Analysis of a single nucleotide polymorphism that controls the cooking quality of rice using a non-gel based assay

The waxy gene encoding granule-bound starch synthase (GBSS) is responsible for the synthesis of amylose in developing grain. Recent work has shown that a G-T polymorphism in the leader intron 5' splice site of GBSS plays a key role in determining the cooking and processing quality of rice. Cultivars with sequence AGGTATA at this location splice GBSS pre-mRNA efficiently and produce relatively large amounts of amylose. These varieties generally a have firm texture when cooked and the grains remain separate. In contrast, GBSS pre-mRNA splicing is temperature sensitive and generally less efficient in cultivars with the sequence AGTTATA. As a result, these cultivars generally have lower amylose content and produce soft and sticky cooked rice. We have used the READIT(TM) assay, a non-gel based assay that uses the ability of DNA polymerase to perform pyrophosphoralysis, the reverse of DNA polymerization, to screen the critical G-T polymorphism in more than 750 samples from U. S. and Asian germplasm. We observed complete concordance between the results obtained using DNA sequencing or restriction enzyme digestion and the READIT(TM) assay. It also gave accurate results with both heterozygous plants and with complex mixtures as might result when grain from advanced generation plants is pooled to obtain larger samples.