• Journal of Ginseng Research
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• v.38 no.2
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• pp.123-129
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• 2014

Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius) are widely used medicinal plants with similar morphology but different medicinal efficacy. Roots, flowers, and processed products of Korean and American ginseng can be difficult to differentiate from each other, leading to illegal trade in which one species is sold as the other. This study was carried out to develop convenient and reliable chloroplast genome-derived DNA markers for authentication of Korean and American ginseng in commercial processed products. One codominant marker could reproducibly identify both species and intentional mixtures of the two species. We further developed a set of species-unique dominant DNA markers. Each species-specific dominant marker could detect 1% cross contamination with other species by low resolution agarose gel electrophoresis or quantitative polymerase chain reaction. Both markers were successfully applied to evaluate the original species from various processed ginseng products purchased from markets in Korea and China. We believe that high-throughput application of this marker system will eradicate illegal trade and promote confident marketing for both species to increase the value of Korean as well as American ginseng in Korea and worldwide.

• Korean Journal of Ichthyology
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• v.34 no.3
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• pp.208-217
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• 2022

We wanted to develop a real-time PCR assay capable of detecting Liobagrus obesus in environmental DNA (eDNA) extracted from freshwater samples using a pair of species-specific primers and probe for the endangered fish, L. obesus. The species-specific primers and probe were designed in consideration of single nucleotide polymorphisms between 65 species of freshwater fish living in the Republic of Korea within the cytochrome b (cytb) gene of mitochondrial DNA. The species-specific primers and probe, in the real-time PCR assay, showed high specificity as only the L. obesus genomic DNA (gDNA) was found to be positive in the specificity verification using 65 species gDNA of freshwater fish in the Republic of Korea. In addition, in the detection limit analysis using the serial dilution concentrations of L. obesus gDNA, it was found that it was possible to detect up to 0.2 pg, showing high sensitivity. Afterwards, using the species-specific primers and probe, real-time PCR assay was performed on freshwater samples obtained from 8 stations in the mid-upper basin of Geum River. As a result, the cytb gene of L. obesus was detected in total 5 stations including all 3 stations where this species was collected at the time of field survey. Therefore, the species-specific primers and probe developed in present study, and the real-time PCR assay using them, can accurately detect the cytb gene of L. obesus from eDNA samples, which can be utilized to monitor the existing habitats of this species and to discover potential new habitats.

• Korean Journal of Medicinal Crop Science
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• v.13 no.2
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• pp.121-125
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• 2005

An analysis of RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) was performed with three Angelica species (A. gigas Nakai, A. sinensis (Olive.) Diels and A. acutiloba Kitag) in an effort to distinguish between members of these three species. Two arbitrary primers (OPC02, OPD11) out of80 primers tested, produced 17 species-specific fragments among the three species. Eight fragments were specific for A. sinensis, four fragments specific for A. gigas, five specific for A. acutiloba. When primers OPC02 and OPD11 were used in the polymerase chain reaction, RAPD-PCR fragments that were specific for each of the three species were generated simultaneously. Primer OPC02 produced eight species-specific fragments: four were specific for A. sinensis, one for A. gigas, and three for A. acutiloba. Primer OPD11 produced nine speciesspecific fragments: four for A. sinensis, three for A. gigas, and two for A. acutiloba. The RAPD-PCR markers that were generated with these two primers should rapidly identify members of the three Angelica species. The consistency of the identifications made with these species-specific RAPD-PCR markers was demonstrated by the observation that each respective marker was generated from three accessions of each species, all with different origins. We also performed the RAPD-PCR analysis with the dried Angelica root samples that randomly collected from marketed and from the OPC02 primer, obtained a A. gigasspecific band and the band were cloned and sequenced.

