• 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.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.474-478
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• 2005

This study was conducted to analyze genetic characteristics and to develop the specific marker for Cheju native horse (Coo) at the level of sequence characterized amplified regions (SCARs). We collected blood samples from Cheju native horse and Thoroughbred horse (Th) and obtained genomic DNA from the blood of 50 individuals randomly selected within the breeds. Seven hundred primers were chosen randomly and were used to examin the polymorphism and 40 kinds of primers showed polymorphic RAPD band patterns between two breeds. Thirty primers of them showed horse specific bands. With the primer MG 30, amplified band of 2.0 kb showed the specificity to Cheju native horse (Cnh). Additionally MG 53 detected the thoroughbred horse (Th) specific markers at size of 2.3 kb. As the next, 2.3 kb band from MG 53 was checked with the all individuals from all the breeds of this study, and it maintained the reproducible breed specificity to thoroughbred horse (Th). With this results, 2.3 kb band was cloned into plasmid vector and sequenced bidirectionally from both ends of the cloned fragment. With the obtained sequences 10 nucleotide extended primers including the original arbitray primer were designed as a SCARs primer. Finally, the primer with extended sequence showed the reproducible breed differentiation pattern and it was possible to identify Cheju native horse (Cnh) from other breeds. The SCARs marker 2.3 kb from MG 53 could be used to identify Cheju native horse (Cnh) for not only registration but also horse breeding programe.

• 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.

• Journal of Life Science
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• v.31 no.6
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• pp.537-542
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• 2021

Schisandra nigra Max., a dioecious plant native to Jeju Island in Korea, is cultivated on a small scale for fruit production. As fruit-producing female individuals are generally considered to be more valuable than male, early identification of plant sex at the seedling stage is important. In this study, a sequence-characterized amplified region (SCAR) marker associated with a female-specific region in the genome of S. nigra was investigated. Of 120 randomly amplified polymorphic DNA (RAPD) primers, one primer (OPB-03) consistently amplified a 749 bp band in female plants. The female-specific PCR product was isolated and cloned, and the nucleotide sequences were then determined. Southern hybridization performed using the female-specific fragment as a probe produced positive signals only in genomic DNA from the female plants. This result revealed that the 749 bp segment of DNA was present in the genome of female plants but absent in the genome of male plants. A SCAR primer pair was designed based on the RAPD marker to amplify a 436 bp fragment in the genomic DNA of female plants. This primer pair amplified the expected size of DNA fragment in female plants and four monoecious individuals collected from a natural population. The SCAR marker identified in this study can be used to distinguish female-flowering individuals at the seedling stage.

• 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.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.34-34
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• 2003

한국산 선발계통 및 일본산 양식계통과 이들 두 계통간 잡종 참돔 집단의 유전적 특성을 분석하기 위하여, RAPD (Random Amplified Polymorphic DNA) marker를 탐색하였다. 10개의 염기로 이루어진 200개의 random primer 분석을 통하여 polymorphic pattern을 나타내는 23개의 random primer를 선발하였으며, 각 primer의 재현성을 확인하였다. 이들 중 OPA-11 primer는 크기가 각각 600 bp, 650 bp 및 750 bp 인 3개의 DNA 단편에 의하여 4개의 genotype을 나타냈으며, 각 genotype의 빈도는 집단간차이를 보였고, 한국산 선발계통 집단에서는 4개의 genotype이 모두 발견되는 반면, 일본산 양식계통 및 일본산 양식계통을 포함한 교배집단에서는 특정 genotype만 발견되었다. OPA-11 primer 유래의 polymorphic DNA 단편을 cloning하고 염기서열을 결정하였으며, SCAR (Sequence Characterized Amplified Region) primer를 제작하고 분석하였다. 본 연구는 참돔집단의 유전적 특성 파악 및 집단 구별에 RAPD marker를 활용하였으며, 참돔 육종시 형질 및 기능관련 DNA marker 탐색에 적용하기 위하여, 이후의 연구에서는 SCAR과 RFLP 분석에 RAPD marker를 이용하여 100% 정확도를 갖는 RFLP maker를 찾고, MAS (Marker-Assisted Selection)에 적용하고자 한다.

