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Title: Genetic marker comparison for the purpose of determining off-type cacao clones within a West African germplasm collection

Author
item Livingstone, Donald
item MOTAMAYOR, JUAN - Mars, Inc
item Schnell Ii, Raymond
item SOUNIGA, OLIVIER - Institute Of Agricultural Research For Development (IRAD)
item EFOMBAGN, BRUNO - Institute Of Agricultural Research For Development (IRAD)
item PADI, FRANCIS - Cocoa Research Institute Of Ghana
item DADZIE, ABU - Cocoa Research Institute Of Ghana
item Kuhn, David

Submitted to: Proceedings of the International Cocoa Producer's Conference
Publication Type: Abstract Only
Publication Acceptance Date: 11/16/2009
Publication Date: 2/28/2010
Citation: Livingstone, D., Motamayor, J.C., Schnell Ii, R.J., Souniga, O., Efombagn, B., Padi, F.K., Dadzie, A.M., Kuhn, D.N. 2010. Genetic marker comparison for the purpose of determining off-type cacao clones within a West African germplasm collection. Proceedings of the International Cocoa Producer's Conference. 1.

Interpretive Summary: Theobroma cacao, the source of cocoa beans for chocolate, is an important tropical agriculture commodity that is affected by a number of fungal pathogens and insect pests, as well as concerns about yield and quality. We are striving to identify and evaluate better molecular genetic markers that are linked to disease resistance and other important economic traits to aid in a marker assisted selection (MAS) breeding program for cacao to ensure a reliable supply of cocoa for the US confectionary industry. Currently there are about 500 molecular genetic markers for cacao, the majority of which are microsatellite markers. The cacao genome sequencing project is expected to discover over 50,000 single-nucleotide polymorphism (SNP) markers. While SNP markers provide several benefits over microsatellites, including ease of analysis, unambiguous results across various platforms, and increased throughput potential, their practical application to the tasks associated with cacao MAS needs to be evaluated. As such, we compare the ability of cacao SNP and microsatellite markers to determine clonal off-types, an essential step to ensure successful propagation of desired traits. We show that the SNPs perform nearly as well as microsatellites while offering significant time and cost savings. 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: Microsatellites are co-dominant PCR-based genetic markers, which are commonly used in modern Marker Aided Selection (MAS) breeding programs. Although many MAS breeding programs have and continue to successfully use microsatellites, various issues with analysis, such as comparing results across different labs, have driven the discovery of new markers. One such marker, Single Nucleotide Polymorphism (SNP), is increasingly being used in crop breeding programs, slowly replacing microsatellites and other markers. SNPs provide several benefits over microsatellites, including ease of analysis and unambiguous results across various platforms. Additionally, SNPs are found in coding and non-coding regions alike, and SNPs, estimated to occur once every 200 nucleotides in Theobroma cacao L., are more prevalent throughout the genome than microsatellites. The higher incidence of SNP markers will allow for more detailed genetic maps, and ultimately more refined breeding efforts. In cacao, a genotype can be fixed by cloning and selected genotypes are often used as parents in breeding programs. Misidentification of clones has been a persistent problem and these misidentified clones (off-types) often lead to the propagation of undesired traits. To compare the usefulness of SNPs to microsatellites for the purpose of determining off-types in clonal collections, screening was performed on 186 trees representing 19 clones of Theobroma cacao L. from Cameroon. DNA extracts from this population were first quantified and normalized using a dual curve Sybr Green I based protocol to improve efficiency of both microsatellite and SNP analysis. Microsatellites representing 11 distinct polymorphic loci from six linkage groups and 12 different SNP markers representing eight distinct loci from seven linkage groups were used to genotype the clones. SNP markers performed as well as microsatellites for the purpose of off-typing, with SNPs identifying 45 off-types called by at least two markers compared to microsatellites which found 46 off-types with the same stringency. Additionally, the SNP assay may be preferred in cacao producer countries where the equipment necessary for microsatellite analysis may be cost prohibitive especially when compared to the equipment costs needed for SNP analysis.