• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.151-155
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• 2021

The application of environmental DNA in the domestic ecosystem is also accelerating, but the processing and analysis of the produced data is limited, and doubts are raised about the reliability of the analyzed and produced biological taxa identification data, and the sample medium (target sample, water, air, sediment, Gastric contents, feces, etc.) and quantification and improvement of analysis methods are also needed. Therefore, in order to secure the reliability and accuracy of biodiversity research using the environmental DNA of the domestic ecosystem, it is a process of actively using the database accumulated through ecological taxonomy and undergoing verification procedures, and experts verifying the resolution of the data increased by gene sequence analysis. This is absolutely necessary. Environmental DNA research cannot be solved only by applying molecular biology technology, and interdisciplinary research cooperation such as ecology-taxa identification-genetics-informatics is important to secure the reliability of the produced data, and researchers dealing with various media can approach it together. It is an area in desperate need of an information sharing platform that can do this, and the speed of development will proceed rapidly, and the accumulated data is expected to grow as big data within a few years.

• Journal of Korean Society of Forest Science
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• v.108 no.2
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• pp.200-207
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• 2019

The aim of this study was to provide the basic information necessary to identify Korean fir (Abies holophylla), momi fir (A. firma), and Nikko fir (A. homolepis), and other fir trees planted in South Korea that are protected by law. Analysis of the morphological characteristics of the needles from each sample was investigated. The shape of the needle-leaf tip from A. holophylla was acute, whereas that from A. firma and A. homolepis was emarginate and that from the protected fir trees was obtuse. The number of stomata on the needles was not significantly different between A. holophylla and A. firma, and the number of stomata on the needles from A. homolepis and the protected fir trees were highly similar. In addition, the genetic differences among the Abies species were analyzed using the sequences of five chloroplast DNA regions-matK, atpF-atpH, rpoC2-rps2, rpoC1, and psbA-trnH.The atpF-atpH and psbA-trnH regions were useful for discriminating A. firma from the other species, but there were no differences among A. holophylla, A. homolepis, and the protected fir trees. The same chloroplast sequences were found in both A. holophylla and A. homolepis, which suggests that additional genetic studies might be necessary to identify the Abies species planted in both South Korea and Japan.

• Korean Journal of Ichthyology
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• v.33 no.4
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• pp.217-225
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• 2021

The family Platycephalidae is a taxonomic group of economically important demersal flathead fishes that predominantly occupy tropical or temperate estuaries and coastal environments of the Indo-Pacific oceans and the Mediterranean Sea. In this study, we for the first time analyzed the complete mitochondrial genome (mitogenome) of the flathead Platycephalus cultellatus Richardson, 1846 from Vietnam by Next Generation Sequencing method. Its mitogenome was 16,641 bp in total length, comprising 13 protein-coding genes (PCGs), two ribosomal RNA genes, and 22 transfer RNA genes. The gene composition and order of the mitogenome were identical to those of typical vertebrates. The phylogenetic trees were reconstructed based on the concatenated nucleotide sequence matrix of 13 PCGs and the partial sequence of a DNA barcoding marker, cox1 in order to determine its molecular phylogenetic position among the order Scorpaeniformes. The phylogenetic result revealed that P. cultellatus formed a monophyletic group with species belonging to the same family and consistently clustered with one nominal species, P. indicus, and two Platycephalus sp. specimens. Besides, the cox1 tree confirmed the taxonomic validity of our specimen by forming a monophyletic clade with its conspecific specimens. The mitogenome of P. cultellatus analyzed in this study will contribute valuable information for further study on taxonomy and phylogeny of flatheads.

• 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.

• Journal of Food Hygiene and Safety
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• v.38 no.6
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• pp.464-475
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• 2023

In this study, based on an analysis of two DNA barcode markers (cytochrome c oxidase subunit I and cytochrome b genes), we performed species identification and monitored labeling compliance for 50 commercial pufferfish products sold in on-line markets in Korea. Using these barcode sequences as a query for species identification and phylogenetic analysis, we screened the GenBank database. A total of seven pufferfish species (Takifugu chinensis, T. pseudommus, T. xanthopterus, T. alboplumbeus, T. porphyreus, T. vermicularis, and Lagocephalus cheesemanii) were identified and we detected 35 products (70%) that were non-compliant with the corresponding label information. Moreover, the labels on 12 commercial products contained only the general common name (i.e., pufferfish), although not the scientific or Korean names for the 21 edible pufferfish species. Furthermore, the proportion of mislabeled highly processed products (n = 9, 81.8%) was higher than that of simply processed products (n = 26, 66.7%). With respect to the country of origin, the percentage of mislabeled Chinese products (n = 8, 80%) was higher than that of Korean products (n = 26, 66.7%). In addition, the market and dialect names of different pufferfish species were labeled only as Jolbok or Milbok, whereas two non-edible pufferfish species (T. vermicularis and T. pseudommus) were used in six commercial pufferfish products described as JolboK and Gumbok on their labels, which could be attributable to the complex classification system used for pufferfish. These monitoring results highlight the necessity to develop genetic methods that can be used to identify the 21 edible pufferfish species, as well as the need for regulatory monitoring of commercial pufferfish products.

• Journal of Food Hygiene and Safety
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• v.39 no.3
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• pp.209-220
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• 2024

Cavier is graded as Beluga, Osetra, and Sevruga based on the species of sturgeon (Acipenser sinensis). In this study, we developed an analytical method for determining the grade of black caviar using DNA barcodes and KASP markers. To identify the sturgeon species, ten black caviar samples were collected, and a reference DNA barcode library was developed using five genes (namely, 16S ribosomal RNA, cytochrome b, cytochrome c oxidase subunit I, cytochrome c oxidase subunit II, and NADH dehydrogenase subunit 5 genes). To develop the KASP markers, we selected 11 markers that could distinguish between the five grades of black caviar. As a result, specific markers for each of the targeted caviars were clustered into FAM-positive sections. DNA barcoding and the KASP assay revealed that one Beluga, six Osetra, and three Sevruga were identified among the ten caviar samples. Moreover, we found that the sturgeon species were mislabeled in two products. Here, we aimed to develop a KASP assay based on SNP that allows rapid and easy identification of caviar grade. These methods are expected to contribute to preventing the distribution of illegal products.

• Horticultural Science & Technology
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• v.35 no.3
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• pp.344-360
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• 2017

Zoysiagrasses are important turf plants used for school playgrounds, parks, golf courses, and sports fields. The two most popular zoysiagrass species are Zoysia japonica and Zoysia sinica. These are widely distributed across different growing zones and are morphologically distinguishable from each other; however, it is phenotypically difficult to differentiate those that grow along the coastal line from those in beach area habitats. A combination of morphological and molecular approaches is desirable to efficiently identify these two plant cultivars. In this study, we used a rapid identification system based on DNA barcoding of the nrDNA-internal transcribed spacer (ITS) regions. The nrDNA-ITS regions of ITS1, 5.8S nrDNA, and ITS2 from Z. japonica, Z. sinica, Agrostis stolonifera, and Poa pratensis were DNA barcoded to classify these grasses according to their molecular identities. The nrDNA-ITS sequences of these species were found at 686 bp, 687 bp, 683 bp, and 681 bp, respectively. The size of ITS1 ranged from 248 to 249 bp, while ITS2 ranged from 270 to 274 bp. The 5.8S coding region ranged from 163 - 164bp. Between Z. japonica and Z. sinica, nineteen (2.8%) nucleotide sites were variable, and the G+C content of the ITS region ranged from 55.4 to 63.3%. Substitutions and insert/deletion (indel) sites in the nrDNA-ITS sequence of Z. japonica and Z. sinica were converted to cleaved amplified polymorphic sequence (CAPS) markers, and applied to the Zoysia grasses sampled to verify the presence of these markers. Among the 62 control and collected grass samples, we classified three groups: 36 Z. japonica, 22 Z. sinica, and 4 Z. japonica/Z. sinica hybrids. Morphological classification revealed only two groups; Z. japonica and Z. sinica. Our results suggest that used of the nrDNA-ITS barcode region and CAPS markers can be used to distinguish between Z. japonica and Z. sinica at the species level.

