DNA Barcode for Identifying Folium Artemisiae Argyi from Counterfeits

Abstract

Folium Artemisiae Argyi is an important herb in traditional Chinese medicine. It is commonly used in moxibustion, medicine, etc. However, identifying Artemisia argyi is difficult because this herb exhibits similar morphological characteristics to closely related species and counterfeits. To verify the applicability of DNA barcoding, ITS2 and psbA-trnH were used to identify A. argyi from 15 closely related species and counterfeits. Results indicated that total DNA was easily extracted from all the samples and that both ITS2 and psbA-trnH fragments can be easily amplified. ITS2 was a more ideal barcode than psbA-trnH and ITS2+psbA-trnH to identify A. argyi from closely related species and counterfeits on the basis of sequence character, genetic distance, and tree methods. The sequence length was 225 bp for the 56 ITS2 sequences of A. argyi, and no variable site was detected. For the ITS2 sequences, A. capillaris, A. anomala, A. annua, A. igniaria, A. maximowicziana, A. princeps, Dendranthema vestitum, and D. indicum had single nucleotide polymorphisms (SNPs). The intraspecific Kimura 2-Parameter distance was zero, which is lower than the minimum interspecific distance (0.005). A. argyi, the closely related species, and counterfeits, except for Artemisia maximowicziana and Artemisia sieversiana, were separated into pairs of divergent clusters by using the neighbor joining, maximum parsimony, and maximum likelihood tree methods. Thus, the ITS2 sequence was an ideal barcode to identify A. argyi from closely related species and counterfeits to ensure the safe use of this plant.

Folium Artemisiae Argyi, the dried leaves of Artemisia argyi Levl. et Van, is an important herb in traditional Chinese medicine and is commonly used in moxibustion, medicine, food, or medicinal bath.^1–4) A. argyi is widely distributed in Korea, Mongolia, Japan, and the Russia Far East.^1,5,6) Folium Artemisiae Argyi was first recorded in “Shi Jing” near 1100 BC and in many clinical and medicine literature, such as “Wu Shi Er Bing Fang,” “Ben Cao Gang Mu,” and “Jin Gui Yao Lve.”^3,7) In traditional Chinese medicine, Folium Artemisiae Argyi was considered to have bitter, pungent, and warm properties and associated with the liver, spleen, and kidney meridians.⁸⁾ Folium Artemisiae Argyi prevents and cures gynecological, respiratory, and dermatological diseases, such as menstruation-related symptoms, infertility, dysmenorrhea, inflammation, hemostasis, tuberculosis, asthma, and eczema.^9–11) Folium Artemisiae Argyi contains complex ingredients, including essential oil, polysaccharide, flavonoid, tannin, sterols, and triterpenes.^12–16)

Unfortunately, identifying A. argyi is difficult because this plant exhibits similar morphological characteristics to closely related species and counterfeits, including congeneric plants Artemisia lavandulifolia, Artemisia anomala, Artemisia princeps, Artemisia indica, Artemisia stolonifera, Dendranthema vestitum, and Dendranthema indicum.^1–3) At present, 112 traditional Chinese patent medicines contained A. argyi in Chinese markets.⁸⁾ The confusion between A. argyi and its adulterants affects the clinical efficacy and safety of using this plant. Traditional classification methods, such as morphologic, microscopic, physical, and chemical identification, are limited in distinguishing A. argyi from closely related species and counterfeits.^3,17) Lee et al.⁵⁾ indicated that A. argyi shares two polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) banding patterns with 17 other Korean Artemisia species and could not be distinguished from other species on the basis of the trnL–F region. Similarly, Wang et al.¹⁸⁾ believed that trnL-F sequences are highly homogeneous in Artemisia L. and have limitations in phylogeny. Lee et al.¹⁹⁾ amplified 254 bp sequence-characterized amplified regions using Fb and R7 primer sets to distinguish A. argyi from four other Korean Artemisia species; however, A. argyi could not be distinguished from A. princeps. Liu et al.²⁰⁾ reported that ITS2, unlike rbcL, matK, and psbA-trnH, could be used as a potential DNA barcode to correctly identify the medicinal plants in Artemisia L. Gao et al.²¹⁾ evaluated the ability of five DNA regions (rbcL, matK, ITS, ITS2, and psbA-trnH) to identify 3490 Asteraceae sequences that represent 2315 species belonging to 494 genera. The results indicated that ITS2 is the best DNA barcode for the Asteraceae family. The ITS2 sequence that was supplemented by the psbA-trnH region was recommended as an ideal DNA barcode to identify medicinal plants.^22–25) Thus, ITS2 and psbA-trnH were used to identify A. argyi from closely related species and counterfeits for its safe use in this study.

