%0 Journal Article %A SONG Jiang-Hua %A WANG Cheng-Gang* %A ZHANG Hui %A ZHANG Ling-Feng %A ZHAO Ying %T Establishment and Optimization of ISSR-PCR Reaction System for Brassica campestris L. var. rosularis %D 2013 %R 10.7525/j.issn.1673-5102.2013.02.020 %J Bulletin of Botanical Research %P 238-242 %V 33 %N 2 %X 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. %U https://bbr.nefu.edu.cn/EN/10.7525/j.issn.1673-5102.2013.02.020