Development of Specific-Barcode from Chloroplast Genome and Its Application in Metabarcoding Molecular Identification of Chinese Patent Medicines Berberidis Radix

Chloroplast genomes have been widely considered as a valuable resource for phylogenetic reconstruction and molecular marker development in plant species. In this study, for the first time, we systematically compared the complete chloroplast genome sequences of the six <em>Berberis</em> species (<em>B. julianae</em>, <em>B. tsienii</em>, <em>B. pruinose</em>, <em>B. thunbergii</em>, <em>B. poiretii</em> and <em>B. wilsoniae</em>), which commonly were used as medicinal herb Berberidis Radix. The <em>ndhD-ccsA</em> as highly divergent region was found and taken as a potential marker for species identification. Combined with DNA metabarcoding technology, the barcode was applied to the Chinese patent medicine containing Berberidis Radix (Sankezhen). The results showed that the six complete chloroplast genomes exhibited a typical quadripartite structure which ranging from 165,934 to 168,828 bp in length. A total of 147 unique genes were identified in each chloroplast genome, comprising 101 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Comparative genome analysis demonstrated that the six chloroplast genomes were highly conserved in genome size, GC contents, and gene organization. The phylogenetic relationships of six <em>Berberis</em> plants were revealed and the results showed that <em>Mahonia</em> was supported as separate clade in the <em>Berberis</em> genera tree, which was coincident with previous studies. The nucleotide diversity analysis revealed seven variable loci in protein coding regions, and four variable loci in gene spacer regions, respectively. The primer pair 1508F-1864R on <em>ndhD-ccsA </em>region was proven to precisely discriminate the six studied <em>Berberis</em> species and recovered the biodiversity of <em>Berberis</em> species in Chinese patent medicines. In general, this study provides meaningful genetic information for <em>Berberis</em> plants, and establishes a method to realize the identification of Berberidis Radix as multi-origin Chinese medicinal materials, which can be applied to Chinese patent medicines containing Berberidis Radix.