Internal Transcribed Spacer Barcoding DNA Region Coupled with High Resolution Melting Analysis for Authentication of Panax Species

INTRODUCTION

Medicinal plants of the Panax genus belonging to the Araliaceae family are well-known rare plants used as tonics in traditional Oriental medicine, and have been described in the Korean Pharmacopoeia. The most commonly used Panax species are Panax ginseng C. A. Meyer (Korean or Asian ginseng), P. quinquefolius L. (American ginseng) and P. notoginseng (Burkill) F. H. Chen (Chinese ginseng). The common name ginseng is somewhat misleading nowadays because several closely related ginseng species exist and are known to have varying phytochemical compositions, and different pharmacological properties (Shaw and But, 1995). Each ginseng species exhibits different pharmacological actions and different clinical indications. Therefore, correct identification of the starting material is an essential part to ensure the quality, safety, and efficacy of herbal products.

Traditionally, subjective methods based on morphological features such as leaf shape, berry skin color, and stem length, were used to discriminate among Panax species (Jo et al., 2014). However, this approach is impractical in many situations because of time constraints, the need for specialized taxonomic knowledge, and the difficulty in species identification in cases where only partial or trace samples are available. Different methods have been used to discriminate among Panax species, including liquid chromatography-tandem mass spectrometry (LC-MS-MS), gas chromatographic-mass spectrometry (GC-MS), NMR spectroscopy, and Fourier transform infrared (FT-NIR) spectroscopy (Li and Fitzloff, 2001; Chan et al., 2000; Cui et al., 1999; Kang et al., 2008; Mao et al., 2014).

Recently, the classification of Panax species using metabolic profiling has been greatly facilitated by the analysis of certain secondary metabolites such as ginseosides, which are considered chemotaxonomic markers of the genus (Yang et al., 2013). However, there are cases where the plant metabolite profile can change because of external factors such as light, temperature, microbial infections, and storage conditions (Yang et al., 2014).

In traditional Oriental medicine, crude plants are usually dried and thus lose their diagnostic features; therefore, the molecular marker is extremely useful for authenticating the composition of herbal medicines. Molecular marker analysis of Panax species has been carried out by comparing the DNA sequence variations within 5.8S ribosomal DNA (rDNA) and internal transcribed spacer (ITS) regions (Choi and Wen, 2000; Wang et al., 2011; Lee et al., 2012). However, this technique requires an agarose gel electrophoresis confirmation step, which is laborious and tends to feature carry-over contamination. Thus, this technique is difficult to utilize for the practical and systematic authentication of material purporting to be comprised of various Panax species.

Recently, high resolution melting (HRM) assays using real-time PCR have been introduced as a powerful tool not only for genome-wide SNP discovery but also for the diagnostic analysis of mutated genes causing human diseases (Stephens et al., 2008; Wittwer et al., 2003; Zhou et al., 2004). This technique is particularly useful for plant cultivar identification, genetic mapping, QTL analysis, the identification of pathogenic species, and gene discovery. Very recently, Jaakola et al. (2010) developed an HRM analysis method targeting DNA barcoding regions, which was intended for use in the verification of the authenticity of berry species, in particular for distinguishing bilberry (Vaccinium myrtillus L.) from other berry species.

The objectives of this study were as follows; (a) to analyze the sequence variations of the ITS1, 5.8S rDNA, and ITS2 regions and test their usefulness for the identification of Panax species; (b) to develop a rapid, simple, and stable barcode DNA high resolution melting (Bar-HRM) assay targeting the ITS region for the identification of Panax species, as an alternative and efficient approach that can be used in molecular taxonomy studies of closely related ginseng species; and (c) to trace the origins of ginseng via Bar-HRM analysis, in particular the ginseng species of high commercial value, that is, P. ginseng and P. quinquefolius.

MATERIALS AND METHODS

1. Plant materials and DNA isolation

P. ginseng and other Panax species (P. quinquefolius and P. notoginseng) were preserved and cultivated at the experimental field of the NIHHS, RDA, Chungbuk Province, Korea. These samples were deposited at Korean medicinal herbarium in NIHHS (Table 1). Fresh leaves of 4-years-old plants from P. ginseng and two Panax species were quickly cut the tissue into small pieces with a sterile razor blade, freeze in liquid nitrogen and grind well using mortar and pestle. Total genomic DNAs were extracted from five leaves of five plants per each specie (P. ginseng, P. quinquefolius and P. notoginseng) using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. The quantity and quality of DNA samples were measured using a NanoDrop ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA) and the final DNA concentration was adjusted to 10 ng/μℓ.

