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Bulletin of Botanical Research ›› 2008, Vol. 28 ›› Issue (4): 402-407.doi: 10.7525/j.issn.1673-5102.2008.04.005

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Optimization for ISSR-PCR System of Freesia refracta Klatt Through Orthogonal Design

ZHOU Ling-Yu;WU Chen-Wei;TANG Dong-Qin;SONG Hui-Shu;LIU Qun-Lu*   

  1. (School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240)
  • Received:1900-01-01 Revised:1900-01-01 Online:2008-07-20 Published:2008-07-20
  • Contact: LIU Qun-Lu
  • Supported by:


The effects of various factors on ISSR-PCR reaction, such as concentration of Mg2+, dNTPs and primers, DNA template and Taq DNA polymerase, were investigated to optimize the ISSR-PCR reaction system of Freesia refracta through orthogonal tests. The optimized ISSR-PCR system of F. refracta incloded 1×Taq DNA polymerase buffer (10 mmol·L-1 KCl, 8 mmol·L-1(NH4)2SO4, 10 mmol·L-1 Tris·HCl, pH 9.0, 0.05% NP-40), 2 mmol·L-1 MgCl2, 0.06 U·μL-1 Taq DNA polymerase, 0.4 μmol·L-1 primer, 4.0 ng·μL-1 DNA template, 0.6 mmol·L-1 dNTPs in 25 μL PCR reaction system. By temperature gradient PCR, the optimum annealing temperature was determined as 51.5℃. The optimized system laid the groundwork for ISSR molecular analysis of F. refracta.

Key words: ISSR-PCR, Freesia refracta Klatt, optimization, orthogonal design

CLC Number: