การพัฒนาวิธีการตรวจสอบความบริสุทธิ์ของพันธุ์ข้าวด้วยโมเลกุลเครื่องหมาย

Abstract

Purity test of rice varietiesy by using molecular markers or DNA fingerprintings is the most accurate method. Molecular marker technology could detect polymorphism between rice varieties. Milled rice seeds are not able to germinate due to loss of the embryo during milling process. The DNA extraction from rice leaves is not possible. Therefore, the varietal purity test requires extraction of DNA directly from a single milled rice seed. This method is difficult because the major composition of milled rice seeds is starch with low DNA content. In this study, we developed a very simple, rapid, convenient and economically feasible DNA extraction method by uang from a single milled rice seed, by comparing three different methods, (1) extraction method by using proteinase K in SDS extraction buffer and 2x CTAB method, (2) extraction method by using a mobile automated DNA extractor (Maxwell) and (3) extraction method by using an automated DNA extractor (Xiril). Yield and quality of DNA extracted from a single milled rice seed was not different from those of leaf samples and also sufficient for DNA fingerprinting. In addition, we had surveyed and analyzed 32 SSR markers for rice allele standard. Twenty-three SSR markers were analyzed with cluster analysis and the results showed 8 groups of makers that can distinguish the rice variety KDML105 from CNT1 variety. Thirteen SSR markers were classified using cluster analysis and revealed 4 groups of makers that can distinguish the rice variety KDML105 from PTT1 variety. All these SSR markers showed 4-13 alleles on 27 rice lines/varieties.