Abstract

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Title:: Establishment and Optimization of ISSR-PCR Reaction System for Meconopsis integrifolia

Author(s):: ZHAO Wanyue; LENG Qiusi; XIONG Jian; OU Zhi; QU Yan; (School of Landscape Architecture,Southwest Forestry University,Kunming,Yunnan 650224)

Keywords:: Meconopsis integrifolia; ISSR; orthogonal design; system establishment and optimization

Abstract:: The M.integrifolia was used as template in this study.And the effects of five PCR reaction components including Mg2+,dNTPs,primers,Taq DNA polymerase,and DNA on the reaction system were studied by using the orthogonal design.The purpose was establishment and optimization of ISSR-PCR reaction system for M.integrifolia,and screened 100 ISSR primers published by UBC with this system.The results showed that the 50 μL reaction system,including 3 mmol·L-1 Mg2+,2 mmol·L-1 dNTPs,0.4 mmol·L-1 primers,1.25 U Taq DNA polymerase,60 ng DNA.And the suitable annealing temperature,extension time and cycle times were determined by gradient PCR.A total of 16 ISSR primers were screened by the system,and 10 individual materials were tested for amplification,which obtained good amplification results.This system could provide some experimental basis for the future study on germplasm identification and genetic diversity analysis of M.Vig.by using ISSR marker.