Discovering SNPs for gene mapping in sugarcane using deep sequencing

As part of a marker program for QTL discovery, 313 sugarcane genes were targeted for the development of SNP markers. However, discovering useful SNPs for mapping these candidates from EST sequences in the public domain was found to be inefficient. As an alternative approach we designed primers to amplify regions of more than 200 of these genes for re-sequencing using 454 Life Sciences Genome Sequencer™ FLX. A region of a four gasket 454 sequencing run was used for the pooled amplicons from each of two mapping population parents. The sequencing yielded 96,755 and 86,241 sequences with perfect matches to a PCR primer used in amplification for the female (IJ76-514) and male (Q165) parents respectively. More than 94% of amplicons from Q165 had a supported SNP, with one SNP every 35 bases and a total of 1,632 SNPs discovered. For IJ76-514, a pure Saccharum officinarum clone, there were significantly fewer SNPs (1,013), with one SNP every 58 bases. More than 200 of these discovered SNPs have been assayed on the Sequenom Mass ARRAY® system with a high proportion being validated. Amplicon re-sequencing using the 454 system resulted in cost effective SNP discovery in a majority of the candidate genes. To map selected genes in sugarcane it is preferable that polymorphisms used for marker development be present on one homeolog only (single dose). Due to the sequence depth attained it has been possible to target assay design to SNPs of low frequency enabling enrichment for single dose markers.