Rapid genetic mapping of ESTs using SNP pyrosequencing and indel analysis

We describe an effective systematic approach to genetic mapping of cDNA clones, including those obtained from EST sequencing. The EST of interest is first partially sequenced from the 3'-end. PCR primers which bracket the 3'-UTR segment of the cDNA are designed. The corresponding gene segment is amplified from the parents of the mapping population, using primers equipped with 3'- and 5'-extensions to facilitate direct sequencing of PCR products. Comparison of the sequences obtained from the mapping parents frequently reveals single nucleotide polymorphisms or insertion / deletion polymorphisms, which can then be genotyped in a mapping population. The genotyping of SNPs is performed by pyrosequencing, a sequencing-by-synthesis method that has been used successfully in SNP diagnostics. SNP analysis of up to 96 samples, a number required to produce meaningful genetic segregation data, can be rapidly accomplished in parallel. The parental genotype of three loci, stearoyl-ACP desaturase, nucleoside-diphosphate kinase and sucrose synthetase-1 were determined by conventional sequencing, and the polymorphism so identified were scored by the pyrosequencing of 94 individuals of a maize recombinant-inbred population. These loci were successfully placed onto chromosomes 3, 7 and 9 respectively. This method is generally applicable to most plant species, which show sufficient sequence diversity in the 3'-UTR region of genes.