Comparative Chloroplast Genome Analysis of Rhubarb Botanical Origins and the Development of Specific Identification Markers

Rhubarb is an important ingredient in traditional Chinese medicine known as Rhei radix et rhizome. However, this common name refers to three different botanical species with different pharmacological effects. To facilitate the genetic identification of these three species for their more precise application in Chinese medicine we here want to provide chloroplast sequences with specific identification sites that are easy to amplify. We therefore sequenced the complete chloroplast genomes of all three species and then screened those for suitable sequences describing the three species. The length of the three chloroplast genomes ranged from 161,053 bp to 161,541 bp, with a total of 131 encoded genes including 31 tRNA, eight rRNA and 92 protein-coding sequences. The simple repeat sequence analysis indicated the differences existed in these species, phylogenetic analyses showed the chloroplast genome can be used as an ultra-barcode to distinguish the three botanical species of rhubarb, the variation of the non-coding regions is higher than that of the protein coding regions, and the variations in single-copy region are higher than that in inverted repeat. Twenty-one specific primer pairs were designed and eight specific identification sites were experimentally confirmed that can be used as special DNA barcodes for the identification of the three species based on the highly variable regions. This study provides a molecular basis for precise medicinal plant selection, and supplies the groundwork for the next investigation of the closely related Rheum species comparing and correctly identification on these important medicinal species.