Development of the primer sets for identification of a rice cultivar, Koshihikari, by PCR

Abstract

As it is obligated, by the revision of the JAS Act, that cultivars, location, and year of production should be discribed on a bag of milled rice grains, the technology to identify them by objective methods is indispensable. Milled rice grains produced at the several Agricultural Experiment Stations as their fundamental cultivars were analyzed to the PCR method for their identification. Promising RAPD primers were used to select the discriminating DNA bands, which were extracted from agarose gels after electrophoreses and cloned into a PCR-XL-TOPO vector followed by sequencing. Forward primers and reverse ones (15 to 28 mers) were designed starting from the RAPD primer portions as sequence-tagged site primers. These STS primers were combined together and suitable primer sets to discriminate Koshihikari from other cultivars were selected as positive and negative sets. By PCR using these primer sets, 33 Koshihikaris from different prefectures showed the same DNA pattern and the other 49 cultivars revealed different DNA patterns. It became possible by this technology not only to identify Koshihikari using a single rice grain sample but also detect different mislabeled cultivar rice grains using these primer sets.