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《Northern Horticulture》 2010-16
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Establishment and Optimization of the ISSR Reaction System for 'Hongyang' Kiwifruit

QIU Li-na1,LIAO Ming-an1,LI Ming-zhang2,WANG Li-hua2,ZHENG Xiao-qin2(1.College of Horticulture,Sichuan Agricultural University,Ya'an,Sichuan 625014;2.Sichuan Institute of Natural Resources,Chengdu,Sichuan 610015)  
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.
【Fund】: 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)
【CateGory Index】: S663.4
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