华北蓝盆花ISSR-PCR引物筛选及反应体系优化

Screening of ISSR-PCR Primers and Optimization of Reaction System for Scabiosa tschiliensis Grun

本研究以内蒙古自治区境内8个自然居群野生华北蓝盆花叶片作为供试材料,进行基因组总DNA的提取检测、ISSR引物筛选和引物退火温度的筛选。针对华北蓝盆花ISSR-PCR反应中的模板DNA浓度和引物浓度2个影响因素,在4个水平上对华北蓝盆花ISSR-PCR反应体系进行优化,确定最佳反应水平,最终建立华北蓝盆花ISSR-PCR扩增的最佳反应体系。结果筛选出了8条多态性较好的华北蓝盆花ISSR引物及其退火温度,平均每条ISSR引物扩增出12.3条带,多态性条带占93.88%;华北蓝盆花ISSR-PCR反应的最佳体系为总体积20μL,DNA模板1μL,DNA模板浓度为60 ng/μL,引物0.6μL,引物浓度为0.8μmol/L,Ex Taq DNA聚合酶10μL,ddH2O 8.4μL,扩增程序为94℃预变性5 min;94℃变性0.5 min,不同引物最佳退火温度下退火1 min,72℃延伸1.5 min,35个循环;循环结束后72℃继续延伸7 min;4℃保存。本研究结果为进一步研究华北蓝盆花居群遗传多样性提供科学依据。

In this study,eight natural populations of wild Scabiosa tschiliensis Grun.in Inner Mongolia Autonomous Region were used as test materials to extract total DNA detection,ISSR primer screening and primer annealing temperature screening.According to the two factors influencing the template DNA concentration and primer concentration in the ISSR-PCR reaction of Scabiosa tschiliensis Grun.,the ISSR-PCR reaction system of Scabiosa tschiliensis Grun.was optimized at four levels to determine the optimal reaction level,and finally,the optimal reaction system of ISSR-PCR amplification of Scabiosa tschiliensis Grun.was established.In the experiment,eight ISSR primers with good polymorphisms and their annealing temperatures were screened out.The average ISSR primers amplified 12.3 bands,and the polymorphic bands accounted for 93.88%;The optimum ISSR-PCR system for Scabiosa tschiliensis Grun.is as follows:The total volume was 20μL,DNA template was 1μL,DNA template concentration is 60 ng/μL,primer concentration was 0.6μL,primer concentration was 0.8μmol/L,ExTaq DNA polymerase was 10μL,ddH2O was 8.4μL.The amplification procedure was pre-denaturation at 94℃for 5 min;denaturation at 94℃for 0.5 min,annealing at different annealing temperatures for 1 min,extension at 72℃for 1.5 min,35 cycles;72℃after the end of the cycle,extension for 7 min;preservation at 4℃.This study provides a theoretical basis for further study on genetic diversity of Scabiosa tschiliensis Grun..