Sequence polymorphism discovery in wheat microsatellite flanking regions using pyrophosphate sequencing

Sequence polymorphisms such as insertion/deletions (indels) and single nucleotide polymorphisms (SNP) are suitable for automated analysis of molecular markers and useful for cultivar identification, genetic mapping and trait association. While they are abundant, their initial discovery, comprising detection, validation and characterisation of sequence polymorphisms, is time consuming and expensive. This is especially true for multi-allellic hexaploid wheat. We investigated simple sequence repeat (SSR) flanking regions as a source for sequence polymorphisms in wheat. SSRs have a potentially high polymorphic frequency, there are a large number of highly characterised markers available, tested primers are published and most are single locus. Of 126 markers investigated, polymorphisms were found in 33 (26%) when tested in 10 wheat varieties. No new primers needed to be designed, the published primer sequences were used as PCR primers and then as sequencing primers. Polymorphism was detected by resequencing using a modification of pyrophosphate sequencing (Pyrosequencing(R)) which yielded quality sequencing from the first base after the primer with up to 80 bases of information. Our method of pyrophosphate sequencing of SSRs, although not suitable for full-length sequencing, is an attractive method to directly find sequence polymorphism in varieties of interest using the abundant, well characterised and published SSR markers.