• 생약학회지
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• 제49권1호
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• pp.55-64
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• 2018

Recently, it has been a big issue to distinguish the dried roots of Cynanchum wilfordii and C. auriculatum in health functional food market. The original plant species of Cynanchi Wilfordii Radix belong to the Asclepiadaceae family is differentially described in the national pharmacopoeia of Korea, China and Japan. Owing to the morphological similarities of the dried roots of this plant to those of C. auriculatum, which is often misidentified in Korean herbal medicine marketplace, distinguishing these two species is exceedingly difficult. The purpose of this study was to compare the conventional-PCR with the real-time PCR for detection of C. wilfordii and C. auriculatum DNA. We also tried to realize a quantitative real-time PCR assay using species-specific matK primers, which allowed us to estimate the ratio of C. willfordii and C. auriculatum using varying ratios of mixed genomic DNA template from the two species. The differentiation of intentional and unintentional mixture in this study would be applied to food safety management and can be helpful for protection of consumer's right and cultivators.

• 원예과학기술지
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• 제32권6호
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• pp.853-863
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• 2014

국내외에서 재배되고 있는 딸기 100품종의 DNA profile 데이터베이스를 구축하기 위하여 다형성이 높은 microsatellite 마커의 선정과 유전적 유사도 분석을 통한 품종식별력 검정 등에 대한 연구를 수행하였다. 딸기 21품종을 274개의 microsatellite 마커로 검정하여 반복 재현성이 높은 25개의 다형성이 높은 마커를 선정하였다. 이들 마커와 국내외에서 재배되고 있는 딸기 100품종을 검정하였을 때 마커당 평균 대립유전자수는 7.50개로 나타났고, 3-13개까지 다양한 분포를 나타내었다. PIC 값은 마커의 유전자형에 따라 0.333-0.841 범위에 속하였으며 평균값도 0.706으로 높게 나타났다. Microsatellite 마커의 대립유전자를 이용하여 딸기 100 품종에 대한 계통도를 작성하였을 때 품종 육성의 계보 및 육성 지역에 따라 7개의 그룹으로 크게 나누어졌으며 2품종을 제외한 98품종이 microsatellite 마커의 유전자형에 의해 식별이 되는 것으로 나타났다. 본 연구에서 얻어진 딸기 품종별 DNA profile 데이터베이스는 품종보호 출원 품종의 재배심사 및 품종진위성과 관련된 종자분쟁을 해결하는 수단으로 유용하게 활용될 수 있을 것이다.

• 생명과학회지
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• 제29권5호
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• pp.524-529
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• 2019

엉겅퀴는 대표적인 다년생의 약용식물이다. 최근 국제적 추세에 따라 자국의 유전자원의 발굴, 보존 등이 강화 됨에 따라 인접국가와 국내 자생 엉겅퀴 계통을 판별 할 수 있는 기준 설정에 관한 연구의 필요성이 대두되고 있지만, 분자생물학적 판별 기술의 개발은 아직 미흡한 실정이다. 본 연구에서는 국내 토종과 해외 유래 엉겅퀴종의 기원을 판별하기 위해 엽록체에 존재하는 trnL-trnF와 MatK 유전자단편에서 SNP를 이용한 판별 프라이머를 확보하였으며 이를 보완하여 보다 신속하게 판별하기 위하여 HRM 분석 기술을 이용한 판별 마커와 그 조건을 확립하였다. 그러므로, 본 연구에서 개발된 SNP 마커는 다양한 지역 또는 국가에서 서식하는 엉겅퀴 종들의 신속한 확인을 위해 매우 유용하게 이용될 것으로 생각된다.

• 대한본초학회지
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• 제36권1호
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• pp.9-17
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• 2021

