Identification of Lonicera japonica and its Related Species Using the DNA Barcoding Method

• Abstract
• Abbreviations
• Introduction
• Material and Methods
• Plant materials
• DNA extraction, amplification, and sequencing
• Sequence alignment and data analysis
• Supporting information
• Results
• Discussion
• Acknowledgements
• References

Abstract

To choose a suitable DNA marker to authenticate the botanical origins of Flos Lonicerae Japonicae and Flos Lonicerae, seven candidate DNA bar codes (i.e., rbcL, matK, psbA-trnH, ITS2, ITS, trnL intron, and trnL‐F intergenic spacer) were tested on forty-four samples of Lonicera japonica and its closely related species using the DNA barcoding method. We found that all seven candidate bar codes yielded 100 % PCR amplification efficiency and that the sequencing efficiency of the five other candidate bar codes was 100 %, with the exception of ITS and ITS2. The highest interspecific divergence was provided by the psbA-trnH intergenic spacer, followed by the trnL‐F intergenic spacer based on six parameters and Wilcoxon signed rank tests. Through the inspection of the histograms of the barcoding gap, the distribution of the psbA-trnH intergenic spacer was well separated; and only this candidate DNA bar code possessed the highest species identification efficiency at 100 % by BLAST1 method. In conclusion, using the psbA-trnH intergenic spacer as a DNA bar code is suitable for the identification of the botanical origins of Flos Lonicerae Japonicae and Flos Lonicerae. This study may provide an important example for the authentication of the botanical origin of medicinal herbs listed in the Chinese Pharmacopoeia.

Abbreviations

BLAST: basic local alignment search tool

CBOL: Consortium for the Barcode of Life

CO1: cytochrome c oxidase subunit 1

IMPLAD: Institute of Medicinal Plant Development

ITS: internal transcribed spacer

K2P: Kimura 2-parameter

MEGA: molecular evolutionary genetics analysis

PCR: polymerase chain reaction

PWG: plant working group

RFLP: restriction fragment length polymorphism

TCM: Traditional Chinese Medicine

Introduction

Flos Lonicerae Japonicae, coming from the flower buds of Lonicera japonica Thunb. (Caprifoliaceae) is widely used in traditional Chinese medicines for its heat-clearing, detoxifying, and anti-inflammatory properties [1]. It is known as the “penicillin” of traditional Chinese medicines [2]. Flos Lonicerae, which comes from L. hypoglauca Miq., L. confusa (Sweet) DC., L. macranthoides Hand.-Mazz., and L. fulvotomentosa Hsu et S. C. Cheng, is another medicinal material described in the Chinese Pharmacopoeia [1]. The botanical origins of these two kinds of medicinal materials are closely related species of Lonicera. At present, the source plants of Flos Lonicerae and many other species of Lonicera have been misused as Flos Lonicerae Japonicae in different areas of China.

To ensure the quality and therapeutic effects of Chinese medicines, differentiating the species that serves as medicinal materials for many traditional Chinese medicine products is critical. Previous studies on the identification of L. japonica and its related species have been focused mainly on traditional classification, chemical methods [3], [4], and various genetic molecular markers, such as RFLP and the diagnostic PCR [5], [6]. However, it is usually hard to identify the closely related species of Lonicera accurately based only on traditional classification owing to their similar morphological characteristics and difficulties to obtain intact herbarium specimens. In addition, the morphological features and chemical components are often unreliable and inconsistent because of the influence of the environment, plant growth phase, harvesting time, testing condition, and various artificial factors.

DNA barcoding is a method of species identification and recognition using specific regions of DNA sequence data [7]. It can identify a species consistently, independent of the influence of the environment, growth phase, and morphological diversity. DNA barcoding has been proposed to be a novel and powerful taxonomic tool [8], which not only facilitates the work of professional taxonomists but also makes it possible for non-experts to identify species rapidly, accurately, and efficiently [9], [10].

