鉴定中药的“真伪优劣”是确保中药质量的关键所在，而其中“真伪”更是中药临床用药安全的重要前提，特别是针对贵重药材以及有毒中药而言。DNA条形码作为一项快速准确鉴定物种的方法在药用植物基原以及药材鉴定中的应用已非常广泛。通过构建标准的DNA条形码参考数据库是该技术应用的主要形式。尽管不同学者提出了诸多用于植物鉴定的DNA条形码，但由于片段长度的限制，单一序列或多序列组合的条形码在近缘种之间的鉴定仍有很大的局限性。植物叶绿体基因组作为用于筛选DNA条形码序列的研究热点，其本身也可作为超级条形码（Super barcode）用于系统进化、亲缘关系以及物种鉴定研究。本研究围绕DNA条形码鉴定技术，首先以《日本药局方》（以下简称“药局方”）中收载的生药材为对象，构建了药局方生药DNA条形码分子鉴定系统；其次，选取人参属药用植物作为名贵药材代表、乌头属药用植物作为有毒中药代表，分别对该技术在这两个属中的应用进行了研究；最后，利用高通量测序技术测定了乌头属叶绿体基因组，为叶绿体基因组作为超级条形码奠定基础，为筛选适合该属的高变异分子标记提供依据。本研究主要内容及结论如下：

1、建立了一套以ITS2序列为主psbA-trnH序列为辅的汉方生药材标准DNA条形码数据鉴定系统。该系统为用户提供了汉方草药信息查询、DNA条形码物种鉴定以及从样品采集到数据分析的标准操作流程三项主要功能。共搜集来自日本及中国不同地区的基原植物和生药材样本共计576份，从中获取的标准序列覆盖了97.3%的药局方（十六版）收载生药品种。另外搜集了100份待测样本，用来检验数据库的鉴定效果。通过BLAST结果显示，100份样品中有71份样品获得的结果与标签上所标注的物种一致，即准确鉴定到物种水平。其余29份样品鉴定到属水平。本课题建立了全世界首个国家级药品法典的DNA条形码分子鉴定在线平台，为全世界其它如中、美、韩等国家的药典提供示范作用。

2、采用ITS2序列对人参属药用植物的SNP位点（single nucleotide polymorphism）进行研究，发现五个稳定的可作为独特标记用来区分人参属不同物种以及人参西洋参混合粉末的种间变异位点。同时，针对人参属ITS2序列的基因组多拷贝进行变异分析，发现ITS2序列在基因组内变异较小时，其在该种的种内变异也较小，反之亦然。再者，使用三个常用DNA条形码（ITS2、psbA-trnH和matK），共计377条序列，对人参属药用植物的种内变异情况进行了分析，发现这三条序列在人参、西洋参和三七中的变异较低，呈现出高度保守的趋势，而在竹节参中的变异相对较大。本课题首次借助SNP位点鉴定人参、西洋参混合粉末，实现了基于DNA条形码方法的人参属珍稀濒危物种及名贵药材的快速准确鉴定。

3、对乌头属19个物种的52份样品的ITS、ITS2、psbA-trnH以及matK序列进行扩增和测序，比较候选序列的扩增及测序成功率、种内种间遗传距离、barcoding gap分析，构建系统发育树用以评估它们在乌头属物种中的鉴定能力。结果显示，ITS和ITS2序列在扩增及测序成功率上表现最优，均为100%。psbA-trnH序列次之，matK由于长度较长且含有poly结构，致其扩增和测序成功率较低。除此以外，ITS及ITS2序列在遗传距离、barcoding gap分析上都较其余两个候选序列有明显优势。通过BLAST方法显示ITS序列在该属的鉴定成功率最高(57.9%)，构建的系统发育树也显示基于ITS序列的乌头属物种呈现的单系性比例最高（57.9%）。综合上述结果，针对本研究所涉及的乌头属物种，ITS序列的表现最为突出，更适合用于该属物种的鉴定。

4、利用高通量测序技术测定了乌头属北乌头（Aconitum kusnezoffii）、牛扁（Aconitum barbatum var. puberulum）以及露蕊乌头（Aconitum gymnandrum）的叶绿体基因组，共含有130个独特的基因，其中包含84个蛋白编码基因，38个tRNA和8个rRNA。通过对它们的比较分析发现大单拷贝区和小单拷贝区的变异大于两个反向重复区。本研究首次测定了植物界中毛茛科乌头属的叶绿体基因组，为其在乌头属系统进化以及物种鉴定等领域的研究提供数据支持。

