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Bulletin of Botanical Research ›› 2013, Vol. 33 ›› Issue (6): 746-751.doi: 10.7525/j.issn.1673-5102.2013.06.018

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Establishment and Optimization of ISSR-PCR Reaction System for Calanthe tsoongiana

QIAN Xin;LIAN Jing-Jing;LIU Fen;NIU Xiao-Ling;WANG Cai-Xia;TIAN Min*   

  1. 1.Research Institute of Subtropical Forestry,Chinese Academy of Forestry,Fuyan 311400;2.South China Botanical Garden,Chinese Academy of science,Guangzhou 510650;3.Administration Bureau of Tianmu Mount National Nature Reserve,Lin’an 311311
  • Received:1900-01-01 Revised:1900-01-01 Online:2013-11-20 Published:2013-11-20
  • Contact: TIAN Min
  • Supported by:

Abstract: Orthogonal design was used to optimize ISSR-PCR amplification system of Calanthe tsoongiana in five factors (Mg2+, dNTP, primer, DNA template and Taq DNA polymerase) at four levels, respectively. The results showed that the suitable ISSR-PCR system was performed in a 25 μL volume containing 3.0 mmol·L-1 Mg2+, 0.3 mmol·L-1 dNTP, 0.4 μmol·L-1 primer, 2.5 ng·μL-1 DNA template, 0.08 U·μL-1 Taq DNA polymerase and 1×PCR buffer. The optimized augmentation procedure was predenaturation at 94℃ for 5 min, followed by 40 cycles of 94℃ for 1 min, annealing at suitable temperature for different primers for 1 min, 72℃ for 1 min, extension at 72℃ for 10 min, and preservation at 12℃. This optimized ISSR-PCR system would play an important role in further research of molecular systematics and genetic diversity in C.tsoongiana.

Key words: Calanthe tsoongiana, ISSR, orthogonal design

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