Objectives : Tetrapanacis Medulla and Akebiae Caulis are one of the most frequently adulterated herbal medicines because of their confusability of terms in the ancient writings and the similarity of morphological features of dried herbal products. The major adulterant is Aristolochia manshuriensis (Guanmutong) which has a serious safety concern with its toxicity. To ensure the safety and quality of the two herbal medicines, it is necessary to discriminate the toxic adulterant from authentic species. The aim of this study is to develop SCAR markers and to establish the multiplex-SCAR assay for discrimination of four plant species related to Tetrapanacis Medulla and Akebiae Caulis. Methods : ITS regions of fifteen samples of four species (Tetrapanax papyrifer, Fatsia japonica, Aristolochia manshuriensis, and Akebia quinata) collected from different sites were amplified and sequenced. Fifteen obtained ITS sequences were aligned and analysed for the detection of species-specific sequence variations. The SCAR markers were designed based on the sequence alignments and then, multiplex-SCAR assay enhancing rapidity was optimized. Results : ITS sequences clearly distinguished the four species at the species level. The developed SCAR markers and multiplex-SCAR assay were successfully discriminated four species and detected the adulteration of commercial product samples by comparison of the amplified DNA fragment sizes. Conclusions : These SCAR markers and multiplex-SCAR assay are a rapid, simple, and reliable method to identify the authentic Tetrapanacis Medulla and Akebiae Caulis from adulterants. These genetic tools will be useful to ensure the safety and to standardize the quality of the two herbal medicines.

• 대한본초학회지
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• 제34권5호
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• pp.13-20
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• 2019

Objectives : To ensure the safety, quality and pharmacological efficacy of Batryticatus Bombyx, it is important to discriminate with adulterants. In Korean Herbal Pharmacopoeias (KHP), the authentic species of Batryticatus Bombyx is defined only Bombyx mori. Therefore, the aim of this study is establishment of PCR assay method using the sequence characterized amplified region (SCAR) marker based on COI DNA barcode for discriminating six species related to Batryticatus Bombyx. Methods : Seventeen samples of six species (Bombyx mori, Bombyx mandarina, Rhodinia fugax, Oberthueria caeca, Actias artemis, and Caligula japponica) were collected from different habitate and nucleotide sequences of cytochrome c oxidase subunit I(COI) barcode regions were analyzed by Sanger sequencing methods. To develop SCAR-based PCR assay method, we designed species-specific primers based on COI sequence variabilities and verified those specificities using 17 samples of six species as well as commercial herbal medicines. Results : In comparative multiple analysis of COI sequences, six species were distinguished by species-specific nucleotides at the species level. To develop rapid and reliable PCR assay method for genetic authentication of Batryticatus Bombyx, therefore, we designed species-specific SCAR primers based on these nucleotide sequences and confirmed those specificities. Using these SCAR primers, We also established simple conventional PCR assay method using these SCAR primers at the species level. Conclusions : The comparative analysis of COI sequences and SCAR-based PCR assay methods represented equal results for distinguishing authentic Batryticatus Bombyx and adulterations at the species level. Therefore, our results are expected protecting adulteration of herbal medicine Batryticatus Bombyx.

• 대한본초학회지
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• 제24권4호
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• pp.115-120
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• 2009

Objectives : Due to the morphological similarity and frequent occurrence of intermediate forms as well as morphological variations of aerial part, the correct identification between Rhei Radix et Rhizoma and Rhei Undulatai Rhizoma is very difficult. To develop a reliable method for correct identification and improving the quality standards of Rhei Radix et Rhizoma and Rhei Undulatai Rhizoma, we analyzed RAPD and developed SCAR marker. Methods : To amplify target DNA at the genomic level, 32 Operon 10-mer random primers were applied with four Rheum species, R. officinale, R. palmatum, R. tanguticum and R. undulatum. The nucleotide sequences were determined and species-specific primers were prepared depending on the species-specific RAPD amplicons after subcloned into the pGEM-Teasy vector. To develop the SCAR markers, species-specific PCR amplification and multiplex-PCR were carried out using the single species-specific primer pairs and combinations of them, respectively. Results : We used RAPD analysis of four Rheum plant species to obtain several species-specific RAPD amplicons. From nucleotide sequences of these RAPD amplicons, we developed two SCAR markers that amplified 314 bp and 390 bp DNA fragments in only R. undulatum but not in R. officinale, R. palmatum, R. tanguticum and R. undulatum, for distinguishing Rhei Undulatai Rhizoma and Rhei Radix et Rhizoma. Furthermore, we established SCAR markers for the simultaneous discrimination of the three species within a single reaction by using multiplex-PCR. Conclusions : These genetic markers can be used for the efficient discrimination of plants species and commercial herbal medicines between Rhei Undulatai Rhizoma and Rhei Radix et Rhizoma, to ultimately prevent indiscriminate distribution and prescription of these herbal medicines.