Abstract

To estimate genetic diversity and to authenticate the three endangered and official genuine species, Rheum officinale, Rheum palmatum and Rheum tanguticum, of herbal medicine of rhubarb, the optimization of DNA isolation methods including modified CTAB and isolation kit, PCR system of inter-simple sequence repeats (ISSRs) and primers screening were investigated in the present work. Modified CTAB was a preferable choice compared to isolation kit, ISSR protocol was optimized based on the use of the concentration of MgCl₂(1.5 mM), lower concentrations of primer (0.4 μM), dNTPs (0.25 mM), Taq DNA polymerase (1.0 U) and 50 ng of template DNA, resulted optimal amplification. Reproducible amplifiable products were observed in all PCR reactions. According to this PCR system, sixteen out of one hundred primers were chosen for their high clarity and repetition. Thus, the results indicated that the optimized protocol for DNA isolation and PCR system was amenable to three genuine species of rhubarb which is suitable for further work on genetic diversity analysis. Furthermore, here the suitable DNA isolation protocol for ISSR-PCR analysis can be used to study the genetic variation in the future in Rheum grown in China.

Key words: Rhubarb, DNA isolation, ISSR-PCR condition, primer screening.