5、利用ClustalW2中的多序列比对功能对乌头属叶绿体基因组的基因以及基因间隔区进行了相似度比较，并从中筛选出了十个变异程度较高的基因间隔区。将筛选出的十个高变异基因间隔区按照统一的顺序进行首尾连接，使其作为一条长度在6 kb左右的长片段分子标记用于乌头属近缘物种鉴定。本研究利用长片段串联序列作为乌头属潜在高变异分子标记，为同属近缘物种分子鉴定提供崭新的研究思路。

The authenticity of the traditional Chinese medicine is the key point of its quality control, as well as the precondition of the clinical medication safety. Especially for the rare and toxic species, authentication between the official species and their adulterants becomes very important. DNA barcoding is a rapid and accurate identification method, which has been widely applied in the authentication of both original plants and crude drugs of the traditional Chinese medicine. Although many different loci have been proposed for plant species identification, none of them could work across all species, especially among closely related ones. In recent years, chloroplast genomes have been advocated as the “super barcode” in the study of phylogenetic analysis, genetic relationships and species identification. Building standard DNA barcoding reference databases is the main form of its application. In this study, we concentrated on the DNA barcoding technic. Firstly, a DNA barcode identification system was set up for the crude drugs in the Japanese pharmacopoeia; secondly, the species of Panax and Aconitum were selected as the representative of the rare and toxic medicines respectively to study the DNA barcoding application; finally, three chloroplast genomes of the Aconitum were sequenced using the high throughput sequencing platforms in order to lay the foundation of super barcode utilization, as well as to discover highly variable loci as novel molecular markers for the Aconitum. In this study, main contents and conclusions include:

1. A standard barcode database based on the ITS2 region and the psbA-trnH region for Kampo crude materials was developed. This tool provides users with the basic information on each crude drug recorded in the Japanese pharmacopoeia, herbal material DNA barcoding identification, and the standard operating procedure (SOP) from sampling to data analysis. A total of 576 samples were collected to establish the database. An additional 100 samples were identified using the database. 71% of test samples were identified at species level as same as the names listed on their label. The remaining 29% were correctly identified at genus level. We report the first database that includes the standard barcode sequences from 97.3% of the crude drugs recorded in the JP (16^th edition), which has a beneficial demonstration effect throughout the other pharmacopoeias.

2. SNPs were detected in ITS2 region from the four Panax species and intra genomic variations were studied. Intraspecific variations were analyzed based on three typical DNA barcodes (ITS2, matK and psbA-trnH). Five stable SNPs were detected in ITS2 region to identify the Panax medicinal species. Results from this study revealed that among the four medicinal plants, greater intragenomic divergence is correlated with larger intraspecific variations. Our findings indicate that SNP-based molecular barcodes could be developed as a routine method for the identification of the Panax genus with closely related species as well as the mixed powder of P. ginseng and P. quinquefolius.

3. Four popular DNA barcodes (ITS, ITS2, matK and psbA-trnH) were selected to test their ability to identify the Aconitum on aspects of successful rates of amplification and sequencing, genetic distances among inter- and intra-species, barcoding gap, and phylogenetic trees. After general analysis, ITS was superior to other loci with the highest successful identification rate of 57.9% and proposed as the best DNA barcode candidate of the four barcodes for the Aconitum.

4. Chloroplast genomes of Aconitum kusnezoffii, Aconitum barbatum var. puberulum and Aconitum gymnandrum were sequenced using Illumina MiSeq and PacBio RS sequencing platforms. There were 130 unique genes in the chloroplast genomes including 84 protein-coding genes, 38 tRNA and eight rRNA. Genome comparison indicated that the large single copy region and the small single copy region were much more variable than the inverted repeat regions. The sequencing of the chloroplast genomes laid the foundation of super barcode utilization in the Aconitum.

5. Ten highly variable intergenic regions were discovered from the three chloroplast genomes based on the multiple sequence alignment of ClustalW2 with the aim to developing unique molecular markers of the Aconitum. The ten intergenic regions were jointed in a certain order to get a highly variable marker with the length of 6kb approximately, which was considered to be a potential molecular marker for the Aconitum identification. This study gave a novel idea on authentication of closely related species.