• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.22 no.1
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• pp.31-44
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• 2017

Pfiesteria piscicida is one of heterotrophic dinoflagellate having toxic metaboliges, and it is difficult to detect and quantify this dinoflagellate via light microscope due to small size and morphological similarity with Pfiesteria-like dinoflagellate (PLD) species. Alternatively, we developed quantitative real-time PCR assay based on EvaGreen and determined field accessibility throughout the investigation of distribution in the entire Korean coastal waters and population dynamics in Shihwa Lake. The P. piscicida-specific primers based on internal transcribed spacer 1 (ITS 1) were designed and the specificity of primers was confirmed by PCR with other genomic DNAs which have genetic similarity with target species. Through real-time PCR assay, a standard curve which had a significant linear correlation between log cell number and $C_T$ value ($r^2{\geq}0.999$) and one informative melting peak ($88^{\circ}C$) were obtained. These results implies that developed real-time PCR can accurately detect and quantify P. piscicida. Throughout the field applications of real-time PCR assay, P. piscicida was distributed in western (Mokpo and Kimje) and easthern (Gangneng) Korean coastal water even though light microscopy failed to identify P. piscicida. In the investigation of population dynamics in Shihwa Lake, the density of P. piscicida was peaked in June, July and August 2007 at St. 1 where salinity (${\leq}15psu$) was lower than the other 2 sites. In this study, we successed to develop EvaGreen bassed real-time PCR for detection and quantification of P. piscicida in fields, so this developed assay will be useful for various ecological studies in the future.

• The Korean Journal of Mycology
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• v.42 no.1
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• pp.1-8
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• 2014

Twelve Universal fungal PCR fingerprint (UFPF) primers that were modified from Universal rice primer (URP) were used to assess genetic diversity of 64 Agaricus strains including 45 A. bisporus strains and other 19 strains of other Agaricus spp. Eight primers, UFPF1, UFPF2, UFPF3, UFPF7, UFPF9, UFPF10, UFPF11, and UFPF12 produced PCR polymorphic bands within and between the Agaricus species. Primer UFPF7 produced specific PCR polymorphic bands that are distinct Korean strain from different strains. Ninety five PCR polymorphic bands were inputted for UPGMA cluster analysis. Forty five strains of A. bisporus are genetically clustered into 8 groups, showing coefficient similarity from 0.75 to 0.9 among them. The varieties, Saea, Saedo, Saejeong and Saeyeon that have recently been developed in Korea were involved in the same group with close genetic relationship of coefficient similarity over 0.96, whereas, other Korean strains were genetically related to A. bisporus strains that were introduced from USA, Eroupe and Chinese.

• The Korean Journal of Mycology
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• v.41 no.1
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• pp.47-51
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• 2013

Yeasts isolated from wild flowers of Gyejoksan in Daejeon city, Oseosan in Chungchungnamdo, and Baekamsan in Jeollabukdo, Korea were identified by comparison of nucleotide sequences for PCR-amplified D1/D2 region of 26S rDNA or internal transcribed spacer (ITS) 1 and 2 including 5.8S rDNA using BLAST. Twelve yeast strains of ten species and seventeen yeast strains of ten species were isolated from wild flowers of Gyejoksan and Oseosan, respectively. And thirty seven yeast strains of twenty four species were isolated from wild flowers of Baekamsan. Total thirty four yeast species were isolated from three different sample collection areas, but only nine species were overlapped from the at least two different sampling areas: Cryptococcus sp., Cryptococcus aureus, Cryptococcus flavescens, Cryptococcus flavus, Metschnikowia sp., Pseudozyma aphidis, Rhodotorula glutinis, Sporobolomyces carnicolor, and Sporobolomyces ruberrimus. Among them only Cryptococcus aureus was occurred from all three different collection sites. Other twenty five species were restricted to specific collection site suggesting that each area has distinctive yeast flora.

