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Authenticity Survey of Cuscutae Semen on Markets Using DNA Barcoding
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Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 Available online at SciVarse ScienceDirect
Chinese Herbal Medicines (CHM) ISSN 1674-6384
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E-mail: [email protected]
Original article
Authenticity Survey of Cuscutae Semen on Markets Using DNA Barcoding Zi-tong Gao, Li-li Wang, Xiao-yue Wang, Yang Liu, Jian-ping Han* Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
ARTICLE INFO
ABSTRACT
Article history
Objective
Received: March, 2016 Revised: June 24, 2016 Accepted: January 18, 2017 Available online: July 17, 2017
DOI: 10.1016/S1674-6384(17)60098-4
Using authentic raw herbal materials is fundamental to herbal medicine
quality. Cuscuta chinensis and C. australis are two important species of Cuscutae Semen recorded in Chinese Pharmacopoeia. Due to having tiny bodies of seeds, it is extremely
difficult to differentiate them from adulterants and closely related species by morphologic characteristics, leading to serious safety problems. Methods
In this study,
we developed a fast and efficient method to identify Cuscutae Semen on the market.
First, a total of 207 ITS2 sequences representing 45 related species of Cuscutae Semen
were collected to construct a standard DNA barcode database, then 33 commercial samples purchased from markets were analyzed by BLAST, and Neighbor-joining tree was used to verify the efficacy of the database. Results
The percentage of counterfeits
and adulterants in the 33 commercial samples were up to 69.7%, and only 10
commercial products were found to be genuine. The adulterated species included 11
species (Amaranthus hybridus, Brassica carinata, Brassica juncea var. megarrhiza, Chenopodium album, Corispermum heptapotamicum, Cuscuta alata, Cuscuta japonica, Cuscuta monogyna, Foeniculum vulgare, Glycine max, and Medicago sativa). Conclusion DNA barcoding is a fast and efficient method to identify Cuscutae Semen on the market.
Key words
Cuscuta australis; Cuscuta chinensis; Cuscutae Semen; DNA barcoding; traditional
Chinese medicine
© 2017 published by TIPR Press. All rights reserved.
1. Introduction The presence of counterfeits in traditional Chinese medicines (TCMs) has been confirmed in commercial markets, leading to adverse effects, drug resistance, and even fatalities (Chen et al, 2012). As the treatment of diseases has been rapidly developed, the increase of TCMs import and export trade in China is followed. However, the frequent
occurrence of serious Chinese medicine incidents has been a bottleneck that impedes progress in TCM trades. Cuscutae Semen called Tusizi in Chinese, is an important Chinese medicinal material recorded in Chinese Pharmacopoeia 2015. As an upper grade drug recorded in Shen Nung’s Classic of Materia Medica, it is commonly used to nourish the liver and kidney, secure essence, reduce urination, prevent miscarriage, improve eyesight, and stop diarrhea (Pharmacopoeia Committee
啊 *
Corresponding author: Han JP
Tel: +86-10-5783 3198
E-mail: [email protected]
Fund: National Natural Science Foundation of China (No. 81673552)
Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 of P. R. China, 2015). Modern pharmacological studies show that flavonoids are the main bioactive ingredients in Cuscutae Semen (Donnapee et al, 2014) with anti-oxidant (Yen et al, 2007; 2008), anti-osteoporotic (Yang et al, 2011; Ye et al, 2014), anti-apoptosis (Sun et al, 2014), immuno-enhancement (Wang et al, 2000), anti-nociceptive, and anti-inflammatory effects (Liao et al, 2014). Cuscutae Semen botanically originates from the seeds of Cuscuta chinensis Lam and C. australis R. Br. However, some species that share similar morphological characteristics have been reported as adulterants (Lin, 2014; Gu and Yu, 2000; Zhang et al, 2000). The application of various adulterants inevitably threatens the safety in the clinical setting. However, specimens with tiny seeds may not be easily identified at species level by morphological characteristics (Liao et al, 2005). Thus, a fast and efficient authentication method for counterfeit or adulterant detection is needed. DNA barcoding is a molecular biological identification method that was first announced in 2003 by Herbert, a Canadian biologist, to identify animals through cytochrome c oxidase subunit 1 (COI) (Hebert et al, 2003a; 2003b). In the following decade, it was quickly developed and given high regard. Researchers compared the global DNA barcode innovation with the human genome project, suggesting that it could be a “big science” breakthrough (Hebert and Gregory, 2005). Miller indicated that DNA barcoding was a “Renaissance of taxonomy” (Miller, 2007). DNA barcoding has become a focus of research globally and a direction for taxonomy using biological methods. In recent years, with the proposal of various candidate markers (Pennisi, 2007; Hollingsworth, 2009), ribosomal internal transcribed spacer 2 (ITS2) was discovered as a core barcode for species determination and has subsequently been widely used (Chen et al, 2010; Yao et al, 2010; Han et al, 2013; Li et al, 2011). To date, this technology has been successfully employed to recognize various species, such as Apiaceae L. (Liu et al, 2014) and Rhodiola L. (Xin et al, 2015), Panax ginseng C. A. Mey. (Chen et al, 2013), P. notoginseng (Burkill) F. H. Chen ex C. Y. Wu & K. M. Feng (Liao et al, 2013), and species of Ganoderma L. (Liao et al, 2015). DNA barcoding has shown to be a very promising and efficient tool for the identification of TCMs (Chen et al, 2013; Shokralla et at, 2014; Yang et al, 2014). All the above provided evidences that DNA barcoding offers convenience and effectiveness in species authentication. In this paper, we reported the results of Cuscutae Semen inspection using DNA barcoding. First, 207 sequences representing 45 species including Cuscutae Semen and its adulterants were collected to build a standard sequence database, and then 33 commercial samples purchased from stores or markets were analyzed. Additional details are described in subsequent sections.