MATERIALS AND METHODS

A total of 146 samples from 16 species were collected from various geographical areas in China (Table 1). A total of 56 samples of A. argyi were collected from Hubei, Hunan, Zhejiang, Jiangsu, Hebei, Henan, Guangdong, Yunnan, Gansu, Liaoning, and Heilongjiang provinces. All specimens were carefully and visually identified by Professor SuyingTian from Guangdong Pharmaceutical University. The voucher specimens were deposited in the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. One ITS2 sequence of Melia azedarach (JF421516) was downloaded from GenBank as outgroup for rooting phylogenetic trees.

DNA Extraction, PCR Amplification, and Sequencing

Genomic DNA was extracted using the DNA barcoding standard operating procedure.^26,27) The samples comprised 20–30 mg of dry leaves that were crushed in powder form in 2 mL clean microcentrifuge tubes at 45 Hz using stainless steel ball milling for 2 min. The total genomic DNA was extracted using a Plant Genomic DNA Kit (Tiangen Biotech Co., Beijing, China). The ITS2 and psbA-trnH barcodes were amplified using the primers described as follows: ITS2F (5′-ATG CGA TAC TTG GTG TGA AT-3′) and ITS3R (5′-GAC GCT TCT CCA GAC TAC AAT-3′) for ITS2 as well as trnH (5′-CGC GCA TGG TGG ATT CAC AAT CC-3′) and psbA (5′-GTT ATG CAT GAA CGT AAT GCT C-3′) for psbA-trnH.^26,27) PCR amplification was performed in a PTC-200 Peltier Thermal Cycler (BioRad Lab Inc., U.S.A.). PCR reactions were performed in a 25 µL volume containing approximately 20–50 ng of genomic DNA template, 12.5 µL of 2×Easy Taq PCR Master Mix (Aidlab Biotechnologies Co., China), and 1 µL of forward and reverse primers (2.5 µmol/L). The PCR conditions were set as previously described.²⁶⁾ The desired PCR products were examined through 0.5% Tris borate ethylenediamine tetraacetic acid (TBE) agarose gel electrophoresis. The purified PCR products were directly sequenced bidirectionally using the ABI 3730 XL sequencer (Applied Biosystems Co., U.S.A.).

Analysis of Data Sets

All sequences, except for the primer regions, were edited and assembled using the CondonCode Aligner V 5.1.3 (CodonCode Co., U.S.A.). HMMer software was used to annotate the ITS2 region.^28–30) The genetic distances were calculated using MEGA 6.0.³¹⁾ Pairwise interspecific and intraspecific distances for each locus were analyzed using the Kimura 2-Parameter (K2P) model. The presence of a “barcode gap” (interspecific divergences that are clearly greater than intraspecific variation) was verified.³²⁾ Three phylogenetic trees, namely, Neighbor–Joining (NJ) tree, maximum parsimony (MP) tree, and maximum likelihood (ML) tree, were constructed to identify A. argyi and its adulterants using the MEGA 6.0 software with 1000 bootstrap replicates.³¹⁾

RESULTS

Universality

DNA was successfully extracted from 146 individuals representing the 16 species of A. argyi and its adulterants. Both the ITS2 and psbA-trnH regions were successfully amplified from all samples by adopting the universality of primers. Bidirectional sequences were obtained from both ITS2 and psbA-trnH regions after assessing the sequence quality and coverage. All the generated sequences in this study were submitted to GenBank (Table 1).