• The Korea Journal of Herbology
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• v.39 no.2
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• pp.1-9
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• 2024

Objectives : Paeng-hwal is described as an insect herbal medicine used for digestive diseases in the Dong-ui-bo-gam. The origin of this herbal medicine is limited to several small crabs, such as Helice tridens. These crab species cohabitat in the same environment and share similar morphological characteristics, making it very difficult to distinguish and collect the individual species for use in dietary supplements or herbal medicines. This study was conducted to develop a genetic identification tool for discriminating among these closely related small crab species. Methods : CO1 DNA barcode regions of 15 samples from 6 species of small crabs were analyzed to obtain the individual sequences. To identify the correct species, comparative analyses were carried out using the database of the NCBI GenBank and the NIBR. SCAR primers were designed to develop simple and rapid assay methods using inter-species specific sequences. Optimal SCAR assay conditions were established through gradient PCR, and the limit of detection (LOD) was determined. Results : Six species of small crabs (Helicana tridens, Macrophthalmus abbreviatus, Helicana tientsinensis, Helicana wuana, Chiromantes dehaani, and Hemigrapsus penicillatus), which are distributed as Paeng-hwal, were identified through CO1 sequences analysis. We also developed SCAR markers to distinguish between six small crabs at the species level. Furthermore, we established the optimal PCR assay methods and the LOD of each individual species. Conclusions : The rapid and simple SCAR-PCR assay methods were developed to identify the species and control the quality of herbal medicines for Paeng-hwal based on the genetic analyses of CO1 DNA barcodes.

• Journal of Korean Society of Forest Science
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• v.105 no.4
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• pp.422-428
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• 2016

Ginkgo (Ginkgo biloba L.) is one of the most appropriate roadside trees because of a good transplantation nature and ability to grow well in urban environment. Ginkgo is a dioecious species. Sex discrimination of ginkgo is possible through comparing morphological characters of reproductive organs. However, it needs more than about twenty years for reproductive organs to appear after sexual maturity. Until now, ginkgo trees for roadside plantation have been planted without discriminating the sex because ginkgo trees have been usually planted before sexual maturity. Ginkgo nuts from the female ginkgo trees planted along the roadside emit a foul odor, and make much pollution on the streets. Thus in this study a novel SCAR marker (SCAR-GBM) for the early sex discrimination was developed. Primers were developed on the basis of the sequence of male-specific RAPD variants reported previously. False-negative problem of SCAR marker, probably caused by dominant nature, was resolved by using multiplex PCR using primers of both the SCAR-GBM and a universal primer set of atp1 region in mitochondria DNA, which resulted in improved discrimination efficiency. The results showed that DNA bands of 1,039 bp were commonly amplified by the atp1 primer set in male and female trees, and SCAR-GBM markers of 675 bp were specifically amplified only in male trees. Reproducible and specific discrimination of the multiplex PCR was finally confirmed by applying multiple male and female individuals.

• Journal of Korean Society of Forest Science
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• v.108 no.3
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• pp.302-310
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• 2019

Precise and fast identification of crop cultivars is essential for efficient breeding and plant breeders' rights. Traditional methods for identification of jujube cultivars are based on the evaluation of morphological characteristics. However, due to time constraints and environmental influences, it is difficult to distinguish cultivars using only morphological traits. In this study, we cloned fragments from improved inter simple sequence repeats (ISSR) analysis, and developed stably diagnostic sequence-characterized amplified region (SCAR) markers. The specific ISSR bands of jujube cultivars from Dalizao and Boeundaechu were purified, cloned, and sequenced. As a result, four clones labeled 827Dalizao550, 827Boeun750, 846Boeun700, and 847Dalizao850 were identified. In order to investigate whether they were specific for the jujube cultivar, four pairs of SCAR primers were then designed and polymerase chain reaction (PCR) amplifications were conducted to analyze 32 samples, including jujube and sour jujube. In the PCR amplification of the 827Dalizao550 SCAR marker, the specific bands with 550 bp were amplified in six samples (Dalizao, Sandonglizao, Dongzao, Yuanlin No. 2, Suanzao 2, Suanzao 4), but unexpected bands (490 bp) were amplified in the others. Moreover, in the PCR amplification of the 847Dalizao850 SCAR marker, the specific bands with 850 bp were found in three samples (Dalizao, Sandonglizao, and Dongzao) and 900 bp unexpected bands were amplified in five samples (Pozao, Suanzao 1, Suanzao 2, Suanzao 3, Suanzao 4). These results showed that newly developed markers could be useful as a fast and reliable tool to identify jujube cultivars. However, further identification of polymorphic information and the development of SCAR markers are required for the identification of more diverse cultivars.

• The Korea Journal of Herbology
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• v.34 no.5
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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.