Molecular Identification of Reynoutria japonica Houtt. and R. sachalinensis (F. Schmidt) Nakai Using SNP Sites

Abstract

Reynoutria japonica and R. sachalinensis have been used as medicinal resources in Korea. However, it is difficult to identify and determine these medicinal herbs correctly because they are usually customized and purchased as the fragmented rhizomes types. To develop molecular markers for distinguishing two species, we analyzed and compared the chloroplast DNA sequences of seven loci (atpB, matK, ccD-psaI, atpF-H, trnL-trnF, psbK-I and rpl32-trnL). Among them, we found two effective SNPs in psbK-I region for R. japonica and atpF-H region for R. sachalinensis. Based on these SNP sites, we designed the new R. japonica- specific primer which is able to amplify 300 bp fragment in psbK-I region. A similar strategy was applied for the atpF-H region of R. sachalinensis. These molecular markers would be successfully applied to recognize R. japonica and R. sachalinensis.

Introduction

Molecular marker usually have several advantages such as reflecting genetic property of species as well as variety, being influenced by any surroundings and being easily processed, stored and shared information. According to the technique, there are five kinds of molecular markers; Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSR) and Single Nucleotide Polymorphism (SNP). Especially, SNP molecular marker can be used easily as well as highly reproducibility and formation of polymorphism because of using technique based on PCR. Therefore, there are many studies of genetic polymorphisms of angiosperms and animals and analysis relationship of species as well as variety (Batley et al., 2002; Deynze et al., 2007; Kim and Misra, 2007). For examples, Korean Ginseng (Panax ginseng)-specific primer was developed based on SNP in ITS region (In et al., 2010). And, Kim et al. (2012) used rbcL and ITS regions to devise the specific primer for identification of Schisandra chinensis with other related species.

Amplification Refractory Mutation System (ARMS) has been usefully applied with SNP as a molecular genetic technique (Kim et al., 2012). For ARMS, when the target DNA does not match the base in 3' end of primer, there is no extension step by DNA polymerase in PCR progress. Then, it is possible to develop the primers based on ARMS-PCR for the species particularity molecular markers. The SNP markers are helpful for the more effectively conformation of identification and origin of medicine herbs.

Polygonaceae Juss. are monophyletic and sister group of Plumbaginaceae Juss. (Chase et al., 1993; Fay et al., 1997; Chase et al., 2002). However, the classification of species within this family remains unclear because of morphological variability. Schuster et al. (2011) used five chloroplast regions and two nuclear genes to reconstruct the relationship within Polygonaceae. Their results showed the clear relationship of Fallopia Adans. and Reynoutria Houtt. which were merged and separated several times during the complex taxonomic history of Polygonaceae. In addition, the recognition of Fallopia as sister to Reynoutria was supported by the different chromosome base numbers which were x=10 for the former (Jaretzky, 1928) and x=11 for the latter (Bailey and Stace, 1992).

In Polygonaceae, Reynoutria comprises of seven species located in temperate region of the Northern Hemisphere including East-Asia, Europe and North America (Conolly, 1977; Hollingsworth et al., 1999; Bailey et al., 2007; Gammon and Kesseli, 2010; Stoll et al., 2012; Dorigo et al., 2012). Five species of Reynoutria; including R. japonica Houtt. (Fallopia japonica=Polygonum cuspidatum; Ho-jang-geun), R. sachalinensis (F. Schmidt) Nakai (F. sachalinensis=P. sachalinensis; Wang-hojang-geun), R. forbesii (Hance) T. Yamaz. (F. forbesii=P. forbesii; Gam-jeol-dae), R. ciliinervis (Nakai) Moldenke (F. ciliinervis=P. ciliinerve; Na-do-ha-su-o) and R. multiflora (Thunb.) Moldenke (F. multiflora=P. multiflorum; Ha-su-o) are distributed in Korea (Baik et al., 1986; Lee et al., 1997; Kim and Park, 2000; Do et al., 2011). Especially, R. sachalinensis can be only found in Ulleungdo island and Dok-do island in Korea. And R. japonica and R. sachalinensis still have taxonomic confusion and are difficult of determining species boundaries (Steward, 1930; Park et al., 2011) because of the complex patterns of variation in morphological characters which are observed according to the habitat and environment as like other Polygonaceae species (Bailey and Stace, 1992). Moreover, those species are highly variable in morphology and chromosome numbers, resulting in notable controversy about differentiation degree, validity of classification, setting of limitation and order (Steward, 1930; Ohwi, 1984; Kim and Park, 2000, Park et al., 2011).

R. japonica and R. sachalinensis have been traditionally used as conventional medicines (Sohn et al., 2003; Kim and Lee, 2013). Especially, R. japonica was used for treatment of joint pains, menstrual cramps, maternity, cystitis, cancer (Kim et al., 2008; Chung et al., 2011), whereas R. sachalinensis was used in a laxative and a diuretic and analgesic treatment (Lajter et al., 2013). However, in the markets, it is very difficult to distinguish these two species correctly because they are usually customized and purchased as the fragmented rhizomes types. Therefore, it is necessary to support the reproducible and easy protocols to evaluate the quality of crude medicinal resources.

