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DNA barcoding of medicinal plant material for identification

Natascha Techen1, Iffat Parveen1, Zhiqiang Pan2 and Ikhlas A Khan1,3

Because of the increasing demand for herbal remedies and for is not affected by external factors and is found in all
authentication of the source material, it is vital to provide a single tissues. Therefore, development of DNA-based markers
database containing information about authentic plant materials is important for authentication of medicinal plants.
and their potential adulterants. The database should provide
DNA barcodes for data retrieval and similarity search. In order to The novel technique of identifying biological specimens
obtain such barcodes, several molecular methods have been using short DNA sequences from either nuclear or orga-
applied to develop markers that aid with the authentication and nelle genomes is called DNA barcoding. The term ‘DNA
identification of medicinal plant materials. In this review, we barcode’ as taxon identifiers was first proposed by Paul
discuss the genomic regions and molecular methods selected to Hebert of University of Guelph in 2003 [1]. He recom-
provide barcodes, available databases and the potential future of mended that the 50 end of cytochrome c oxidase 1 (CO1)
barcoding using next generation sequencing. from the mitochondrial genome was sufficient to generate
Addresses DNA barcodes for the identification of animals [1–4]. On
1
National Center for Natural Products Research and Research Institute the basis of this initial success with animals, CO1 was
of Pharmaceutical Sciences, Department of Pharmacognosy, School of suggested as the locus that could provide recognition tags
Pharmacy, University of Mississippi, P.O. Box 1848, MS 38677, USA
2 for all organisms. They further emphasized that DNA
USDA-ARS-NPURU, P.O. Box 8048, University, MS 38677, USA
3
School of Pharmacy, King Saud University, Saudi Arabia barcoding not only helps in the identification of species
but can also define species boundaries, flagging of new
Corresponding author: Khan, Ikhlas A (ikhan@olemiss.edu) species and species delimitation [2,3]. However, in plants
the mitochondrial genes are slowly evolving, with very
low substitution rates and were not suitable for barcoding.
Current Opinion in Biotechnology 2014, 25:103–110
Therefore, the search for plant barcode shifted to chlor-
This review comes from a themed issue on Analytical biotechnology oplast and nuclear genomes with high substitution rates.
Edited by Frank L Jaksch and Savaş Tay Following initial in-silico and laboratory-based assessment
of different loci from chloroplast and nuclear genomes led
to the conclusion that no single locus plant barcode exists,
and soon it was realized that multi-locus barcodes are
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
requisite for plant barcoding. Subsequently a number of
loci were being tested for their suitability as plant bar-
http://dx.doi.org/10.1016/j.copbio.2013.09.010
codes and many multi-locus combinations were
suggested. The Consortium for the Barcode of Life Plant
Working Group (CBOL) [5] evaluated seven chloroplast
Introduction genomic regions across the plant kingdom and proposed a
The traditional system of medicine utilizes medicinal combination of matK and rbcL as plant barcodes. High
plants to cure various ailments but the herbal industry universality but less species resolution is provided by rbcL
suffers from substitution and adulteration of medicinal whereas matK affords high resolution but less universal-
herbs with closely related species. The efficacy of the ity. A combination of these two can help to achieve
drug decreases if it is adulterated, and in some cases, can maximum species discrimination. Nevertheless, in clo-
be lethal if it is substituted with toxic adulterants. Hence, sely related species, the discriminating ability of these
the correct formulation is important for the medicinal two markers is low [6,7]. Therefore, the China Plant BOL
herb to be effective. The traditional methods of medic- Group [8] proposed the addition of nuclear ITS (Internal
inal plant identification include organoleptic methods Transcibed Spacer) to the matK + rbcL combination as
(identification by the senses: taste, sight, smell, touch), plant barcode in order to achieve maximum identification
macroscopic and microscopic methods (identification by rates even in closely related species.
shape, colour, texture) and chemical profiling (e.g. TLC,
HPLC-UV, HPLC-MS). However, neither method can The aim of this review is to assess the progress made so far
identify the related species easily in processed products in the field of DNA barcoding in relation to the identifi-
because the former method requires trained personal for cation of botanicals. In the current paper, we review the
macroscopic and microscopic examinations. In the latter genomic regions selected as possible barcodes for medicinal
method, chemical profiles or markers may be affected by plants and the emerging new methods for rapid generation
physiological and storage conditions. Authentication at of barcodes. We also discuss the challenges of barcoding
the DNA level provides more reliability because, in and what databases are available to retrieve barcodes of
contrast to RNA, DNA is a stable macromolecule that medicinal plants, their substitutes and adulterants.

