• 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.

• Korean Journal of Ichthyology
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• v.34 no.3
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• pp.186-200
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• 2022

In order to understand the species composition and distribution characteristics of larval assemblages in the East Sea, Korea, larvae were collected at 13 stations every other month from February to December 2018. Fish larvae were identified through DNA barcoding along with morphological methods, and as the result, a total of 104 taxonomic groups appeared during the survey. Among these, Engraulis japonicus, which accounted for 76.2% of the total population, was the most dominant species, followed by Maurolicus japonicus, which accounted for 15.0%. In addition, Ammodytes personatus, Champsodon snyderi, Scomber japonicus and Echelus uropterus appeared more frequently than other taxa. The above six species accounted for 93.2% of the total catch. The number of taxa and the amount of larvae collected in the survey area were higher during the high water temperature periods (June to October) than during the low water temperature periods (February, April and December). Analysis of similarity (ANOSIM) showed a statistically significant difference in monthly larval assemblages. The results of the canonical correspondence analysis (CCA) show that the distributions of larval community were mainly affected by sea surface temperature during low water temperature periods, and various environmental factors such as salinity, dry weight of zooplankton, and the concentrations of nutrients during high water temperature periods.