2. Materials and methods 2.1 Plant materials Sixty-one specimens were collected from different
219
habitats in China. All the voucher specimens were identified by Prof. Yu-lin Lin (Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Science and Peking Union Medical College) and deposited at the herbarium of IMPLAD, Chinese Academy of Medical Sciences and Peking Union Medical College. Other sequences were downloaded from GenBank. A total of 207 sequences representing 45 species were used to establish a barcode database. Thirty-three batches of commercial Cuscutae Semen were purchased from the medicinal material markets, drug stores, and hospitals to carry out a species survey (Table 1). Photographs of some seeds were taken using a Digital Microscope System (SZX-ILLB2−200, Olympus Corporation, Japan, Figure 1).
2.2 DNA extraction, PCR amplification and sequencing Specimens: Specimens (20 mg each) were powdered using Mixer Mill MM400 (Retsch GmbH, Haan, Germany) at 30 r/s for 2 min. Total genomic DNA was extracted using a Plant Genomic DNA kit (Tiangen Biotech Co., Ltd.). ITS2 was amplified following DNA barcoding standard operating procedure (SOP) developed by Chen et al (Chen et al, 2010; 2014). Then, agarose gel electrophoresis was applied to detect the results preliminarily. All the PCR products were sent to Chinese Academy of Agricultural Sciences for sequencing. Commercial samples: DNA from commercial samples was amplified using LA Taq polymerase (Takara). The 25 µL reaction contained 0.25 µL TaKaRa LA Taq®, 2.5 µL 10 × LA Taq Buffer II (Mg2+ plus/free), 2 µL dNTP mix, 1 µL primers (2.5 µmol/L, ITS2F/3R), 1 µL genomic DNA template and 17.25 µL ddH2O. The PCR procedure was the same as above. 1 × TAE Gel Agarose Gel Electrophoresis was used for gel extraction, and each well contained all 25 µL of the PCR product together with 3 µL 6 × loading buffer. DNA was recovered using a Gel Extraction Kit (Tiangen Biotech Co., Ltd.), cloned to the pMD® 18-T vector (TaKaRa Bio, Japan) and transformed into Escherichia coli (Tiangen Biotech Co., Ltd., China) following the protocol described in Molecular Cloning: A Laboratory Manual (Russell, 2005). The final bacterial suspension was sent for sequencing.
2.3 Data analysis CodonCode Aligner 4.2.7 (CodonCode Corporation, USA) was employed to assemble raw data, trim vectors and delete low-quality regions. All ITS2 sequences obtained from GenBank were extracted from published articles or verified by BLAST in GenBank and the TCM database (http://tcmbarcode.cn/china/). The fungal sequences and the sequences in length of less than 100 bp were removed. Then, the ITS2 region was annotated based on the Hidden Markov model (HMM) (Keller et al, 2009) to delete 5.8S and 28S regions at the two ends of the sequence. The consensus sequences were compared to the sequence database built in
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Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 Table 1
Species composition of commercial Cuscutae Semen
Sample No.
BLAST results
Locations in China
C001
Amaranthus hybridus, Brassica carinata, Chenopodium, Cuscuta australis
Hebei
C002
Amaranthus hybridus, Cuscuta australis
Hebei
C003
Amaranthus hybridus, Cuscuta australis
Hebei
C010
Amaranthus hybridus, Cuscuta australis
Beijing
C011
Amaranthus hybridus, Cuscuta australis
Inner Mongolia
C012
Cuscuta australis, Amaranthus
Inner Mongolia
C013
Amaranthus hybridus, Cuscuta australis, Medicago sativa, Setaria
Inner Mongolia
C014
Amaranthus hybridus
Inner Mongolia
C015
Amaranthus hybridus, Cuscuta australis
Inner Mongolia
C016
Amaranthus hybridus, Setaria, Glycine max
Inner Mongolia
C017
Amaranthus hybridus, Cuscuta australis
Inner Mongolia
C018
Amaranthus hybridus, Cuscuta australis
Inner Mongolia
C019
Amaranthus hybridus
Sinkiang
C030
Cuscuta australis, Brassica carinata, Brassica juncea var. megarrhiza
Inner Mongolia
C031
Chenopodium album
Jiangsu
C032
Cuscuta australis, Chenopodium album
Guangxi
C033
Chenopodium, Cuscuta australis, Cuscuta, Foeniculum vulgare,
Shanxi
C034
Chenopodium, Cuscuta australis,
Hebei
C035
Chenopodium, Cuscuta australis,
Shanxi
Cuscuta australis, Corispermum heptapotamicum, Cuscuta chinensis, C036
Cuscuta alata
Inner Mongolia
C043
Cuscuta chinensis
Anhui
C044
Cuscuta chinensis
Inner Mongolia
C046
Cuscuta chinensis
Anhui
C047
Cuscuta australis
Anhui
C048
Cuscuta japonica
Sichuan
C050
Cuscuta australis, Glycine max
Inner Mongolia
C051
Cuscuta australis
Inner Mongolia
C052
Cuscuta australis
Beijing
C053
Cuscuta australis, Cuscuta, Foeniculum vulgare, Umbelliferae
Inner Mongolia
C072
Cuscuta australis
Chongqing
C073
Cuscuta australis
Henan
C074
Cuscuta australis
Sinkiang
C075
Cuscuta australis
Beijing
Figure 1
Morphological structures of commercial Cuscutae Semen and adulterants
1−4, 6 and 8: Cuscuta australis; 5: Amaranthus hybridus; 7: Cuscuta epilinum Seeds with different morphological characteristics were screened out of samples using naked eyes, and photographed using Digital Microscope System.
Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 this study for sample identification using a Blast search. Mega 6.0 (Tamura et al, 2013) was employed for sequence characteristic analysis. G + C content was analyzed. Based on the p-distance model, genetic distances were calculated. Haplotypes in the database and commercial samples were selected to construct a Neighbor-joining tree (NJ tree). The NJ tree was built on the basis of p-distance with a bootstrap test (1000 replicates) to verify the identification efficacy of the standard DNA barcode database. SNP sites were detected with CodonCode Aligner 4.2.7.