www.sciencedirect.com Current Opinion in Biotechnology 2014, 25:103–110

104 Analytical biotechnology

Loci suggested as plant barcodes SCAR markers (17 references) have been developed from
At the Fourth International Barcode of Life Conference RAPD, ISSR and a variety of genomic regions.
(http://www.dnabarcodes2011.org/) the option of a three-
locus barcode (matK + rbcL + psbA-trnH) versus a two- Zuo et al. [14] analyzed 12 genomic regions of 95 ginseng
locus barcode was discussed. The two-locus barcode samples, representing all the species in the genus. They
was preferred to avoid the increased costs of sequencing demonstrated that the combination of psbA-trnH and ITS
three loci rather than two in very large sample sets, and was sufficient for the identification of all species and
to prevent further delays in implementing a standard species clusters in the genus. For various samples ana-
barcode for land plants. The barcode combination lyzed, the combination of up to three genomic regions
rbcL + matK was the preferred choice. (matK, rbcL, ITS, psbA-trnH) provided the required infor-
mation for identification [15,35,38].
A search of the literature in SciFinder (a chemical
abstracts service database) from 2010 to 2013 resulted Chen et al. [11] analyzed >6600 plant samples belonging
in 60 publications (Figure 1 and Table 1). In the literature to 4800 species from 753 distinct genera using the geno-
analyzed in this review, a total of 17 barcode regions mic regions psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS, and
(matK, rbcL, ITS, ITS2, psbA-trnH, atpF-atpH, ycf5, psbK- ITS2. Their data suggested that the second internal
I, psbM trnD, rps16, coxI, nad1, trnL-F, rpoB, rpoC1, atpF- transcribed spacer (ITS2) of nuclear ribosomal DNA
atpH, rps16) of medicinal plants were reported to aid in represents the most suitable region for DNA barcoding
the authentication and identification of medicinal plant applications. The percentage of successful identification
materials. The majority of barcoding regions mentioned with ITS2 at the species level was 92.7%. He et al. and
in the literature were the ITS region (26 references), Selvaraj et al. [9,18] also analyzed multiple genomic
psbA-trnH (21 references), matK (19 references), and rbcL barcode regions and came to a similar conclusion that
(14 references). Further genomic regions used for barcod- ITS or ITS2 showed the highest discrimination rate
ing were ITS2 (9 references), rpoC1 (6 references), rpoB among the samples analyzed. In contrast, the highest
(4 references), and trnL-F (3 references). discrimination rate was found to be from psbA-trntrnH
[10,12,39] or from matK [41,48].
Besides using known genomic regions, other PCR-based
methods have been applied to develop markers that help Theodoridis et al. [36] analyzed medicinal plants of the
with the authentication and identification of medicinal Labiatae (Lamiaceae) family from Chios Island (Greece)
plant material: RAPD, RFLP, microsatellites, ISSRs, and the adjacent Cesme-Karaburun Peninsula (Turkey)
SNPs, and ARMS (12,3,1,2,2,2 publications, respectively). using all three barcode loci matK, rbcL, psbA-trnH and

Figure 1

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26
25
21
20 19
17

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12

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3 3
2 2 2
1
2 2
0

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Current Opinion in Biotechnology

Literature search in SciFinder (a Chemical Abstracts Service database) using the keywords ‘DNA barcod* medicinal plant’ (excluding endophytic fungi,
including patents) from 2010 to 2013 resulted in 60 publications. Various genomic regions and PCR-based methods were used to develop barcodes.

