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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION AND DIVERSITY ASSESSMENT OF COCOA GERMPLASM IN THE AMERICAS
2009 Annual Report


1a.Objectives (from AD-416)
The goal of this project is to develop and implement scientific approaches for managing and facilitating the use of cacao germplasm. Over the next 5 years we will focus on the following objectives:

Objective 1: Rationalize ex situ cacao collections in international and non-U.S. national genebanks.

Sub-objective 1.A. Maximize diversity coverage and reduce mislabeling and redundancy in international and non-U.S. national collections. Sub-objective 1.B. Improve the integration and expand the scope of phenotype, pedigree, and molecular data in International Cacao Databases. This objective will assure that germplasm collections are representative of the diversity available and that breeders are using correctly labeled and described breeding parents.

Objective 2: Develop complementary conservation methods, including in situ and on-farm conservation methods that can improve cacao productivity while maintaining or enhancing genetic diversity. This will be carried out with partners in developing country centers of diversity.

Objective 3: Characterize and evaluate targeted economic/agronomic traits of cacao and identify likely sources of new genes for breeders. This will serve as a guide to breeders as to what germplasm may contain the traits in which they are interested.


1b.Approach (from AD-416)
The project will continue to genotype cacao samples from ex situ collections from various cocoa producing countries in Latin America. These genotypes developed with a standard set of 15 SSR markers will continue to be used to fingerprint the cacao collections from Ecuador, Colombia, Bolivia, and several small collections in Central America and the Caribbean, following our established protocol of DNA extraction, SSR analysis and allele sizing. The generated multi-locus SSR data, together with those previously obtained from the two International genebanks and other non-U.S. national collections will be compiled and used for the identification of duplicates within and among collections. After the elimination of mislabeled and duplicate accessions, summary statistics for measuring genetic diversity will be conducted to analyze the geographical distribution of cacao germplasm and identify “hotspots” and geographical areas that have complementary levels of diversity. A minimum of two gap-collecting expeditions will be carried out in areas lacking representation in the ex situ collections. The information on genetic identities obtained in Sub-objective 1.A will serve as the foundation for correcting the existing nomenclatures in the two international databases, as well as the International Cocoa Quarantine Centre, Reading (ICQC, R), which serves as the source for the dissemination of disease-free (post-quarantine) accessions for the global cacao community. Comparison of on-farm diversity with the natural populations in the Peruvian Amazon will be implemented using the populations previous collected from Ucayali, Huallaga, and Mararon River valleys to develop complementary conservation methods, including on-farm conservation methods. Finally phenotypic and genetic analyses will be conducted to characterize and evaluate targeted economic/agronomic traits of cacao and identify likely sources of new genes for breeders.


3.Progress Report
The first expedition to collect wild cacao germplasm from Upper Amazon was completed in July – August 2008 by a team of researchers from the Instituto de Cultivos Tropicales (ICT), the Sustainable Perennial Crops Laboratory (SPCL), and the Systematic Mycology and Microbiology Laboratory (SMML) at Beltsville. A total of 198 samples from seven rivers were collected and genetic diversity analysis revealed new allelic diversity which does not exist in previously reported wild germplasm from Peru. Completed the characterization of the earliest cacao collection from the Peruvian Amazon, which was collected in the 1930's-1940's. This collection represents the most widely used cacao populations in the International Cacao Genebank in Trinidad. This collection is composed of a large number of related family members collected from a relatively small geographical area, which exposed the fact that much of the Upper Peruvian Amazon has not been sufficiently sampled. Completed the identification of 240 landraces and farmer cultivars from Mexico, Honduras and Nicaragua. This achievement provides a strong backstopping to the cacao on-farm conservation activities in Mesoamerica. A pilot experiment was completed using a new molecular marker system (Single Nucleotide Polymorphism). The results show that 30 high polymorphic SNPs would be sufficient to differentiate all tested cultivars and the 30 SNPs can be multiplexed in a single well thus leading to an increase in the efficiency of genetic diversity analysis. This method is highly suitable for detecting intra-clone mislabeling (off-type) in the field genebank, where examination of large number of trees with a small set of molecular markers is required.


4.Accomplishments
1. New cacao germplasm found in the Peruvian Amazon. Inadequate representation of genetic diversity in the cacao genebanks remains a major vulnerability for cacao conservation. Rapid deforestation has put the wild cacao populations in Amazon under eminent risk of genetic erosion. A plan of systematic sampling of the wild cacao germplasm from Peruvian Amazon was developed by SPCL scientists and the collaborators in Peru. The first expedition took place in July – August 2008 by a team of researchers from Instituto de Cultivos Tropicales (ICT), Peru, the Sustainable Perennial Crops Laboratory, and the Systematic Mycology and Microbiology Laboratory at Beltsville. A total of 198 samples from seven rivers were collected and 120 living trees were propagated in the ICT facilities in Tarapoto, Peru. Analysis of genetic diversity using 15 microsatellite DNA markers revealed new allelic diversity which does not exist in previously reported wild germplasm from Peru. These new germplasm will potentially provide new genes or alleles for resistance to cacao diseases and pests.

2. An earliest cacao collection from Peruvian Amazon was characterized using molecular marker. The river basins in Upper Amazon harbor a large number of diverse cacao populations. Since the 1930s, several populations have been collected from the Peruvian Amazon and maintained as ex situ germplasm repositories in various countries, with the largest held in the International Cacao Genebank in Trinidad. In the present study, we assessed the individual identity, sibship, and population structure in 612 wild cacao trees collected from Peru in the 1930-1940s using microsatellite DNA marker. The results show that in spite of the high level of allelic diversity, this collection was composed of a large number of related family members collected from a relatively small area. The vast majority of the Peruvian Amazon, especially the upper Maranon River and its tributaries, have not been sampled by any collecting expeditions. The improved understanding of the individual identities, genealogical relationships and geographical origin of cacao germplasm in this collection will contribute to more efficient conservation and utilization of these germplasm. Additionally, this study also provides more baseline information to help guide future collecting expeditions in the Peruvian Amazon.


6.Technology Transfer

Number of the New/Active MTAs (providing only)1
Number of Other Technology Transfer3

Review Publications
Motilal, L., Zhang, D., Umaharan, P., Mischke, B.S., Boccara, M., Pinney, S.M. 2008. Increasing accuracy and throughput in large-scale microsatellite fingerprinting of cacao field germplasm collections. Tropical Plant Biology. 2:23-37.

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.

Zhang, D., Boccara, M., Motilal, L., Mischke, B.S., Johnson, E.S., Butler, D., Bailey, B.A., Meinhardt, L.W. 2009. Molecular characterization of an earliest cacao (Theobroma cacao L.) collection from Peruvian Amazon using microsatllite DNA markers. Tree Genetics and Genomes. http://dx.doi.org/10.1007/s11295-009-0212-2.

Last Modified: 10/23/2014
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