3. Results 3.1 Establishment of standard Cuscutae Semen DNA barcode database For
all
61
specimens,
the
efficiency
of
Table 2
Items
Sequences
G+C content / %
PCR
221
amplification and sequencing was 100%. The other 146 ITS2 sequences of genus Cuscuta Linn. and adulterants were downloaded from GenBank. A total of 207 sequences representing 45 species were used to establish a standard Cuscutae Semen DNA barcode database. The sequence lengths were 181 to 237 bp (Table 2). The average G + C content was 55.6%, and the average interspecific distance between the 45 species was 0.444. Thirty-six sequences of C. australis and 15 sequences of C. chinensis were contained in the database. The sequence length of C. australis was 211 bp. There were 10 variable sites in C. australis that were used to divide the 36 sequences into six haplotypes. The sequence lengths of C. chinensis were 208–216 bp. There were seven variable sites in C. chinensis. The average and maximum intraspecific distances of C. chinensis were 0.005 and 0.024, respectively, and the average and maximum intraspecific distances of C. australis were 0.019 and 0.043, respectively.
Sequencing information of ITS2
Sequence length / bp
Average
Average
interspecific
intraspecific
distance
distance
Max intraspecific distance
Database
207
55.60
181−237
0.444
−
−
Commercial Cuscutae
329
56.40
189−231
−
−
−
36
53.89
211
−
0.019
0.043
15
51.93
208−216
−
0.005
0.024
Semen C. australis in database C. chinensis in database “−” represents lack of related information.
3.2 Identification of commercial Cuscutae Semen with BLAST method A total of 33 commercial samples labeled “Tusizi” were purchased from medical markets (10 specimens), drug stores (22 specimens) and hospitals (one specimen; Table 1). The PCR amplification success rate of ITS2 region was 100%. Fifteen clones per sample were selected for sequencing. A total of 329 sequences were obtained. The sequence lengths ranged from 189 to 231 bp (Table 2). Using BLAST and the database built in this study, five of 33 samples were found to be substituted with other species. Two batches were Amaranthus hybridus from Inner Mongolia and Jiangsu province, China. One batch was Cuscuta japonica, one batch was Chenopodium album, and one batch was a mixture of Amaranthus hybridus, Setaria spp. and Glycine max. Eighteen samples were detected to be adulterated with one to three other species. The adulterant species were mainly Amaranthus hybridus, Brassica carinata, Brassica juncea var. megarrhiza, Chenopodium album, Corispermum heptapotamicum, Cuscuta alata, Cuscuta japonica, Foeniculum vulgare, Glycine max and Medicago sativa. The percentage of counterfeits and adulterants in the 33 commercial samples was up to 69.7%. The other 10 commercial products were found to be identical
to the description on their labels.
3.3 Identification of commercial samples using NJ tree and single nucleotide polymorphisms (SNPs) To verify the BLAST identification results, an NJ Tree was constructed using haplotypes of species in the database and the commercial Cuscutae Semen (Figure 2). The results demonstrated the two original plant species of Cuscutae Semen (C. australis and C. chinensis) clustered in genus Cuscuta Linn. C. obtusiflora is the synonym of C. australis in the Scientific Database of China Plant Species (DCP) (http://www.plants.csdb.cn/eflora/help/shouye.aspx); Therefore, C. obtusiflora and its variants were both clustered in C. australis clade. In addition, C. applanata is the synonym of C. chinensis (Costea et al, 2011), and their sequences clustered within one clade. All the other species were clustered into their own clades, except for C. australis, C. pentagona, and C. campestris. By analyzing the haplotypes of the three species, we found two SNP sites among them; i.e., 126 bp T/C and 172 bp T/G sites could distinguish C. australis from the other two (Table 3). The SNP analysis was a complementary method for DNA barcoding identification of Cuscutae Semen in this study.
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Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225
Figure 2 NJ tree for all haplotypes of database and commercial Cuscutae Semen All ITS2 sequences from commercial Cuscutae Semen and database were aligned separately according to species using Codoncode Aligner 4.2.7 and finally haplotypes were determined. All haplotypes were used to construct NJ tree based on p-distance. The bootstrap scores (1000 replicates) were shown (≥ 50%) for each branch. Sequence number marked red and end with letters “CM” represent sequences harvest from commercial Cuscutae Semen, while sequence number marked black and end with letters “DB” represent sequences using for database construction.
Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 Table 3 Haplotypes
223
Variable sites in haplotypes of C. australis, C. campestris and C. pentagona Positions / bp
5
21
22
36
93
115
126
133
158
172
173
194
196
199
C. australis H1
T
G
T
A
T
T
T
T
G
T
C
G
T
G
C. australis H2
C
G
T
A
G
A
T
G
A
T
T
T
T
T
C. australis H3
C
G
T
A
G
A
T
G
A
T
T
T
T
K
C. australis H4
C
G
T
A
T
A
T
G
A
T
T
T
T
C
C. australis H5
T
G
T
A
T
A
T
G
A
T
T
G
T
T
C. australis H6
T
R
K
A
T
T
T
A
G
T
C
G
T
G
C. campestris
T
G
T
A
T
T
C
T
G
G
C
G
T
G
C. pentagona H1
T
G
T
A
T
T
C
T
G
G
C
G
T
G
C. pentagona H2
T
G
T
G
T
T
C
T
G
G
C
G
T
G
C. pentagona H3
C
G
T
A
G
T
C
T
G
G
C
G
C
G
4. Discussion 4.1 Why did we select ITS2 to identify Cuscutae Semen? Recovery of sequences is such an important process in DNA barcode identification (Hebert et al, 2004; Stoeckle et al, 2011) that qualified DNA is foundational. However, once Cuscuta seeds are soaked in water, mucilage will emerge (Pharmacopoeia Committee of P. R. China, 2015). This is a practical identification characteristic of the genus Cuscuta L. However, the mucilage is also an obstacle for DNA extraction. CTAB extraction buffer causes genome DNA to be released from the cell, and it also makes the mucilage release simultaneously, which leads to the solution becoming extremely sticky and impossible to centrifuge in the subsequent procedures. Therefore, we decreased the sample quantity to approximately 20 mg while the volume of extraction buffer was kept consistent in the protocols. By this way, mucilage could be eliminated more sufficiently. To obtain enough DNA for amplification, we increased the isopropanol precipitate time to a half an hour in a -20 ℃ refrigerator and decreased the volume of water in the solution to 30 µL. Finally, qualified DNA was obtained. Gao et al confirmed that ITS (approximately 650 bp) was suitable for Cuscutae Semen identification (Gao et al, 2006). However, due to low PCR amplification efficiency and the lack of a universal primer, ITS was excluded as a universal land plant barcode in the earlier stages (Chen et al, 2010). In the present study, to resolve all the adulterants in the commercial “Tusizi”, we selected the universal medicinal barcode ITS2 as the core barcode to guarantee a high rate of species identification. After verification, ITS2 served as a more universal and efficient DNA barcode with a rate of successful identification of 92.7% at the species level, which was superior to other candidate barcodes (Chen et al, 2010). The relatively short sequence ITS2 (450 bp) made amplification easier in the Cuscuta genus. Based on the above, we finally selected ITS2 as a core barcode. In the current study, the NJ tree showed C. australis had a closer relationship with C. pentagona and C. campestris, and SNPs should be used in a complementary identification procedure. Therefore, DNA barcoding
accompanied with SNP sites could successfully identify Cuscutae Semen from its adulterants and closely related species.
