In this study, orthogonal design was used to optimize ISSR amplification system on Brassica campestris ssp. chinensis in four factors (Mg~(2+), dNTP, primers, glycerine) at three levels respectively. A suitable ISSR reaction system was established, namely 20 μL reaction system containing 1×PCR buffer, 1.5 mmol·L~(-1) MgCl_2, 200 μmol·L~(-1) dNTP, 0.5 μmol·L~(-1) primer, 1% glycerine, 30 ng DNA template and 1U TaqDNA polymerase. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient...
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