Biotechnology Bulletin ›› 2014, Vol. 0 ›› Issue (8): 65-69.

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Optimization of ISSR-PCR Reaction System of Anise（Illicium verum Hook. f.）Based on Orthogonal Design

Feng Dan1,2, Ning Delu1, Chen Shaoyu1,2, Li Yongjie1, Zhang Yanli1, Wu Tao1,2, Chen Haiyun1   

  1. 1. Yunnan Academy of Forestry, Kunming 650201;
    2. Yunnan Provincial Key Laboratory of Cultivation and Exploitation of Forest Plants, Yunnan Laboratory for Conservation of Rare, Endangered & Endemic Forest Plants, Public Key Laboratory of the State Forestry Administration, Kunming 650201
  • Revised:2013-11-15 Online:2014-08-15 Published:2014-08-01
  • Contact: 通讯作者：陈海云，女，副研究员，研究方向：经济林良种选育及栽培；E-mail：kmchenhy@163.com
    吴涛，男，博士，助理研究员，研究方向：林木遗传育种；E-mail：ynafwt@126.com
  • Supported by:

    云南省“十一五”科技攻关项目(2006NG26);云南省自然科学基金项目(2010ZC234)

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Abstract:

Based on orthogonal design experiments, five main factors in ISSR-PCR amplification system, including Mg2+ concentration, Taq DNA polymerase, DNA template, primer（UBC 868）and dNTPs, were studied to optimize the system for Illicium verum Hook. f.. The results showed that the optimized system was a total volume of 25 μL containing 3 mmol/L Mg2+, 0.5 U Taq DNA polymerase, 0.016 ng DNA template, 0.8 μmol/L primer and 0.2 mmol/L dNTPs. The optimal amplified procedure was started with predegeneration at 94℃ for 5 minutes, followed by 40 cycles of 30 seconds for denaturalization at 94℃, 45 seconds of annealing at 46℃, 60 seconds of extension at 72℃；7 minutes of extension at 72℃ and indefinitely remain at 4℃.

Key words: Illicium verum Hook. f., ISSR-PCR, Orthogonal design, Reaction system