4.2 DNA barcoding has potential power for detecting counterfeits in TCM marketplace With the great success of DNA barcoding, it has been highly praised as producing a renaissance in authenticating herbal medicine (Chen et al, 2014). Its application has expanded to commercial dietary identifications (Xin et al, 2015; Little et al, 2013; Zhao et al, 2015), and it has been successfully used to identify various commercial TCMs. Zhao et al used ITS2 as a core barcode and successfully detected five adulterants in the nine Acanthopanacis Cortex specimens available to customers. Among the adulterants, a toxic Periplocae Cortex was found most often with a percentage of 80% (Zhao et al, 2015). Xin et al built a reference database with ITS2 sequences of authoritative species in Rhodiola L. Using this database, they found that 60% of commercial Rhodiolae Crenulatae Radix et Rhizoma decoction samples were counterfeits (Xin et al, 2015). What is more, Chinese caterpillar fungus (Xiang et al, 2013) and medicinal species in Panax L. (Chen et al, 2013) have been successfully authenticated by DNA barcoding. In the present study, we first used ITS2 to detect the species composition of commercial Cuscutae Semen, and 11 species (Amaranthus hybridus, Brassica carinata, Brassica juncea var. megarrhiza, Chenopodium album, Corispermum heptapotamicum, Cuscuta alata, Cuscuta japonica, Cuscuta monogyna, Foeniculum vulgare, Glycine max, and Medicago sativa) were found to be an adulterant or substitute for the commercial Cuscutae Semen (Table 1). Five of 33 commercial “Tusizi” were found to be substituted by other species. Eighteen were detected to be adulterated with one to three other species. The percentage of counterfeits and adulterants in the 33 commercial samples was up to 69.7%, and only 10 commercial products were found to be genuine. These results indicated that the ratio of adulteration was surprisingly high and is a great threat in quality supervision of TCM. DNA barcoding can serve as an efficient, simple and convenient method to supervise and administrate applications for TCM (Xin et al, 2013; Chen et al,
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2013). During the collection of TCMs, we found that C. australis (34 specimens) comprised the majority on market, while C. chinensis (eight specimens) was relatively rare.
4.3 DNA barcoding technology can meet demands of rapid customs clearance. In addition to medical use in TCM, Cuscutae Semen is also an important inspected and quarantined weed that is distributed around the globe (Costea et al, 2011). In China, according to the governmental list promulgated by the General Administration of Quality Supervision, Inspection and Quarantine of P. R. China, all the species in genus Cuscuta L. are banned from entering China. In 2009 to 2011 alone, a total of 63 quarantined weeds (in 12 families and 37 genera) were intercepted by the inspection institutions in China (Hu et al, 2014). Among them, seven species in the genus Cuscuta were detected from 37 batches of goods, such as soybeans. Considering the tiny seeds of Cuscuta Linn., morphological identification is limited (Liao et al, 2005), and the identification time is long. With the frequency and ease of international communications, monitoring situations of inspection and quarantine has become increasingly important. In the present study, DNA barcoding could be used to successfully detect Setaria spp. and Glycine max in commercial Cuscutae Semen, and it would be possible for the Cuscuta L. species to be mixed in the seeds of Glycine max and Setaria spp., which could also be successfully detected. The DNA barcoding method built and used in the current study undoubtedly could serve as an accurate, rapid, simple method for rapid quarantined clearance that is required by several quarantine departments.