• Journal of Life Science
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• v.27 no.11
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• pp.1331-1339
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• 2017

DNA barcoding is the identification of a species based on the DNA sequence of a fragment of the cytochrome C oxidase subunit I (COI) gene in the mitochondrial genome. It is widely applied to assist with the sustainable development of fishery-product resources and the protection of fish biodiversity. This study attempted to verify horse-head fish (Branchiostegus japonicus) and fake horse-head fish (Branchiostegus albus) species, which are commonly consumed in Korea. For the validation of the two species, a real-time PCR method was developed based on the species' mitochondrial DNA genome. Inter-species variations in mitochondrial DNA were observed in a bioinformatics analysis of the mitochondrial genomic DNA sequences of the two species. Some highly conserved regions and a few other regions were identified in the mitochondrial COI of the species. In order to test whether variations in the sequences were definitive, primers that targeted the varied regions of COI were designed and applied to amplify the DNA using the real-time PCR system. Threshold-cycle (Ct) range results confirmed that the Ct ranges of the real-time PCR were identical to the expected species of origin. Efficiency, specificity and cross-reactivity assays showed statistically significant differences between the average Ct of B. japonicus DNA ($21.85{\pm}3.599$) and the average Ct of B. albus DNA ($33.49{\pm}1.183$) for confirming B. japonicus. The assays also showed statistically significant differences between the average Ct of B. albus DNA ($22.49{\pm}0.908$) and the average Ct of B. japonicus DNA ($33.93{\pm}0.479$) for confirming B. albus. The methodology was validated by using ten commercial samples. The genomic DNA-based molecular technique that used the real-time PCR was a reliable method for the taxonomic classification of animal tissues.

• Korean Journal of Plant Taxonomy
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• v.49 no.3
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• pp.224-239
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• 2019

Species identification for the Korean tribe Desmodieae was conducted using the DNA barcoding genes rbcL, matK (from chloroplast DNA) and ITS (from nuclear ribosomal DNA). A total of 25 taxa (n = 75) in five genera were sequenced, and neighbor-joining trees were constructed using different combinations of DNA barcodes. When comparing these phylogenetic trees, a tree with all loci combined (rbcL + matK + ITS) showed the highest rate of identification success (72%). On this tree, two subtribes and five genera within the tribe were supported as monophyletic. In the Desmodiinae clade, Desmodium and Hylodesmum were more closely related to each other than to Ohwia. In the Hylodesmum clade, H. oldhamii was found to be a sister to H. podocarpum complex, and all taxa within the complex were identified successfully. Subsp. fallax, regarded as a variety of subsp. oxyphyllum, is closely clustered with subsp. podocarpum. Although var. mandshuricum has been regarded as a synonym of var. oxyphyllum, this taxon is supported as a distinct variety. For the Lespedezinae clade, all species of Kummerowia were monophyletic, while nine of 16 Lespedeza taxa were identified successfully. In particular, the resolution of Macrolespedeza (28.5%) was lower than that of Junceae (77.8%). Among the Lespedeza taxa, L. cuneata was distinguishable from L. lichiyuniae, despite morphological similarities. It has been suggested that both L. maritima and L. inschanica are hybrids. The former is thought to be an independent species. While it is difficult to determine whether the latter originated via hybridization, this study showed that it is closely related to L. juncea.

• Korean Journal of Ecology and Environment
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• v.56 no.1
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• pp.36-44
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• 2023

Environmental DNA (eDNA) can exist in both intracellular and extracellular forms in natural ecosystems. When targeting harmful cyanobacteria, extracellular eDNA indicates the presence of traces of cyanobacteria, while intracellular eDNA indicates the potential for cyanobacteria to occur. However, identifying the "actual" potential for harmful cyanobacteria to occur is difficult using the existing sediment eDNA analysis method, which uses silica beads and cannot distinguish between these two forms of eDNA. This study analyzes the applicability of a density gradient centrifugation method (Ludox method) that can selectively analyze intracellular eDNA in sediment to overcome the limitations of conventional sediment eDNA analysis. PCR was used to amplify the extracted eDNA based on the two different methods, and the relative amount of gene amplification was compared using electrophoresis and Image J application. While the conventional bead beating method uses sediment as it is to extract eDNA, it is unknown whether the mic gene amplified from eDNA exists in the cyanobacterial cell or only outside of the cell. However, since the Ludox method concentrates the intracellular eDNA of the sediment through filtration and density gradient, only the mic gene present in the cyanobacteria cells could be amplified. Furthermore, the bead beating method can analyze up to 1 g of sediment at a time, whereas the Ludox method can analyze 5 g to 30 g at a time. This gram of sediments makes it possible to search for even a small amount of mic gene that cannot be searched by conventional bead beating method. In this study, the Ludox method secured sufficient intracellular gene concentration and clearly distinguished intracellular and extracellular eDNA, enabling more accurate and detailed potential analysis. By using the Ludox method for environmental RNA expression and next-generation sequencing (NGS) of harmful cyanobacteria in the sediment, it will be possible to analyze the potential more realistically.