Ho Chi Minh City University of Food Industry 140 Le Trong Tan Street,
Tan Phu district, Ho Chi Minh City 700000, Vietnam.

Corresponding author email: thehv@hufi.edu.vn

Article Publishing History

INTRODUCTION

Ginseng is the common name of the species in the Panax genus Araliaceae. This plant has been used as medicine for a long time as its outstanding medicinal properties include stimulating metabolism in the body, regulating blood sugar in the body, and protecting the activity of the central nervous system, counteract the formation and inhibit the growth of tumors, increase the body’s resistance to diseases (Ali et al., 2012; Peng et al., 2012; Lee et al., 2015).

High medicinal activity of ginseng is due to the plant’s high ginsennoside content, it is also rich in other valuable ingredients such as anti-oxidant compounds, peptides, polysaccharides, and vitamins (Lee et al., 1995; Hong et al., 2012). So far, at least 17 Panax species have been reported worldwide (Zhang et al., 2000) and medicinal compounds are variable from species to species (Harkey et al., 2001; Liu et al., 2016, Hyeonah et al 2021 ).

Currently, ginseng identification and classification is mainly based on morphological characteristic since it is straightforward to perform and feasible to carry out on the field at a low cost (Proctor et al., 2011; Hong et al., 2012). Nevertheless, significant limitations of this method have been addressed such as low number, complex inheritance pattern, and vulnerable to changes in the environment (Ahmedand and Mohamed, 2014). Consequently, ginseng authentication and cultivar identification based on this method are unreliable.

With the rapid development in molecular biology, DNA-based markers have been used intensively for characterizing the genetic relationships among plant species due to their fast, specific, reliable, and sensitive features (Shim et al., 2003; Serrone et al., 2006; Kim et al., 2016). Recently, the DNA barcode has been used intensively for classifying at the species level (CBOL Plant Working Group, 2009). This marker type has been applied to discriminate at species level for different organism such as plants, animal, fungi and bacteria (Hollingsworth et al., 2011; Barcaccia et al., 2016; Hu et al., 2019).

Among several DNA barcode loci, Internal Transcribed Spacer (ITS) region has been proposed as a standard region for plant identification because the ITS region is highly conserved among interspecies but variable between interspecies and is easy to amplify even from limited DNA quantity (Chase et al., 2007; Giudicelli et al., 2015). Numerous researchers reported that discrimination power of ITS is higher in the comparison to other plastid markers (Muellner et al., 2011; Yang et al., 2012; Zhang et al., 2014).So far, several studies have exploited the ITS-based DNA barcode in the investigating the genetic composition and developing protocols to differentiate species in Panax genus. In 2011, a study in India showed the effectiveness of ITS in clustering analysis of Panax assamicus populations according to geographic locations (Panday and Ali, 2011).

In Saudi Arabia, Ali and colleagues applied ITS2 sequence to control ginseng-derived products (Ali et al., 2012). Bang and colleagues used ITS to differentiating and authenticating Panax species in Korea (Bang et al., 2015). More recently, this barcode region was also applied to characterize genetic diversity of 24 ginseng samples collected at Lai Chau province of Vietnam (Pham et al., 2018). The present study describes the effectiveness of ITS-based DNA barcode in classifying 23 Panax samples belong to different species collected in different geographical locations in Vietnam. The obtained data could be applied for classification, identification, and authentication activities in Panax plants of Vietnam.

MATERIAL AND METHODS

Sample collection:Total of 23 Panax samples were collected from North to South of Vietnam, samples were collected from different sources such as Panax traders, research institutes, universities, and companies in different locations (Figure 1 and Table 1). Collected samples were then store in cool and dry condition and targeted for DNA extraction.

Figure 1: Map showing locations for collecting Panax samples.

Table 1. List of  collected Panax samples used in this study.

No.	Sample ID	Local name	Collection location
1	TTB1	Tam that bac	Quang Nam province
2	TTB2	Tam that bac	Quang Nam province
3	TTH1	Tam that hoang	Lao Cai province
4	TTH2	Tam that hoang	Yen Bai province
5	TT1	Tam that	Quang Nam province
6	TT2	Tam that	Quang Nam province
7	TT3	Tam that	Quang Nam province
8	TT4	Tam that	Quang Nam province
9	TT5	Tam that	Quang Nam province
10	TT6	Tam that	Quang Nam province
11	TT7	Tam that	Quang Nam province
12	TT8	Tam that	Quang Nam province
13	TT9	Tam that	Quang Nam province
14	TT10	Tam that	Quang Nam province
15	SLC1	Sam Lai Chau	Lai Chau province
16	SLC2	Sam Lai Chau	Lai Chau province
17	SNL1	Sam Ngoc Linh	Ho Chi Minh city
18	SNL2	Sam Ngoc Linh	Quang Nam province
19	SNL3	Sam Ngoc Linh	Quang Nam province
20	SNL4	Sam Ngoc Linh	Quang Nam province
21	SVD1	Sam vu diep	Yen Bai province
22	SVD2	Sam vu diep	Lao Cai province
23	SVD3	Sam vu diep	Yen Bai province

