• Journal of Plant Biotechnology
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• 제44권2호
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• pp.135-141
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• 2017

Cudrania tricuspidata Bureau is a widely used medicinal perennial woody plant. For conservation and germplasm utilization of the plant, it is imperative to obtaining information regarding the genetic diversity of the plant populations. Although C. tricuspidata is an important medicinal plant registered in South Korea, no molecular markers are currently available to distinguish Korean-specific ecotypes from other ecotypes of different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from chloroplast genomic sequences to identify distinct Korean-specific ecotypes of C. tricuspidata via the amplification refractory mutation system (ARMS)-PCR analyses. Molecular authentication of twelve C. tricuspidata ecotypes from different regions was performed, using DNA sequences in the trnL-F chloroplast intergenic region. The SNP markers developed in this study are useful for rapidly identifying specific C. tricuspidata ecotypes from different regions.

• Korean Journal of Agricultural Science
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• 제44권4호
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• pp.495-503
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• 2017

Genotyping-by-sequencing (GBS) is an outstanding technology for genotyping and single nucleotide polymorphism (SNP) discovery compared to next generation sequencing (NGS) because it can save time when analyzing large-scale samples and carries a low cost per sample. Recently, studies using GBS have been conducted on major crops and, to a greater extent, on fruit crops. However, many researchers have some problems due to low GBS efficiency resulting from low quality GBS libraries. To overcome this limitation, we developed an efficient GBS library construction method that regulates important conditions such as restriction enzymes (RE) digestion and a PCR procedure for grapevine. For RE digestion, DNA samples are digested with ApeKI (3.6U) at $75^{\circ}C$ for 5 hours and adapters are ligated to the ends of gDNA products. To produce suitable PCR fragments for sequencing, we modified the PCR amplification conditions; temperature cycling consisted of $72^{\circ}C$ (5 min), $98^{\circ}C$ (30 s), followed by 16 cycles of $98^{\circ}C$ (30 s), $65^{\circ}C$ (30 s), $72^{\circ}C$ (20 s) with a final extension step. As a result, we had obtained optimal library construct sizes (200 to 400 bp) for GBS analysis. Furthermore, it not only increased the mapping efficiency by approximately 10.17% compared to the previous method, but also produced mapped reads which were distributed equally on the19 chromosomes in the grape genome. Therefore, we suggest that this system can be used for various fruit crops and is expected to increase the efficiency of various genomic analysis performed.

• The Plant Pathology Journal
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• 제22권1호
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• pp.36-45
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• 2006

Twenty isolates of Didymella bryoniae were isolated from infected cucurbit plants in various growing areas of southern Korea in 2001 and 2002. Random Amplified Polymorphic DNA (RAPD) group [RG] I of D. bryoniae was more virulent than RG IV to watermelon. Virulence of the RG I isolate was strong to moderate to cucumber, whereas that of the RG IV varied from strong, moderate to weak. Two hundred seventy-three amplified fragments were produced with 40 primers, and were analyzed by a cluster analysis using UPGMA method with an arithmetic average program of NTSYSPC. At the distance level of 0.7, two major genomic DNA RAPD groups were differentiated among 20 isolates. The RG I included 7 isolates from watermelon and one isolate from melon, whereas the RG IV included 12 isolates from squash, cucumber, watermelon and melon. Amplification of internal transcribed spacer (ITS) region and small subunit rRNA region from the 20 isolates yielded respectively a single fragment. Restriction pattern with 12 restriction enzymes was identical for all isolates tested, suggesting that variation in the ITS and small subunit within the D. bryoniae were low. Amplification of the genomic DNAs of the tested isolates with the sequence characterized amplified regions (SCAR) primer RG IF-RG IR specific for RG I group resulted in a single band of 650bp fragment for 8 isolates out of the 20 isolates. Therefore, these 8 isolates could be assigned into RG I. The same experiments done with RG IIF-RG IIR resulted in no amplified PCR product for the 20 isolates tested. An about 1.4 kb-fragment amplified from the RG IV isolates was specifically hybridized with PCR fragments amplified from genomic DNAs of the RG IV isolates only, suggesting that this PCR product could be used for discriminating the RG IV isolates from the RG I isolates as well other fungal species.

• Asian-Australasian Journal of Animal Sciences
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• 제17권7호
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• pp.910-913
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• 2004

Insulin-like growth factor binding protein-3 (IGFBP-3) gene is a structural gene associated with the growth and development of the animals. The present investigation was carried out to unravel nucleotide sequence and polymerase chain reactionrestriction fragment polymorphism (PCR-RFLP) of IGFBP-3 gene in buffalo. Genomic DNA was isolated from a total of 157 animals belonging to Murrah, Surti, Jaffarabadi and Nagpuri breeds of Indian riverine buffalo. A 655 bp of IGFBP-3 gene was amplified in all the breeds and amplicons were digested with Hae III, Taq I and Msp I restriction enzymes. On digestion with Hae III yielded single restriction pattern of 8 fragments of sizes 201, 165, 154, 56, 36, 19, 16 and 8 bp in all the animals studied. Similarly Taq I and Msp I also revealed single restriction pattern yielding fragments of sizes 240 and 415 bp and 145 and 510 bp, respectively. This shows nonpolymorphic nature of restriction sites in buffalo. Nucleotide sequencing of 587 bp of IGFBP-3 gene in Murrah buffalo was done and submitted to the GenBank (Accession No. AY304829). Nucleotide sequencing revealed an addition of 4 bases in the intronic region as compared to cattle.