Current Opinion in Biotechnology 2014, 25:103–110 www.sciencedirect.com

 Medicinal plant barcoding Techen et al. 105

Table 1

Overview of DNA based methods and technologies investigated for medicinal plant identification.

Ref. Analyzed material Genomic regions analyzed/molecular methods applied

[9] Angelica spp. matK rbcL ITS ITS2 psbA-trnH
[10] Rhododendron spp. matK rbcL ITS ITS2 psbA-trnH
[11] 753 genera matK rbcL ITS ITS2 psbA-trnH
[12] Lonerica spp. matK rbcL ITS psbA-trnH trnL-F
[13] Solanum spp and adulterants matK rbcL ITS psbA-trnH trnL-F
[14] Ginseng genus matK rbcL ITS psbA-trnH rpoB rpoC1
[15] Astragalus spp. and adulterants matK rbcL ITS
[16] Various medicinal roots matK ITS psbA-trnH rpoC1
[17] Various medicinal plants ITS ITS2
[18] Boerhavia spp. Astragalus spp. and adulterants ITS ITS2 psbA-trnH
[19] Sedum spp. Astragalus spp. and adulterants ITS ITS2
[20] Various (old) medicinal plants ITS ITS2
[21] Rubus spp. ITS psbA-trnH trnL-F SCAR SNP
[22] Hypericum spp. ITS SCAR
[23] Ochradenus spp. ITS rpoB rpoC1
[24] Rehmannia spp. ITS RAPD SCAR
[25] Medicinal vines (22 genera) ITS
[26] Dipsacus spp. ITS
[27] Dendrobium spp. ITS ARMS
[28] Paris spp. ITS RFLP
[29] Citrus spp. ITS SNP
[30] Dendrobium spp. ITS
[31] Ruta spp. ITS rpoB rpoC1 SCAR
[32] Astragalus spp. ITS
[33] Various medicinal plants ITS
[34] Meconopsis spp. ITS
[35] Scutellaria spp. Astragalus spp. and adulterants matK rbcL psbA-trnH
[36] Lamiaceae matK rbcL psbA-trnH
[37] Various medicinal plants matK rbcL psbA-trnH
[38] Sabia spp. matK rbcL psbA-trnH
[39] Pteridophytes matK rbcL psbA-trnH rpoB rpoC1
[40] Vitex spp. matK psbA-trnH RFLP
[41] Sideritis spp. matK psbA-trnH
[42] Paris spp. and adulterants psbA-trnH
[43] Senna spp. psbA-trnH
[44] Smilax spp. psbA-trnH
[45] Phyllanthus spp. psbA-trnH
[46] Cistance spp. psbA-trnH
[47] Galpemia spp. matK rbcL rpoC1
[48] Dendrobium spp. matK rbcL
[49] Rhubarb matK SCAR
[50] Pueraria candollei, Butea superb, Mucuna collettii matK RFLP
[51] Orobanche spp. and adulterants ITS2
[52] Taraxacum and adulterants ITS2 SCAR
[53] Cuscuta spp. and adulterants RAPD SCAR
[54] Artemisia spp. RAPD SCAR
[55] Liriope spp, Ophiopogon spp. RAPD SCAR
[56] Rheum spp. RAPD SCAR
[57] Glycerrhiza spp. SCAR
[58] Zizyphus spp. RAPD SCAR
[59] Crocus spp. and adulterants RAPD SCAR
[60] Prunella spp. RAPD SCAR
[61] Viola spp. and adulterants RAPD SCAR
[62] Srocphularia spp. SCAR ISSR
[63] Panax ginseng RAPD SCAR
[64] Uncaria spp. RAPD
[65] Marsdeniae and adulterants RAPD
[66] Phyllanthus spp. ISSR
[67] Asparagus and adulterants ARMS
[68] Dendrobium spp. MS

MS = microsatellites.