5. Conclusion In this article, a standard Cuscutae Semen DNA barcode database was established and used to analyze the composition of commercial Cuscutae Semen. Among the 33 commercial samples analyzed, the percentage of counterfeits and adulterants was up to 69.7%. The results provide a useful tool for herbal market supervision and rapid customs clearance. Acknowledgments We are grateful to Prof. Yu-lin Lin for the identification of voucher specimens. Conflict of interest statement The authors declare no conflict of interest. References Chen SL, Pang XH, Song JY, Shi LC, Yao H, Han JP, Leon C, 2014. A renaissance in herbal medicine identification: From morphology to DNA. Biotechnol Adv 32: 1237-1244. Chen SL, Yao H, Han JP, Liu C, Song JY, Shi LC, Zhu YJ, Ma XY, Gao T, Pang XH, 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PloS One
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Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 floral variations of the genus Cuscuta in Taiwan. Taiwania 50: 123-130. Liao JC, Chang WT, Lee MS, Chiu YJ, Chao WK, Lin YC, Lin MK, Peng WH, 2014. Antinociceptive and anti-inflammatory activities of Cuscuta chinensis seeds in mice. Am J Chin Med 42: 223-242. Lin HB, 2012. Identification studies on different hosts of Semen Cuscutae to use gel electrophoresis. J Sichuan Tradit Chin Med 30: 62-63. Little DP, Jeanson ML, 2013. DNA barcode authentication of saw palmetto herbal dietary supplements. Sci Rep 3(15): 3518. Liu JX, Shi LC, Han JP, Li G, Lu H, Hou JY, Zhou XT, Meng FY, Downie SR, 2014. Identification of species in the angiosperm family Apiaceae using DNA barcodes. Mol Ecol Resour 14: 1231-1238. Miller SE, 2007. DNA barcoding and the renaissance of taxonomy. Proc Natl Acad Sci 104: 4775-4776. Pennisi E, 2007. Wanted: A barcode for plants. Science 318: 190-191. Pharmacopoeia Committee of P. R. China, 2015. Pharmacopoeia of People’s Republic of China. Beijing: China Medical Science and Technology Press. Russell JSDW, 2005. Molecular Cloning: A Laboratory Manual. Science Press: Beijing. Shokralla S, Gibson JF, Nikbakht H, Janzen DH, Hallwachs W, Hajibabaei M, 2014. Next generation DNA barcoding: Using next-generation sequencing to enhance and accelerate DNA barcode capture from single specimens. Mol Ecol Resour 14: 892-901. Stoeckle MY, Gamble CC, Kirpekar R, Young G, Ahmed S, Little DP, 2011. Commercial teas highlight plant DNA barcode identification successes and obstacles. Sci Rep 1(7): 42. Sun SL, Guo L, Ren YC, Wang B, Li RH, Qi YS, Yu H, Chang ND, Li MH, Peng HS, 2014. Anti-apoptosis effect of polysaccharide isolated from the seeds of Cuscuta chinensis Lam on cardiomyocytes in aging rats. Mol Biol Rep 41: 6117-6124. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 2725-2729. Wang Z, Fang JN, Ge DL, Li XY, 2000. Chemical characterization and immunological activities of an acidic polysaccharide isolated
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Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 Available online at SciVarse ScienceDirect
Chinese Herbal Medicines (CHM) ISSN 1674-6384
Journal homepage: www.tiprpress.com
E-mail: [email protected]
Original article
Authenticity Survey of Cuscutae Semen on Markets Using DNA Barcoding Zi-tong Gao, Li-li Wang, Xiao-yue Wang, Yang Liu, Jian-ping Han* Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
ARTICLE INFO
ABSTRACT
Article history
Objective
Received: March, 2016 Revised: June 24, 2016 Accepted: January 18, 2017 Available online: July 17, 2017
DOI: 10.1016/S1674-6384(17)60098-4
Using authentic raw herbal materials is fundamental to herbal medicine
quality. Cuscuta chinensis and C. australis are two important species of Cuscutae Semen recorded in Chinese Pharmacopoeia. Due to having tiny bodies of seeds, it is extremely
difficult to differentiate them from adulterants and closely related species by morphologic characteristics, leading to serious safety problems. Methods
In this study,
we developed a fast and efficient method to identify Cuscutae Semen on the market.
First, a total of 207 ITS2 sequences representing 45 related species of Cuscutae Semen
were collected to construct a standard DNA barcode database, then 33 commercial samples purchased from markets were analyzed by BLAST, and Neighbor-joining tree was used to verify the efficacy of the database. Results
The percentage of counterfeits
and adulterants in the 33 commercial samples were up to 69.7%, and only 10
commercial products were found to be genuine. The adulterated species included 11
species (Amaranthus hybridus, Brassica carinata, Brassica juncea var. megarrhiza, Chenopodium album, Corispermum heptapotamicum, Cuscuta alata, Cuscuta japonica, Cuscuta monogyna, Foeniculum vulgare, Glycine max, and Medicago sativa). Conclusion DNA barcoding is a fast and efficient method to identify Cuscutae Semen on the market.
Key words
Cuscuta australis; Cuscuta chinensis; Cuscutae Semen; DNA barcoding; traditional
Chinese medicine
© 2017 published by TIPR Press. All rights reserved.
1. Introduction The presence of counterfeits in traditional Chinese medicines (TCMs) has been confirmed in commercial markets, leading to adverse effects, drug resistance, and even fatalities (Chen et al, 2012). As the treatment of diseases has been rapidly developed, the increase of TCMs import and export trade in China is followed. However, the frequent
occurrence of serious Chinese medicine incidents has been a bottleneck that impedes progress in TCM trades. Cuscutae Semen called Tusizi in Chinese, is an important Chinese medicinal material recorded in Chinese Pharmacopoeia 2015. As an upper grade drug recorded in Shen Nung’s Classic of Materia Medica, it is commonly used to nourish the liver and kidney, secure essence, reduce urination, prevent miscarriage, improve eyesight, and stop diarrhea (Pharmacopoeia Committee
啊 *
Corresponding author: Han JP
Tel: +86-10-5783 3198
E-mail: [email protected]
Fund: National Natural Science Foundation of China (No. 81673552)
Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 of P. R. China, 2015). Modern pharmacological studies show that flavonoids are the main bioactive ingredients in Cuscutae Semen (Donnapee et al, 2014) with anti-oxidant (Yen et al, 2007; 2008), anti-osteoporotic (Yang et al, 2011; Ye et al, 2014), anti-apoptosis (Sun et al, 2014), immuno-enhancement (Wang et al, 2000), anti-nociceptive, and anti-inflammatory effects (Liao et al, 2014). Cuscutae Semen botanically originates from the seeds of Cuscuta chinensis Lam and C. australis R. Br. However, some species that share similar morphological characteristics have been reported as adulterants (Lin, 2014; Gu and Yu, 2000; Zhang et al, 2000). The application of various adulterants inevitably threatens the safety in the clinical setting. However, specimens with tiny seeds may not be easily identified at species level by morphological characteristics (Liao et al, 2005). Thus, a fast and efficient authentication method for counterfeit or adulterant detection is needed. DNA barcoding is a molecular biological identification method that was first announced in 2003 by Herbert, a Canadian biologist, to identify animals through cytochrome c oxidase subunit 1 (COI) (Hebert et al, 2003a; 2003b). In the following decade, it was quickly developed and given high regard. Researchers compared the global DNA barcode innovation with the human genome project, suggesting that it could be a “big science” breakthrough (Hebert and Gregory, 2005). Miller indicated that DNA barcoding was a “Renaissance of taxonomy” (Miller, 2007). DNA barcoding has become a focus of research globally and a direction for taxonomy using biological methods. In recent years, with the proposal of various candidate markers (Pennisi, 2007; Hollingsworth, 2009), ribosomal internal transcribed spacer 2 (ITS2) was discovered as a core barcode for species determination and has subsequently been widely used (Chen et al, 2010; Yao et al, 2010; Han et al, 2013; Li et al, 2011). To date, this technology has been successfully employed to recognize various species, such as Apiaceae L. (Liu et al, 2014) and Rhodiola L. (Xin et al, 2015), Panax ginseng C. A. Mey. (Chen et al, 2013), P. notoginseng (Burkill) F. H. Chen ex C. Y. Wu & K. M. Feng (Liao et al, 2013), and species of Ganoderma L. (Liao et al, 2015). DNA barcoding has shown to be a very promising and efficient tool for the identification of TCMs (Chen et al, 2013; Shokralla et at, 2014; Yang et al, 2014). All the above provided evidences that DNA barcoding offers convenience and effectiveness in species authentication. In this paper, we reported the results of Cuscutae Semen inspection using DNA barcoding. First, 207 sequences representing 45 species including Cuscutae Semen and its adulterants were collected to build a standard sequence database, and then 33 commercial samples purchased from stores or markets were analyzed. Additional details are described in subsequent sections.