Related studies on DNA barcoding have been carried out in different groups currently. In animals, a mitochondrial gene cytochrome c oxidase subunit 1 (CO1) is used as a DNA bar code and varies enough to distinguish most animal lineages [11], [12]. In plants, several candidate DNA bar codes or their combinations have been proposed, such as the psbA-trnH intergenic spacer region [13], [14], [15], [16], [17], ITS2 region [18], [19], matK gene [10], [15], rbcL gene [14], [15], trnL intron [20], and others. In particular, the use of the two-locus combination of rbcL + matK as the standard DNA bar code for differentiating plants has been recently recommended by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG). Certain noncoding plastid regions, such as the psbA-trnH intergenic spacer and trnL intron, as well as the internal transcribed spacers of nuclear ribosomal DNA (ITS) were proposed as supplementary loci in individual projects [21].

In this study, seven candidate DNA bar codes (i.e., rbcL, matK, psbA-trnH, ITS2, ITS, trnL intron, and trnL‐F intergenic spacer) were employed in accordance with the principle of population sampling to identify L. japonica and its related species. Our objective is to choose a suitable DNA marker to accurately and efficiently authenticate the botanical origins of Flos Lonicerae Japonicae and Flos Lonicerae.

Material and Methods

Plant materials

Forty-four samples that belong to eight species and one variety of Lonicera, which were used as Flos Lonicerae Japonicae and Flos Lonicerae in Chinese markets, were collected from large geographical areas in China between 2008 and 2009 ([Table 1]). All plant species were identified by Professor Yulin Lin from the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences, and Professor Fengqin Zhou from the Shandong University of Traditional Chinese Medicine (TCM). The voucher samples were deposited in the herbarium of IMPLAD.

DNA extraction, amplification, and sequencing

Genomic DNA was extracted from silica gel-dried leaves or flower buds bought from the markets in accordance with the protocol of the Plant Genomic DNA Kit (Tiangen Biotech Co.). Polymerase chain reaction (PCR) amplifications of the DNA bar code regions of the seven candidates were carried out in a Peltier Thermal Cycler PTC0200 (BioRad) using approximately 30 ng of genomic DNA as a template in a 25 µL reaction mixture (1 × PCR buffer without MgCL₂, 2.0 mM MgCL₂, 0.2 mM of each dNTP, 0.1 µM of each primer [synthesized by Sangon Co.], and 1.0 U of Taq DNA Polymerase [Biocolor BioScience & Technology Co., China]). Primers and reaction conditions used in the present study were obtained from previous studies [10], [13], [18], [22], [23], [24]. The PCR products were run on a 1.0 % agarose gel in 0.5 × TBE buffer and purified using the TIANGel Midi Purification Kit (Tiangen Biotech Co.). The purified PCR products were sequenced in both directions with the primers used for PCR amplification on a 3730XL sequencer (Applied Biosystems).

Sequence alignment and data analysis

Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner V 3.0 (CodonCode Co.). The boundary of the psbA-trnH intergenic spacer was determined according to the annotations of similar sequences in GenBank. The trnL intron and the trnL‐F intergenic spacer were delimited by primers C and D, E and F, respectively [23]. The matK and the rbcL regions were delimited by alignment with known sequences in the databases using the CodonCode Aligner. To perform a phylogenetic study, the sequences of the DNA regions were aligned using Clustal W and the genetic distances were computed using MEGA 4.0 in accordance with the Kimura 2-Parameter (K2P) model [25], [26]. The all interspecific distance, theta prime, and minimum interspecific distance were used to represent interspecific divergence. Meanwhile, the all intraspecific distance, theta, and coalescent depth were calculated to evaluate the intraspecific variation using the K2P model [18], [27], [28]. The distribution of intra versus interspecific variability was compared using DNA barcoding gaps [10], [28]. Wilcoxon signed rank tests and two-sample tests were performed as described previously [10], [14]. The BLAST1 method was used to evaluate the species identification efficiency as described previously [29].

Supporting information

Data on Wilcoxon signed rank tests of interspecific divergence among loci, Wilcoxon signed rank tests of intraspecific divergence among loci, Wilcoxon two-sample tests for distribution of intra- vs. interspecific divergences, and multiple sequence alignment of psbA-trnH intergenic spacer of forty-four samples of L. japonica and its related species are available as Supporting Information.

