Inter-simple sequence repeats (ISSR) molecular fingerprinting markers for authenticating the genuine species of rhubarb

Abstract

Rhubarb is prescribed as the roots and rhizomes of Rheum officinale Baill., Rheum palmatum L., and Rheum tanguticum Maxim. ex Balf. in Chinese Pharmacopoeia. These three species are difficult to discriminate due to the morphological and anatomical similarity of the aerial parts and herbal medicines. In the present paper, inter-simple sequence repeats (ISSR) molecular fingerprinting markers have been employed to authenticate three genuine species of rhubarb using 15 primers to discriminate R. officinale, R. palmatum, andR. tanguticum. A total of 155 DNA fragments were amplified, of which 132 were polymorphic (85.2% of all bands). Four specific authentication markers have been found to authenticate three species of rhubarb. The UBC807 and UBC811 primers each generated one species-specific fragment that was clearly amplified in R. officinale (400bp from UBC807 and 248bp from UBC811). The application of the UBC807 primer also produced a 520bp DNA fragment from R. palmatum and R. tanguticum, and UBC816 primer generated a 620bp fragment that was specific for R. palmatum and R. officinale. Therefore, three loci combinations from primer UBC816 with any one of the primer UBC807and UBC811, that is UBC816-620bp with UBC807-520bp, UBC816-620bp with UBC807-400bp, and UBC816-620bp with UBC811-248bp, served the purpose of distinguishing three genuine species of rhubarb. To enhance the efficiency of authentication, ISSR fingerprinting codes have been constructed using four polymorphic bands for authenticating three genuine species of rhubarb. The present study could be applied to distinguish the genuine species of rhubarb at the molecular level. Key words: Rhubarb, inter-simple sequence repeats-polymerase chain reaction, molecular marker, authentication.