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106 Analytical biotechnology

tested them either as single-region or as multi-region Figure 2

barcodes. They showed that matK and psbA-trnH were
as useful in discriminating species of the Labiatae as any
multi-region combination. For the Labiatae species ana-
PCR
lyzed matK and psbA-trnH could serve as single-region Sequencing
Hybridization methods:

Marker
SCAR
barcodes. Schori and Showalter [37] analyzed the rbcL, /Microarray of RAPD
matK, psbA-trnH loci of fourteen species of medicinal methods genomic ISSR/SSR
plants from Pakistan and found that, depending on plant regions etc.
to be identified, one region was preferred over the other to
aid with species identification.

Single barcode-regions for identification have been

reported for (i) matK [41,49,50], (ii) ITS [22,25–30,32–
34], (iii) ITS2 [17,20,51,52], and (iv) psbA-trnH [42–46].
Barcoding of Botanicals

Not only genomic regions, but also the usefulness of

Future ?
RAPD-based DNA markers has been reported for med-
icinal plant identification. RAPD and SCAR markers have
been developed for the identification of several medicinal
plant materials [53–56,58–61,62].
Next
 Generation
The review by Heubl [69 ] focused on the most com- Sequencing:
monly used DNA-based technologies (RAPD, RFLP, Transcriptome
ARMS, CAPS, AFLP, DAF, ISSR, SSR, sequencing, Genome
hybridization and microarrays) and new achievements
in the field of DNA barcoding and DNA chip technology
Current Opinion in Biotechnology
used to identify traditional Chinese medicinal materials.
This particular review provides a critical view of the
Current and proposed barcoding applications of botanicals.
methods presented concerning sensitivity, reproducibil-
ity, and dependability.
transcriptome of medicinal plants to provide a robust
Future developments resource for gene discovery and downstream metabolic
Although the traditional DNA barcoding techniques pathway discovery [74–76]. Since medicinal plants tend
remain an effective DNA method for identification of to have a large genome, the combination of targeted
medicinal plants, the more advanced and newly devel- genomic enrichment [77] and NGS could ultimately
oped high throughput sequencing, specifically next- make the technologies applicable in DNA barcoding of
generation sequencing (NGS) technologies [70], could medicinal plant material (Figure 2).
be adopted and potentially revolutionize the process.
Even though DNA barcoding usually targets short regions Challenges and limitations of barcoding
of DNA molecule within the genome and does not The isolation of pure, high molecular weight DNA is
require full genome-scale data, the potential of using critical for the successful application of molecular
NGS to simultaneously identify multiple species or bulk methods. This can be quite a challenge since in processed
samples of organisms by sequencing DNA barcodes is medicinal plant material the DNA is often highly
enormous. degraded or the plant material contains high amounts
of polysaccharides, polyphenols and other secondary
The continuing improvement in NGS technologies and metabolites, such as, alkaloids and flavonoids. Various
the massive expansion of reference sequence databases commercial kits and modified traditional methods are
have made the NGS approach promising. However, only available to yield in good quality DNA from raw and
one publication using NGS for medicinal plant DNA powdered medicinal plant material, herbarium speci-
barcoding has appeared to date in which it was utilized mens, capsules, tablets, or tinctures for downstream appli-
to identify potential nuclear genomic regions for barcod- cations [22,78,79,80,81]. Särkinen et al. [79] found a
ing [71]. But most recently, there have been a number strong negative correlation between amplicon size and
of cases published in which NGS was employed, PCR success, indicating that shorter fragments are easier
for example, to explore the intragenomic divergence to amplify from herbarium DNA. The relatively large
of Dikarya [72], and the taxonomic characterization of genomic region rbcL (670 bp) was amplified only in 10%
freshwater diatom communities [73]. Presently NGS of the analyzed samples, the medium sized LEAFY
and bioinformatics are used to probe and deduce the intron 3 (260 bp) amplified in 24%, while the small

