A high-throughput SNuPE assay for genotyping SNPs in the flanking regions of Zea mays sequence tagged simple sequence repeats

Information on genetic relationships among individuals is of importanceto maize breeders for line and hybrid development. Estimates on the geneticsimilarity of breeding materials is best obtained using DNA markers. SingleNucleotide Polymorphisms and small insertions/deletions are both emerging as anew generation of markers, due to their abundance and amenability to fullyautomated genotyping. Application of SNPs, for example in genetic mappingprojects or breeding programs, involves the analysis of a large number ofsamples, and therefore requires rapid, inexpensive, and highly automatedmethodsto genotype the sequence variants. Towards this, we have applied a highthroughput Single Nucleotide Primer Extension assay to assess 23 polymorphicbase variations at five microsatellite loci in 22 inbred maize lines, as wellasin a mapping population of two of the inbred lines. Using a MegaBACE automatedgenotyper, we are able to assay more than 1248 (96 × 13) samples in asingle 75 minute run. The SNuPE method successfully genotyped the base pairvariations of interest in all maize lines. It was also found that primerscontaining polymorphisms themselves could be used to genotype the samples. Thistechnique allows the rapid production of valuable high throughput informationongenetic relationships among maize varieties. We further demonstrate the utilityof this method, using it to successfully map one of the microsatellite loci.