Abstract
In the present study, 4 sets of multiplex PCR primers were developed to identify 130 varieties of rice-paddy cultivated in Japan (12 of which being suitable for sake brewery,) covering approximately 99% of the domestic rice planted area. Gene arrangements to distinguish each of these samples were defined, and 15 sets of STS (Sequence-Tagged Sites) primers were designed. Each of these sets was used for PCR analysis of 130 varieties to detect discriminating bands that are unique to each variety. These 15 sets of primers were integrated into 4 multiplex PCR primer sets by examining their base sequence, combination of primers and PCR conditions, so that they would not interfere with each other. Multiplex PCR was then performed to confirm that they could identify each of the 130 varieties chosen. The rice varieties that are cultivated in several prefectures in Japan, namely Koshihikari, Hitomebore, Hinohikari, Akitakomachi, Kinuhikari, Nihonbare, Sasanishiki, Hanaechizen, Matsuribare and Asahinoyume were analyzed by PCR using the 4 multiplex PCR primer sets. The results showed that there were no differences in the discrimination of the bands among the samples of a variety collected from different prefectures. The present study suggests that the multiplex PCR primer sets developed were effective in distinguishing the rice varieties cultivated in Japan.
