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Title: Development of Single Nucleotide Polymorphism markers in Theobroma cacao and comparison to Simple Sequence Repeat markers for genotyping of Cameroon clones.

item Livingstone, Donald
item MOTAMAYOR, JUAN - Mars, Inc
item Schnell Ii, Raymond
item Martinez, Kathleen
item Freeman, Barbara - Barbie
item Meerow, Alan
item Brown, James
item Kuhn, David

Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2009
Publication Date: 3/11/2010
Citation: Livingstone, D., Motamayor, J.C., Schnell Ii, R.J., Cariaga, K.A., Freeman, B.L., Meerow, A.W., Brown, J.S., Kuhn, D.N. 2011. Development of Single Nucleotide Polymorphism markers in Theobroma cacao and comparison to Simple Sequence Repeat markers for genotyping of Cameroon clones. Molecular Breeding. DOI 10.1007/s1 1032-010-9416-2

Interpretive Summary: Theobroma cacao, the source of cocoa beans for chocolate, is an important tropical agriculture commodity. 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. Currently there are about 500 molecular genetic markers for cacao, the majority of which are simple sequence repeat (SSRs) 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 convert thirteen previously developed cacao single-strand conformational polymorphism (SSCP) markers into SNP markers. These SNP markers are among the first published for cacao, and are added to a genetic map of cacao segregating for resistance to witch’s broom. Furthermore, we compare the ability of cacao SNP and SSR 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 SSRs 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: Single Nucleotide Polymorphism (SNP) markers are increasingly being used in crop breeding programs, slowly replacing Simple Sequence Repeats (SSR) and other markers. SNPs provide many benefits over SSRs, including ease of analysis and unambiguous results across various platforms. We have identified and mapped SNP markers in the tropical tree crop Theobroma cacao, and here we compare SNPs to SSRs for the purpose of determining off-types in clonal collections. Clones are used as parents in breeding programs and the presence of mislabeled clones (off-types) can lead to the propagation of undesired traits and limit genetic gain from selection. Screening was performed on 186 trees representing 19 Theobroma cacao clones from the Institute of Agricultural Research for Development (IRAD) breeding program in Cameroon. Our objectives were to determine the correct clone genotypes and off-types using both SSR and SNP markers. SSR markers that amplify 11 highly polymorphic loci from six linkage groups and 12 SNP markers that amplify eight loci from seven linkage groups were used to genotype the 186 trees and the results from the two different marker types were compared. The SNP assay identified 98% of the off-types found via SSR screening. SNP markers spread across multiple linkage groups may serve as a more cost effective and reliable method for off-type identification, especially in cacao producing countries where the equipment necessary for SSR analysis may not be available.