Results

The PCR amplification efficiency of the seven candidate bar codes was 100 %. Except for the ITS and ITS2 regions, the sequencing efficiency of the other five candidate bar codes was 100 %. ITS2 and ITS were excluded in the further analysis of this study because of the lower success rate of sequencing. The sequence length, GC content, indels, and variable sites of the five regions based on the results of the CodonCode Aligner and Clustal W alignment were presented ([Table 2]). The length range of the psbA-trnH intergenic spacer was the shortest, whereas the number of variable sites and indels was greater than in the other regions. Besides, from the alignment of psbA-trnH intergenic spacer, four specific sites in the sequence of L. japonica were noticeably different from other studied species of Lonicera. Meanwhile, a consistent single nucleotide difference was found between L. japonica and its variety, L. japonica var. chinensis (Wats.) Bak. (Fig. 1S, Supporting Information). All sequences generated in this study were submitted to GenBank ([Table 1]).

We estimated genetic divergence using six parameters ([Table 3]). In the comparisons of interspecific genetic distances of the five candidate bar codes among L. japonica and its related species, the psbA-trnH intergenic spacer exhibited the highest interspecific divergence according to all three parameters (i.e., all interspecific distance; theta prime; and minimum interspecific distance), followed by the trnL-F intergenic spacer. At the intraspecific level, the lowest divergence was found for the rbcL, and the highest belonged to the psbA-trnH intergenic spacer, while there was no noticeable difference in divergences among the trnL intron, the trnL‐F intergenic spacer, and the matK gene. The result also showed that the minimum interspecific divergence of the psbA-trnH intergenic spacer and the trnL‐F intergenic spacer was greatly higher than their corresponding maximal intraspecific divergence (i.e., coalescent depth).

Wilcoxon signed rank tests confirmed that the psbA-trnH intergenic spacer provided the highest interspecific divergence, which was significantly higher than those of other regions. The second highest interspecific divergence belonged to the trnL‐F intergenic spacer, whereas the lowest divergence belonged to the rbcL (Table 1S, Supporting Information). In addition, the psbA-trnH intergenic spacer also had the highest intraspecific divergence; the divergence of matK was the same as that of the trnL‐F intergenic spacer; and the rbcL possessed the lowest variation (Table 2S, Supporting Information).

Bar codes should generally demonstrate a “barcoding gap” between intra- and interspecific distances. However, the overlap of genetic divergence increases when closely related species are increased [28]. Although the inspection of the histogram did not show a clear gap between intraspecific variation and interspecific divergence in the distributions of the five tested loci ([Fig. 1]), it does suggest a clearly defined range, where the intraspecific variation is considerably lower than the distribution of interspecific divergence. Furthermore, among them, the distribution of the psbA-trnH intergenic spacer was well separated. Wilcoxon two-sample tests confirmed that the mean of the interspecific divergences of the five loci (i.e., psbA-trnH intergenic spacer, matK, rbcL, trnL intron, and trnL‐F intergenic spacer) was significantly higher than that of the corresponding intraspecific variations. The most significant difference was observed in psbA-trnH intergenic spacer and matK gene (Table 3S, Supporting Information).

Finally, the BLAST1 method was used to test the applicability of different regions for species identification. The results indicated that only the psbA-trnH intergenic spacer possessed the highest species identification efficiency at 100 %, whereas the rbcL possessed the lowest species identification efficiency at 37.5 %. Moreover, the efficiency of species identification using the combination of matK and rbcL data, which the CBOL PWG recommended as core bar codes, was lower than psbA-trnH alone at 75 % ([Table 2]).