Table 1. Sample Informations of Artemisia argyi and Counterfeits

Species	Collecting locality	Number	Sample number	GenBank accession number
				ITS2	psbA
Artemisia argyi	Ningbo, Zhejiang, China	10	A01001-10	KU855072-81	KU855207-16
	Hangzhou, Zhejiang, China	5	A01011-15	KU855082-86	KU855217-21
	Kunming, Yunnan, China	3	A01016-18	KU855087-89	KU855222-24
	Shengyang, Liaoning, China	1	A01019	KU855090	KU855225
	Fushun, Liaoning, China	1	A01020	KU855091	KU855226
	Dalian, Liaoning, China	6	A01021-26	KU855092-97	KU855227-32
	Benxi, Liaoning, China	1	A01027	KU855098	KU855233
	Nanjing, Jiangsu, China	5	A01028-32	KU855099-103	KU855234-38
	Ning, Hunan, China	1	A01033	KU855104	KU855239
	Shengnongjia, Hubei, China	2	A01034-35	KU855105-06	KU855240-41
	Qichun, Hubei, China	7	A01036-42	KU855107-13	KU855242-48
	Danjingkou, Hubei, China	1	A01043	KU855114	KU855249
	Haerbing, Heilongjiang, China	3	A01044-46	KU855115-17	KU855250-52
	Queshan, Henan, China	1	A01047	KU855118	KU855253
	Miyang, Henan, China	1	A01048	KU855119	KU855254
	Anguo, Hebei, China	3	A01049-51	KU855120-22	KU855255-57
	Nanxiong, Guangdong, China	1	A01052	KU855123	KU855258
	Guangzhou, Guangdong, China	1	A01053	KU855124	KU855259
	Lanzhou, Gansu, China	3	A01054-56	KU855125-27	KU855260-62
Artemisia lavandulifolia	Nanning, Guangxi, China	5	A02001-05	KU855155-59	KU855295-99
Artemisia indica	Chongqing, China	1	A03001	KU855141	KU855281
	Nanning, Guangxi, China	5	A03002-06	KU855142-46	KU855282-86
	Hongkong, China	1	A03007	KU855147	KU855287
	Zhongshan, Guangdong, China	3	A03008-10	KU855148-50	KU855288-90
	Ning, Hunan, China	2	A03011-12	KU855151-52	KU855291-92
	Jiaocheng, Shanxi, China	2	A03013-14	KU855153-54	KU855293-94
Artemisia capillaris	Beijing, China	6	A04001-06	KU855134-39	KU855269-74
	Yantai, Shandong, China	1	A04007	KU855140	KU855275
Artemisia anomala	Chongqing, China	10	A05001-10	KU855062-71	KU855197-206
Artemisia annua	Nanning, Guangxi, China	3	A06001-03	KU855054-56	KU855189-91
	Shengnongjia, Hubei, China	3	A06004-06	KU855057-59	KU855192-94
	Guilin, Guangxi, China	2	A06007-08	KU855060-61	KU855195-96
Artemisia aurata	Kunming, Yunnan, China	6	A07001-06	KU855128-33	KU855263-68
Artemisia maximowicziana	Haerbing, Heilongjiang, China	2	A08001-02	KU855160-61	KU855300-01
	Daqing, Heilongjiang, China	3	A08003-05	KU855162-64	KU855302-04
Artemisia vexans	Kunming, Yunnan, China	6	A09001-06	KU855340-45	KU855320-25
Artemisia tangutica	Guangzhou, Guangdong, China	4	A10001-04	KU855176-79	KU855316-19
Artemisia princeps	Taiwan, China	4	A11001-04	KU855165-68	KU855305-08
Artemisia igniaria	Nanyang, Henan, China	5	A12001-05	KU855335-39	KU855276-80
Artemisia stolonifera	Wenshan, Yunnan, China	4	A13001-04	KU855172-75	KU855312-15
Artemisia sieversiana	Shengnongjia, Hubei, China	3	A14001-03	KU855169-71	KU855309-11
Dendranthema vestitum	Dalian, Liaoning, China	3	A15001-03	KU855185-87	KU855331-33
	Beijing, China	1	A15004	KU855188	KU855334
Dendranthema indicum	Dalian, Liaoning, China	5	A16001-05	KU855180-84	KU855326-30

