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Title: Cacao single-nucleotide polymorphism (SNP) markers: A discovery strategy to identify SNPs for genotyping, genetic mapping and genome wide association studies (GWAS)

item Kuhn, David
item Schnell Ii, Raymond
item MOTAMAYOR, JUAN - M & M Mars Company - United States
item Livingstone, Donald
item MAY, GREG - National Center For Genome Resources
item RIGOUTSOS, ISIDORE - International Business Machines Corporation (IBM)
item HAIMINEM, NIINA - International Business Machines Corporation (IBM)

Submitted to: Proceedings of the International Cocoa Producer's Conference
Publication Type: Proceedings
Publication Acceptance Date: 11/16/2009
Publication Date: 2/28/2011
Citation: Kuhn, D.N., Schnell Ii, R.J., Motamayor, J.C., Livingstone, D., May, G., Rigoutsos, I., Haiminem, N. 2011. Cacao single-nucleotide polymorphism (SNP) markers: A discovery strategy to identify SNPs for genotyping, genetic mapping and genome wide association studies (GWAS). Proceedings of the International Cocoa Producer's Conference. pp. 135-142.

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 trying to find 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 and we are taking advantage of the cacao genome sequencing project to expand that to greater than 50,000 single-nucleotide polymorphism (SNP) markers. We will use these markers to improve the resolution of our current genetic maps and to find associations between specific SNPs and advantageous traits such as disease resistance or higher yield. Our results are important to scientists trying to understand the mechanism of disease resistance and, eventually, to cacao farmers who will benefit from superior disease resistant and more productive cultivars produced through our MAS breeding program.

Technical Abstract: Single-nucleotide polymorphisms (SNPs) are the most common genetic markers in Theobroma cacao, occurring approximately once in every 200 nucleotides. SNPs, like microsatellites, are co-dominant and PCR-based, but they have several advantages over microsatellites. They are unambiguous, so that a SNP called on any platform will be identical, which makes them particularly useful for international genotype databases, as there are no problems in normalizing the data as with microsatellites. Because the SNP assay does not require electrophoresis, SNPs can be assessed rapidly on relatively inexpensive equipment and without the need for expensive analysis software or highly trained technical support personnel to carry out the analysis. SNPs are practically unlimited in number for the genome and are found equally in both coding and non-coding regions, unlike microsatellites. As a direct consequence of the cacao genome sequencing project, we sequenced total RNA isolated from 1-2 g of young green leaf tissue of two accessions from each of the ten diverse groups of cacao identified by Motamayor et al (PLoS ONE 3(10): e3311 doi:10.1371/journal.pone.0003311, 2008). The RNA was sequenced using an Illumina Genome Analyzer which generated ~2 Gb of sequence data for each RNA, representing >50x coverage of all transcribed loci. Sequencing was performed by NCGR and SNPs identified by NCGR and IBM, using different sequence assembly and SNP detection methods. We expect to identify >50,000 SNPs by this method. Transcripts containing SNPs will be mapped against the available genome sequence scaffolds to identify SNP markers that cover the entire genome at a spacing of 1 cM or less between markers. The expected 50,000 SNPs will be used to saturate the recombinant genetic map using an Illumina Infinium chip or the Illumina GoldenGate bead array to genotype the individuals in every mapping population. The same SNP assay tool will be used to screen the cacao germplasm collection to provide genotypes for association mapping of traits where populations are not available. SNP markers that map to known QTL regions for disease resistance, yield traits and other phenotypes of interest will be converted to a TaqMan or similar type individual SNP assay for use in marker-assisted selection in West Africa, Central and South America, and Oceania (Papua New Guinea).