Discussion

The accurate identification of the sources of botanical material is the first step in assuring the quality of herbal medicines. DNA barcoding as a sequence-based method for species identification is rapidly increasing in popularity. The psbA-trnH intergenic spacer is among the most variable regions in the angiosperm chloroplast genome. It is a popular tool for plant population genetics and species level phylogenetics and has been proposed suitable for DNA barcoding studies [13], [30]. Moreover, at the Third International Barcoding Conference, the CBOL proposed that the plastid region psbA-trnH should be considered as the second level of standard bar code since they are “effective in distinguishing among closely related species”. In our study, firstly we found that the average length of the psbA-trnH intergenic spacer is rather short at 332–357 base pairs. Therefore, the psbA-trnH sequences are relatively easy to be amplified using one pair of universal primers. Secondly, examination of the genetic divergences using six parameters and statistical tests confirmed that the psbA-trnH intergenic spacer possesses high interspecific divergence. Analyses of the DNA barcoding gap and Wilcoxon two-sample tests support the notion that the mean interspecific divergence of the psbA-trnH intergenic spacer is significantly higher than its mean intraspecific variation. Thirdly, according to the BLAST1 method, identification accuracy using the psbA-trnH intergenic spacer is 100 %. In particular, based on the alignment of the psbA-trnH intergenic spacer, L. japonica, which serves as the only source of Flos Lonicerae Japonicae, is different from the plant origins of Flos Lonicerae and other studied Lonicera species obviously with several specific sites in the sequences. Besides, in comparison with other regions, only the psbA-trnH intergenic spacer has enough variation to discriminate Lonicera macranthoides and L. hypoglauca, which serve as the members of plant origins of Flos Lonicerae. It is also worth noting that the sequence data for psbA-trnH do differentiate L. japonica and its variety, L. japonica var. chinensis, based on a consistent single nucleotide difference, so it demonstrates that psbA-trnH intergenic spacer exhibits excellent discrimination power at species level or lower. In summary, using the psbA-trnH intergenic spacer as a DNA bar code is suitable for the identification of L. japonica and its related species. In other words, it can authenticate the botanical origins of Flos Lonicerae Japonicae and Flos Lonicerae and meanwhile discriminate the multi-origin species of Flos Lonicerae.

In contrast, the two-locus combination of rbcL + matK, which the CBOL PWG recommends as the standard bar code, does not contain enough variable sites to distinguish the closely related species of Lonicera, and its identification efficiency is only 75 %. Nevertheless, based on the histogram of DNA barcoding gap and species identification, matK was proven to be better than the other regions, with the exception of psbA-trnH in our study. In addition, Chen et al. proposed that the ITS2 region should be the most promising universal DNA bar code for authenticating medicinal plants [18]. Unfortunately, because of the low success of the sequencing of the ITS2 region in our study, this region was not analyzed in depth herein.

The trnL‐F region, which is comprised of the trnL intron and the trnL‐F intergenic spacer, has become one of the most widely used chloroplast markers for phylogenetic analysis in plants [31]. The plastid trnL intron has been suggested as a possible plant bar code and does have conserved priming sites [20]. However, the limited interspecific sequence divergence of this region makes it an unlikely universal marker for species level identification [14]. From our study, the comparison of the trnL intron and the trnL‐F intergenic spacer suggests that the trnL‐F intergenic spacer regions are more variable than the trnL introns and have a broader range of variance, which is in agreement with the opinion of Shaw et al. [32]. We think that the trnL‐F intergenic spacer, when used as a DNA bar code, has better potential capacity for species level identification than the trnL intron, even though the identification efficiency of the trnL‐F intergenic spacer is at 62.5 % in our study.

In conclusion, our study shows that using the psbA-trnH intergenic spacer as a DNA bar code is an efficient tool for the identification of L. japonica and its closely related species. Not only does it distinguish the botanical origins of Flos Lonicerae Japonicae and Flos Lonicerae, it also discriminates the multi-origin species of Flos Lonicerae. This study may provide an important example for the authentication of the botanical origin of medicinal herbs in the Chinese Pharmacopoeia. Furthermore, this study is also valuable for the resource protection of medicinal plants.

Acknowledgements

We thank Prof. J. Y. Song and Prof. C. Liu for their valuable comments and careful revision of an early version of the manuscript. This work was supported by the International Cooperation Project (2007DFA30990) of the Ministry of Science and Technology of China and the Special Funding of the Ministry of Health (200802043) granted to Prof. S. L. Chen. We also thank the support of the National Natural Science Foundation of China (30973879) to Prof. Q. M. Guo.