Current Opinion in Biotechnology 2014, 25:103–110 www.sciencedirect.com

 Medicinal plant barcoding Techen et al. 107

genomic region trnL P6 loop (10–143 bp) was successfully is a website that includes DNA sequences and infor-
amplified in 78% of the samples. For heavily damaged mation and key references of the medicinal materials
DNA, it is therefore recommended to develop con- recorded in the Pharmacopoeia of the People’s
ditions to produce very short amplicons, which are easier Republic of China, American Herbal Pharmacopoeia
to amplify, or perform DNA ‘repair reactions’ [79,82], and other related references. This database, updated
and that are also available as commercial kits. Särkinen in May 2012 with 1658 species and 31,468 sequences
et al. [79] believes that fragmented DNA of medicinal available, provides information material for disting-
plants is going to be far less of a problem in the near uishing medicinal materials (plant, animal, and fungi)
future using the NGS approach since this technique from their common substitutes and adulterants [88].
requires fragmented (and ligated) DNA as starting (vi) The GDR (Genome Database for Rosaceae), founded
material. in 2009, provides genetic markers and ESTs of
Rosaceae. A large number of species in Rosaceae or
rose family have a medicinal value (http://www.
Availability of data rosaceae.org/).
It is desirable to have access to a single barcode library for
all medicinal material used (including fungal and animal Conclusion
species). Currently, however several barcode libraries Molecular barcoding methods are reliable tools for the
are freely accessible (see also review by Taylor and identification of medicinal plants, their substitutes and
Harris [83]): adulterants at the genus and species level. The methods
discussed provide consistent and reliable results regard-
(i) BOLD (The barcode of life data system) [84] (http:// less of the age, plant part, or environmental factors of the
www.barcodinglife.com) was created and is main- sample.
tained by the University of Guelph in Ontario. It offers
researchers a way to collect, manage, and analyze DNA Based on the literature analyzed in this review, it appears
barcode data. The goal is, over the next 20 years, to that, although the Barcode of Life Plant Working Group
provide a barcode library for all eukaryotic life. [5] recommends the genomic regions rbcL + matK for
(ii) CBOL (Consortium for the barcode of life) (http:// barcoding, often other genomic regions could be more
www.barcodeoflife.org/) is a public reference library useful for medicinal material identification. Furthermore,
of species identifiers which could be used to assign depending on the material analyzed, one or the combi-
unknown specimens to known species. CBOL was nation of up to three genomic regions was necessary to
founded in 2004 and promotes barcoding through provide the required information for identification.
working groups, networks, workshops, conferences,
outreach, and training. CBOL has 200 member Because of the increasing demand for herbal remedies,
organizations from 50 countries and operates from a authentication of the medicinal plant material is import-
Secretariat Office located in the Smithsonian ant; therefore it is vital to provide a sole, extensive
Institution’s National Museum of Natural History database with DNA data for easy identification.
in Washington, DC [5].
(iii) iBOL (International Barcode of Life project) (http:// Future sequencing advances to analyze large scale
www.ibol.org/) consists of a group of hundreds of nucleic acid sequences have a great potential for geno-
scientists from 25 nations working together to typing and taxon identification, even for damaged or
construct a DNA barcode reference library that will fragmented template DNA. In the near future, full geno-
be the foundation for a DNA-based identification mic sequence data from medicinal plants will be avail-
system for all multi-cellular life. Their five-year able. The challenge will be to provide sufficient storage
(2010–2015) goal is to barcode five million specimens space for all the data produced.
representing 500,000 species.
(iv) The GenBank online genetic sequence database Acknowledgements
(http://www.ncbi.nlm.nih.gov/genbank/) [85] is This research was funded in part by the Food and Drug Administration and
possibly one of the most important repositories of USDA. We thank Jon Parcher for his revision of the manuscript and
suggestions.
genetic information. GenBank contains over
108 million entries for over 260,000 named organ- References and recommended reading
isms and is one of the most frequently used databases Papers of particular interest, published within the period of review,
for genomic authentication [86]. With the BLAST have been highlighted as:
program [87] an unknown DNA sequence can be  of special interest
rapidly and accurately compared to known and well  of outstanding interest
characterized sequences.
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www.sciencedirect.com Current Opinion in Biotechnology 2014, 25:103–110

108 Analytical biotechnology

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