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Bulletin of Botanical Research ›› 2013, Vol. 33 ›› Issue (2): 238-242.doi: 10.7525/j.issn.1673-5102.2013.02.020

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Establishment and Optimization of ISSR-PCR Reaction System for Brassica campestris L. var. rosularis

SONG Jiang-Hua;ZHAO Ying;WANG Cheng-Gang*;ZHANG Ling-Feng;ZHANG Hui   

  1. College of Horticulture,Anhui Agricultural University,Hefei 230036
  • Received:1900-01-01 Revised:1900-01-01 Online:2013-03-20 Published:2013-03-20
  • Contact: WANG Cheng-Gang
  • Supported by:

Abstract: For optimizing ISSR-PCR reaction system of Brassica campestris L. var. rosularis, single factor gradient and orthogonal design experiments were conducted. The main factors affecting ISSR-PCR amplification i.e. suitable concentration of primer, dNTP, Mg2+ and Taq DNA polymerase were studied. Furthermore, the annealing temperature and cycling numbers were optimized on the base of the above tests. An ideally ISSR-PCR reaction system was established, namely 20 μL reaction system containing template DNA 30 ng, 0.50 μmol·L-1 primer, 0.25 mmol·L-1 dNTP, 1 mmol·L-1 Mg2+ and Taq DNA polymerase 1.0 U. The optimal PCR amplification program was:3 min at 94℃ for predenaturation, followed by 35 cycles of 30 sec at 94℃ for denaturation, 1 min at 50℃ for annealing, 90 sec at 72℃ for extension, finally extension at 72℃ for 7 min and holding the samples at 4℃. This optimized ISSR-PCR reaction system would provide the basis for the analysis of germplasm classification and identification in B.campestris.

Key words: Brassica campestris L. var. rosularis, ISSR-PCR, reaction system, orthogonal design

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