Genotyping of single nucleotide polymorphisms in sugarcane using mass spectrometry and CEL I polymorphism detection

Genetic linkage maps of sugarcane have to date been largely based on one or a combination of RAPD, RFLP, SSR and AFLP markers. However high throughput and potentially low-cost genotyping based on single nucleotide polymorphisms (SNPs) along with the possibility of perfect markers has made SNPs based on expressed sequences such as ESTs an attractive option for marker development in sugarcane. To test the feasibility of SNP marker based genotyping in sugarcane we have developed a system for SNP detection based on Ecotilling using CEL I polymorphism detection. Partial CEL I digests of labelled PCR products generated from the DNA of individuals can be used for both discovery and determining segregation patterns. However genotyping with this approach is not high throughput or cheap but to this end SNPs segregating as single dose in progeny sub-samples can be genotyped cheaply and with high-throughput using mass spectrometry (Sequenom). The strategy of utilising Ecotilling for SNP confirmation and determination of segregation pattern followed by mass spectrometry based genotyping on full progeny sets has been tested and confirmed using members of the sucrose-phosphate synthase gene family in sugarcane. In addition to genotyping for mapping, mass spectrometry should also be a useful method of allele frequency determination for LD based association studies.