CAJ | 학술논문

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：20ng模板DNA，0．2mmol／LdNTP，0．8μmol／LISSR引物，1．0UTaqDNA聚合酶，2．5μL10×PCR Buffer，2．0mmol／LMgCl2。PCR扩增程序为：94℃预变性2rain，94℃变性30s，55℃退火45s，72℃延伸1rain，共30个循环；72℃延伸7min，4℃保温。
고가색삼협초（Trifolium ambiguum Bieb．）시허다간한화한랭지구가이충식병재각방면표현우량적삼협초충，건립괄용우고가색삼협초적ISSR반응체계，장위ISSR표기기술재삼협초속품충자원연구중적응용제공삼고。본시험통과우화영향고가색삼협초ISSR—PCR적주요삼수，건립괄우고가색삼협초적ISSR반응체계화확증정서。재25μL체계중각반응물적최괄함량위：20ng모판DNA，0．2mmol／LdNTP，0．8μmol／LISSR인물，1．0UTaqDNA취합매，2．5μL10×PCR Buffer，2．0mmol／LMgCl2。PCR확증정서위：94℃예변성2rain，94℃변성30s，55℃퇴화45s，72℃연신1rain，공30개순배；72℃연신7min，4℃보온。
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℃.