Min-Kyeoung Kim

Molecular authentication of Panax ginseng and ginseng products using robust SNP markers in ribosomal external transcribed spacer region

Panax ginseng and Panax quinquefolius are the most widely used Panax species, but they are known to have different properties and medicinal values. The aim of this study is to develop a robust and accurate DNA marker for identifying P. ginseng and the origins of ginseng products. Two single nucleotide polymorphism (SNP) sites specific to P. ginseng were exploited from nuclear ribosomal external transcribed spacer (ETS) region. Based on the SNP sites, two specific primers were designed for P. ginseng and P. quinquefolius respectively. P. ginseng can be easily discriminated from P. quinquefolius by amplifying the two specific alleles using multiplex allele-specific PCR. Favorable results can also be obtained from commercial ginseng products. The established method is highly sensitive and can detect 1% of intentional adulteration of P. quinquefolius into P. ginseng down to the 0.1ng level of total DNA. Therefore this study provides a reliable and simple DNA method for authentication of the origins and purities of ginseng products.

Molecular authentication of the Oriental medicines Pericarpium Citri Reticulatae and Citri Unshius Pericarpium using SNP markers

Pericarpium citri reticulatae and Citri unshius pericarpium are important Oriental medicinal materials used in many prescriptions. Among the Citrus species, the dried peels of C. japonica, C. maxima, and C. trifoliata are found to be adulterants and substitutes of Pericarpium citri reticulatae and Citri unshius pericarpium. In order to develop a simple and reliable DNA method for authentication of these two medicinal materials, nuclear ribosomal internal transcribed spacer (ITS) region was targeted for molecular analysis. A host of single nucleotide polymorphism (SNP) sites were detected among ITS sequences of five Citrus species. From two SNP sites, two modified specific primers were designed for authentication of Pericarpium citri reticulatae and Citri unshius pericarpium using multiplex PCR. The established multiplex allele-specific PCR system was proven to be effective for simultaneous authentication of Pericarpium citri reticulatae and Citri unshius pericarpium. The scheme used in this study could be adapted for determination of the botanical identity and origin of other medicinal materials.

Expression of the ginseng PgPR10-1 in Arabidopsis confers resistance against fungal and bacterial infection.

Korean ginseng (Panax ginseng C. A. Meyer) consists of nine cultivars from three Jakyung, Chungkyung, and Hwangsook lines. Among three previously identified PR-10 homologs from ginseng (PgPR10-1, PgPR10-2, and PgPR10-3), we found that the exact same sequence of PgPR10-2 exist in all tested nine cultivars. But a deletion and SNP was found in American ginseng (Panax quinquefolius). PR-10 proteins are known to be small and cytosolic, and showed similar three-dimensional structure. Here we show that the heterologous overexpression of PgPR10-1 in Arabidopsis showed enhanced resistance against Pseudomonas syringe, Fusarium oxysporum, and Botrytis cinerea and in-frame tagging with fluorescent protein showed its cytoplasm and nucleus localization. Protein-protein interaction of PgPR10-2 with PgPR10-1, PgPR10-2 and PgPR10-3 suggests that the PgPR10 proteins might form multimeric complexes in different cellular compartments to function in development and in defense-related mechanism. Differential response of PgPR10-1 and PgPR10-2 against different sets of biotic stresses in ginseng plant supports this notion.

Authentication of Medicinal Plants by SNP-Based Multiplex PCR

Highly variable intergenic spacer and intron regions from nuclear and cytoplasmic DNA have been used for species identification. Noncoding internal transcribed spacers (ITSs) located in 18S-5.8S-26S, and 5S ribosomal RNA genes (rDNAs) represent suitable region for medicinal plant authentication. Noncoding regions from two cytoplasmic DNA, chloroplast DNA (trnT-F intergenic spacer region), and mitochondrial DNA (fourth intron region of nad7 gene) are also successfully applied for the proper identification of medicinal plants. Single-nucleotide polymorphism (SNP) sites obtained from the amplification of intergenic spacer and intron regions are properly utilized for the verification of medicinal plants in species level using multiplex PCR. Multiplex PCR as a variant of PCR technique used to amplify more than two loci simultaneously.

