Progress 06/25/04 to 08/25/08
Outputs
Progress Report Objectives (from AD-416) To characterize genetic identities of cocoa germplasm and reduce redundancy and misidentifications in international cocoa genebanks; To assess genetic diversity in the Latin American cocoa gene pool and identify possible gaps for potential future collection or acquisition by specific genebanks. Approach (from AD-416) Through a collaborative consortium, cocoa samples will be collected from various cocoa producing countries in Latin America. These samples will be genotyped using DNA fingerprinting technology, including both dominant and co-dominant markers. The generated fingerprints, in combination with germplasm passport data, will be used to verify the genetic identities of cocoa accessions. Analytical tools of population genetics and bioinformatics will be applied to assess the level and pattern of cocoa genetic diversity within and among cocoa genebanks, in farmer's fields, and in natural populations. The resulted data and information will be used to improve the accuracy and efficiency in cocoa germplasm conservation and utilization. Significant Activities that Support Special Target Populations This progress contributes to National Program 301 (Plant Genetic Resources, Genomics, and Genetics Improvement), Component 1: Plant and Microbial Genetic Resource Management. Completed the assessmenet of the ancestry and population structure in 220 farmer selections from the Huallaga river valley in the Peruvian Amazon using DNA markers. A high level of genetic diversity in these farmer selections is the result of native Huallaga valley cacao combining with other introduced international clones. The distinctive population profile of the native cacao identified highlights the need for assessing on-farm genetic diversity and identifying conservation gaps in the Peruvian Amazon. Completed the survey of genetic diversity of semi-wild and cultivated cacao from Beni river valley in Bolivia. Molecular analysis of the germplasm collected revealed a new population that is completely different from the known cacao groups from the rest part of South America. This finding demonstrates that there remains many diversity gaps that need to be filled in the International cacao collections. Intra clonal variation was identified in cacao germplasm using both morphological traits and DNA fingerprints. This experiment, supported by a Vavilov-Frankel Fellowship, compared cacao accessions (305 trees) belonging to five duplicates groups in the CATIE International collection using DNA fingerprints, morphological characteristics and agronomical traits. The result supported the hypothesis that there is intra-clonal variation in cacao. This variation may not be identified by using a limited set of DNA markers, but is useful from the perspective of germplasm utilization. In the International cacao genebank in Trinidad (ICG, T), a total of 504 accessions were genotyped in 2008, which completed the verification of all major germplasm groups in Marper Farm � the site where majority of the original cacao germplasm are maintained. Admixture mapping on 350 Refractario cacao accessions was carried out to establish the association between DNA fingerprints and cocoa butter fat content, pod number, fresh bean weight and other major morphological descriptors. So far the 350 accessions have been genotyped by 70 SSR markers. This project started in 2004 and will be terminated this year. During the project period, we genotyped a total of 5312 cacao accessions from South and Central America, as well as the Caribbean region using a standard set of SSRs. Mislabeling and duplications in the two International Collections held in CATIE, Costa Rica and the University of West Indies, Trinidad have been identified. Genetic structure in these collections has been analyzed. In addition to the International collections, molecular characterization were also carried out for local collections in Bolivia, Ecuador, French Guiana, Honduras, Pueto Rico, Peru, Mexico, and Nicaragua, which provides important base-line information for the rationalization of the cacao germplam collections.
Impacts
(N/A)
Publications
- Zhang, D., Mischke, B.S., Goenaga, R.J., Nicholas, C., Ford, C., Hemeida, A.A., Saunders, J.A. 2006. Accuracy and reliability of high-throughput microsatellite genotyping for cacao cultivar identification. Crop Science. 46:2084-2092.
- Johnson, E.S., Mora, A., Schnell Ii, R.J. 2007. Field guide efficacy in the identification of reallocated clonally propagated accessions of cacao (theobroma cacao l.). Genetic Resources and Crop Evolution. 54:1301-1313.
- Zhang, D., Mischke, B.S., Johnson, E.S., Mora, A., Phillips-Mora, W., Meinhardt, L.W. 2008. Molecular characterization of an International cocoa collection using microsallte markers. Tree Genetics & Genomics. doi: http://dx.doi.org/10.1007/s11295-008-0163-z.
