Comparative Analysis of the Complete Chloroplast Genomes of Six Berberis Species and Development of Specific DNA Barcode and its Application in Chinese Medicinal Material 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.

叶绿体基因组（chloroplast genome）已被广泛视为植物物种系统发育重建与分子标记开发的宝贵资源。本研究首次系统比较了6种常用作药用药材三颗针根（Berberidis Radix）的小檗属（Berberis）植物完整叶绿体基因组序列，分别为B. julianae、B. tsienii、B. pruinose、B. thunbergii、B. poiretii及B. wilsoniae。研究发现高度变异的ndhD-ccsA区域，可作为物种鉴定的潜在分子标记。结合DNA宏条形码（DNA metabarcoding）技术，该标记条形码被应用于含三颗针根的中成药‘三颗针（Sankezhen）’中。结果显示，6条完整叶绿体基因组均呈现典型的四分体结构，基因组长度介于165,934 bp至168,828 bp之间。每个叶绿体基因组中共鉴定出147个独特基因，其中包括101个蛋白编码基因、38个转运RNA（tRNA）基因以及8个核糖体RNA（rRNA）基因。比较基因组分析结果表明，6个叶绿体基因组在基因组大小、GC含量以及基因组织结构方面均高度保守。本研究解析了6种小檗属植物的系统发育关系，结果显示十大功劳属（Mahonia）在小檗属系统发育树中被支持为独立进化支，该结论与既往研究结果一致。核苷酸多样性分析分别在蛋白编码区鉴定出7个变异位点，在基因间隔区鉴定出4个变异位点。针对ndhD-ccsA区域设计的引物对1508F-1864R经证实可精准区分6种供试小檗属物种，且可检出中成药中小檗属物种的生物多样性。总体而言，本研究为小檗属植物提供了极具价值的遗传信息，并建立了一套可实现多基原中药材三颗针根鉴定的方法，该方法可应用于含三颗针根的中成药质量鉴定与管控。