Table 1.

Details of plant materials used in the high resolution meting (HRM) analysis.

4. Primer design and high resolution melting analysis

According to the sequence information from the barcoding analyses, three primer pairs were designed from the rDNA ITS region (Table 2). Primers were designed using the following criteria: (1) a minimum primer length of 18 bp, (2) melting temperature between 58°C and 62°C with a maximum discrepancy of 2°C among primers, and (3) PCR product size ranging from 100 to 200 bp. The HRM analysis PCR amplification was performed in 10μℓ on a Bio-Rad CFX96 RealTime PCR System (Bio-Rad, Hercules, CA, USA). A standard PCR was performed in 10μℓ reaction volumes with 50 ng of genomic DNA as template (five samples per species), 10 pmol of reverse and forward primers, and 5μℓ 2X Precision Melt Supermix (Bio-Rad, Hercules, CA, USA). HRM was performed using a CFX96 real-time PCR detection system and the cycling conditions for HRM followed the manual of Precision Melt Supermix, 95°C for 2 min, 40 cycles of denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s. To assess product specificity, amplicons were systematically checked by the melting curve analysis. Melting curves were generated from 65°C to 95°C with increments of 0.2°C/cycle. Melting profiles were analyzed with the Bio-Rad Precision Melt Software version 1.0, as described in the following paragraphs.

Table 2.

Information of primer sequence used in Bar-HRM analysis.

RESULTS

1. DNA sequencing and ITS sequence comparison

The DNA sequences in the ITS1-5.8S-ITS2 of rDNA were PCR-amplified from the leaves of three Panax species (Fig. 1); P. ginseng, P. quinquefolius and P. notoginseng. We obtained PCR products approximately 750 bp long from the Panax species tested. The length of the amplified products was determined to be 746 bp via sequencing, and the pure ITS1-5.8S-ITS2 region was determined to be 682 bp long, via a comparison with sequence data in the NCBI GenBank nucleotide databases (AY548192, FJ606755, and U41685). The amplified DNA of the three plant samples was sequenced as described in Figure 1. As expected, the sequences of the species showed a very high degree of homology (97.9 - 99.4%) in the ITS1-5.8S-ITS2 region (Fig. 2). The ITS and 5.8s rDNA sequences of the three Panax species had a G + C content of 59.5 - 59.7%. SNPs were observed at the 27 bp, 48 bp, 70 bp, 81 bp, 83 bp, 117 bp, 129 bp, 130 bp, and 248 bp locations of ITS1; at 328 bp and 423 bp of the 5.8s rDNA gene; and at 427 bp, 429 bp, 438 bp, 441 bp, 535 bp, 602 bp, and 613 bp of the ITS2 region (Fig. 2 and Table 2).

Fig. 1.

A diagram of the nuclear ITS (Internal Transcribed Spacer) region and the positions of the primers used for PCR.

Fig. 2.

DNA sequences in the ITS1-5.8S-ITS2 region for three Panax species. Arrows indicate the ranges of ITS1, 5.8S rRNA, and ITS2. Primer binding regions and directions were indicated with dot arrows. The areas enclosed by the boxes indicates SNP variations.

2. HRM curves analysis

Three Panax species (P. ginseng, P. quinquefolius and P. notoginseng) were tested with real-time PCR using three primer sets: ITS1HRM, 5.8SHRM, and ITS2HRM (Table 3). The resulting melting curves showed that the three Panax species could be clearly discriminated between by using the ITS1, ITS2, and 5.8S rDNA barcoding region in combination with HRM analysis. Distinct HRM peaks were observed for each SNP genotype of the Panax species, each of which is represented by a different color (Fig. 3). In all the samples evaluated, the five replicates always had similar curves and peaks, and were assigned to the same group, demonstrating the consistency and reproducibility of the HRM assay.

Table 3.

Nucleotide variations in the ITS region among Panax species tested.

Fig. 3.

Normalized high resolution melting (HRM) curve profiles of PCR amplicons using the primers. A; ITS1HRM, B; 5.8SHRM, and C; ITS2HRM.