Fifty six samples of A. argyi were collected from Hubei, Hunan, Zhejiang, Jiangsu, Hebei, Henan, Guangdong, Yunnan, Gansu, Liaoning, and Heilongjiang provinces. ITS2 sequences and psbA-trnH sequences were submit to get the GenBank accession number.

Genetic Divergence Analysis

The sequence length, GC content, and K2P genetic distance of the ITS2 and psbA-trnH regions of A. argyi and its adulterants were analyzed and summarized (Table 2, Fig. 1). The ITS2 sequence length of A. argyi and 12 other closely related species and counterfeits was 225 bp, except for Artemisia capillaris, Artemisia maximowicziana, and Artemisia sieversiana, whose sequence lengths were 226, 228, and 227 bp, respectively. The average GC content of 56 ITS2 sequences of A. argyi was 56.4%. The counterfeits, namely, Dendranthema vestitum and Dendranthema indicum, had the lowest average GC content of 53.3% among the 16 species. No variable site existed in the 56 ITS2 sequences of A. argyi from 11 provinces. No variable site was detected in the 12 other species, including A. indica, A. anomala, Artemisia annua, Artemisia aurata, Artemisia vexans, Artemisia tangutica, A. princeps, Artemisia igniaria, A. stolonifera, A. sieversiana, D. vestitum, and D. indicum (Fig. 1). However, both A. lavandulifolia and A. maximowicziana had two variable sites, whereas A. capillaris had one variable site (Fig. 1). Single nucleotide polymorphism (SNP) identification is an efficient, rapid, and accurate method for identifying species.³³⁾ After multiple alignment, A. capillaris had 10 SNPs; A. anomala and A. annua had three SNPs; A. igniaria had two SNPs; and A. maximowicziana, A. princeps, D. vestitum, and D. indicum had one SNP (Fig. 1).

Table 2. Sequence Characters and K2P Genetic Distances of Artemisia argyi and Counterfeits

