Data_Sheet_1_Development of Mini-Barcode Based on Chloroplast Genome and Its Application in Metabarcoding Molecular Identification of Chinese Medicinal Material Radix Paeoniae Rubra (Chishao).docx

Radix Paeoniae Rubra (Chishao), a typical multi-origin Chinese medicinal material, originates from the dried roots of Paeonia lactiflora or P. veitchii. The previous study suggested that these two commonly used Chishao showed variation in their chemical compositions and clinical efficacies. Therefore, accurate identification of different Chishao species was of great significance for the guide of clinical medication, and timely treatment of patients. In this study, the chloroplast genome sequences of P. lactiflora and P. veitchii were obtained by next-generation sequencing (NGS) technology, and then the hypervariable regions were selected to design two mini-barcode candidates for species identification. Combined with DNA metabarcoding technology, we performed qualitative and quantitative analysis on the artificially mixed samples of P. lactiflora and P. veitchii and evaluated the identification ability of these mini-barcode candidates. Furtherly, the mini-barcode with good performance was applied to distinguish the Chinese patent medicine “cerebral thrombosis tablets” containing Chishao. The results indicated that the chloroplast genomes of P. lactiflora and P. veitchii were 152,750 and 152,527 bp, respectively. As published previously, they exhibited a typical quadripartite structure including a large single-copy region (LSC), a small single-copy region (SSC) and a pair of inverted repeat regions (IRs). The nucleotide polymorphism analysis revealed seven variable protein-coding regions as petL, psaI, psbJ, rpl16, ycf1b, psaC, and ndhF, and two mini-barcodes were developed from ycf1b and ndhF respectively. The result suggested that both two mini-barcodes performed well distinguishing P. lactiflora from P. veitchii. Besides, P. lactiflora was the only raw material of Chishao in all collected “cerebral thrombosis tablets” samples. In general, this study has established a method to realize the qualitative and quantitative identification of Chishao as multi-origin Chinese medicinal materials, which can be applied to Chinese patent medicines containing Chishao.

红花丹皮（赤芍），一种典型的多产地中国药用材料，源自芍药属植物白芍（Paeonia lactiflora）或川芍（P. veitchii）的干燥根部。既往研究指出，这两种常用的赤芍在化学成分和临床疗效上存在差异。因此，对不同赤芍品种的准确鉴定对于临床用药指导和患者及时治疗具有重要意义。在本研究中，通过下一代测序（NGS）技术获得了白芍和川芍的叶绿体基因组序列，并选取高变区设计了两款物种鉴定候选迷你条形码。结合DNA条形码技术，我们对人工混合的白芍和川芍样本进行了定性和定量分析，并评估了这些迷你条形码的鉴定能力。进一步地，性能优良的迷你条形码被应用于区分含有赤芍的中药专利药品“脑血栓通片”。结果显示，白芍和川芍的叶绿体基因组长度分别为152,750和152,527碱基对。如前所述，它们表现出典型的四分体结构，包括一个大型单拷贝区（LSC）、一个小型单拷贝区（SSC）和一对反转重复区（IRs）。核苷酸多态性分析揭示了七个可变蛋白编码区域，分别为petL、psaI、psbJ、rpl16、ycf1b、psaC和ndhF，并分别从ycf1b和ndhF中开发了两个迷你条形码。研究结果表明，这两个迷你条形码在区分白芍和川芍方面均表现良好。此外，在收集的所有“脑血栓通片”样品中，白芍是赤芍的唯一原材料。总体而言，本研究建立了一种实现多产地药用材料赤芍的定性和定量鉴定方法，该方法可应用于含有赤芍的中药专利药品。