In this study, we applied the ARMS method to establish the molecular identification methodology of R. japonica and R. sachalinensis. For this purpose, we analyzed and compared the sequence variation of seven chloroplast loci (matK, atpB, atpF-H, trnF-trnL, accD-psal, psbK-I and rpl32-trnL) among the two species. Based on the results, we developed the new molecular markers inferred from cpDNA for recognizing both species based on SNP sites.

 

Materials and Methods

Plant materials

The collection information for the used samples was summarized in Table 1. R. japonica was collected from four different areas of South Korea. Also, seven individuals of R. sachalinensis were sampled at Ulleung-do island. Voucher specimens were deposited at GCU (Herbarium, Gachon University). And we also extracted DNAs from dried specimens of KH (Herbarium of Korea National Arboretum), MPRB (Medicinal Plant Resources Bank for Immune and Metabolic Disease) and PDBK (Plant DNA Bank in Korea).

Table 1.Plant materials of this study

DNA extraction and amplification of each loci

Total genomic DNA was extracted by following modified 2X CTAB buffer method (Doyle and Doyle, 1987). Lipids and other metabolites were removed using SEVAG solution [chloform (24) : isoamylalcohol (1)], and the DNA was precipitated with isopropanol at −20℃. The extracted DNA was then melted in 1X TE buffer and kept at −20℃. The DNA products were checked by 1% agarose gel electrophoresis with ethidium bromide staining and measured the concentration by spectrophotometer (biospec-nano; Shimadzu).

The information of primers used for amplification of each loci was shown in Table 2. PCR performed in 25 μl volume comprising 80 ng of template DNA, 0.1 U of e-Taq DNA polymerase (Solgent, Korea), 2 μl of 10X reaction buffer (100 mM Tris-HCl, 500 mM KCl, and 15 mM MgCl2), 0.25 mM dNTPs, 2.5 mM MgCl2, and 0.5 μM forward and reverse primers using a Perkin-Elmer 9700 machine (Perkin Elmer Applied Biosystems, Waltham, MA, USA). The thermo cycling profile consisted of 4 mins at 94℃, followed by 30-35 cycles of 2-3 mins at 94℃, 1 min 52-55℃, 1 min at 72℃, and an additional 7 mins extension at 72℃. The products were checked by 1% agarose gel electrophoresis with ethidium bromide staining and visualization under UV light.

Table 2.Primer sequences used for PCR in this study

Sequencing and alignment

All PCR products were purified using MeGa quick-spin total fragment DNA purification kit (Intron Biotechnology, Inc), and sequenced using cycle-sequencing BigDye Terminator Kit (V3.1, Life Technologies) based on the manufacturer’s protocol. The high quality sequences were assembled and aligned using geneious v.7.1.8 (Biomatters Ltd., New Zealand).

The amplified sequences were aligned for finding the SNP sites in seven regions (Appendix 1). These regions were selected based on NCBI and CBOL Plant Working Group (2009).

Design of specific primers and PCR protocol

In psbK-I region, two internal primers (psbK, psbI) were designed to be used as a positive control, and a specific primer (psbKI-SNP-C) for the identification of R. japonica and its allies were designed on the basis of SNP. A similar strategy was applied for the atpF-H region in which a specific primer (atpFH-SNP-C) was designed. The reactions were run in a 25 μl volume containing 60-80 ng of template DNA, 0.1 U of e-Taq DNA polymerase (Solgent, Korea), 2.5 μl of 10X reaction buffer and 0.5 μl of 10 mM dNTPs. The amplification process consisted of 1 min at 94℃, followed by 25 cycles of 30 sec at 94℃, 15 sec at 60℃, and 30 sec at 72℃, with an additional 1 min extension at 72℃ for psbK-I and atpF-H.

 

Results

Sequences alignment

After seven loci or regions were sequenced, we measured the AT contents and counted the variable sites within each species and between species. Additionally, the percentages of variable sites on the loci were calculated (Table 3).

Table 3.The information of investigated sequences

We found ten variable sites between the species and thirty-nine variable sites within populations of R. japonica and R. sachalinensis, respectively. But most of sites between the species were unuseful because those were located in high AT rich regions and terminals of regions. Therefore, we could selected two SNP sites from psbK-I and atpF-H regions which are effective to create molecular marker for identification of R. japonica and R. sachalinensis, respectively.

SNP primer specific to R. japonica of psbK-I

A PCR product of about 520 bp from psbK-I were amplified and sequenced. Within the populations of R. japonica, there was no sequence variation among individuals. Nevertheless, compared to individuals of two species including R. sachalinensis, we found two variable sites. And one of them could be used as species-specific SNP for R. japonica (Fig. 1).

Fig. 1.(A) Schematic diagram of psbK-I and atpF-H region including the positions and size of the primers used for check reproducibility and availability of the species identification (psbK & psbKI_SNP_C; specific for R. japonica and atpF & atpF_SNP_C; specific for R. sachalinensis). (B) PCR products using newly designed primers (a) psbK & psbKI_SNP_C, (b) atpF & atpF_SNP_C. M: 100 bp DNA ladder; JA1-JA5: R. japonica; SA1-SA5 ; R. sachalinensis.