References

• 1 Chinese Pharmacopoeia Commission .The Pharmacopoeia of the People's Republic of China, Vol. 1. Beijing; Chemical Industry Press 2010 28-29 205-206
  PubMedSearch in Google Scholar
• 2 Geng S L, Xu H H. Research survey of good agriculture practice of Flos Lonicerae.  Chin Tradit Herbal Drugs. 2003;  34 F14-17
  PubMedSearch in Google Scholar
• 3 Ren M T, Cheng J, Song Y, Sheng L S, Li P, Qi L W. Identification and quantification of 32 bioactive compounds in 4 Lonicera species by high performance liquid chromatography coupled with time-of-flight mass spectrometry.  J Pharm Biomed Anal. 2008;  48 1351-1360
  CrossrefPubMedSearch in Google Scholar
• 4 Song Y, Li S L, Wu M H, Li H J, Li P. Qualitative and quantitative analysis of iridoid glycosides in the flower buds of Lonicera species by capillary high performance liquid chromatography coupled with mass spectrometric detector.  Anal Chim Acta. 2006;  564 211-218
  CrossrefPubMedSearch in Google Scholar
• 5 Wang C Z, Li P, Ding J Y, Fishbein A, Yung C S. Discrimination of Lonicera japonica THUNB. from different geographical origins using restriction fragment length polymorphism analysis.  Biol Pharm Bull. 2007;  30 779-782
  CrossrefPubMedSearch in Google Scholar
• 6 Peng X X, Li W D, Wang W Q, Bai G B. Identification of Lonicera japonica by PCR-RFLP and allele-specific diagnostic PCR based on sequences of internal transcribed spacer regions.  Planta Med. 2009;  75 1-3
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Hebert P D N, Cywinska A, Ball S L, DeWaard J R. Biological identification through DNA barcodes.  Proc R Soc B Biol Sci. 2003;  270 313-321
  CrossrefPubMedSearch in Google Scholar
• 8 Hebert P D N, Gregory T R. The promise barcoding for taxonomy.  Syst Biol. 2005;  54 852-859
  CrossrefPubMedSearch in Google Scholar
• 9 Schindel D E, Miller S E. DNA barcoding, a useful tool for taxonomists.  Nature. 2005;  435 17
  PubMedSearch in Google Scholar
• 10 Lahaye R, van der Bank M, Bogarin D, Warner J, Pupulin F, Gigot G. DNA barcoding the floras of biodiversity hotspots.  Proc Natl Acad Sci USA. 2008;  105 2923-2928
  CrossrefPubMedSearch in Google Scholar
• 11 Hebert P D N, Ratnasingham S, de Waard J R. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species.  Proc Biol Sci. 2003;  270 (Suppl. 1) S96-S99
  CrossrefPubMedSearch in Google Scholar
• 12 Marshall E. Taxonomy – will DNA barcodes breathe life into classification?.  Science. 2005;  307 1037
  CrossrefPubMedSearch in Google Scholar
• 13 Kress W J, Wurdack K J, Zimmer E A, Weigt L A, Janzen D H. Use of DNA barcodes to identify flowering plants.  Proc Natl Acad Sci USA. 2005;  102 8369-8374
  CrossrefPubMedSearch in Google Scholar
• 14 Kress W J, Erickson D L. A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region.  PLoS ONE. 2007;  2 e508
  CrossrefPubMedSearch in Google Scholar
• 15 Newmaster S G, Fazekas A J, Steeves R A D, Janovec J. Testing candidate plant barcode regions in the Myristicaceae.  Mol Ecol Res. 2008;  8 480-490
  CrossrefPubMedSearch in Google Scholar
• 16 Song J Y, Yao H, Li Y, Li X W, Lin Y L, Liu C, Han J P, Xie C X, Chen S L. Authentication of the family Polygonaceae in Chinese pharmacopoeia by DNA barcoding technique.  J Ethnopharmacol. 2009;  124 434-439
  CrossrefPubMedSearch in Google Scholar
• 17 Yao H, Song J Y, Ma X Y, Liu C, Li Y, Xu H X, Han J P, Duan L S, Chen S L. Identification of Dendrobium species by a candidate DNA barcode sequence: the chloroplast psbA-trnH intergenic region.  Planta Med. 2009;  75 667-669
  Thieme ConnectPubMedSearch in Google Scholar
• 18 Chen S L, Yao H, Han J P, Liu C, Song J Y, Shi L C, Zhu Y J, Ma X Y, Gao T, Pang X H, Luo K, Li Y, Li X W, Jia X C, Lin Y L, Leon C. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species.  PLoS ONE. 2010;  5 e8613
  CrossrefPubMedSearch in Google Scholar
• 19 Pang X H, Song J Y, Zhu Y J, Xie C X, Chen S L. Using DNA barcoding to identify species within Euphorbiaceae.  Planta Med. 2010;  DOI: 10.1055/s-0030-1249806 , advance online publication April 8, 2010
  