Species	Sample No.	ITS2				psbA-trnH				ITS2+psbA-trnH
		Length	G+C content (%)	Intraspecific distance (mean)	Interspecific distance (mean)	Length	G+C content (%)	Intraspecific distance (mean)	Interspecific distance (mean)	Length	G+C content (%)	Intraspecific distance (mean)	Interspecific distance (mean)
A. argyi	56	225	56.4	0	0.005–0.086 (0.032)	362	24.9	0–0.008 (0.002)	0–0.022 (0.007)	587	37	0–0.005 (0.001)	0.002–0.046 (0.017)
A. lavandulifolia	5	225	55.3	0–0.009 (0.005)	0.018–0.056 (0.029)	362	24.9	0	0–0.020 (0.004)	587	36.5	0–0.003 (0.002)	0.005–0.046 (0.013)
A. indica	14	225	56.9	0	0.005–0.081 (0.020)	362	25.1	0–0.003 (0.001)	0–0.020 (0.005)	587	37.2	0–0.002 (0.001)	0.002–0.042 (0.011)
A. capillaris	7	226	59.2	0–0.004 (0.002)	0.061–0.113 (0.082)	362–363	24.6	0	0.020–0.022 (0.020)	588–589	37.9	0–0.002 (0.001)	0.035–0.055 (0.043)
A. anomala	10	225	56.4	0	0.027–0.080 (0.045)	362	24.6	0	0.008–0.022 (0.014)	587	36.8	0	0.017–0.040 (0.026)
A. annua	8	225	54.7	0	0.023–0.076 (0.047)	353	25.2	0	0.003–0.017 (0.009)	578	36.7	0	0.014–0.039 (0.024)
A. aurata	6	225	56.9	0	0.005–0.071 (0.026)	362	24.6	0	0.003–0.017 (0.005)	587	37	0	0.003–0.037 (0.013)
A. maximowicziana	5	228	55.7	0–0.009 (0.004)	0–0.0081 (0.040)	362	25	0–0.003 (0.002)	0–0.020 (0.010)	590	36.9	0–0.003 (0.002)	0–0.040 (0.021)
A. vexans	6	225	57.3	0	0.005–0.076 (0.028)	362	24.9	0	0–0.020 (0.004)	587	37.3	0	0.002–0.041 (0.013)
A. tangutica	4	225	55.2	0	0.014–0.066 (0.031)	366	24.6	0	0–0.020 (0.005)	591	36.2	0	0.007–0.037 (0.014)
A. princeps	4	225	55.5	0	0.014–0.092 (0.034)	360–362	24.8	0	0–0.020 (0.005)	585–587	36.6	0	0.005–0.046 (0.016)
A. igniaria	5	225	54.3	0	0.014–0.113 (0.049)	362	25.1	0	0–0.022 (0.008)	587	36.3	0	0.007–0.055 (0.023)
A. stolonifera	4	225	55.1	0	0.005–0.102 (0.046)	362	24.9	0	0–0.020 (0.006)	587	36.4	0	0.002–0.050 (0.021)
A. sieversiana	3	227	55.6	0	0–0.076 (0.050)	362	25.1	0	0–0.017 (0.010)	589	36.8	0	0–0.039 (0.025)
D. vestitum	4	225	53.3	0	0.009–0.092 (0.038)	367	24.6	0	0–0.022 (0.005)	592	35.5	0	0.003–0.050 (0.017)
D. indicum	5	225	53.3	0	0.009–0.091 (0.062)	367	24.6	0	0–0.022 (0.010)	592	35.5	0	0.003–0.048 (0.029)

The sequence length, GC content, and K2P genetic distance of the ITS2 and psbA-trnH regions of A. argyi and its adulterants were analyzed.

Fig. 1. The Variable Site of of Artemisia argyi and Counterfeits (A: ITS2; B: psbA-trnH)

The highlighting of the bases with different colors represented the SNP sites.

For the psbA-trnH sequences, the sequence length of A. argyi and nine other species was 362 bp. A. capillaris and A. princeps had one and two insertions or deletions, respectively. The counterfeits D. vestitum and D. indicum had a maximum sequence length of 367 bp among the 16 species. The average GC content of A. argyi was 24.9%. Five variable sites were detected in the 56 psbA-trnH sequences of A. argyi. A. indica, A. capillaris, and A. maximowicziana had one variable site. After multiple alignment, A. capillaris had five SNPs at sites 79, 112, 142, 261, and 338. A. anomala had two SNPs at sites 114 and 363. A. annua had an SNP at site 114 and 13 bp deletions at site 304. A. tangutica had four insertions at site 309 (Fig. 1).

Genetic Distance and Barcoding Gap

The intraspecific divergence calculated using the K2P model indicated that the ITS2 sequence of A. argyi and 12 other closely related species and counterfeits exhibited zero intraspecific distance, which was the lowest (Table 2). The maximum intraspecific distance of all the species was lower than the minimum interspecific distance, except that of A. maximowicziana and A. sieversiana. In other words, A. argyi and 13 other closely related species and counterfeits can be distinguished from each other on the basis of the ITS2 sequence by using the distance method.

