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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
2012 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:
In FY 2012, progress was made in identification of traditional varieties and assessment of on-farm diversity of cacao. In Nicaragua and Honduras, ancient Criollo and Amelonado cacao types were identified among local farmer selections using Single Nucleic Polymorphism (SNP) markers. Parentage analysis shows that Amelonado made the most parentage contribution to the local Trinitario type varieties in this region. Progress was also made in southern Peru, where ‘Chuncho Cacao’ is an important traditional variety with great potential for the production of fine-flavored chocolates. Surveys were conducted in five traditional farms around Urubamba, Peru. Morphological evaluation and molecular characterization confirmed that the ‘Chuncho Cacao’ was a cultivated form of the wild population distributed in southern Peru. This study provides a new example of cacao domestication in the Amazon. In Indonesia, SNP analysis showed that only one third of the farmer selections in Aceh had parentage contribution from Upper Amazon Forastero. The result suggests that the selection for superior clones may have over-emphasized on yield and morphological appearance such as pod and bean size, whereas disease-resistance did not receive sufficient attention. In the International cacao genebank, eighteen SSR loci were used to clarify the extent of genetic redundancy in the Refracterio germplasm group. A synonymous rate of 7.5% was found in the collection and there were 56 full-sib families nestled within 189 half-sib families, revealing a high level of genetic redundancy in the collection. A new collecting expedition was under taken in Peru in 2012. Eighty four wild trees and 26 farmer varieties were collected from the rivers of Madre de Dios and Ucayali, which filled a major geographic gap in the cacao gene pool.


4.Accomplishments
1. Identification of an important traditional cacao variety from Peru. “Cacao Chuncho” is an important traditional variety in Peru but this variety has been diminishing due to rapid cultivar replacement in recent years. A survey was conducted in traditional farms in the Urubamba region in 2012 to assess the genetic identity and origin of this traditional variety. A total of 130 farmer varieties were examined morphologically and genotyped using 100 Single Nucleic Polymorphism (SNP) markers. Their DNA profiles were compared with wild cacao populations in Peru. The result of the analysis showed that the ‘Cacao Chuncho’ has a distinguished genetic profile that has not been reported before and it matches with the wild population recently discovered in southern Peru. This study thus confirmed the indigenous origin of “Cacao Chuncho”and provides a new example of direct domestication of cacao in Peruvian Amazon. The ‘Chuncho Cacao’ has a distinguished flavor profile and thus has a great potential for the production of fine-flavored chocolates.

2. Parentage analysis of landrace cacao from Honduras and Nicaragua. Understanding genetic diversity in farmer varieties is essential for sustainable production of fine-flavored cacao beans and contributes to in situ/on-farm conservation of cacao germplasm. Based on 100 Single Nucleic Polymorphism (SNP) DNA markers, we analyzed 84 fine-flavored landrace varieties collected from traditional cacao farms in Honduras and Nicaragua. Synonymous groups, including 14 Criollo and two Amelonado varieties were identified among the 84 farmer selections. The well known traditional variety “Indio” in this region was identified as synonymous with Amelonado. Parentage analysis showed that the majority of the Trinitario type varieties in this region have parentage from “Indio” (or Amelonado). The present study further depicts the diverse origins and parentage in farmer varieties from Mesoamerica. This information thus will be highly useful for conservation and utilization of cacao germplasm from this region.

3. Collection of wild cacao from the Madre de Dios and Ucayali river basins of Peru. The Peruvian Amazon is the heart of the center of diversity for cacao and holds great potential for finding new genetic variations for cacao breeding. However the wild populations are not well represented in the national and international cacao collections. A new expedition was under taken in 2012 in southern Peru. Eighty four wild trees were collected from the Madre de Dios and Ucayali watersheds, which filled a major geographic gap in the cacao gene pool. Most of these collected trees were free of disease symptoms. We also collected soil samples and endophytes (fungi that grow within the bark of trees) for these wild cacao germplasm. These wild cacaos have been transported to the ICT cacao germplasm bank at Tarapoto, Peru and grafted onto rootstock. These wild cacao trees will be evaluated for resistance to major cacao diseases.

4. Analysis of the on-farm diversity of cacao in Indonesia. Indonesia is the 3rd largest cocoa producer in the world but the genetic composition of on-farm diversity in Indonesia remained unknown. Single Nucleic Polymorphism (SNP) fingerprinting was used to assess to what extent the Upper Amazon Forastero (UAF) germplasm, particularly those harboring disease resistance, have been incorporated into the farmer selections. We assessed 92 famer selections and found that only 30 of them had parentage of Upper Amazon Forastero. The result suggests that in spite of the common use of disease resistant UAF parental clones in the seeds gardens and breeding program in Indonesia, the selection may have over-emphasized yield and morphological appearance such as pod and bean size, whereas disease-resistance did not receive sufficient attention. Therefore, clone trial in disease infected area is needed to fully evaluate the agronomic performance, including disease resistance, of these farmer selections.


Review Publications
Susilo, A., Zhang, D., Motalal, L., Meinhardt, L.W. 2011. Assessing genetic diversity in java fine-flavor cocoa (theobroma cacao l.) Germplasm by simple sequence repeat (ssr) markers. Tropical Agriculture (Trinidad). 55:84-92.

Kane, N., Sveinsson, S., Dempewolf, H., Yang, J., Zhang, D., Engels, J., Cronk, Q. 2012. Ultra-barcoding in cacao (Theobroma spp.; malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA. American Journal of Botany. 99:320-329.

Ji, K., Zhang, D., Motilal, L., Boccara, M., Lachenaud, P., Meinhardt, L.W. 2012. Genetic diversity and parentage in farmer varieties of cacao (Theobroma cacao L.) from Honduras and Nicaragua as revealed by Single Nucleotide Polymorphism (SNP) markers. Genetic Resources and Crop Evolution. DOI: 10.1007/s10722-012-9847-1.

Sitther, V., Zhang, D., Harris, D., Okie, W.R., Dhekney, S., Yadav, A. 2012. Cultivar identification, pedigree verification, and diversity analysis among Peach (Prunus persica L. Batsch) Cultivars based on Simple Sequence Repeat markers. Journal of the American Society for Horticultural Science. 137:114-121.

Zhang, D., Figueira, A., Motilal, L., Lachenaud, P., Meinhardt, L.W. 2011. Theobroma. In: Kole, C., editor. Wild Crop Relatives: Genomic and Breeding Resources, Plantation and Ornamental Crops. New York, NY: Springer-Verlag Berlin Heidelberg. p. 277-296.

Last Modified: 11/21/2014
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