2. Materials and methods 2.1 Plant materials Sixty-one specimens were collected from different
219
habitats in China. All the voucher specimens were identified by Prof. Yu-lin Lin (Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Science and Peking Union Medical College) and deposited at the herbarium of IMPLAD, Chinese Academy of Medical Sciences and Peking Union Medical College. Other sequences were downloaded from GenBank. A total of 207 sequences representing 45 species were used to establish a barcode database. Thirty-three batches of commercial Cuscutae Semen were purchased from the medicinal material markets, drug stores, and hospitals to carry out a species survey (Table 1). Photographs of some seeds were taken using a Digital Microscope System (SZX-ILLB2−200, Olympus Corporation, Japan, Figure 1).
2.2 DNA extraction, PCR amplification and sequencing Specimens: Specimens (20 mg each) were powdered using Mixer Mill MM400 (Retsch GmbH, Haan, Germany) at 30 r/s for 2 min. Total genomic DNA was extracted using a Plant Genomic DNA kit (Tiangen Biotech Co., Ltd.). ITS2 was amplified following DNA barcoding standard operating procedure (SOP) developed by Chen et al (Chen et al, 2010; 2014). Then, agarose gel electrophoresis was applied to detect the results preliminarily. All the PCR products were sent to Chinese Academy of Agricultural Sciences for sequencing. Commercial samples: DNA from commercial samples was amplified using LA Taq polymerase (Takara). The 25 µL reaction contained 0.25 µL TaKaRa LA Taq®, 2.5 µL 10 × LA Taq Buffer II (Mg2+ plus/free), 2 µL dNTP mix, 1 µL primers (2.5 µmol/L, ITS2F/3R), 1 µL genomic DNA template and 17.25 µL ddH2O. The PCR procedure was the same as above. 1 × TAE Gel Agarose Gel Electrophoresis was used for gel extraction, and each well contained all 25 µL of the PCR product together with 3 µL 6 × loading buffer. DNA was recovered using a Gel Extraction Kit (Tiangen Biotech Co., Ltd.), cloned to the pMD® 18-T vector (TaKaRa Bio, Japan) and transformed into Escherichia coli (Tiangen Biotech Co., Ltd., China) following the protocol described in Molecular Cloning: A Laboratory Manual (Russell, 2005). The final bacterial suspension was sent for sequencing.
2.3 Data analysis CodonCode Aligner 4.2.7 (CodonCode Corporation, USA) was employed to assemble raw data, trim vectors and delete low-quality regions. All ITS2 sequences obtained from GenBank were extracted from published articles or verified by BLAST in GenBank and the TCM database (http://tcmbarcode.cn/china/). The fungal sequences and the sequences in length of less than 100 bp were removed. Then, the ITS2 region was annotated based on the Hidden Markov model (HMM) (Keller et al, 2009) to delete 5.8S and 28S regions at the two ends of the sequence. The consensus sequences were compared to the sequence database built in
220
Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 Table 1
Species composition of commercial Cuscutae Semen
Sample No.
BLAST results
Locations in China
C001
Amaranthus hybridus, Brassica carinata, Chenopodium, Cuscuta australis
Hebei
C002
Amaranthus hybridus, Cuscuta australis
Hebei
C003
Amaranthus hybridus, Cuscuta australis
Hebei
C010
Amaranthus hybridus, Cuscuta australis
Beijing
C011
Amaranthus hybridus, Cuscuta australis
Inner Mongolia
C012
Cuscuta australis, Amaranthus
Inner Mongolia
C013
Amaranthus hybridus, Cuscuta australis, Medicago sativa, Setaria
Inner Mongolia
C014
Amaranthus hybridus
Inner Mongolia
C015
Amaranthus hybridus, Cuscuta australis
Inner Mongolia
C016
Amaranthus hybridus, Setaria, Glycine max
Inner Mongolia
C017
Amaranthus hybridus, Cuscuta australis
Inner Mongolia
C018
Amaranthus hybridus, Cuscuta australis
Inner Mongolia
C019
Amaranthus hybridus
Sinkiang
C030
Cuscuta australis, Brassica carinata, Brassica juncea var. megarrhiza
Inner Mongolia
C031
Chenopodium album
Jiangsu
C032
Cuscuta australis, Chenopodium album
Guangxi
C033
Chenopodium, Cuscuta australis, Cuscuta, Foeniculum vulgare,
Shanxi
C034
Chenopodium, Cuscuta australis,
Hebei
C035
Chenopodium, Cuscuta australis,
Shanxi
Cuscuta australis, Corispermum heptapotamicum, Cuscuta chinensis, C036
Cuscuta alata
Inner Mongolia
C043
Cuscuta chinensis
Anhui
C044
Cuscuta chinensis
Inner Mongolia
C046
Cuscuta chinensis
Anhui
C047
Cuscuta australis
Anhui
C048
Cuscuta japonica
Sichuan
C050
Cuscuta australis, Glycine max
Inner Mongolia
C051
Cuscuta australis
Inner Mongolia
C052
Cuscuta australis
Beijing
C053
Cuscuta australis, Cuscuta, Foeniculum vulgare, Umbelliferae
Inner Mongolia
C072
Cuscuta australis
Chongqing
C073
Cuscuta australis
Henan
C074
Cuscuta australis
Sinkiang
C075
Cuscuta australis
Beijing
Figure 1
Morphological structures of commercial Cuscutae Semen and adulterants
1−4, 6 and 8: Cuscuta australis; 5: Amaranthus hybridus; 7: Cuscuta epilinum Seeds with different morphological characteristics were screened out of samples using naked eyes, and photographed using Digital Microscope System.
Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 this study for sample identification using a Blast search. Mega 6.0 (Tamura et al, 2013) was employed for sequence characteristic analysis. G + C content was analyzed. Based on the p-distance model, genetic distances were calculated. Haplotypes in the database and commercial samples were selected to construct a Neighbor-joining tree (NJ tree). The NJ tree was built on the basis of p-distance with a bootstrap test (1000 replicates) to verify the identification efficacy of the standard DNA barcode database. SNP sites were detected with CodonCode Aligner 4.2.7.
3. Results 3.1 Establishment of standard Cuscutae Semen DNA barcode database For
all
61
specimens,
the
efficiency
of
Table 2
Items
Sequences
G+C content / %
PCR
221
amplification and sequencing was 100%. The other 146 ITS2 sequences of genus Cuscuta Linn. and adulterants were downloaded from GenBank. A total of 207 sequences representing 45 species were used to establish a standard Cuscutae Semen DNA barcode database. The sequence lengths were 181 to 237 bp (Table 2). The average G + C content was 55.6%, and the average interspecific distance between the 45 species was 0.444. Thirty-six sequences of C. australis and 15 sequences of C. chinensis were contained in the database. The sequence length of C. australis was 211 bp. There were 10 variable sites in C. australis that were used to divide the 36 sequences into six haplotypes. The sequence lengths of C. chinensis were 208–216 bp. There were seven variable sites in C. chinensis. The average and maximum intraspecific distances of C. chinensis were 0.005 and 0.024, respectively, and the average and maximum intraspecific distances of C. australis were 0.019 and 0.043, respectively.
Sequencing information of ITS2
Sequence length / bp
Average
Average
interspecific
intraspecific
distance
distance
Max intraspecific distance
Database
207
55.60
181−237
0.444
−
−
Commercial Cuscutae
329
56.40
189−231
−
−
−
36
53.89
211
−
0.019
0.043
15
51.93
208−216
−
0.005
0.024
Semen C. australis in database C. chinensis in database “−” represents lack of related information.
3.2 Identification of commercial Cuscutae Semen with BLAST method A total of 33 commercial samples labeled “Tusizi” were purchased from medical markets (10 specimens), drug stores (22 specimens) and hospitals (one specimen; Table 1). The PCR amplification success rate of ITS2 region was 100%. Fifteen clones per sample were selected for sequencing. A total of 329 sequences were obtained. The sequence lengths ranged from 189 to 231 bp (Table 2). Using BLAST and the database built in this study, five of 33 samples were found to be substituted with other species. Two batches were Amaranthus hybridus from Inner Mongolia and Jiangsu province, China. One batch was Cuscuta japonica, one batch was Chenopodium album, and one batch was a mixture of Amaranthus hybridus, Setaria spp. and Glycine max. Eighteen samples were detected to be adulterated with one to three other species. The adulterant species were mainly Amaranthus hybridus, Brassica carinata, Brassica juncea var. megarrhiza, Chenopodium album, Corispermum heptapotamicum, Cuscuta alata, Cuscuta japonica, Foeniculum vulgare, Glycine max and Medicago sativa. The percentage of counterfeits and adulterants in the 33 commercial samples was up to 69.7%. The other 10 commercial products were found to be identical
to the description on their labels.
3.3 Identification of commercial samples using NJ tree and single nucleotide polymorphisms (SNPs) To verify the BLAST identification results, an NJ Tree was constructed using haplotypes of species in the database and the commercial Cuscutae Semen (Figure 2). The results demonstrated the two original plant species of Cuscutae Semen (C. australis and C. chinensis) clustered in genus Cuscuta Linn. C. obtusiflora is the synonym of C. australis in the Scientific Database of China Plant Species (DCP) (http://www.plants.csdb.cn/eflora/help/shouye.aspx); Therefore, C. obtusiflora and its variants were both clustered in C. australis clade. In addition, C. applanata is the synonym of C. chinensis (Costea et al, 2011), and their sequences clustered within one clade. All the other species were clustered into their own clades, except for C. australis, C. pentagona, and C. campestris. By analyzing the haplotypes of the three species, we found two SNP sites among them; i.e., 126 bp T/C and 172 bp T/G sites could distinguish C. australis from the other two (Table 3). The SNP analysis was a complementary method for DNA barcoding identification of Cuscutae Semen in this study.
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Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225
Figure 2 NJ tree for all haplotypes of database and commercial Cuscutae Semen All ITS2 sequences from commercial Cuscutae Semen and database were aligned separately according to species using Codoncode Aligner 4.2.7 and finally haplotypes were determined. All haplotypes were used to construct NJ tree based on p-distance. The bootstrap scores (1000 replicates) were shown (≥ 50%) for each branch. Sequence number marked red and end with letters “CM” represent sequences harvest from commercial Cuscutae Semen, while sequence number marked black and end with letters “DB” represent sequences using for database construction.
