高加索三叶草ISSR-PCR反应体系的建立与优化

ESTABLISHMENT AND OPTIMIZATION OF ISSR REACTION SYSTEM FOR CAUCASIAN CLOVER

高加索三叶草(Trifolium ambiguum Bieb.)是许多干旱和寒冷地区可以种植并在各方面表现优良的三叶草种,建立适用于高加索三叶草的ISSR反应体系,将为ISSR标记技术在三叶草属品种资源研究中的应用提供参考。本试验通过优化影响高加索三叶草ISSR-PCR的主要参数,建立适于高加索三叶草的ISSR反应体系和扩增程序。在25μL体系中各反应物的最适含量为:20 ng模板DNA,0.2 mmol/L dNTP,0.8μmol/L ISSR引物,1.0 U TaqDNA聚合酶,2.5μL 10×PCR Buffer,2.0mmol/L MgCl2。PCR扩增程序为:94℃预变性2 min,94℃变性30s,55℃退火45s,72℃延伸1 min,共30个循环;72℃延伸7min,4℃保温。

The Caucasian clover which can be grown in many arid and cold regions is unique in clover species. Establishment of Caucasian dover for ISSR reaction system will lay the foundation for the study of ISSR makers in clover cultivars. The optimal ISSR - PCR reaction conditions in Caucasian clover were established by studying the main parameters. The results showed that the optimum concentrations of seven reactants in 25μL reaction mixture were as follows： 20ng genomic DNA, 0.2mmol L-1 dNTP, 0.8μmol L-1 ISSR primer, I. 0U Taq DNA Polymcrase, 2.5μL 10 × PCR Buffer, 2.0mmol L - IMgC12. The suitable PCR procedure was one preliminary denaturation at 94℃ for 2 rain; 30 cycles each involved denaturation at 94℃ for 30s, anneal at 55℃ for 45s, extended at 72℃ for lmin, and a final extension at 72℃ for 7min, then keep the temperature at 4℃.