Assessment Of Candidate DNA Barcodes For Chinese Medicinal Plants From Genus Rhododendron

DNA barcoding is a new biological tool for accurately and objectively identifying species by using short and standard DNA regions that can be amplified easily by PCR. One of the problems in plant DNA barcode reseaches was that the previous studies were mainly carried out on a large scale with only a few species within one genus and rarely on some specific genus with much sister species, which result in a superficially high rate of discrimination and low credibility of the reference database. Rhododendron is a group of famous landscape plants with high medicinal and ornamental value. However, there is no simple and universal way to discriminate various species of this group. DNA barcoding technique is a new biological tool that can accurately and objectively identify species by using short and standard DNA regions. The CBOL PWG established the use of matK+ rbcL as core barcodes and ITS2 as one of the supplementary loci for differentiating plants at the Third International Barcoding Conference. To choose a suitable DNA marker to authenticate Rhododendron species, four candidate DNA barcodes (rbcL, matK, psbA-trnH, ITS2 intergenic spacer) were tested on sixty-eight samples of thirty-eight species. We found that the psbA-trnH candidate barcode yielded 86.8% sequencing efficiency. The highest interspecific divergence was provided by the psbA-trnH intergenic spacer based on six parameters and Wilcoxon signed rank tests. Although there was not a clear barcoding gap, Wilcoxon two sample tests indicated that the interspecific divergence of the psbA-trnH intergenic spacer was significantly higher than the relevant intraspecific variation. The psbA-trnH DNA barcode possessed the highest species identification efficiency at 100% by BLAST1 method, followed by rbcL only 59%. The present results showed that psbA-trnH intergenic spacer was the most promising one of the four markers for barcoding Rhododendron species. To further evaluate the ability of the psbA-trnH marker to discriminate closely related species, the samples were expanded to ninety-four samples of fifty-three species in the genus and the rate of successful identification was 93.6%. Among the unidentifiable individuals of three pair samples, every pair comes from a same subgenus or section with complicated background in taxa and this shows that the psbA-trnH sequence could narrow the sample to a small taxa area and reveal the existing of complicated group in taxa.In conclusion, the psbA-trnH intergenic region is a valuable DNA marker for identifying Rhododendron species.Since the genus was established by Linnaeus, various methods have been constantly used to revise its classification system on the basis of morphology, cytology, chemotaxonomy and molecular taxonomy. However, the Rhododendron genus still has some problems at various systematic classification levels; there is no simple and universal way to discriminate various species within the genus. Phylogenetic relationships within the genus Rhododendron were studied using psbA-trnff intergenic spacer seqences. In the analysis, thirty-five Rhododendron species, which belong to seven subgenus according to taxonomy system of Sleumer in "Flora of China" were analyzed. The results indicated that:all species are grouped into seven clades and all samples come from every same subgenus formed into a monophyletic clade with higher bootstrap value which supported the taxonomy system of Sleumer in "Flora of China". Pseudorhodorastrum as an independent subgenus was supported. The opinion of Chamberlian proposed the subg. Pseudazalea should be placed in subg. Rhododendron as a subsection Trichoclada was not supported by the reconstructed phylogenetic tree.The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. To discriminate ten medicinal plants in Rhododendron us ing ITS2 sequence, twenty-three sequences were analyzed by means of CLUSTRAL X and MEGA softwares and the phylogenetic tree was constructed using UPGMA and in the cluster dendrogram ten species of Rhododendron showed monophyletic. The result showed that ITS2 sequence can be used to correctly identify ten medicinal plants in Rhododendron, and it has certain application value in the identification of traditional Chinese medicine in Rhododendron.