DNA extraction:DNA from Panax samples extracted by cetyltrimethyl ammonium bromide (CTAB) method followed Li et al. (2013). After extraction, DNA quality was examined by electrophoresis on 1% agarose then spectrophotometer (Optima SP 3000 nano UV-VIS, Japan) was used to determine DNA concentrations. The DNA samples were then kept at -20 °C freezer until use for PCR reactions.

PCR reactions and DNA sequencing:The composition of PCR reactions to amplify ITS region as follows: 7.5 μL 2X Mytaq Red Mix (Bioline, UK), 20 ng DNA, 0.2 μM of each primer (C26A 5’ AGGAGAAGTCGTAACAAG3’ and N-nc18S10 5’ GTTTCTTTTCCTCCGCT 3’) (Wen and Zimmer, 1996), and PCR water for a final volume of 15 μl. The PCR cocktails were run in thermal cyclers SureCycler 8800 Thermal Cycler (Agilent, USA) with following conditions: initial denaturation at 94 °C for 2 minutes; then repeated by 35 cycles of 30 seconds at 94 °C, 30 seconds at 55 °C, 50 seconds at 72 °C, and finally one minute at 72 °C to complete the reaction. The PCR products were visualized on gel electrophoresed and use 1 kb ladder (Bioline, UK) to determine amplification length.

Correct PCR products were sequenced by Sanger methods at Nam Khoa Company (Ho Chi Minh City, Vietnam). Each sample were sequenced for both sense and antisense directions. Nucleotide sequences of both DNA strands were obtained and compared the forward and reverse sequence to ensure accuracy. The finally assembled sequences were submitted to NCBI GenBank to obtained accession number (Table 2) and then used for following analysis.

Data analysis: The homology identification of each sequence were implemented by using Basic Local Alignment Tools (BLAST) function in NCBI (National Center for Biotechnology Information, USA) database. To evaluate the capacity of ITS based DNA barcode in distinguish Panax accessions from Vietnam and abroad, additional ten ITS sequences of related Panax species retrieved from Genbank. All sequences were aligned using the Clustal method in Molecular Evolutionary Genetics Analysis (MEGA) 6 software (https://www.megasoftware.net).

Evolutionary trees were constructed based on two methods consisting of Maximum Likelihood (ML) and Neighbour Joining (NJ) since they represent for discrete character methods and distance methods, respectively. For satisfactory reliability in phylogenetic construction, bootstrap value was set at 1000 replicates and bootstrap support was classified as Kress et al. (2002).

RESULTS AND DISCUSSION

PCR and DNA sequence: Although three are few study reported the difficulty in amplifying and sequencing ITS region (Gonzalez et al., 2009; Wang et al., 2016), all PCR and sequencing reactions in this study were performed successfully. The sequences length is 693 bp on average, varying from 631 bp to 707 bp. The similarity of obtained sequences to homologous sequences in NCBI Genbank ranges from 99.52 to 100%. The Latin names of collected samples were identified and presented in Table 2.

Table 2. Genbank accession numbers of samples and corresponding Latin names.

