紫花苜蓿ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR Reaction System for Alfalfa

通过优化影响紫花苜蓿（Medicago sativa L．）ISSR—PCR的主要参数，建立适于紫花苜蓿的ISSR反应体系和扩增程序。在20μL体系中各反应物的最适含量为：15ng模板DNA，0．2mmol／LdNTP，0．4μmol／LISSR引物，0．8U Taq DNA聚合酶，2μL 10×PCR Buffer，1．5mmol／L MgCl2，2．5％去离子甲酰胺。PCR扩增程序为：94℃预变性4min，94℃变性30s，62℃（62℃～58℃）退火45s，72℃延伸1min45s，共11个循环，每个循环退火温度降1℃；94℃变性30s，52℃（52℃～48℃）退火45s，72℃延伸1min45s，共24个循环；72℃延伸5min，25℃保温。

Alfalfa （Medicago sativa. L. ） was subjected to ISSR （Inter-simple sequence repeats）- PCR to investigate the genetic diversity and the specific molecular markers linked to specific characters. By studying the main parameters we established the optimal ISSR-PCR reaction conditions in alfalfa. Results show that the optimum concentrations of seven reactants in 20 μL reaction mixture are as follows： 15 ng genomic DNA, 0.2 mmol L^-1dNTP, 0.4 μmol L^-1ISSR primer, 0.8 U Taq DNA Polymerase, 2 μL 10×PCR Buffer, 1. 5 mmol L^-1 MgC12, 2. 5% deionized formamide. The suitable PCR procedure is one preliminary denaturation at 94℃ for 4 min; 11 cycles each involved denaturation at 94℃ for 30 s, anneal at 62℃（62℃～58℃） for 45 s, with 1℃ declined each cycle, extended at 72℃ for 1 min 45 s; then followed by 24 cycles each with denaturation at 94℃ for 30 s, anneal at 52℃ for 45 s, extended at 72℃ for 1 min 45 s; and a final extension at 72℃ for 5 min, then keep the temperature at 25℃.