  PubMedSearch in Google Scholar
• 20 Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C, Valentini A, Vermat T, Corthier G, Brochmann C, Willerslev E. Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding.  Nucleic Acids Res. 2007;  35 e14
  CrossrefPubMedSearch in Google Scholar
• 21 CBOL Plant Working Group . A DNA barcode for land plants.  Proc Natl Acad Sci USA. 2009;  106 12794-12797
  CrossrefPubMedSearch in Google Scholar
• 22 Sass C, Little D P, Stevenson D W, Specht C D. DNA barcoding in the Cycadales: testing the potential of proposed barcoding markers for species identification of Cycads.  PLoS ONE. 2007;  2 e1154
  CrossrefPubMedSearch in Google Scholar
• 23 Taberlet P, Gielly T J, Pautou G, Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA.  Plant Mol Biol. 1991;  17 1105-1109
  CrossrefPubMedSearch in Google Scholar
• 24 Wang Y J, Liu J Q. A preliminary investigation on the phylogeny of Saussurea (Asteraceae: Cardueae) based on chloroplast DNA TrnL-F sequences.  Acta Phytotaxon Sin. 2004;  42 136-153
  PubMedSearch in Google Scholar
• 25 Thompson J D, Higgins D G, Gibson T J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.  Nucleic Acids Res. 1994;  22 4673
  CrossrefPubMedSearch in Google Scholar
• 26 Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.  Mol Biol Evol. 2007;  24 1596-1599
  CrossrefPubMedSearch in Google Scholar
• 27 Meier R, Zhang G Y, Ali F. The use of mean instead of smallest inter-specific distances exaggerates the size of the “Barcoding Gap” and leads to misidentification.  Syst Biol. 2008;  57 809-813
  CrossrefPubMedSearch in Google Scholar
• 28 Meyer C P, Paulay G. DNA barcoding: Error rates based on comprehensive sampling.  PLoS Biol. 2005;  3 2229-2238
  PubMedSearch in Google Scholar
• 29 Ross H A, Murugan S, Li W L S. Testing the reliability of genetic methods of species identification via simulation.  Syst Biol. 2008;  57 216-230
  CrossrefPubMedSearch in Google Scholar
• 30 Storchová H, Olson M S. The architecture of the chloroplast psbA-trnH non-coding region in angiosperms.  Plant Syst Evol. 2007;  268 235-256
  CrossrefPubMedSearch in Google Scholar
• 31 Quandt D, Müller K, Stech M, Frahm J P, Frey W, Hilu K W, Borsch T. Molecular evolution of the chloroplast trnL-F region in land plants.  Monogr Syst Bot Mo Bot Gard. 2004;  98 13-37
  PubMedSearch in Google Scholar
• 32 Shaw J, Lickey E B, Schilling E E, Small R L. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III.  Am J Bot. 2007;  94 275-288
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Shilin Chen

Institute of Medicinal Plant Development
Chinese Academy of Medical Sciences
Peking Union Medical College