- Zhang, D., Boccara, M., Motilal, L., Butler, D.R., Umaharan, P., Mischke, B.S., Meinhardt, L.W. 2006. �Refractario� cocoa � genetic identity and genetic structure assessed using microsallite markers. Conservation Genetics. 9(2):327-337.
- Lachenaud, P., Zhang, D. 2008. Genetic diversity and population structure in wild stands of cacao trees (Theobroma cacao L.) in French Guiana. Forest Science. 65(3):310.
- Khan, N., Motilal, L.A., Sukha, D.A., Bekele, F.L., Iwaro, A.D., Bidaisee, P., Umaharn, L.H., Grierson, L.H., Zhang, D. 2008. Variability of butterfat content in cacao (Theobroma cacao L.): combination and correlation with other seed-derived traits at the International Cocoa Genebank, Trinidad. Plant Genetic Resources. http://dx.doi.org/10. 1017/S1479262108994132.
- Guiltinan, M.J., Verica, J., Zhang, D., Figueira, A. 2008. Genomics of Theobroma cacao, �the Food of the Gods�. In: Moore, P.H. & Ming, R. editors Genomics of Tropical Crop Plants. 1st edition. New York, NY: Springer. p. 145-170.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) To characterize genetic identities of cocoa germplasm and reduce redundancy and misidentifications in international cocoa genebanks; To assess genetic diversity in the Latin American cocoa gene pool and identify possible gaps for potential future collection or acquisition by specific genebanks. Approach (from AD-416) Through a collaborative consortium, cocoa samples will be collected from various cocoa producing countries in Latin America. These samples will be genotyped using DNA fingerprinting technology, including both dominant and co-dominant markers. The generated fingerprints, in combination with germplasm passport data, will be used to verify the genetic identities of cocoa accessions. Analytical tools of population genetics and bioinformatics will be applied to assess the level and pattern of cocoa genetic diversity within and among cocoa genebanks, in farmer's fields, and in natural populations. The resulted data and information will be used to improve the accuracy and efficiency in cocoa germplasm conservation and utilization. Significant Activities that Support Special Target Populations The reference molecular profiles for cacao generated in this project are being used to verify the genetic identity of cacao germplasm from around the world. We have completed the analysis of genetic diversity in the international collections, which will provide the baseline information for sustainable conservation and utilization of cacao genetic diversity. The genetic profile of the Refractario group from Ecuador has been analyzed and the results show that the Refractario group has a unique genetic profile among the known cacao germplasm groups. They are most likely a hybrid progenies derived from the Nacional cocoa and multiple other parental varieties. Criollos from Nicaragua were analyzed and while they shared the same genetic profiles as cacao those from Mexico, Honduras and Belize and other Central American countries, they have a broader genetic backgrounds. A core set of cacao from one of the international collections (ICG,T) was evaluated for bean number, bean size, bean mass, butterfat content and pod index. Based on our analyses several cacao accessions were identified as potential parents for the breeding of new varieties with multiple desirable traits. Accomplishments Mislabeling Evaluations Mislabeling of cacao accessions is a significant problem for the international cocoa community and hinders the efficient conservation and use of cocoa germplasm worldwide. To date, a total of 5124 cocoa accessions maintained in the two international genebanks International Cacao Genebank in Trinidad (ICG, T) and CAITE in Costa Rica, and in several other national collections in the Americas, have been genotyped or DNA fingerprinted. Duplicated accessions, both within and among different collections, were identified through genotype matching. The reference molecular profiles generated in this project 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 baseline information for sustainable conservation and utilization of cacao genetic diversity. This accomplishment is contributes to the National Program 301 Component 1: Plant and Microbial Genetic Resource Management and this accomplishment is within the scope of Problem Statement 1A: Efficiently and Effectively Manage Plant and Microbial Genetic Resources. Evaluation of Refractario germplasm �Refractario� cacao is a large group of Ecuadorian germplasm selected for its potential resistance to witches� broom disease and its high quality cocoa beans. Despite the superior economic traits, the Refractario germplasm has not been widely used for cacao genetic improvement due to insufficient information on the nomenclature, origin, genetic composition, and population structure of this Ecuadorian cacao germplasm. With the ICG, T, we characterized 482 accessions collected from farms in Ecuador, using DNA fingerprinting techniques. The results showed that the Refractarios are unique among the known cacao germplasm groups and are most likely hybrids derived from the Nacional cacao and multiple other parental varieties. The improved understanding of Refractario cacao will contribute to a more efficient conservation and use of this germplasm. This accomplishment contributes to the National Program 301 Component 1: Plant and Microbial Genetic Resource Management and is within the scope of Problem Statement 1B: Assess the Systematic Relationships and Genetic Diversity of Crop Genetic Resources. Evaluation landrace cacao from Nicaragua Nicaragua has a long history of cacao cultivation that can be traced back to the Aztec. The landrace cacao (Criollo) is descended from ancient times and remains one of the best cacao for premium market. Molecular characterization of these landraces was carried out