【摘要】 22个虉草基因组DNA为ISSR-PCR扩增模板,采用单因素试验方法,对影响PCR扩增体系中dNTP、引物浓度、Taq酶和模板DNA用量4个因素及引物退火温度进行梯度试验,优化得到最佳的ISSR-PCR反应体系,即20μL反应体系中分别加入0.3μL Taq DNA聚合酶(5U/μL),2μL 10×PCR Buffer(mg2+plus),1.5μL dNTP(2.5mmol/L),1.5μL引物(10pmol/μL),50ng模板DNA,ddH2O补足体积。以此体系对24条引物进行筛选,最终获得了多态性高,重复性好的引物12条。引物UBC808、809、811、815、818、820、826的适宜退火温度为55℃,引物835,841和842的适宜退火温度为56℃,而引物810和834的适且退火温度分别为52℃和54℃。12条引物共扩增总条带数192条,其中,多态性条带数173条,多态位点百分率89.81%。更多还原

【Abstract】 Genomic DNA extracted from 22 Phalaris arundinacea materials was used as template for ISSRPCR to study the impact of dNTPs concentration,template DNA,primer and Taqpolymerase in PCR system by using a single factor experiment method in order to establish the optimized ISSR-PCR reaction system.The results showed that the best reaction system was 2μL 10×PCR Buffer（mg2+plus）,1.5μL dNTPs（2.5mmol/L）,1.5μL primer（10pmol/μL）,0.3μL Taqpolymerase（5 U/μL）,50 ng template,and ddH₂O to 20μL at last.Base on this PCR system,24 primers were selected,in which,12 primers showed high polymorphism and repeatability.The suitable annealing temperature of primer 808,809,811,815,818,820,826 was 55℃,and it was 56℃for primer 835,841 and 842,52℃and 54℃for primer 810 and 834respectively.A total of 192 bands were amplified from 12 ISSR primers,in which,173 were polymorphic loci,the average percentage of polymorphic band was 90.62%.The reaction system would provide the basis for the diversity analysis of Phalaris arundinacea with ISSR markers.更多还原