1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
This project falls under National Program 301- Plant Microbial and Insect Genetic Research, Genomics and Genetics. The project is designed to accurately identify the germplasm accessions of Theobroma cacao in the international and national genebanks and assess genetic diversity in the Latin America gene pool.

Cacao germplasm, and its efficient use, is of major importance to the U.S. chocolate industry as well as developing countries. Cacao germplasm must be maintained as collections of live trees in the field, because storage of the recalcitrant seeds is impossible. Incorrect labeling of accessions has been a problem in national or international collections around the world. Many cacao accessions (perhaps as high as 40%) held in these collections are known to be mislabeled, duplicated, or have no assignment of identity at all. The level and structure of the genetic diversity in these collections, as well as in the overall Latin America genepool, are poorly understood. These constraints limit the ability of both U.S. and collaborating foreign scientists to acquire and exchange cacao germplasm, reduce efficiency in using cacao germplasm for breeding, and hinder the development of a rational strategy for the conservation of cacao genetic diversity in the Americas. This project is addressing the critical need to correctly identify individual accessions in and across these collections for utilization in breeding programs and provide this information to international databases. Results will contribute to more efficient organization and management of field genebanks and better use of scarce resources for genebank maintenance. We are also assessing genetic variation in the cacao collections and in the Latin America genepool using DNA markers, with the intent to identify gaps for which new cacao germplasm could be collected.

Theobroma cacao doesn’t grow in the continental U.S. but, together with major quantities of milk, sugar, almonds and peanuts produced by U.S. farms, represents an 8.6 billion dollar chocolate industry. The confectionary industry in the U.S. would be severely affected by a shortage of cacao supply. Therefore, the information gained during this project directly aids U.S. agriculture and will be used by plant breeders, plant pathologist, and extension personnel to help small landholders in developing countries.

2.List by year the currently approved milestones (indicators of research progress)
Year 1 (2004) The extent of mislabeling in the CATIE collection and the original reference population in the Trinidad collection estimated. The effectiveness and accuracy of the 15 SSR loci for individual identification evaluated.

Year 2 (2005) Original reference trees in Trinidad and CATIE collections fingerprinted and mislabeling in the reference trees identified and corrected. Mislabeling in the USDA collection at Mayaguez, Puerto Rico identified and correct name re-assigned. On-farm diversity in Huallaga and Ucayali area of Peru assessed.

Year 3 (2006) Mislabeling and off-types within each clone identified in CATIE and Trinidad International collections. Intra- and inter- population variation in the wild populations from the Camopi and Tanpok basins of French Guiyana analyzed. A survey of on farm diversity in the Yungas, Bolivia started.

Year 4 (2007) The INIAP collection in Ecuador genotyped. Genetic background of the Ecuadorian population “National” clarified. On-farm diversity in Ecuador assessed. Linkage disequilibrium estimated in different cocoa populations and association mapping tested in cocoa.

Year 5 (2008) Taxonomic relationship among the Theobroma taxa clarified and gene flow between cultivated cocoa and the wild compartment analyzed. Phylogeography of cocoa in the Upper Amazon region analyzed. Existing gaps in national and international collections identified. Recommendation for on farm conservation of cocoa genetic resources proposed.

4a.List the single most significant research accomplishment during FY 2006.
Identification of mislabeled cocoa germplasm from the Upper Amazon region

This accomplishment aligns with Component I (Genetic Resources Management) of the National Program 301- Plant Microbial and Insect Genetic Research, Genomics and Genetics. Cocoa germplasm collected from the rainforest of upper Amazon formed the foundation of the cocoa breeding activities in the world. This accomplishment address the problem of significant mislabeling within existing cocoa genebanks. Using microsatellitte DNA analysis, we identified 274 mislabeled accessions out of the 1124 upper Amazon cocoa accessions held in this collection. This accomplishment has significantly improved accuracy and reduced redundancy in the International Cocoa Genebank. The developed stand DNA profiles are also being used to identify mislabeling in other cocoa collections in Asia and Africa.

4b.List other significant research accomplishment(s), if any.
The wild cocoa populations in French Guiana. This accomplishment aligns with Component I (Genetic Resources Management) of the National program 301 - Plant Microbial and Insect Genetic Research, Genomics and Genetics. The wild cocoa populations collected from southeast French Guiana were analyzed using 15 SSR DNA markers. We detected a low level of genetic diversity and high level of inbreeding in most of the populations. However, the SSR analysis also showed that the French Guiana cocoa differed greatly from the upper Amazon cocoa. Substantial pollen exchanges among trees in neighboring populations were detected. These results provided important baseline information for further collecting and conservation of the Guiana wild cocoa germplasm.

4c.List significant activities that support special target populations.
None.

4d.Progress report.
None.

5.Describe the major accomplishments to date and their predicted or actual impact.
This accomplishment aligns with Component I (Genetic Resources Management) of the National Program 301- Plant Microbial and Insect Genetic Research, Genomics and Genetics. Incorrect labeling of cocoa accessions is a significant problem in the international cocoa community that has hindered the efficient conservation and use of cocoa germplasm. To date, a total of 4027 cocoa accessions maintained in the two international genebanks (ICG, T and CAITE), and in several other national collections in Latin America, have been genotyped with a set of 15 standard microsallite (SSR) loci. Based on the multi-locus SSR profiles, duplicated accessions - both within and among different collections - were unambiguously identified through genotype matching. Mislabeled accessions were detected through assignment tests, kinship inference, and cross-genebank comparisons. Hidden pedigree and population structure were reconstructed for important international clones. SSR profiles of 2047 accessions have been submitted to the International Cocoa Databases (ICG, D) and CocoaGeneDB. Cross-laboratory comparison of reference fingerprints, together with data generated from other genotyping systems, has been proved successful. These reference profiles can be used to verify the genetic identity of cocoa germplasm maintained in Asia and Africa. The on-going analysis of genetic diversity in these collections will provide critical baseline information for sustainable conservation and utilization of cocoa genetic diversity. This information will be used by cocoa germplasm curators and plant breeders and could potential help all future cocoa breeding programs.

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
• The SSR fingerprinting data of 1038 cacao accessions from the international cocoa germplasm collections, as well as the identified mislabeling, have been submitted to the international database (CocoaGeneDB) hosted by CIRAD in France.

• The standard reference DNA profiles for 446 international clones are being used by collaborating institutions such as CRU in Trinidad and CATIE in Costa Rica, for the correction of the off-types.

• Mislabeled accessions in the Tingo Maria cocoa collection, Peru were identified, which will benefit Peruvian cacao breeding programs.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
none

Review Publications
Rincones, J., Mazotti, G.D., Griffith, G.W., Pomela, A.W., Figueira, A., Queiroz, M.V., Pereira, J.F., Azevedo, R.A., Pereira, G.A., Meinhardt, L.W. 2006. Genetic variability and chromosome-length polymorphisms of the witches’ broom pathogen crinipellis perniciosa from various plant hosts in south america. Mycological Research. 110:821-832.