151 Malianwa North Road, Haidian District

Beijing 100193

People's Republic of China

Phone: +86 10 62 89 97 00

Fax: +86 10 62 89 97 76

Email: slchen@implad.ac.cn

References

• 1 Chinese Pharmacopoeia Commission .The Pharmacopoeia of the People's Republic of China, Vol. 1. Beijing; Chemical Industry Press 2010 28-29 205-206
  PubMedSearch in Google Scholar
• 2 Geng S L, Xu H H. Research survey of good agriculture practice of Flos Lonicerae.  Chin Tradit Herbal Drugs. 2003;  34 F14-17
  PubMedSearch in Google Scholar
• 3 Ren M T, Cheng J, Song Y, Sheng L S, Li P, Qi L W. Identification and quantification of 32 bioactive compounds in 4 Lonicera species by high performance liquid chromatography coupled with time-of-flight mass spectrometry.  J Pharm Biomed Anal. 2008;  48 1351-1360
  CrossrefPubMedSearch in Google Scholar
• 4 Song Y, Li S L, Wu M H, Li H J, Li P. Qualitative and quantitative analysis of iridoid glycosides in the flower buds of Lonicera species by capillary high performance liquid chromatography coupled with mass spectrometric detector.  Anal Chim Acta. 2006;  564 211-218
  CrossrefPubMedSearch in Google Scholar
• 5 Wang C Z, Li P, Ding J Y, Fishbein A, Yung C S. Discrimination of Lonicera japonica THUNB. from different geographical origins using restriction fragment length polymorphism analysis.  Biol Pharm Bull. 2007;  30 779-782
  CrossrefPubMedSearch in Google Scholar
• 6 Peng X X, Li W D, Wang W Q, Bai G B. Identification of Lonicera japonica by PCR-RFLP and allele-specific diagnostic PCR based on sequences of internal transcribed spacer regions.  Planta Med. 2009;  75 1-3
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Hebert P D N, Cywinska A, Ball S L, DeWaard J R. Biological identification through DNA barcodes.  Proc R Soc B Biol Sci. 2003;  270 313-321
  CrossrefPubMedSearch in Google Scholar
• 8 Hebert P D N, Gregory T R. The promise barcoding for taxonomy.  Syst Biol. 2005;  54 852-859
  CrossrefPubMedSearch in Google Scholar
• 9 Schindel D E, Miller S E. DNA barcoding, a useful tool for taxonomists.  Nature. 2005;  435 17
  PubMedSearch in Google Scholar
• 10 Lahaye R, van der Bank M, Bogarin D, Warner J, Pupulin F, Gigot G. DNA barcoding the floras of biodiversity hotspots.  Proc Natl Acad Sci USA. 2008;  105 2923-2928
  CrossrefPubMedSearch in Google Scholar
• 11 Hebert P D N, Ratnasingham S, de Waard J R. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species.  Proc Biol Sci. 2003;  270 (Suppl. 1) S96-S99
  CrossrefPubMedSearch in Google Scholar
• 12 Marshall E. Taxonomy – will DNA barcodes breathe life into classification?.  Science. 2005;  307 1037
  CrossrefPubMedSearch in Google Scholar
• 13 Kress W J, Wurdack K J, Zimmer E A, Weigt L A, Janzen D H. Use of DNA barcodes to identify flowering plants.  Proc Natl Acad Sci USA. 2005;  102 8369-8374
  CrossrefPubMedSearch in Google Scholar
• 14 Kress W J, Erickson D L. A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region.  PLoS ONE. 2007;  2 e508
  CrossrefPubMedSearch in Google Scholar
• 15 Newmaster S G, Fazekas A J, Steeves R A D, Janovec J. Testing candidate plant barcode regions in the Myristicaceae.  Mol Ecol Res. 2008;  8 480-490
  CrossrefPubMedSearch in Google Scholar
• 16 Song J Y, Yao H, Li Y, Li X W, Lin Y L, Liu C, Han J P, Xie C X, Chen S L. Authentication of the family Polygonaceae in Chinese pharmacopoeia by DNA barcoding technique.  J Ethnopharmacol. 2009;  124 434-439
  CrossrefPubMedSearch in Google Scholar
• 17 Yao H, Song J Y, Ma X Y, Liu C, Li Y, Xu H X, Han J P, Duan L S, Chen S L. Identification of Dendrobium species by a candidate DNA barcode sequence: the chloroplast psbA-trnH intergenic region.  Planta Med. 2009;  75 667-669
  Thieme ConnectPubMedSearch in Google Scholar
• 18 Chen S L, Yao H, Han J P, Liu C, Song J Y, Shi L C, Zhu Y J, Ma X Y, Gao T, Pang X H, Luo K, Li Y, Li X W, Jia X C, Lin Y L, Leon C. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species.  PLoS ONE. 2010;  5 e8613
  CrossrefPubMedSearch in Google Scholar
• 19 Pang X H, Song J Y, Zhu Y J, Xie C X, Chen S L. Using DNA barcoding to identify species within Euphorbiaceae.  Planta Med. 2010;  DOI: 10.1055/s-0030-1249806 , advance online publication April 8, 2010
  