• Genomics & Informatics
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• v.21 no.2
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• pp.24.1-24.7
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• 2023

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

• The Korea Journal of Herbology
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• v.37 no.5
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• pp.9-16
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• 2022

Objectives : The purpose of this study is to report that applying the genetic discrimination method to Pinelliae Tuber is suitable as a countermeasure for the limitations of morphological identification announced publicly in the Ministry of Food and Drug Safety(MFDS). Methods : Randomly selected fifty samples in Pinelliae Tuber imported from China were used for morphological and genetic identification. The morphological identification was applied method announced publicly by the MFDS. The traits of morphological identification were classified as Pinellia ternata, P. tripartita, Pinellia pedatisecta, and Typhonium flagelliforme, according to the formation of tuberous root and tuber morphology. The genetic identifications were conducted by Sequence Characterized Amplified Region(SCAR) marker and DNA barcoding analysis for cross-validation, respectively. SCAR marker was verified according to the presence or absence of amplicon through PCR amplification using species-specific primers. DNA barcoding analysis used sequence information of the matK region. Results : As a result of the morphological identification, 27 out of 50 samples were identified as original species 'P. ternata' of genuine 'Pinelliae Tuber', and 23 were identified as adulterant species 'P. pedatisecta'. Unlike this, the genetic identification was identified as the original species 'P. ternata' in all 50 samples in the SCAR marker and matK regional sequence analysis. Conclusions : Pinelliae Tuber of morphological mutant that can not be classified by morphological identification is imported from China. The SCAR marker would be used as accurate and efficient assays for species identification of the morphological mutant.

• Development and Reproduction
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• v.15 no.4
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• pp.315-324
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• 2011

This study was conducted to detect the specific expressing genes by using RAPD-PCR and RFLP method in the Korea Native Brindled Cattle, Korean Native cow and Holstein cattle. And then, the specific marker gene was investigated by the analysis of the genes for detection significance according to the expressing pattern. We found the specific expression gene by the RAPD-PCR analysis in Korea Native Brindled Cattle. It was detected the differences of the species in the colour and external section. The Korea Native Brindled Cattle were vary low compare to the Korean Native cow and Holstein cattle by analysis result of polymorphism and distribution. And there were a found the specific marker gene by sequencing in the R9B gene fragment of Korea Native Brindled Cattle. And the sequencing result of the R9B was different between Korean Native cow and Holstein cattle. Thus, this gene can be apply as the specific marker gene in the Korea Native Brindled Cattle.

• The Korea Journal of Herbology
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• v.29 no.6
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• pp.45-53
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• 2014

Objectives : Due to the morphological similarity of in the roots of herbal medicine, the official herbal medicine is very difficult to authenticate between the original plants of Patriniae Radix and two adulterant Patrinia species. Therefore, we introduced DNA barcode analysis to establish a powerful tool for the authentication of Patriniae Radix from its adulterants. Methods : To analyze DNA barcode regions, genomic DNA was extracted from twenty-nine specimens of Patrinia scabiosaefolia, Patrinia villosa, Patrinia saniculifolia, and Patrinia rupestris, and internal transcribed spacer 2(ITS2), matK and rbcL genes were amplified. For identification of species specific sequences, a comparative analysis was performed by the ClastalW based on entire sequences of ITS2, matK and rbcL genes, respectively. Results : In comparison of three DNA barcode sequences, we identified 22, 22, and 12 species-specific nucleotides enough to distinguish each four species from ITS2, matK and rbcL gene, respectively. The sequence differences at the corresponding positions were available genetic marker nucleotides to discriminate the correct species among analyzed four species. These results indicated that comparative analysis of ITS2, matK and rbcL genes were useful genetic markers to authenticate Patriniae Radix. Conclusions : The marker nucleotides enough to distinguish P. scabiosaefolia, P. villosa, P. saniculifolia, and P. rupestris, were obtained at 22 SNP marker nucleotides from ITS2 and matK DNA barcode sequences, but they were confirmed at 12 SNP marker nucleotides from rbcL. These differences could be used to authenticate Patriniae Radix from its adulterants as well as discriminating each four species.

• The Korea Journal of Herbology
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• v.37 no.4
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• pp.9-16
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• 2022