To establish a molecular identification method for R. japonica and its related species, we designed a species-specific primer pair based on a SNP in the sequence that the second nucleotide from the 3'-end of the original sequence was modified. Specifically, the reverse primer psbKI-SNP-C (5'-CTC ACA AGG TCT TTC ACG GCG-3') was designed to amplify the PCR products of R. japonica by replacing A with C. This modified primer did not work in R. sachalinensis (Fig. 1, Table 4).

Table 4.The sequence of species-specific primer pair based on an SNPs

SNP primer specific to R. sachalinensis of atpF-H

The complete atpF-H sequences from two species, 615 bps, were aligned. There were twenty two variation sites within R. sachalinensis, as well as thirty one variable sites among two species including twenty seven sites of insertion/deletion. From these results, we could find four species- specific SNPs sites for R. sachalinensis (Fig. 1).

Based on the atpF-H sequence variations, we designed a species-specific primer pair modifying an SNP located 300 bp which is a specific to R. sachalinensis. And the reverse primer atpFH-SNP-C (5'-TCG CAA TTT ACA CGA AAA CCC GCC-3') was designed to amplify R. sachalinensis by replacing G with C at the second nucleotide position from the 3' end of the primer. Also, this primer did not work for R. japonica (Fig. 1, Table 4).

 

Discussion

SNP is a specific site that has a higher variation rate resulting from small deletions or insertions in DNA sequences (Kim and Misra, 2007). Therefore, molecular marker using SNP sites contributes the development of an effective method for identification of close relative taxa. For example, Kim et al. (2013) reported the utilization of multiplex PCR to distinguish Cynanchum wilfordii (Maxim.) Hemsl. (白首烏; Eun-jo-rong, in Korean and ge shan xiao in Chinese), C. auriculatum Royle ex Wight (異葉牛皮消; niu pi xiao in Chinese) and R. multiflora (Thunb.) Moldenke (何首烏; Ha-su-o in Korean and he shou wu in Chinese). Similarly, the specific molecular marker for S. chinensis, P. ginseng, Tomato, Zea mays were developed (Batley et al., 2002; Deynze et al., 2007; In et al., 2010; Kim et al., 2012).

In this study, we have compared the seven loci (atpB, matK, accD-psal, atpF-H, trnL-trnF, psbK-I and rpl32-trnL) from chloroplast DNA sequences to establish the molecular markers for distinguishing R. japonica, R. sachalinensis and related taxa. Generally, the sequence variations are more frequent in non-coding and intron regions than protein- coding sequences (Ching et al., 2002; Van Deynze et al., 2007). However, intron regions are conserved among species, therefore, it is more effective to design primers within non-coding regions than intron regions for SNP study (Fourmann et al., 2002). Based on previous studies, five intergenic spacer regions of accD-psaI, atpF-H, trnL-F, psbK-I, rpl32-trnL and two highly variable coding regions of atpB and matK were selected in this study (Taberlet et al., 1991; Chiang et al. 1998; Shaw et al., 2007; Lahaye et al., 2008; Yan et al., 2008). The results showed different numbers of variable sites within R. japonica and R. sachalinensis (Table 3). Especially, coding region of matK showed the highest number of different sites (12 sites in R. japonica and 9 sites in R. sachalinensis) followed by the rpl32-trnL region (6 sites in R. japonica and 2 sites in R. sachalinensis).

Among the investigated sequence regions, we found two species-specific SNPs in psbK-I and atpF-H regions. Although the variations were observed within species of R. japonica and R. sachalinensis, it was not so different between these two species except the atpF-H, psbK-I and rpl32-trnL (Table 3). This result suggested the close relationship of R. japonica and R. sachalinensis. Additionally, it was successful to design SNP primers specific to R. japonica with psbK-I and to R. sachalinensis with atpF-H (Fig. 1). These specific primers, which were designed based on ARMS, amplified 300 bp specific fragment in each region. Previously, the circumscription of R. japonica and R. sachalinensis remained controversy because of their complex morphological characters (Steward 1930; Bailey and Stace, 1992; Kim and Park 2000; Park et al., 2011). Furthermore, R. japonica and R. sachalinensis have been used confusedly for the treatment of menstrual cramps, cancer, chronic bronchitis and so on (Kim et al., 2008; Chung et al., 2011). Therefore, it is necessary to establish the distinguishing protocols using molecular biology technique for methodical identification of useful medicinal herbs.

In this study, it was able to identify R. japonica and R. sachalinensis based on molecular marker from psbK-I and atpF-H regions, respectively. And these newly designed molecular markers could be applied effectively to recognize both species. These molecular markers will be useful information for detection of genotyping maker for protection as well as digitalization for identification of cultivars. Furthermore, the protocol described in this study should be useful for further studies focusing an identification of economically important species. Generally, it can be applied to promote the value of plant resources and to increase the needs on modernized discrimination protocol of natural products.