Gao ZT et al. Chinese Herbal Medicines, 2017, 9(3): 218-225 Table 3 Haplotypes
223
Variable sites in haplotypes of C. australis, C. campestris and C. pentagona Positions / bp
5
21
22
36
93
115
126
133
158
172
173
194
196
199
C. australis H1
T
G
T
A
T
T
T
T
G
T
C
G
T
G
C. australis H2
C
G
T
A
G
A
T
G
A
T
T
T
T
T
C. australis H3
C
G
T
A
G
A
T
G
A
T
T
T
T
K
C. australis H4
C
G
T
A
T
A
T
G
A
T
T
T
T
C
C. australis H5
T
G
T
A
T
A
T
G
A
T
T
G
T
T
C. australis H6
T
R
K
A
T
T
T
A
G
T
C
G
T
G
C. campestris
T
G
T
A
T
T
C
T
G
G
C
G
T
G
C. pentagona H1
T
G
T
A
T
T
C
T
G
G
C
G
T
G
C. pentagona H2
T
G
T
G
T
T
C
T
G
G
C
G
T
G
C. pentagona H3
C
G
T
A
G
T
C
T
G
G
C
G
C
G
4. Discussion 4.1 Why did we select ITS2 to identify Cuscutae Semen? Recovery of sequences is such an important process in DNA barcode identification (Hebert et al, 2004; Stoeckle et al, 2011) that qualified DNA is foundational. However, once Cuscuta seeds are soaked in water, mucilage will emerge (Pharmacopoeia Committee of P. R. China, 2015). This is a practical identification characteristic of the genus Cuscuta L. However, the mucilage is also an obstacle for DNA extraction. CTAB extraction buffer causes genome DNA to be released from the cell, and it also makes the mucilage release simultaneously, which leads to the solution becoming extremely sticky and impossible to centrifuge in the subsequent procedures. Therefore, we decreased the sample quantity to approximately 20 mg while the volume of extraction buffer was kept consistent in the protocols. By this way, mucilage could be eliminated more sufficiently. To obtain enough DNA for amplification, we increased the isopropanol precipitate time to a half an hour in a -20 ℃ refrigerator and decreased the volume of water in the solution to 30 µL. Finally, qualified DNA was obtained. Gao et al confirmed that ITS (approximately 650 bp) was suitable for Cuscutae Semen identification (Gao et al, 2006). However, due to low PCR amplification efficiency and the lack of a universal primer, ITS was excluded as a universal land plant barcode in the earlier stages (Chen et al, 2010). In the present study, to resolve all the adulterants in the commercial “Tusizi”, we selected the universal medicinal barcode ITS2 as the core barcode to guarantee a high rate of species identification. After verification, ITS2 served as a more universal and efficient DNA barcode with a rate of successful identification of 92.7% at the species level, which was superior to other candidate barcodes (Chen et al, 2010). The relatively short sequence ITS2 (450 bp) made amplification easier in the Cuscuta genus. Based on the above, we finally selected ITS2 as a core barcode. In the current study, the NJ tree showed C. australis had a closer relationship with C. pentagona and C. campestris, and SNPs should be used in a complementary identification procedure. Therefore, DNA barcoding
accompanied with SNP sites could successfully identify Cuscutae Semen from its adulterants and closely related species.
4.2 DNA barcoding has potential power for detecting counterfeits in TCM marketplace With the great success of DNA barcoding, it has been highly praised as producing a renaissance in authenticating herbal medicine (Chen et al, 2014). Its application has expanded to commercial dietary identifications (Xin et al, 2015; Little et al, 2013; Zhao et al, 2015), and it has been successfully used to identify various commercial TCMs. Zhao et al used ITS2 as a core barcode and successfully detected five adulterants in the nine Acanthopanacis Cortex specimens available to customers. Among the adulterants, a toxic Periplocae Cortex was found most often with a percentage of 80% (Zhao et al, 2015). Xin et al built a reference database with ITS2 sequences of authoritative species in Rhodiola L. Using this database, they found that 60% of commercial Rhodiolae Crenulatae Radix et Rhizoma decoction samples were counterfeits (Xin et al, 2015). What is more, Chinese caterpillar fungus (Xiang et al, 2013) and medicinal species in Panax L. (Chen et al, 2013) have been successfully authenticated by DNA barcoding. In the present study, we first used ITS2 to detect the species composition of commercial Cuscutae Semen, and 11 species (Amaranthus hybridus, Brassica carinata, Brassica juncea var. megarrhiza, Chenopodium album, Corispermum heptapotamicum, Cuscuta alata, Cuscuta japonica, Cuscuta monogyna, Foeniculum vulgare, Glycine max, and Medicago sativa) were found to be an adulterant or substitute for the commercial Cuscutae Semen (Table 1). Five of 33 commercial “Tusizi” were found to be substituted by other species. Eighteen were detected to be adulterated with one to three other species. The percentage of counterfeits and adulterants in the 33 commercial samples was up to 69.7%, and only 10 commercial products were found to be genuine. These results indicated that the ratio of adulteration was surprisingly high and is a great threat in quality supervision of TCM. DNA barcoding can serve as an efficient, simple and convenient method to supervise and administrate applications for TCM (Xin et al, 2013; Chen et al,
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2013). During the collection of TCMs, we found that C. australis (34 specimens) comprised the majority on market, while C. chinensis (eight specimens) was relatively rare.
4.3 DNA barcoding technology can meet demands of rapid customs clearance. In addition to medical use in TCM, Cuscutae Semen is also an important inspected and quarantined weed that is distributed around the globe (Costea et al, 2011). In China, according to the governmental list promulgated by the General Administration of Quality Supervision, Inspection and Quarantine of P. R. China, all the species in genus Cuscuta L. are banned from entering China. In 2009 to 2011 alone, a total of 63 quarantined weeds (in 12 families and 37 genera) were intercepted by the inspection institutions in China (Hu et al, 2014). Among them, seven species in the genus Cuscuta were detected from 37 batches of goods, such as soybeans. Considering the tiny seeds of Cuscuta Linn., morphological identification is limited (Liao et al, 2005), and the identification time is long. With the frequency and ease of international communications, monitoring situations of inspection and quarantine has become increasingly important. In the present study, DNA barcoding could be used to successfully detect Setaria spp. and Glycine max in commercial Cuscutae Semen, and it would be possible for the Cuscuta L. species to be mixed in the seeds of Glycine max and Setaria spp., which could also be successfully detected. The DNA barcoding method built and used in the current study undoubtedly could serve as an accurate, rapid, simple method for rapid quarantined clearance that is required by several quarantine departments.
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