in collaboration with Penn State University and the Nicaraguan National Agricultural Research Institute. The result showed that the Criollo from Nicaragua shared the same genetic profiles as cacao those from Mexico, Honduras and Belize and other Central American countries. However, many other varieties in this collection, with broader genetic backgrounds, also shared the superior quality. The results suggest that there is a high level of genetic diversity in the Nicaragua premium cacao varieties. This accomplishment contributes to the National Program 301 Component 1: Plant and Microbial Genetic Resource Management and is within the scope of Problem Statement 1B: Assess the Systematic Relationships and Genetic Diversity of Crop Genetic Resources. Evaluation of economic traits of cacao Cocoa butterfat and cocoa powder are key economic products from the cacao tree. Association analysis was initiated to explore the genetic diversity of cocoa butterfat, cocoa powder, as well as other related economic traits in the ICG, T. A core set of 326 germplasm accessions were selected from the whole collection based on known variations. This core set was evaluated for bean number, bean size, bean mass, butterfat content and pod index. The range of variation for each trait and their relationship between the traits were analyzed. Several cacao accessions were identified as potential parents for the breeding of new varieties that would combine multiple desirable traits. This accomplishment contributes to the National Program 301 Component 1: Plant and Microbial Genetic Resource Management and is within the scope of Problem Statement 1B: Assess the Systematic Relationships and Genetic Diversity of Crop Genetic Resources.
Impacts
(N/A)
Publications
- Zhang, D., Arevalo, E.G., Mischke, B.S., Zuniga, L.C., Barreto, A.C. 2006. Genetic diversity and population structure of cocoa (theobroma cacao l.) in the hullaga and ucayali valleys of peru. Annals Of Botany. 98:647-655
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 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 doesnt 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. 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.
Impacts
(N/A)
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.
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Progress 10/01/04 to 09/30/05
Outputs
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? What does it matter? This project continued its focus on the accurate and consistent identification of the germplasm accessions of Theobroma cacao in the international and national genebanks and the assessment of 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 will first address 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 will assess 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 US 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. Thus, the information gained during this project directly aids substantial U.S. agriculture. The research to be undertaken falls under National Program 301- Plant Microbial and Insect Genetic Research, Genomics and Genetics and address goal 2.1 described in the National Program Action Plan. Specifically these are: 2.1.2.1 Plant Molecular and Cellular Genetics- Acquire knowledge concerning wide crosses, tissue culture, identification, location, and means to manipulate and transfer genes that regulate and control traits of economic importance in plant production/product quality to enhance modification of plant germplasm and achieve genetic progress and, 2.1.2.3 Plant Genome Mapping - Develop gene maps, gene markers, and
cytogenetic knowledge of economically important plant species to enhance breeding programs. 2. List the milestones (indicators of progress) from your Project Plan. 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. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Original reference trees in Trinidad and CATIE collections fingerprinted and mislabeling in the reference trees identified and corrected Milestone Fully Met 2. On-farm
diversity in Huallaga and Ucayali area of Peru assessed. Milestone Fully Met 3. Mislabeling in the USDA collection at Mayaguez, Puerto Rico identified and correct name re-assigned Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY2006 Mislabeling and off-types within each clone identified in CATIE and Trinidad International collections. All the trees at the International Cocoa Genebank, Trinidad will be assessed by comparison with the original tree. Initially fruit and flower morphology will be compared and, in cases of doubt, DNA fingerprints will be compared using the SSR profile of the reference trees as standard. The off-types then will be identified with confidence. This work will be carried in collaboration with the Cocoa Research Unit in the University of West Indies, Trinidad. Completion of this step will fully
clear the intra-clone mislabeling in the international cacao genebanks in Trinidad, which is the major source of distributed cocoa germplasm around the world. A verification of genetic identity across three major International Gene banks - CATIE, Trinidad and the University of Reading, UK, will be carried out. This is an essential step to correct mislabeling in the International cocoa databases. Intra- and inter- population variation in the wild populations from the Camopi and Tanpok basins of French Guiyana will be assessed. The resulted information will include, but not limited to, mating system, pollen dispersal and gene flow patterns. This information is critical for in situ and on farm conservation of cacao genetic resources. A survey of on farm diversity in the Yungas, Bolivia started. This effort is being delayed due to lack of access to the required plant material. Access is being sought through collaborators in Peru. FY2007 The INIAP collection in Ecuador genotyped.