No.	Sample ID	Length (bp)	Accession number	Latin name	Percent identity (%)
1	TTB2	704	MZ149934	Panax notoginseng	100
2	TTB1	707	MZ149935	Panax notoginseng	99.86
3	TTH1	703	MZ149936	Panax stipuleanatus	99.72
4	TTH2	631	MZ149937	Panax stipuleanatus	99.68
5	TT1	705	MZ149938	Panax japonicus var. bipinnatifidus	99.43
6	TT2	702	MZ149939	Panax japonicus var. bipinnatifidus	99.57
7	TT3	702	MZ149940	Panax japonicus var. bipinnatifidus	99.57
8	TT4	702	MZ149941	Panax japonicus var. bipinnatifidus	99.57
9	TT5	702	MZ149942	Panax japonicus var. bipinnatifidus	99.57
10	TT6	702	MZ149943	Panax japonicus var. bipinnatifidus	99.57
11	TT7	701	MZ149944	Panax japonicus var. bipinnatifidus	99.43
12	TT8	702	MZ149945	Panax japonicus var. bipinnatifidus	99.57
13	TT9	702	MZ149946	Panax japonicus var. bipinnatifidus	99.57
14	TT10	702	MZ149947	Panax japonicus var. bipinnatifidus	99.57
15	SLC1	700	MZ149948	Panax vietnamensis var. fuscidiscus	99.86
16	SLC2	630	MZ149949	Panax vietnamensis var. fuscidiscus	99.52
17	SNL1	704	MZ149950	Panax vietnamensis	100
18	SNL2	701	MZ149950	Panax vietnamensis	99.71
19	SNL3	702	MZ149951	Panax vietnamensis	99.72
20	SNL4	705	MZ149952	Panax vietnamensis	99.86
21	SVD1	698	MZ149953	Panax stipuleanatus	100
22	SVD2	703	MZ149954	Panax stipuleanatus	99.72
23	SVD3	642	MZ149955	Panax stipuleanatus	99.53

After identification, 23 samples were found belonging to four species consisting of Panax notoginseng, Panax stipuleanatus, Panax japonicus var bipinnatifidus, Panax vietnamensis var. fuscidiscus, and Panax vietnamensis Thus, two different plant groups in Vietnam local names consisting of “Tam that hoang” including TTH1 and TTG2 samples and “sam vu diep” including SVD1, SVD2 and SVD3 samples were identified as Panax stipuleanatus. This confirm the low accuracy in traditional method for Panax classification due to several uncontrollable factors such as changes of environmental conditions and developmental stages (Ali et al., 2012).

Sequence alignment and Phylogenetic analysis: Together with 23 obtained sequences from this study, additional 10 ITS sequences from different species in Panax genus were downloaded from NCBI Genbank and combined for sequence alignment and the alignment summary is presented in Table 3. Totally, up to three 33 variation sites were detected within analyzed sequences. Previously, several research groups have employed the nucleotide variations to develop molecular makers for plant identification such as in Taxus (Liu et al., 2011) or onion (Ipek et al., 2014). Thus, these variations are valuable for developing specific markers or diagnosis KIT for discriminating different Panax species.

Table 3. Variable sites in 33 ITS sequences from different Panax species.

(*: sequences were retrieved from NCBI Genbank)

The genetic distances among 23 Panax samples were calculated based on Kimura-2 parameter (K2P) method of MEGA 6 software. The lowest genetic distance is 0.000 indicating the sequence similarity among samples, while the highest is 0.052 this result showing the close genetic relationship among these accessions when using ITS regions. The phylogenetic trees constructed by either neighbor-joining (NJ) or Maximum Likelihood methods are shown in Figure 2 and Figure 3, respectively.

Figure 2:  Neighbor-Joining tree with 1000 bootstrap replicated based on ITS sequences (Star symbols indicating sequences retrieved from NCBI Genbank and the availability of common names are shown in parentheses).

Figure 3: Maximum Likelihood tree with 1000 bootstrap replicated based on ITS sequences (Star symbols indicating sequences retrieved from NCBI Genbank and the availability of common names are shown in parentheses).

In general, the results show the high similarity between Neighbor-Joining and Maximum Likelihood methods. Panax samples collected from Vietnam in this study is clustered into several groups corresponding to its identified species using NCBI BLAST. Interestingly, Vietnam ginsengs are also separated from different ginseng species collected from different countries. The grouping of Panax species in obtained phylogenetic trees of this study is slightly different with previous study of Yang and colleagues in Korea in which they reported the closer clustering of P. notogingsen and P. pseudoginseng as well as P. omeiensis and P. zingiberensis (Yang et al., 2001).

CONCLUSION

The obtained data from this study confirms the effectiveness of using ITS marker to classify Panax samples in Vietnam. Furthermore, this marker also shows high discrimination power to distinguish Panax species from Vietnam and from abroad. Future study should focus on develop specific primer pairs or diagnosis KIT to serve for identification and authentication activities of Panax plants.

Ethics approval and consent to participate: Not applicable.

Conflict of Interests: The author has no conflict of interest.

Funding statement: This work was sponsored and funded by Ho Chi Minh City University of Food Industry under Contract No. 53/HD-DCT.

ACKNOWLEDGEMENTS

The authors would like to thank Ho Chi Minh University of Food Industry for providing research fund and laboratory facility.

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