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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

Location: Sustainable Perennial Crops

2010 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
A cacao genotyping kit based on 100 Single Nucleotide Polymorphism (SNP) markers has been validated for large scale genotyping of cacao germplasm using the Sequenom platform. The validation was performed using 280 cacao accessions sampled from seven different genetic groups. The genotyping kit was able to unambiguously discriminate all the tested accessions, including known siblings, parent-offspring, and duplicated genotypes. Moreover, high correlation was detected between genetic distances based on 30 microsatellite (SSR) and SNP markers, suggesting that these SNP markers are excellent for cacao diversity analysis as well. This genotyping kit is now being used to detect intra-clone mislabeling (off-type) in the International Cacao Germplasm Bank in Trinidad (ICG,T).

Genetic diversity in ancient Criollo varieties in Belize was analyzed and their contribution to the origin of Trinitario cacao was assessed. The Belizean Criollo is a group of relic varieties that are thought to be similar to those used by the Olmecs and Mayans during early domestication. A population of Belize Criollo collected from the Maya Mountains in Belize was characterized. In contrast to the commonly accepted notion that the ancient Criollo is one of the parents of Trinitario cacao, results of the new study shows that the contribution of Criollo cacao to the Trinitario is small. It appeared that there were different kinds of landraces grew in Trinidad before the 18th century, in addition to the Central America Criollo. The result provides new insight into the origin of the Trinitario cacao, which will be useful for conservation of cacao landraces from Central America and Caribbean region.

The second expedition to collect wild cacao germplasm from Upper Amazon was taken in July – August 2009. The objective was to collect wild cacao in areas lacking representation in the ex situ cacao germplasm collections in the world. The geographical focus of this expedition includes Rio Santiago, Rio Morona and Rio Tigre in northern Peru. A total of 140 wild trees were collected and were propagated in the ICT facilities in Tarapoto, Peru. High level of allelic diversity was observed in these accessions based on the results of DNA fingerprinting. These new materials are being analyzed using DNA fingerprinting technology. These new germplasm will potentially provide new genes or alleles for resistance to cacao diseases and pests.

The extent of intra-accessions mislabeling in the International Cacao Genebank, Trinidad (ICG, T) was assessed using the established reference genotypes. A total of 972 individual trees sampled across locations by field, by accession, and by trees within each plot were genotyped using nine microsatellite loci. Based on their DNA fingerprints, mislabeling were identified within and among fields and accessions. The study found a mislabeling rate ranging from 7 – 62 % within field sections, with an overall rate of mislabeling about 30%. These results will help direct the Cocoa Research Unit to prioritize additional fingerprinting of accessions, which will use more cost effective SNP genotyping.


4.Accomplishments
1. Two hundred and twenty six farmer selections from Bolivia, Nicaragua, Honduras, Mexico and Peru were assessed using 15 SSR and 100 single nuclei polymorphism (SNP) markers. The identification of rare populations of cacao (chocolate) trees is of increasing importance to industry. A total of 120 known accessions with population membership in the International Cocoa Gene banks were used as references. Cluster analysis and Bayesian assignment tests successfully identified traditional varieties based on their affiliation with known reference genotypes, including ‘Amelonado’, Comum’, ‘Central American Criollo’, ‘Indio Rojo’, ‘Venezuela Porcelana and ‘Nacional’. Traditional farmers’ varieties suitable for the production of fine flavor chocolate still exist in farmers’ fields in the Americas. Accurate identification of these rare and valuable landraces is essential for sustainable production of fine flavored cacao beans, contributes to in situ/on-farm conservation of these premium varieties and is important for the fine flavor chocolate industry.

2. Re-examination of the relationship between ancient Criollo cacao from Belize and the Trinitario cacao from Trinidad & Tobago provides new insight into the origin of the Trinitario cacao. In contrast to the commonly accepted notion that the ancient Criollo from Mesoamerica is one of the parents of Trinitario cacao, results of the new study shows that the contribution of Criollo cacao to the Trinitario is small. The ICS (acronym for “Imperial College Selections”) cacao from Trinidad possessed a more varied parentage than had been previously envisaged. It appeared that there were different kinds of landraces growing in Trinidad before the 18th century, in addition to the Central America Criollo. The result of the present study will be highly useful for the ex situ and in situ conservation of cacao landraces from Central America and Caribbean region. These findings are also helping to track the origin of fine flavored cacao.


Review Publications
Motilal, L., Zhang, D., Umaharan, P., Mischke, B.S., Mooleedhar, V., Meinhardt, L.W. 2010. The relic Criollo cacao in Belize- genetic diversity and relationship with Trinitario and other cacao clones held in the International Cocoa Genebank, Trinidad. Plant Genetic Resources: Characterization and Utilization. Available at http://dx.doi.org/10.1017/S1479262109990232.

Last Modified: 9/2/2014
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