Mislabeled germplasm in the INIAP collection, Ecuador verified. Genetic background of the Ecuadorian population "National" clarified. Linkage disequilibrium estimated in different cocoa populations and association mapping tested in cocoa. Linkage disequilibrium estimated in different cocoa populations and association mapping tested using the fingerprinting data generated by this project and the phenotypic data collected by the genebank cuators. On-farm diversity in Ecuador assessed. On-farm diversity in Ecuador assessed using SSR markers. Genetic diversity will be compared using Pound's collection in the 1930's and 1940's as reference. This analysis will improve our understanding the diversity changes in Ecuador in the past 70 years. 2008 Taxonomic relationship among the Theobroma taxa clarified and gene flow between cultivated cocoa and the wild compartment analyzed. A comprehensive analysis of Theobroma taxa will be carried out using previously collected data from farmer
selection studies and international collections. 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. Data from Upper Amazon collections and studies will be used to determine the continuity of available plant material and to identify areas for additional collections. 4a What was the single most significant accomplishment this past year? Genetic Identity International Cocoa Genebanks. Genetic identity of 2100 cocoa Genetic Identity International Cocoa Genebanks. Genetic identity of 2100 cocoa accessions from two international cocoa genebanks, the cocoa germplasm collection in Costa Rica and the collection in Trinidad, were verified using microsatellite DNA markers. The multi-locus fingerprints unambiguously identified 348 duplicates and 79 homonymous mislabeling from the two gene banks. Population admixture was detected in the Upper
Amazonian and Trinitario genetic groups via assignment tests. Pedigree relationships were reconstructed for the wild cocoa populations collected from the Peruvian Amazon forest in the 1930's. This data will be used by breeders and cocoa researchers to identify and develop promising new material for release to farmers. 4b List other significant accomplishments, if any. On-farm diversity in Peru. A survey of on-farm variety and gene diversity were carried out in Huallaga valley, Peru, in collaboration with ICT - a local non-government organization. A high level of on-farm diversity was observed in this region. This high level on-farm diversity was due to the introduction of hybrids families derived from various indigenous and international clones. This finding demonstrates that the large number of farmer's selections in this region offers a good opportunity for rapid dissemination of promising clones in northeastern Peru. It also suggests that identification of productive trees
through farmer participatory selection is a cost-effective approach for cocoa improvement. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. To date, this project has generated DNA profiles for approximately 2600 "reference trees" cocoa accessions maintained in the international cocoa collections. These reference profiles are essential for accurate identification of cocoa germplasm around the world. Based on the DNA profiles, we have also analyzed the population structure and reconstructed the pedigree relationship for several widely distributed cocoa populations. Incorrect labeling of accessions and lack of understanding on population structure have been major problems impedes the efficient management of cocoa germplasm. The identification of individual accessions and populations will significantly improve the accuracy and efficiency in cocoa germplasm conservation and utilization in breeding new varieties. 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 fingerprints data of about 2600 cacao accessions from various international and national cocoa germplasm collections, as well as the identified mislabeling, have been transferred to our collaborators. This information is being used by our collaborating institutions for the correction of the off-types. 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). There is no non-peer reviewed publication printed in the current reporting period.