Development of a single-nucleotide-polymorphism marker for specific authentication of Korean ginseng (Panax ginseng Meyer) new cultivar “G-1”

Background Korean ginseng (Panax ginseng) is a well-known medicinal plant of Oriental medicine that is still in practice today. Until now, a total of 11 Korean ginseng cultivars with unique features to Korean ginseng have been developed based on the pure-line-selection method. Among them, a new cultivar namely G-1 with different agricultural traits related to yield and content of ginsenosides, was developed in 2012. Methods The aim of this study was to distinguish the new ginseng cultivar G-1 by identifying the unique single-nucleotide polymorphism (SNP) at its 45S ribosomal DNA and Panax quinquefolius region than other Korean ginseng cultivars using multiplex amplification-refractory mutation system–polymerase chain reaction (ARMS-PCR). Results A SNP at position of 45S ribosomal DNA region between G-1, P. quinquefolius, and the other Korean ginseng cultivars was identified. By designing modified allele-specific primers based on this site, we could specifically identified G-1 and P. quinquefolius via multiplex PCR. The unique primer for the SNP yielded an amplicon of size 449 bp in G-1 cultivar and P. quinquefolius. This study presents an effective method for the genetic identification of the G-1 cultivar and P. quinquefolius. Conclusion The results from our study shows that this SNP-based approach to identify the G-1 cultivar will be a good way to distinguish accurately the G-1 cultivar and P. quinquefolius from other Korean ginseng cultivars using a SNP at 45S ribosomal DNA region.

Development of a multiplex amplification refractory mutation system for simultaneous authentication of Korean ginseng cultivars “Gumpoong” and “Chungsun”

Background and aims. Molecular authentication of Korean ginseng cultivars was investigated using the mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 7 (nad7) intron 3 region. Materials and methods. A mutation site specific to Panax ginseng “Gumpoong” and “Chungsun” cultivars was detected within the sequence data. Based on this mutation site and the “Gumpoong”-specific single nucleotide polymorphism site reported in 26S rDNA, two modified allele-specific primer pairs were designed and a multiplex amplification refractory mutation system (MARMS) was applied to identify “Gumpoong” and “Chungsun.” Results. The results showed that “Gumpoong” and “Chungsun” can be clearly discriminated from the other Korean ginseng cultivars by simultaneously identifying the haplotype of “Gumpoong” and the specific allele of “Chungsun” by applying the MARMS. Conclusion. This study, therefore, provides a simple and reliable method for simultaneous authentication of “Gumpoong” and “Chungsun” cultivars.

Discrimination of Korean ginseng (Panax ginseng Meyer) cultivar Chunpoong and American ginseng (Panax quinquefolius) using the auxin repressed protein gene

Background Korean ginseng (Panax ginseng) is one of the most important medicinal plants in the Orient. Among nine cultivars of P. ginseng, Chunpoong commands a much greater market value and has been planted widely in Korea. Chunpoong has superior quality “Chunsam” (1st grade ginseng) when made into red ginseng. Methods A rapid and reliable method for discriminating the Chunpoong cultivar was developed by exploiting a single nucleotide polymorphism (SNP) in the auxin repressed protein gene of nine Korean ginseng cultivars using specific primers. Results An SNP was detected between Chunpoong and other cultivars, and modified allele-specific primers were designed from this SNP site to specifically identify the Chunpoong cultivar and P. quinquefolius via multiplex polymerase chain reaction (PCR). Conclusion These results suggest that great impact to prevent authentication of precise Chunpoong and other cultivars using the auxin repressed protein gene. We therefore present an effective method for the authentication of the Chunpoong cultivar of P. ginseng and P. quinquefolius.