• Journal of Plant Biotechnology
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• v.47 no.1
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• pp.15-25
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• 2020

Allium L. is one of the largest genera of the Amaryllidaceae family, with more than 920 species including many economically important species used as vegetables, spices, medicines, or ornamental plants. Currently, DNA barcoding tools are being successfully used for the molecular taxonomy of Allium. A total of 46 Allium species were collected from their native areas, and DNA was extracted using the IBRC DNA extraction kit. We used specific primers to PCR amplify matK. DNA sequences were edited and aligned for homology, and a phylogenetic tree was constructed using the neighbor-joining method. The results show thymine (38.5%) was the most frequent and guanine (13.9%) the least frequent nucleotide. The matK regions of the populations were quite highly conserved, and the amount of C and CT was calculated at 0.162 and 0.26, respectively. Analysis of the nucleotide substitution showed C-T (26.22%) and A-G (8.08%) to have the highest and lowest percent, respectively. The natural selection process dN/dS was 1.16, and the naturality test results were -1.5 for Tajima's D and -1.19 for Fu's Fs. The NJ dendrogram generated three distinct clades: the first contained Allium austroiranicum and A. ampeloprasum; the second contained A. iranshahrii, A. bisotunense, and A. cf assadi; and the third contained A. rubellum and other species. In this study, we tested the utility of the matK region as a DNA barcode for discriminating Allium. species.

• The Plant Pathology Journal
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• v.18 no.4
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• pp.187-191
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• 2002

This paper describes the cloning and sequence analysis of the 5'-terminal region and full-length cDNA production of genomic RNA of Lily symptomless virus (LSV), a Species Of the genus Carlavirus. A sing1e DNA band about 600 bp harboring the 5'-end of genomic RNA of the virus was successfully amplified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), and was cloned for nucleotide sequence determination. Sequence analysis of selected RACE cDNA clones revealed that the LSV 5'non-translated region consists of 67 nucleotides long of AT rich stretch followed GC rich from the 5'-end. To produce full-length cDNA products for the viral genomic RNA, a set of LSV-specific primers could be designed based on the obtained sequence in this study and the known sequences of 3'-terminal region for the virus. Full-length cDNA copies of LSV, an 8.4 kb long, were directly amplified by the long-template RT-PCR technique from the purified viral genomic RNA samples. This full-length cDNA copies were analyzed by restriction mapping. The molecules produced in this study can be useful for the production of in vitro infectious cDNA clone, as well as, for the completion of genomic RNA sequence and genome structure for the virus.

• Mycobiology
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• v.31 no.2
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• pp.68-73
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• 2003

Roots of Glycine max and Miscanthus sinensis and soil samples were collected from various field sites at Goesan, Chungbuk in Korea. Microscopic observations of the roots indicated high colonization rates of both arbuscular mycorrhizal fungi(AMF) and other fungi. The partial small subunit of ribosomal DNA genes were amplified with the genomic DNA extracted from their roots by nested polymerase chain reaction(PCR) with universal primer NS1 and fungal specific primers AML Restriction fragment length polymorphism(RFLP) was analyzed using the combinations of three restriction enzymes, HinfI, AluI and AsuC21. Nucleotides sequence analysis revealed that ten sequences from Miscanthus sinensis and one sequence from Glycine max were close to those of arbuscular mycorrhizal fungi. Also, 33% of total clones amplified with NS31-AM1 primers from M. sinensis and 97% from G. max were close to Fusarium oxysporum or other pathogenic fungi, and they were successfully distinguished from AME Results suggested that these techniques could help to distinguish arbuscular mycorrhizal fungi from root pathogenic fungi in the plant roots. Especially, DNA amplified by these primers showed distinct polymorphisms between AMF and plant pathogenic species of Fusarium when digested with AsuC21.

• The Korea Journal of Herbology
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• v.20 no.4
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• pp.151-161
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• 2005

Objectives : Our research purpose is to establish the standard identification analysis on C. officinale and L. chuanxiong in Korea and China by PCR-mediated fingerprinting. Methods : The Restriction Fragment Length Polymorphism (RFLP) and Randomly Amplified Polymorphic DNA (RAPD) method was used on Internal Transcribed Spacer (ITS) regions and rbcL regions to compare and discriminate genes extracted from crude drugs as C. officinale and L. chuanxiong in Korea and China. Results : L. chuanxiong Korea and China have very similar polymorphism, whereas L. chuanxiong in Korea and C. officinale have very different polymorphism in RFLP. And restriction enzymes AluI and SacI forms the specific fragment band only in C. officinale, they can be used as RFLP marker on ITS regions to discriminate among the species. Conclusions : The results could be applied in discriminating crude drugs among C. officinale and L. chuanxiong in Korea and China. Also they could be used in controlling drug quality, preserving medicinal plants, and improving plant description.

