Abstract:

In order to establish the optimized RAPD-PCR experiment system for Mallotus oblongifolius (Miq.) Muell.-Arg., the single factor experiments were at first used to detect the suitable concentration ranges of the different influential factors, and then the orthogonal design was used to optimize RAPD-PCR amplification system of M. oblongifolius in 5 factors (Mg2+, dNTPs, primer, Taq polymerase, DNA template) at 4 levels, respectively. And then the data were analyzed by Soft Ware SAS. A optimal RAPD reaction system was finally established. Namely, 2.5 μL 10×PCR buffer, 2.5 mmol Mg2+, 0.2 mmol dNTPs, 1.5 U Taq polymerase, 0.48 μmol/L primers, and 80 ng DNA template were contained in 25 μL reaction solution. The PCR amplification program was that pre-denaturing at 94℃ for 4 min, then denaturing at 94℃ for 30 s, annealing at 38℃ for 45 s, extension at 72℃ for 120 s, for 45 cycles, at last extension at 72℃ for 10 min. The productions were stored at 16℃. The optimized RAPD-PCR reaction system was suitable for the study of relationship and genetic diversity of M. oblongifoliu in the different populations.