On the basis of the psbA-trnH sequences, the intraspecific distance of 13 species was zero (Table 2). The intraspecific and interspecific distances of A. argyi were 0–0.008 and 0–0.022, respectively. The maximum intraspecific distance of A. capillaris, A. anomala, A. annua, and A. aurata was lower than the minimum interspecific distance. For ITS2+ psbA-trnH, the maximum intraspecific distance of A. argyi was 0.005, which was higher than the minimum interspecific distance 0.002.

A barcode gap exists if the minimum interspecific variation is larger than the maximum intraspecific variation. Thus, the species can be identified affectively. The distribution of the intraspecific and interspecific variations of ITS2 exhibited distinct gaps at a scale of 0.001 distance units in A. argyi and 13 closely related species and counterfeits. All the 14 species fell above the 1 : 1 line, indicating the presence of a “barcode gap.” However, most of the species, except for A. capillaris, A. anomala, A. annua, and A. aurata, fell under or on the 1 : 1 line on the basis of the psbA-trnH sequences. For ITS2+ psbA-trnH, A. argyi fell under the 1 : 1 line, indicating that A. argyi could not be distinguished from other species (Fig. 2).

Fig. 2 Barcode Gap between Interspecific versus Intraspecific Divergences in Artemisia argyi and Counterfeits

The genetic distances were calculated using MEGA 6.0. Pairwise interspecific and intraspecific distances for each locus were analyzed using the Kimura 2-Parameter (K2P) model. The presence of a “barcode gap” was verified.

Thus, A. argyi could be distinguished from all the closely related species and counterfeits using the ITS2 sequence but not using the psbA-trnH and ITS2+ psbA-trnH sequences on the basis of the distance and barcode gap results.

Phylogenetic Tree Analysis

Phylogenetic trees were constructed using the ITS2, psbA-trnH, and ITS2+psbA-trnH sequences in accordance with the NJ, MP, and ML tree methods. Significant differences in the tree topologies were observed among the MP, ML, and NJ trees on the basis of the ITS2, psbA-trnH, and ITS2+psbA-trnH combinations. For the ITS2 sequence, the topologies of the ML, ME, and NJ trees were the same (Fig. 3). A. argyi and all the closely related species and counterfeits, except for A. maximowicziana, and A. sieversiana, were separated into pairs of divergent clusters in the ML, ME, and NJ trees (Fig. 3). Unfortunately, A. argyi and the closely related species and counterfeits could not be distinguished from each other on the basis of the psbA-trnH sequence using the tree methods. For the ITS2+psbA-trnH combination, A. argyi could be distinguished from the closely related species and counterfeits in the ML tree but not in the ME and NJ trees. Thus, A. argyi could be distinguished from the 15 closely related species and counterfeits on the basis of the ITS2 sequence by using the tree method.

Fig. 3. Neighbor–Joining (NJ) Tree Based on ITS2 Sequences of Artemisia argyi and Counterfeits

Neighbor–Joining (NJ) tree based on ITS2 sequences of Artemisia argyi and counterfeits calculated by MEGA 6.0 software with 1000 bootstrap replicates.

DISCUSSION

Artemisia is a large, diverse genus with over 300 species belonging to the family Asteraceae, most of which are mainly distributed in the Northern Hemisphere, as well as in Africa, Australia, and Central, and South America.^1,6,7) Artemisia comprises hardy herbaceous plants and shrubs, which are known for the powerful chemical constituents in their essential oils. A total of 186 Artemisia species (82 endemic) exist in China, 67 species and 8 varieties of which are medicinal plants.^1,34) Many species are rich in polyacetylenes, flavonoids, terpenoids, and cyanogenic glycosides and are well-known medicinal plants.^35–37) Notable species include A. annua, A. argyi, Artemisia dracunculus, and A. capillaris. A. annua is currently the only commercial source of the sesquiterpene lactone artemisinin, which is particularly important for the treatment of chloroquine-resistant strains of malaria.^38,39) A. argyi is used mostly for moxibustion^3,40) A. dracunculus is widely used as a culinary herb, particularly in French cuisine.^37,41) A. capillaris has been widely used to treat various hepatic disorders in traditional Oriental medicine.^42,43) Some species are used to stabilize sand in desert or semidesert areas or as herbage for feeding livestock.¹⁾

