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United States Department of Agriculture

Agricultural Research Service


Location: Subtropical Horticulture Research

2009 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.

3.Progress Report
An investigation into the use of our SNP markers to determine offtypes within a clonal population from Cameroon was completed. Microsatellite markers flanking QTLs for black pod resistance were converted to single nucleotide polymorphism (SNP) markers for ease of use in West Africa. 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, Ecuador, and Ghana were trained in molecular marker technology in the Miami lab this year.

A new Trust agreement was signed with M&M Mars to completely sequence the genome of cacao. This agreement includes collaboration with IBM on genome annotation and assembly. Significant progress has been made towards this objective using the additional funds provided by Mars. Several 454 runs have been made on the accession Matina 1-6 and the sequences have been annotated. Genome assembly will begin next year. The Physical Map has been completed including placing over 200 microsatellite and candidate gene markers from the linkage map onto the physical map. Bacterial Artifical Chromosome (BAC) end sequences for the minimum tiling path are being sequenced and will aid in genome assembly. A new website ( has been designed and contains all the information for the cacao genome sequencing project.

1. Marker Assisted Selection (MAS). The MAS breeding program continues to move forward with the release of new cultivars in Ecuador and Costa Rica. Phenotypic and molecular data continued to be collected for all the breeding projects. A regional coordinator for West Africa was hired through CRIG to coordinate all our projects with the four NARS. SNP markers have been developed from our microsatellite flanking sequences that allow identification of favorable alleles at Quantitative Trait Loci (QTL) for black pod (BP) and witches broom (WB) resistance. The use of this technique is being implemented at CRIG. An additional 30,000 SNP markers are in the process of being identified for cacao to increase the saturation of our genetic map and for association studies. Three BAC libraries have been made and fingerprinted from Matina 1-6 and a physical map constructed. This map contains the position of markers from our linkage map allowing the alignment of the linkage and physical maps. Shotgun sequencing has been accomplished on the genome of Matina 1-6 and annotation of the sequences is underway. A new webpage has been developed for the sequencing project with much of the early data available through this site.

6.Technology Transfer


Review Publications
Argout, X., Fouet, O., Wincker, P., Gramacho, K., Legavre, T., Sabau, X., Risterucci, A.M., Da Silva, C., Cascardo, J., Allegre, M., Kuhn, D.N., Verica, J., Courtois, B., Loor, G., Babin, R., Sounigo, O., Ducamp, M., Guiltinan, M.J., Ruiz, M., Alemanno, L., Machado, R., Phillips, W., Schnell Ii, R.J., Gilmour, M., Rosenquist, E., Butler, D., Maximova, S., Lanaud, C. 2008. Towards the understanding of the cocoa transcriptome: Production and analysis of an exhaustive dataset of ESTs of Theobroma cacao L. generated from various tissues and under various conditions.. Biomed Central (BMC) Genomics. 2008.9:512.

Araujo, I.S., De Souza Filho, G.A., Faleiro, F.G., Queiroz, V.T., Moreira, M.A., De Barros, E.G., Guimaraes, C.T., Machado, R.C., Pires, J.L., Lopes, U.V., Schnell Ii, R.J. 2008. Mapping of quantitative trait loci for butter content and hardness in cocoa beans (Theobroma cacao L.). Plant Molecular Biology Reporter. DOI 10.1007/s11105-008-0069-9

Efombagn, M., Sounigo, O., Eskes, A.B., Motamayor, J.C., Manzanares-Dauleux, J.J., Schnell Ii, R.J., Nyasse, S. 2009. Parentage analysis and outcrossing patterns in cacao (Theobroma cacao L.) farms in Cameroon. Heredity. p.1-8.

Kuhn, D. N., J. C. Motamayor, A. W. Meerow, J. W. Borrone and R. J. Schnell. SSCP markers provide a useful alternative to microsatellites in genotyping and estimating genetic diversity in populations and germplasm collections of plant specialty crops. Electrophoresis29: 4096–4108 . 2008.

Motamayor, J.C., Lachenaud, P., Wallace, J., Loor, G., Kuhn, D.N., Brown, J.S., Schnell Ii, R.J. 2008. Geographic and genetic population differentiation of the Amazonian chocolate tree. PLoS One. Available: DOI: 10.1007/s12042-008-9011-4

Johnson, E.S., Bekele, F., Brown, J.S., Song, J.H., Motamayor, J., Zhang, D., Schnell II, R.J., Meinhardt, L.W. 2009. Population Structure and Genetic Diversity of the Trinitario Cacao (Theobroma Cacao L.) from Trinidad and Tobago. Crop Science. 49:564-572.

Aikpokpodion, P.O., Kolesnikova-Allen, M., Adetimirin, V.O., Motamayor, J., Adu-Ampomah, Y., Eskes, A.B., Schnell Ii, R.J. 2009. Genetic diversity assessment of sub-samples of cacao, Theobroma cacao L. collections in West Africa using simple sequence repeats marker.. Tree Genetics & Genomics. DOI 10.1007/s11295-009-0221-1

Last Modified: 4/19/2014
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