Objectives : In the Korean Pharmacopoeia 12th edition (KP 12) and the Korean Herbal Pharmacopoeia (KHP), two authentic herbal medicines are described, namely Bang-gi (Cheong-pung-deung) and Mok-bang-gi, respectively. In China, Bun-bang-gi is also used as herbal medicine. This study was conducted to develop a molecular authentication tool for distinguishing the three herbal medicine used as Bang-gi, which are Sinomeni Caulis et Rhizoma (Rhizome of Sinomenium acutum), Stephaniae Tetrandrae Radix (Root of Stephania terandra), and Cocculi Radix (Root of Cocculus trilobus). Methods : Twelve samples of three species (four samples of S. acutum, five samples of S. tetrandra, and three samples of C. trilobus) were collected from different habitats. The sequences of internal transcribed spacer (ITS) regions were obtained and comparatively analyzed to design the species-specific sequence characterized amplified region (SCAR) primers. The specificity of each pair of SCAR primers that amplified species-specific amplicon was evaluated for establishing the singleplex and multiplex PCR assay tools. Results : The singleplex SCAR markers show discriminability in C. acutum, S. tetrandra, and C. trilobus. These SCAR markers were also efficiently authenticated three species in the multiplex SCAR amplification using single PCR reaction. Furthermore, these PCR assay methods were applicable to authenticate dried herbal medicines distributed in the markets. Conclusions : The SCAR markers and PCR assay tools help discriminate the three herbal medicines used as Bang-gi at the species levels and provide a reliable genetic method to prevent the inauthentic distribution of these herbal medicines.

• The Korea Journal of Herbology
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• v.30 no.3
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• pp.41-47
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• 2015

Objectives : Due to the morphological similarity of the pericarp and description of multi-species in National Pharmacopoeia of Korea and China, the Zanthoxylum Pericarpium is difficult to authenticate adulterant in species levels. Therefore, we introduced the sequence analysis of DNA barcode and identification of single nucleotide polymorphism(SNP) to establish a reliable tool for the distinction of Zanthoxylum Pericarpium from its adulterants. Methods : To analyze DNA barcode region, genomic DNA was extracted from twenty-four specimens of authentic Zanthoxylum species and inauthentic adulterant and the individual internal transcribed spacer regions (rDNA-ITS and ITS2) of nuclear ribosomal RNA gene were amplified using ITS1, ITS2-S2F, and ITS4 primer. For identification of species-specific sequences, a comparative analysis was performed using entire DNA barcode sequences. Results : In comparison of four Zanthoxylum ITS2 sequences, we identified 16, 4, 6, and 4 distinct species-specific nucleotides enough to distinguish Z. schinifolium, Z. bungeanum, Z. piperitum, and Z. simulans, respectively. The sequence differences were available genetic marker to discriminate four species. Futhermore, phylogenetic relationship revealed a clear classification between different Zanthoxylum species showing 4 different clusters. These results indicated that comparative analysis of ITS2 DNA barcode was an useful genetic marker to authenticate Zanthoxylum Pericarpium in species levels. Conclusions : The marker nucleotides, enough to distinguish Z. schinifolium, Z. piperitum, Z. bungeanum, and Z. simulans, were obtained at 30 SNP marker nucleotides from ITS2 sequences. These differences could be used to authenticate official Zanthoxylum Pericarpium from its adulterants as well as discriminating each four species.

• Journal of Plant Biotechnology
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• v.50
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• pp.190-199
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• 2023

Date palm (Phoenix dactylifera L.: Arecaceae) is a dioecious species where only female trees bear fruits. In their natural state, date palms produce dates once a year. However, in Thailand, some trees were observed to produce dates during the off-season, despite no variations in morphology. The availability of such off-season fruits can significantly increase their market value. Interestingly, most female off-season date palms investigated in this study were obtained through micropropagation. Hence, there is an urgent need for genetic markers to distinguish female offseason flowering plantlets within tissue culture systems. In this study, we aimed to develop random amplification of polymorphic DNA-sequence characterized amplified region (RAPD-SCAR) markers for the identification of female off-season flowering date palms cultivated in Thailand. A total of 160 random decamer primers were employed to screen for specific RAPD markers in off-season flowering male and female populations. Out of these, only one primer, OPN-02, generated distinct genomic DNA patterns in female off-season flowering (FOFdp) individuals compared to female seasonal flowering genotypes. Based on the RAPD-specific sequence, specific SCAR primers denoted as FOFdpF and FOFdpR were developed. These SCAR primers amplified a single 517-bp DNA fragment, predominantly found in off-season flowering populations, with an accuracy rate of 60%. These findings underscore the potential of SCAR marker technology for tracking offseason flowering in date palms. Notably, a BLAST analysis revealed a substantial similarity between the SCAR marker sequence and the transcript variant mRNA from Phoenix dactylifera encoding the SET DOMAIN GROUP 40 protein. In Arabidopsis, this protein is involved in the epigenetic regulation of flowering time. The genetic potential of the off-season flowering traits warrants further elucidation.