journal6 ›› 2014, Vol. 35 ›› Issue (2): 73-78.DOI: 10.3969/j.issn.1007-2985.2014.02.017

• Biological resources • Previous Articles     Next Articles

Establishment and Optimization of ISSR Reaction System for Ficus Pumila L. var. Awkeotsang

 SHI  Hui, HUANG  Qi-Mei, YE  Mei-Na, RUAN  Shao-Jiang, CHEN  Yong   

  1. (1.Department of Biology,Ningde Normal University,Ningde Fujian 352100,China;2.Fujian Provinces University Project Research Center of Biological Resources with Mingdong’s Unique Feature,Ningde 352100,Fujiang China)
  • Online:2014-03-25 Published:2014-05-09

Abstract: The factors affecting the ISSR-PCR amplification on Ficus pumila L. var. awkeotsang such as dosage of template DNA,Taq DNA polymerase,dNTPs,primers and extension time and cycle times were optimized through the combination of single factor and orthogonal design experiment.The results showed that the optimal reaction system for ISSR-PCR of Ficus pumila were as follows:In the 20 μL reaction volume,1ng template DNA,1.0U Taq DNA polymerase,0.4 μmol/L primers,0.18 mmol/L dNTPs.The optimal PCR amplification process was:4 minutes at 94 ℃ for predenaturation,then followed by 35 cycles,each with 45 seconds at 94 ℃ for denaturation,45 seconds at 53 ℃ for annealing,2 at 72 ℃ for extension,finally extension at 72 ℃ for 7 minutes and holding the samples at 4 ℃.The system was applied in the amplification of 14 varieties of indicated the suitability and stability of the system.

Key words: Ficus pumila L. var. awkeotsang, inter-simple sequence repeat(ISSR), reaction system, orthogonal design

WeChat e-book chaoxing Mobile QQ