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ARS Home » Southeast Area » Miami, Florida » Subtropical Horticulture Research » Research » Publications at this Location » Publication #257642
Title: Optimization of a SNP assay for Genotyping Theobroma cacao under field conditions

Author
item Livingstone, Donald
item Freeman, Barbara - Barbie
item MOTAMAYOR, JUAN - M & M Mars Company - United States
item Schnell Ii, Raymond
item Royaert, Stefan
item TAKRAMA, JEMMY - Cocoa Research Institute Of Ghana
item Meerow, Alan
item Kuhn, David

Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2011
Publication Date: 7/1/2011
Citation: Livingstone, D., Freeman, B.L., Motamayor, J.C., Schnell Ii, R.J., Royaert, S.E., Takrama, J., Meerow, A.W., Kuhn, D.N. 2011. Optimization of a SNP assay for Genotyping Theobroma cacao under field conditions. Molecular Breeding. DOI 10.1007/s11032-011-9596-4.

Interpretive Summary: Theobroma cacao L. (cacao) is grown commercially for its beans, which are used in the production of cocoa butter and chocolate. We are striving to identify and evaluate better molecular genetic markers that are linked to important economic traits to aid marker assisted selection (MAS) breeding programs for cacao to ensure a reliable supply of cocoa for the US confectionary industry. Roughly 70% of the world’s supply of cacao beans currently comes from small farms in West Africa. While breeding programs in cacao producer nations are the source of improved planting material, modern marker-based breeding is difficult to perform due to the lack of genotyping facilities in these countries. We describe a 5' Nuclease (Taqman) based Single Nucleotide Polymorphism (SNP) assay to successfully genotype cacao plants under conditions similar to those found in most cacao producing areas. Additionally, we outline the identification of additional SNP markers, and evaluate the use of SNP markers for paternal analysis. The simple, effective method we describe to genotype cacao seedlings in the field should allow for more efficient resource management of seed gardens, and is currently being implemented in Ghana. These findings will be of interest to scientists considering investing in SNP markers, and will provide cacao breeders with a robust, simple, and less expensive tool to assist in the improvement of cacao germplasm.

Technical Abstract: The tropical tree crop Theobroma cacao L. is grown commercially for its beans, which are used in the production of cocoa butter and chocolate. Although the upper Amazon region is the center of origin for cacao, 70% of the world’s supply of cacao beans currently comes from small farms in West Africa. While cacao breeding programs in producer nations are the source of improved planting material, modern marker-based breeding is difficult to perform due to the lack of genotyping facilities in these countries. While DNA extraction can be routinely performed, the equipment needed to analyze Simple Sequence Repeats (SSR) is seldom available, forcing the outsourcing of genotyping to foreign laboratories and delaying the breeding process. We describe a 5' Nuclease (Taqman) based Single Nucleotide Polymorphism (SNP) assay to successfully genotype cacao plants under conditions similar to those found in most cacao producing areas. The assay was tested under field conditions by planting open pollinated seeds of seven pods from four different maternal plants. The resulting 171 seedlings were successfully genotyped with 18 SNP markers representing 12 loci. The ability to use temperature stable reagents and rapid DNA extraction methods is also explored. Additionally, by examining the seedling genotypes for the SNP markers and 14 additional SSR markers, we investigate whether seeds in a pod are the result of single or multiple pollination events. This simple, effective method to genotype cacao seedlings in the field should allow for more efficient resource management of seed gardens, and is currently being implemented in Ghana.