大花萱草ISSR-PCR最佳反应体系的建立

Establishment of the Optimum ISSR-PCR Reaction System in Hemerocallis spp.

ISSR标记能揭示出种间、种内遗传变异情况,更好地揭示亲缘关系和遗传多样性。以大花萱草的5个品种为试材,研究了影响大花萱草ISSR-PCR扩增效果的各项因素,建立了适合大花萱草ISSR-PCR的最佳反应体系:20μL反应体系中,10×buffer 2μL,dNTP 0.2 mmol/L,Mg2+浓度1.5 mmol/L,引物浓度0.5 mmol/L,Taq酶10.002 nkat,DNA模板20 ng。大花萱草的最佳ISSR扩增反应程序为:94℃预变性5 min;94℃变性40 s,50℃退火45 s,72℃延伸1.5 min,共40个循环;最后72℃延伸10 min。通过该反应程序进行的大花萱草ISSR扩增可获得清晰、稳定的条带。

Inter-simple sequence repeat（ISSR） method was applied to detect inter-and intra-specific genetic variation,and better reveal genetic relationship and genetic diversity.The factors which affect the inter-simple sequence repeat（ISSR） analysis were studied through 5 daylily cultivars.The optimal ISSR-PCR reaction conditions for daylily were established as follows： in a volume of 20 μL containing 2 μL 10×buffer,0.2 mmol/L dNTP,1.5 mmol/L Mg2＋,0.5 mmol/L primer,10.002 nkat Taq DNA polymerase,and 20 ng template DNA.The optimal amplified procedure was as follows： after a pre-denaturing of 5 min at 94 ℃,40 cycles were performed with denaturing of 40 s at 94 ℃,annealing of 45 s at 50 ℃,extension of 1.5 min at 72 ℃,a final extension of 10 min at 72 ℃.The clear and stable amplified bands might be obtained with the above ISSR-PCR reaction conditions.