ISSR-PCR和链亲和素磁珠吸附法开发白菜SSR引物

SSR Primers Development in Non-heading Chinese Cabbage by ISSR-PCR and Streptavidin-coated Magnetic Beads Adsorption

分别利用ISSR-PCR和链亲和素磁珠吸附法分离白菜基因组微卫星,共得到41对SSR引物。ISSR-PCR法运用巢式PCR,分别设计微卫星两侧引物IP2和IP3,SSR引物得率为12%。磁珠吸附法首先利用生物素标记的SSR探针与文库杂交,链亲和素磁珠吸附富集含SSR的片段。然后用接头引物扩增,克隆。根据测序结果,设计SSR两侧引物PL和PR,引物得率为9.6%。

Two methods of ISSR-PCR and streptavidin-coated magnetic beads adsorption were used to isolate microsatellites from genomic DNA of non-heading Chinese cabbage （Brassica campestris ssp. chinensis） and a total of 41 SSR primers were successfully developed in this study. SSR primers （IP2 and IP3 ） were designed on the base of the determined terminal sequence of nested PCR products in the ISSR-PCR method with the efficiency of 12% for SSR primer development. As to the method of streptavidin-coated magnetic beads adsorption, firstly the biotin-labeled SSR probe was hybridized with digested genomic DNA and SSR fragments were enriched by absorption with strepavidin-coated magnetic beads and then they were amplified, cloned and sequenced. On the base of the sequence flanking microsatellites SSR primers （ PL and PR） were designed with an efficiency of 9.6%.