Optimization of a SNP assay for genotyping Theobroma cacao under field conditions

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 (SSRs) 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 for genotyping 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 investigated whether seeds in a pod are the result of single or multiple pollination events. This simple, effective method of genotyping cacao seedlings in the field should allow for more efficient resource management of seed gardens and is currently being implemented in Ghana.