. Genetic diversity, population structure, conservation and utilization of Theobroma cacao L., genetic resources in the Dominican Republic. Tree Genetics and Genomes. Available:http://www.bionity.com/en/publications/424313/genetic-diversity-conservation-and-utilization-of-theobroma-cacao-l-genetic-resources-in-the-dominican-republic.html.
Takrama, J., Dadzie, A., Opoku, S.Y., Padi, F., Adomako, B., Adu-Ampomah, Y., Livingstone, D., Motamayor, J.C., Schnell II, R.J., Kuhn, D.N. 2012. Applying SNP marker technology in the cacao breeding program at the Cocoa Research Institute of Ghana. African Crop Science Journal. 20(1):67-75.