Development of multiplexed perfect markers for screening key rice traits

Next Generation allows rapid acquisition of genome wide sequence data for a range of key reference varieties which differ by important phenotypes. These data can be quickly converted to high throughput genotyping assays allowing rapid analysis of populations derived from the reference varieties. These data also can be used for variety identification and important quality controls. We have optimized a high-throughput multiplexed SNP assay for eight polymorphisms which explain two agronomic and three grain quality traits in rice. Gene fragments coding for the agronomic traits plant height (sd-1) and blast disease resistance (Pi-ta) and the quality traits amylose content (waxy), gelatinisation temperature (alk) and fragrance (fgr) were amplified in a multiplex PCR. A single base extension reaction carried out at the polymorphism responsible for each of these phenotypes within these genes generated extension products which were quantified by a MALDI–TOF Mass Spectrometry system, the Sequenom® MassARRAY®. The assay detects both SNPs and indels and is co-dominant, simultaneously in one 5 µL reaction, detecting both homozygous and heterozygous samples in a multiplex system. We are extending this approach to the analysis of rice starch biosynthesis genes and their association studies.