Discovery of single nucleotide polymorphisms in sugarcane for gene mapping using 454 sequencing

Sugarcane is genetically complex due to polyploidy, aneuploidy and hybridisation which have led to a large but variable number of copies of each chromosome and a large number of chromosomes overall in the genomes of commercial varieties. To map genes of interest, ‘single dose’ SNPs are desirable because they are fully informative across the mapping population. However discovery of these SNPs requires considerable depth of sequencing to find the single copy alleles and confirm the polymorphisms. To achieve this depth, 454 sequencing of pooled PCR amplicons was utilised on the parents of a QTL mapping population. Three hundred pooled amplicons from each parent were sequenced yielding 96,755 and 86,241 sequences from the two parents, with average sequence depth of approximately 300 and average read length of 220 bases. In the more polymorphic parent, 94% of amplicons analysed (227/242) had evidence of a reliable SNP – an average of a SNP every 35 bases. Candidate single dose SNPs were validated and genotyped for mapping across the progeny using the Sequenom MassARRAY (MALDI-TOF mass spectrometer) system. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom system. Genotyping across the mapping population was carried out for 197 SNPs. Amplicon re-sequencing using the 454 system enables cost effective SNP discovery that can be targeted to genes of interest. This approach should be useful for the detection of SNPs in polyploid species generally - for linkage mapping, association studies and for population & ecological genetics.