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《Journal of Anhui Agricultural University》 2012-03
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Establishment and optimization of an ISSR reaction system for Polygonatum cyrtonema Hua using orthogonal design

ZHANG Hong-mei1,SHAO Yuan-hua1,LONG Tian-tian1,XIANG Yan1,WANG Jin2,TANG Feng2(1.School of Forestry and Landscape Agriculture,Anhui Agricultural University,Hefei 230036; 2.International Centre for Bamboo and Rattan,Beijing 100102)  
In this paper,the orthogonal design of five factors(Taq DNA polymerase,buffer(Mg2+),DNA tem-plate,dNTP,and primer) in four levels was used to optimize the ISSR-PCR amplification system on Polygonatum sibiricum.Finally,a satisfactory ISSR(Inter Simple Sequence Repeat) reaction system for Polygonatum sibiricum with desirable repeatability and polymorphic bands was established.It contained 1.0 U Taq DNA polymerase,3.5 mmol?L-1 buffer(Mg2+),80 ng DNA template,0.08 mmol?L-1 dNTP and 0.16 μmol?L-1 primer respectively in a 25 μL ISSR reaction system.The optimal annealing temperature of every prime for ISSR-PCR reaction was proposed by gradient PCR with established ISSR reaction,the optimal annealing temperature was 54.8℃ for primer UBC841,and different primers had different annealing temperatures.This optimized ISSR reaction system would provide the basis for the analysis of classification,map construction and gene localization of Polygonatum sibiricum.
【Fund】: 国家林业公益性行业科研专项(201004055)资助
【CateGory Index】: S567.239
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