  PubMedSearch in Google Scholar
• 20 Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C, Valentini A, Vermat T, Corthier G, Brochmann C, Willerslev E. Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding.  Nucleic Acids Res. 2007;  35 e14
  CrossrefPubMedSearch in Google Scholar
• 21 CBOL Plant Working Group . A DNA barcode for land plants.  Proc Natl Acad Sci USA. 2009;  106 12794-12797
  CrossrefPubMedSearch in Google Scholar
• 22 Sass C, Little D P, Stevenson D W, Specht C D. DNA barcoding in the Cycadales: testing the potential of proposed barcoding markers for species identification of Cycads.  PLoS ONE. 2007;  2 e1154
  CrossrefPubMedSearch in Google Scholar
• 23 Taberlet P, Gielly T J, Pautou G, Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA.  Plant Mol Biol. 1991;  17 1105-1109
  CrossrefPubMedSearch in Google Scholar
• 24 Wang Y J, Liu J Q. A preliminary investigation on the phylogeny of Saussurea (Asteraceae: Cardueae) based on chloroplast DNA TrnL-F sequences.  Acta Phytotaxon Sin. 2004;  42 136-153
  PubMedSearch in Google Scholar
• 25 Thompson J D, Higgins D G, Gibson T J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.  Nucleic Acids Res. 1994;  22 4673
  CrossrefPubMedSearch in Google Scholar
• 26 Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.  Mol Biol Evol. 2007;  24 1596-1599
  CrossrefPubMedSearch in Google Scholar
• 27 Meier R, Zhang G Y, Ali F. The use of mean instead of smallest inter-specific distances exaggerates the size of the “Barcoding Gap” and leads to misidentification.  Syst Biol. 2008;  57 809-813
  CrossrefPubMedSearch in Google Scholar
• 28 Meyer C P, Paulay G. DNA barcoding: Error rates based on comprehensive sampling.  PLoS Biol. 2005;  3 2229-2238
  PubMedSearch in Google Scholar
• 29 Ross H A, Murugan S, Li W L S. Testing the reliability of genetic methods of species identification via simulation.  Syst Biol. 2008;  57 216-230
  CrossrefPubMedSearch in Google Scholar
• 30 Storchová H, Olson M S. The architecture of the chloroplast psbA-trnH non-coding region in angiosperms.  Plant Syst Evol. 2007;  268 235-256
  CrossrefPubMedSearch in Google Scholar
• 31 Quandt D, Müller K, Stech M, Frahm J P, Frey W, Hilu K W, Borsch T. Molecular evolution of the chloroplast trnL-F region in land plants.  Monogr Syst Bot Mo Bot Gard. 2004;  98 13-37
  PubMedSearch in Google Scholar
• 32 Shaw J, Lickey E B, Schilling E E, Small R L. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III.  Am J Bot. 2007;  94 275-288
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Shilin Chen

Institute of Medicinal Plant Development
Chinese Academy of Medical Sciences
Peking Union Medical College

151 Malianwa North Road, Haidian District

Beijing 100193

People's Republic of China

Phone: +86 10 62 89 97 00

Fax: +86 10 62 89 97 76

Email: slchen@implad.ac.cn

• Supplementary Material