披碱草属物种AFLP体系优化

Optimization of AFLP in Elymus(Poaceae)

试验对内切酶EcoRⅠ和MseⅠ的用量和酶切时间、预扩和选扩过程中模板稀释倍数以及扩增引物浓度分别进行了优化,并采用了Bassam法和Sanguinetti银染法进行了比较分析。结果表明:采用3UEcoRⅠ和MseⅠ这两种酶在37℃下酶切3 h,预扩模板稀释10倍和1.5 ng/μL引物进行预扩,选扩模板稀释20倍和2 ng/μL引物进行选扩,采用Bassam法进行银染,能够得到较好的试验结果,为AFLP进一步在披碱草属中的应用提供了参考。

The key factors affecting the AFLP analysis in Elymus were optimized,including the digesting time and the content of the EcoRⅠ and MseⅠ,diluted ratio and concentrations of primers in the pre-amplification and selective amplification.Furthermore,the methods of Bassam and Sanguinetti silver staining were also compared in the staining quality.The optimal reaction system of AFLP were established,which including digest with 3 units of each enzymes at 37 ℃ for 3 h,pre-amplification performed with templates diluted 10 times and 1.5 ng/μL primers,selective amplification performed with templates diluted 20 times and 2 ng/μL primers,as well as stained with Bassam silver staining.The results could apply for the further study on AFLP in Elymus.