不结球白菜SSR引物的高效开发及其通用性研究

利用改进的ISSR-PCR分离不结球白菜基因组微卫星,进行两步PCR,分别设计出SSR两端引物IP2和IP3（即SSR引物对）,SSR引物产率为12%,明显高于传统方法.不结球白菜SSR序列以（GA）n为主,占70%.将69对SSR引物用于芸苔属其它8种作物的扩增,97%引物有显著扩增,扩增率最高的为四倍体白菜和甘蓝（85.5%）,最低的为青花菜（49.3%）.10对为通用引物,在所检测的8种作物中片段大小一致,其余的扩增片段和数目差异较大,表现出较为丰富的多态性.尤其是在C基因组的甘蓝和花椰菜上,最多可检测到5个位点,表明运用lSSR-PCR开发出的引物多态性较高.

Development of SSR Primers of Non-heading Chinese Cabbage and Transferability among Closely Related Species

Modified ISSR-PCR method was presented for isolating microsatellites from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) genomic DNA. The method was carried out through a two-step PCR,and then primers (IP2 and IP3) were designed according to the flanking sequence of microsatellite. The success rate was 12%, which is significantly higher than that of traditional method. The SSR sequence was mainly (GA/CT)n, which was in the majority of 70%. 69 pairs of SSR primers were amplified among eight Brassica types, of which 97% primers had evident amplified bands. The highest amplification rate was 85.5%, occurring in non-heading Chinese cabbage with tetraploid and Brassica oleracea L. And the lowest was 49.3% in Brassica var. botrytis L. of above 69 pairs, 10 pairs were transferable among eight Brassica types, whose amplified fragments were the same,whereas others were diverse in size and number, showing higher polymorphic. Especially in Brassica oleracea L. and Brassica var. botrytis L., which were typical C chromosome types, five loci were tested at most, suggesting these primers developed by ISSR-PCR had rich polymorphic.