Genotyping of SNPs in a Polyploid Genome by Pyrosequencing™

Abstract

Single-nucleotide polymorphisms (SNPs) are the most frequent DNA sequence variations, and they have become increasingly popular markers for association studies. Allelic discrimination of the mostly binary SNPs has been reported for diploid species, mainly the human, but not for polyploid genomes such as the agriculturally important crops. In the present study, we analyzed the applicability of pyrosequencing to genotyping SNPs in tetraploid potatoes. Out of 94 polymorphic loci tested, 76 (81%) proved to be amenable to allelic discrimination by pyrosequencing. An additional locus could be genotyped by the addition of an ssDNA binding protein to the pyrosequencing reaction. Of the remaining 17 loci, two failed because of the presence of paralogs in the genome, while in the other cases, self-annealing of the primer or template at the low reaction temperature (28°C) employed in pyrosequencing rendered allelic discrimination impossible. The quantitative precision of pyrosequencing was found to be similar to that of conventional dideoxy sequencing and single-nucleotide primer extension. Except for some sequence-specific limitations, pyrosequencing appears to be an appropriate method for genotying SNPs in polyploid species because it is possible to distinguish not only between homo- and heterozygosity but also between the different heterozygous states.