• International Journal of Industrial Entomology and Biomaterials
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• v.15 no.2
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• pp.123-130
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• 2007

The Hsp 20.8 and Hsp 90 cDNA sequence retrieved from NCBI database and consists of 764 bp and 2582 bp lengths respectively. The corresponding cDNA homologus sequences were BLAST searched in Bombyx mori genomic DNA database and two genomic contigs viz., BAAB01120347 and AADK01011786 showed maximum homology. In B. mori Hsp 20.8 and Hsp 90 is encoded by single gene without intron. Specific primers were used to amplify the Hsp 20.8 gene and Hsp 90 variable region from genomic DNA by using the PCR. Obtained products were 216 bp in Hsp 20.8 and 437 bp in Hsp 90. There was no variation found in the six silkworm races PCR products size of contrasting response to thermal tolerance. The comparison of the sequenced nucleotide variations through multiple sequence alignment analysis of Hsp 90 variable region products of three races not showed any differences respect to their thermotolerance and formed the clusters among the voltinism. The comparison of aminoacid sequences of B. mori Hsps with dipteran and other insect taxa revealed high percentage of identity growing with phylogenetic relatedness of species. The conserved domains of B. mori Hsps predicted, in which the Hsp 20.8 possesses ${\alpha}-crystallin$ domain and Hsp 90 holds HATPase and Hsp 90 domains.

• The Plant Pathology Journal
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• v.16 no.1
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• pp.36-42
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• 2000

The coat protein gene of iris severe mosaic potyvirus, which was isolated in Korea, ISMV-K, from iris plant was cloned and its nucleotide sequence was determined. The coat protein of the virus contained 252 amino acid residues, including five potential N-glyxosylation site motifs. The coat protein of ISMV-K has 99.1% and 98.4% sequence identities with those of the Netherlands isolate of ISMV (ISMV-Ne) form crocus for the nucleotide and amino acids, respectively. The coat protein of ISMV-K has 50.4% to 60.3% nucleotide sequence identities and 47.3% to 55.7% amino acid identities with those of other 21 potyviruses, indicating ISMV to be a distinct species of the genus. The coat protein of ISMV-K was closely related with bean yellow mosaic virus and clover yellow vein virus in the phylogenetic tree analysis among the potyviruses analyzed. ISMV was easily and reliably detected from virus-infected iris leaves by RT-PCR with a set of the virus-specific primers.

• Biomedical Science Letters
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• v.8 no.1
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• pp.29-37
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• 2002

Listeria monocytogenes poses an increasing health risk, which in part is due to increasing health risk, consumption of ready-to-eat food products and the introduction of increasing numbers of food products from regions with different dietary habits. L. monocytogenes can be present in meat, shellfish, vegetables, unpasteurised milk and soft cheese and poses a risk if food containing these products is stored at refrigeration temperature and is not properly heated before consumption, as L. monocytogenes is psychrophilic. Amplified-fragment length polymorphism (AFLP) analysis is the method of genotypic techinique in which adaptor oligonucleotides are ligated to restriction enzyme fragments and then used as target sites for primers in a PCR amplification. The amplified fragments are electrophoretically separated to give strain-specific band profiles. Single-enzyme approach that did not require costly equipment or reagents for the fingerprinting of strains of Listeria monocytogenes was developed. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis was used to perform species and strain identification of Salmonella, Shigella, Yersinia and E. coli. By careful selection of AFLP primers, it was possible to obtain reproducible and sensitive identification to strain level. The AFLP patterns of L. monocytogenes are divided by the kinds of specimens in which were isolated. SE-AFLP fragments can be analyzed using standard gel electrophoresis, and can be easily scored by visual inspection, due to the low complexity of the fingerprint obtained by this method. These features make SE-AFLP suitable for use in either field or laboratory applications.