The Artemisia genus has many characteristics, such as abundant species, diverse species types, and complex genetic relationship.⁶⁾ Therefore, species identification of Artemisia is difficult because of their similar morphological characteristics. A. argyi is also hard to distinguish from closely related species and counterfeits on the basis of morphological characteristics alone.³⁾ In this study, we distinguished A. argyi from closely related species and counterfeits on the basis of ITS2, psbA-trnH, and ITS2+psbA-trnH sequences. In an ideal DNA barcode, PCR fragments should be easily amplified, and sequence-based identification should be accurate, rapid, cost effective, universally accessible, and useful for non-experts.^22,23,44) In the present study, high-quality DNA was easily extracted from all samples, and both ITS2 and psbA-trnH fragments can be easily amplified. Unlike that of the psbA-trnH and ITS2+psbA-trnH sequences, the genetic variation of the ITS2 sequence demonstrated separate and non-overlapping distributions between intraspecific and interspecific samples. Thus, ITS2 was an ideal barcode to identify A. argyi from closely related species and counterfeits in 146 samples. A. maximowicziana could be distinguished from A. sieversiana using SNP identification but not using the distance and tree methods. Thus, A. argyi and the 15 other closely related species and counterfeits could be distinguished from each other on the basis of the ITS2 sequences. However, the variation in psbA-trnH fragments was insufficient at the species level. Thus, A. argyi could not be distinguished from closely related species and counterfeits. The same outcome occurs in the ITS2+psbA-trnH combination. Our results are consistent with several previous studies in which ITS2 is the best DNA barcode to identify Artemisia species.^20,21)

A. argyi cv. qiai, a cultivar named by Yourun Lin, is distributed in Qichun County, Hubei Province. The variety exhibits the features of a large plant, has a strong aroma and high leaf thickness, and is suitable for moxibustion.^3,12,13) The ITS2 sequences of the seven samples of A. argyi cv. qiai collected from Qichun County were same as those of the other A. argyi samples from 10 provinces. For psbA-trnH sequence, two informative sites were not unique for the samples of A. argyi cv. qiai. Thus, both the ITS2 and psbA-trnH sequences could not be used to distinguish A. argyi cv. qiai from the other samples of A. argyi.

CONCLUSION

Folium Artemisiae Argyi is an important herb in traditional Chinese medicine and is commonly used. However, identifying A. argyi is difficult and the confusion with adulterants threatens the clinical efficacy and safety of this herb. In this paper, high-quality DNA was easily extracted from all samples, and both the ITS2 and psbA-trnH fragments can be easily amplified. Using the ITS2 sequence is a better approach to distinguish A. argyi from closely related species and counterfeits than using the psbA-trnH and ITS2+psbA-trnH combinations. No variable site was detected for the 56 ITS2 sequences of A. argyi. The intraspecific K2P distance (0) was lower than the minimum interspecific distance (0.005). All the ITS2 sequences of A. argyi clustered together in the NJ, MP, and ML trees. Thus, the ITS2 sequence was an ideal barcode to identify A. argyi from closely related species and counterfeits to ensure the safe use of this plant.

Acknowledgment

This research was supported by the Major Scientific and Technological Special Project for “Significant New Drugs Creation” (No. 2014ZX09304307).

Conflict of Interest

The authors declare no conflict of interest.

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