紫花苜蓿ISSR-PCR反应体系的建立与优化

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:15 ng模板DNA,0.2 mmol/L dNTP,0.4μmol/L ISSR引物,0.8 U Taq DNA聚合酶,2μL 10×PCR Buffer,1.5 mmol/L MgCl₂,2.5%去离子甲酰胺。PCR扩增程序为:94℃预变性4 min,94℃变性30 s,62℃(62℃~58℃)退火45 s,72℃延伸1 min 45 s,共11个循环,每个循环退火温度降1℃;94℃变性30 s,52℃(52℃~48℃)退火45 s,72℃延伸1 min 45 s,共24个循环;72℃延伸5 min,25℃保温。

Establishment and Optimization of ISSR Reaction System for Alfalfa

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^-1 dNTP,0.4 μmol L^-1 ISSR primer,0.8 U Taq DNA Polymerase,2 μL 10×PCR Buffer, 1.5 mmol L^-1 MgCl₂,2.5% 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℃.