Impacts
(N/A)
Publications
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Progress 10/01/03 to 09/30/04
Outputs
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? What does it matter? The main emphasis of this project is to provide a means to accurately and consistently identify germplasm accessions of Theobroma cacao in the international and national genebanks and to evaluate 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 will first address 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 will assess 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 a 8.6 billion dollar chocolate industry. The confectionary industry in the U.S. would be severely affected by a shortage of cacao supply. Thus, the information gained during this project directly aids substantial U.S. agriculture. 2. List the milestones (indicators of progress) from your Project Plan. 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. 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 Huanuco and Ucayali area of Peru assessed. 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 Guiana analyzed. - A survey of on-farm diversity in the Yungas, Bolivia assessed. 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. 2008 - Taxonomic relationship among the Theobroma taxa clarified and gene flow between cultivated cocoa and its wild realtives 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. 3. Milestones: A. 2004 milestones fully met: - 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. B. 2005 - All original reference trees in Trinidad and CATIE collections will be genotyped. Mislabeling in the original reference trees will be identified through inter-population and inter-genebank comparison. The generated DNA fingerprint data, as well as the derived analytical results, will be incorporated into TROPGENE, ICGD and other relevant databases. - Using the original tree in Trinidad collection as reference, the DNA profile of accessions in the USDA collection at Mayaguez, Puerto Rico will be compared. Mislabeling in this collection will be identified and correct names will be assigned to the mislabeling accessions. - SSR genotyping of Peruvian farmer accessions completed. On-farm diversity in Huanuco and Ucayali area of Peru assessed. Genetic diversity will be compared
between the wild population in Upper Amazon and farmer's varieties. 2006 - Using the DNA profile of the reference trees as standard, all suspected mislabeling within each clone will be genotyped. Completion of this step will thoroughly clear the intra-clonal mislabeling in the two international cacao genebanks in Trinidad and CATIE. - Intra- and inter- population variation in the wild populations from the Camopi and Tanpok basins of French Guiana will be assessed. The resulted information will include, but not limited to, mating system, pollen dispersal and gene flow patterns. This information is critical for in situ and on-farm conservation of cacao genetic resources. - A survey of on-farm diversity in the Yungas, Bolivia will be carried out, based on both molecular data and farmer's knowledge. The resulted information is essential for on-farm conservation of cacao genetic diversity in the Andes. 2007 - Mislabeled germplasm in the INIAP collection, Ecuador verified. Genetic
background of the Ecuadorian population "National" clarified. - Linkage disequilibrium estimated in different cocoa populations and association mapping tested using the fingerprinting data generated by this project and the phenotypic data collected by the genebank curators. - On-farm diversity in Ecuador assessed using SSR markers. Genetic diversity will be compared using Pound's collection in the 1930's-1940's as reference. This analysis will improve our understanding of the diversity changes in Ecuador in the past 70 years. 4. What were the most significant accomplishments this past year? A. The effectiveness and accuracy of the 15 SSR loci for cacao cultivar identification has been evaluated in different cacao populations. The results showed that sufficient statistical power could be achieved using this set of SSR loci. Using these 15 primers, we assessed the extent of mislabeling in two reference populations in the International Cacao Genebank in Trinidad. Our results
showed that only 2-3% of the original reference trees were mislabeled in the two reference populations. Moreover, we were able to reconcile each of the mislabeled trees by comparing the SSR profile of the mislabeled one with its neighboring trees in the field. This result shows that there is a trustworthy reference that can serve as a baseline for individual identification. B. None. C. None. D. None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This new CRIS started in June, 2004. The project is a continuation of the previous CRIS 1275-21000-1275-21000 152-00D entitled "Analysis of genetic diversity and biochemistry of cacao and drug crops", which expired in 2004. However, in this new project, both objectives and approaches have been significantly revised and expanded. The predicted impact of this project will be the corrected misidentification and improved understanding of genetic relationships for the cocoa
germplasm in genebanks as well as in the Latin America genepool. The resulted information will lead to a rationalized germplasm conservation and higher efficiency in terms of the use of cacao germplasm in breeding. 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 fingerprints data of about 1300 cacao accessions from Trinidad and CATIE, as well as the identified mislabeling, have been transferred to CATIE, Costa Rica, and the Cacao Research Unit in the University of West Indies, Trinidad, for correcting the off-types in their collection.
Impacts
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Publications
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