X Pang, Shilin Chen*. Cladistics, 2011, 27
Comparative analysis of a large dataset indicates that ITS should be incorporated into the core barcode for seed plants China Plant BOL Group, Li Dezhu et al., PNAS, 2011 We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants. (建议ITS/ITS2应成为种子植物的核心 条形码) In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful back-up as it is easier to amplify and sequence this subset of the marker. (ITS难以扩增和测序时， ITS2可以有效地弥补该缺陷)
Identification efficiency for ITS2 and psbA-trnH loci using different methods
Marker Method of species identification (Ross et al. 2008 Syst Biol 57: 216-230) BLAST Distance psbA-trnH BLAST Distance Correct identification Species level % 92.7 90.3 67.6 72.8 Genus level % 99.8 99.7 95.4 96.5 Incorrect identification Species level % 0 0 0 0 Genus level % 0 0 0 0 Ambiguous identification Species level % 7.3 9.7 32.4 27.2 Genus level % 0.2 0.3 4.6 3.5
Identification of Lycium barbarum and its closely related species using ITS2 barcode
Table 1 Analysis of inter/intra-specific divergence in ITS2 barcode of the three Lycium species
首次Байду номын сангаас证 ITS2为药用植物鉴定通用条形码
• 通过研究比较psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2 和 ITS等 七个候选DNA条形码，发现ITS2是最佳条形码序列
• ITS2长度约230bp，易于扩增和测序，物种鉴定能力强，适
合中药材等存在DNA降解材料的鉴定 • 人参、西洋参、羌活、宽叶羌活DNA条形码鉴定方法正在纳 入国家药典
中药DNA条形码鉴定方法体系的建立与推广
陈士林
中国中医科学院中药研究所
中药鉴定领域寻求变革与突破
经典中药鉴定方法难以满足现代需求
鉴定方法缺失导致严重安全性事件 -- 关木通误当木通 广防己误当防己 导致急性肾功能衰竭 （马兜铃酸肾病事件）
--土三七误当三七服d-rank tests of inter-specific divergence among loci.
W+ ITS2 ITS2 ITS2 matK matK rbcL
WmatK rbcL rpoC1 rbcL rpoC1 rpoC1
Relative Ranks, n, P value W+ = 703, W- = 0, n = 37, P ≤1.139 × 10-7 W+ = 703, W- = 0, n = 37, P ≤ 1.137 × 10-7 W+ = 703, W- = 0, n = 37, P ≤ 1.133 × 10-7 W+ = 620, W- = 46, n = 36, P ≤ 6.479 × 10-6 W+ = 655, W- = 11, n = 36, P ≤ 4.174 × 10-7 W+ = 359, W- = 106, n = 30, P ≤ 0.009
木通
中药材天地网
Hoang et al. Science Translational Medicine, 2013, 5: 197ra102. Nortier J L, Martinez M C M, Schmeiser H H, et al. New England Journal of Medicine, 2000, 342(23): 1686-1692.
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PNAS 2009 Science 2009
Three genomes in plants and candidate barcodes
focused on genes and introns of the chloroplast genome
Identification efficiency for ITS2: 92.7% and 99.8% at species and genus level, respectively
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Plant Working Group recommended rbcL + matK as the plant barcode, successfully discriminate at the species level --72%. Far from perfect due to the low identification rate
mean±SD (range) Average interspecific distance Average intraspecific distance Intraspecific distances of L. barbarum 0.0535±0.0114 (0.0269-0.0892) 0.0043±0.0064 (0.0000-0.0844) 0.0038±0.0041 (0.0000-0.0132) 0.0085±0.0133 (0.0000-0.0386) 0.0458±0.0263 (0.0000-0.0844)
DNA条形码鉴定冬虫夏草及其伪品
背景：分布范围狭窄，生态环境脆弱，资源蕴藏量有限（产量每年80-100吨）； 市场产不足销，价格已超黄金 (Guo, 2012) 混伪品泛滥，如古尼虫草，蛹虫草，人造伪品滇紫菀等
样品：冬虫夏草与13种混伪品及近缘种，冬虫夏草采自四川、青海、西藏等主产区 185 条ITS/ITS2序列 (冬虫夏草样品53份，伪品45份，GenBank序列87 条) 结果：基于BLAST/DISTANCE的鉴定效率：100% 基于NJ tree 的鉴定结果：所有的冬虫夏草序列单独聚为一支， 并能与所有的混伪品及近缘种区分开 冬虫夏草及其混伪品/近缘种
A：正品：冬虫夏草 B：伪品： 亚香棒虫草/古尼虫草 C：人造伪品： 滇紫菀 D：伪品 凉山虫草 E：伪品：蝉花 F：伪品：下垂虫草 G：伪品： Cordyceps robertsii（新西兰）
冬虫夏草
混伪品及近缘种
Xiang Li, Chen Shilin et al. FEMS Microbiology Letters, 2013 人造伪品：滇紫菀
Shilin Chen* et al PLoS ONE 2010,5(1):e8613
课题组首次提出并验证ITS2条形码，引起国际广泛关注
该文发表后 3年引文319 次
国际期刊PLoS ONE关注药用植物DNA条形码。该文提出ITS2序列 为药用植物通用的条形码序列，为陆地植物DNA条形码序列筛选提出 了新的视角，由此引起同行的关注和讨论。
中药鉴定技术瓶颈
一万余种中药材物种鉴定 品种混乱 传统四大鉴定方法体系的局限 路在何方？ • • • • 基原鉴定 形态鉴定 显微鉴定 理化鉴定
DNA barcoding can achieve a rapid and accurate identification for herbs
DNA barcoding technology uses a short and standardized gene region to identify species One sequence One species


The ITS2 locus is a favorable universal barcode and possesses a perfect prospect in authenticating species used for a wide set of plant taxa. The psbA-trnH spacer may be a complementary barcode to be used.
Result ITS2 > matK ITS2 > rbcL ITS2 > rpoC1 matK > rbcL matK > rpoC1 rbcL > rpoC1
Relative distribution of inter-specific divergence between congeneric species and intra-specific variation for four loci. The two bars in each box represent inter-specific (above) and intra-specific (below) genetic distances.