The melting characteristics of the ITS1 amplicons of P. ginseng, P. quinquefolius and P. notoginseng were assessed by plotting three different curves (Fig. 3A). The melting curves were characterized by peaks of 85.60°C in profile 1 (P. quinquefolius), 86.00°C in profile 2 (P. ginseng), and 86.20°C in profile 3 (P. notoginseng). The three Panax species tested generated distinctive HRM profiles and normalized HRM profiles, allowing for the discrimination of each species. They were easily discriminated because they produced different HRM patterns because of the differences in the nucleotide sequences of the ITS1 fragments amplified in this assay.

The analysis of the normalized HRM curves using the barcode marker 5.8SHRM is shown in Fig. 3B. It shows that each genotype was represented by three peaks. The first peak (profile 1, P. ginseng) ranged from 84.20 to 84.60°C, the second peak (profile 2, P. quinquefolius) from 84.60 to 85.00°C, and the third peak (profile 3, P. notoginseng) was at 84.60°C (Fig. 3B). Analysis of the normalized HRM curves produced using the 5.8SHRM marker revealed that Panax species can easily be distinguished from each other. The primer ITS2HRM produced a single melt peak, and the melt temperature for each sample at 89.20°C (Fig. 3C). Sequence variations in the ITS1 region and 5.8s rDNA genes allowed for very clear and reproducible HRM curve analysis differentiation among the Panax species analyzed in this study.

DISCUSSION

The Bar-HRM method allows the fast analysis of genetic variation in various plans using universal barcoding regions (ITS or chloroplast) while at the same time is a close tube post PCR method reducing the risk of contamination.

The ITS nuclear ribosomal DNA is considered one of the best barcoding regions available (Bladwin, 1992). This region has a number of valuable characteristics, such as conserved regions that can be used for designing a universal primer, the ease with which it can be amplified, and sufficient variability to distinguish between even closely related species (Yao et al., 2010).

Thus, several researchers have tried to detect variations in the ITS regions in order to carry out phylogenetic studies of the genus Panax (Wen and Zimmer, 1996). Studies for distinguishing among Panax species were performed using the cleaved amplified polymorphic sequences (CAPS) marker system (Kim et al., 2007), and species-specific PCR based on SNPs detected in the ITS regions (Park et al., 2006; Bang et al., 2012). However, this technique requires the inclusion of a confirmation step using agarose gel electrophoresis, which is laborious and tends to feature carry-over contamination. Thus, they are difficult to utilize for the practical and systematic authentication of Panax species.

Recently, a new methodology known as plant DNA barcoding with high resolution melting (plant Bar-HRM) analysis has been developed (Kalivas et al., 2014). Plant Bar-HRM has been used for species identification, evolution studies, and forensics as it targets small conserved DNA regions such as the chloroplast, mitochondria, and ITS (Ganopoulos et al., 2013; Madesis et al., 2012; Bosmali et al., 2012). It is a closed tube assay that does not employ additional fluorescent probes and simply utilizes a DNA melting assay and computerized analysis of the results to produce a graphic output, thus decreasing the risk of sample contamination.

According to Ganopoulos et al. (2012) the use of DNA markers to authenticate legume species requires polymorphic and high-copy analytical targets such as the universal nuclear plant DNA barcoding region ITS, which has been used to discriminate among several plant species. Similarly, we used the PCR-amplified universal barcoding region as an analytical target for the HRM curve assay in order to discriminate among Panax species (Fig. 3). A melting curve analysis using ITS barcoding regions was sufficient to distinguish among three Panax species (Table 1). The authentication of Panax species is of great importance, especially when seeds are mixed with species that can be a source of seed purity management and quality control problems in the manufacture of ginseng products.

In conclusion, we have successfully developed a Bar- HRM method coupled with real-time PCR, which can be used for the rapid identification of three Panax species. The Bar-HRM method has been proven to be a universal method, as it allowed for discrimination among three species using two barcoding regions, that is, ITS1 and 5.8S rDNA (Fig 1). The ITS1HRM and 5.8SHRM primers could be used to differentiate among three Panax species in conjunction with HRM curve analysis.

ACKNOWLEDGEMENTS

This work was carried out with the support of Cooperative Research Program for Agriculture Science and Technology Development(PJ01047301), Rural Development Administration, Republic of Korea.

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