正交设计对‘红阳’猕猴桃ISSR反应体系的优化研究  

Establishment and Optimization of the ISSR Reaction System for 'Hongyang' Kiwifruit

作　　者：邱利娜[1] 廖明安[1] 李明章[2] 王丽华[2] 郑晓琴[2] 

机构地区：[1]四川农业大学园艺学院,四川雅安625014 [2]四川省自然资源科学研究院,四川成都610015

出　　处：《北方园艺》2010年第16期125-128,共4页Northern Horticulture

基　　金：国际科技合作资助项目(2009DFA30870);四川省"十一五"攻关资助项目(04FB013-016)

摘　　要：以‘红阳’猕猴桃及其杂交F1代为试材,通过正交设计对ISSR-PCR扩增反应的影响因子进行优化,初步确立了适合‘红阳’猕猴桃ISSR-PCR反应体系：总体积20μL,10×PCR buffer2.0μL,Mg2＋0.75 mmol/L,Taq酶1.5 U,模板DNA 60 ng,引物1.25μmol/L,dNTPs 0.15mmol/L。扩增程序：94℃预变性7 min,94℃变性30 s,50-58℃退火45 s,72℃延伸2 min,45个循环,72℃延伸7 min。引物UBC841的最佳退火温度为58.5℃。应用该优化反应体系,用UBC835对35份杂交F1代DNA进行ISSR-PCR扩增,结果显示优化后的反应体系具有较高的稳定性。The orthogonal design was used to optimize ISSR-PCR amplification system on ＇hongyang＇kiwifruit,in five level of five factors which were Mg2＋,Taq DNA polymerase,primer,dNTPs,DNA template respectively.The results showed that the 20 μL reaction system consisted of 10×PCR buffer 2.0 μL,1.5 U Taq DNA polymerase,60 ng DNA template,1.25 μmol/L primer,0.15 mmol/L dNTPs and 0.75 mmol/L Mg2＋.The optimal PCR amplification process was as following：1 cycle initial denaturalization at 94℃ for 7 min,followed by 45cycles,which included denaturalization at 94℃ for 30 s,annealing for 45 s,and extension at 72℃ for 2 min,and then extension at 72℃ for 7 min,and finally holding the samples at 4℃ for ever.The reaction condition of ISSR experiment of kiwifruit was discussed,which was a basis for study on genetic diversity among kiwifruit populations.58.5℃ was the most suitable annealing temperature of UBC841 primer 35 F1 individuals of ＇RedSun＇ kiwifruit were used to tested the stabilization of the optimized reaction system.The results showed that the optimized reaction system was stable.

关 键 词：'红阳’猕猴桃 正交设计 ISSR 反应体系 

分 类 号：S663.4[农业科学—果树学]