• Journal of Animal Science and Technology
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• 제46권6호
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• pp.897-902
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• 2004

To improve the methods used for the identification of Hanwoo, we performed a PCR using 3 -tailed primer associated with single nucleotide polymorphism(SNP) in Melanocortin 1 receptor(MCIR) gene. MCIR plays an important role in melanin synthesis and the SNP within MCIR was used as a target for PCRRFLP studies previously. A forward 3 -tailed primer, which matches with the template DNA of Hanwoo but not with others(blackhaired; Holstein and Black angus) at the site of 594th base sequence, and one reverse primer were designed for this study. When use this primer set, a size of 343bp was amplified by PCR only in Hanwoo, not in Holstein and Black angus. This result suggests that the PCR using our 3 -tailed primer would be very accurate, easy, reproducible and economic method to discriminate between Hanwoo meat and other black-haired ones.

• Food Science of Animal Resources
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• 제32권2호
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• pp.241-246
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• 2012

This report outlines the development of a rapid, simple, and sensitive detection system for pathogenic bacteria using a capillary electrophoresis-based, single strand conformation polymorphism (CE-SSCP) combined with PCR. We demonstrate that this method, used with primers targeting the V4 region of the16S rRNA gene, is capable of the simultaneous detection of 10 microbes that could be associated with foodborne illness, caused by animal-derived foods: Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7, Campylobacter jejuni, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Yersinia enterocolitica, Vibrio parahaemolyticus, and Enterobacter sakazakii. The traditional detection techniques are time-consuming and labor-intensive, due to the necessary task of separate cultivation of each target species. As such, the CE-SSCP-PCR method, that we have developed, has the potential to diagnose pathogens rapidly, unlike the traditional technique, in order to prevent foodborne illness in a much more efficient manner.

• Journal of Microbiology and Biotechnology
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• 제27권7호
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• pp.1223-1232
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• 2017

Cordyceps militaris, a member of Ascomycota, a mushroom referred to as caterpillar Dong-chung-ha-cho, is commercially valuable because of its high content of bioactive substances, including cordycepin, and its potential for artificial cultivation. Cordycepin (3'-deoxyadenosine) is highly associated with the pharmacological effects of C. militaris. C. militaris is heterothallic in that two mating-type loci, idiomorph MAT1-1 and MAT1-2, exist discretely in two different spores. In this study, nine C. militaris strains were mated with each other to prepare newly bred strains that produced a larger amount of cordycepin than the parent strains. Nine strains of C. militaris were identified by comparing the internal transcribed spacer sequence, and a total of 12 single spores were isolated from the nine strains of C. militaris. After the MAT idiomorph was confirmed by PCR, 36 mating combinations were performed with six single spores with MAT1-1 and the others with MAT1-2. Eight mating combinations were successfully mated, producing stroma with perithecia. Cordycepin content analysis of all strains by high-performance liquid chromatography revealed that the KASP4-bred strain produced the maximum cordycepin among all strains, regardless of the medium and stroma parts. Finally, universal rice primer-PCR was performed to demonstrate that the bred strains were genetically different from the parental strains and new C. militaris strains. These results may be related to the recombination of genes during mating. The newly produced strains can be used to meet the industrial demand for cordycepin. In addition, breeding through mating suggests the possibility of producing numerous cordycepin-producing C. militaris strains.

• Korean Journal of Agricultural Science
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• 제38권4호
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• pp.689-693
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• 2011

The purpose of this study was to investigate the Cytochrome P450 (CYP2A6) gene as a candidate gene for the traits related with meat fatty acid composition traits in pigs. Porcine CYP2A6 polymorphisms were detected and PCR-RFLP was performed for genotyping of Korean native pig (n=14), Landrace (n=3), Duroc (n=3), Berkshire (n=3), Yorkshire (n=8) and F2 population composed of 202 individuals from an intercross between Korean Native pig and Yorkshire. PCR primer set amplified a 612 bp fragment of CYP2A6 and digestion of the PCR products was performed with the restriction enzymes SchI. The CYP2A6 SchI polymorphism was only found in the KNP breed. The genotype frequencies of TT, TC and CC genotypes were 0.36, 0.56 and 0.08 in the KNP respectively and the other pig breeds were fixed with CC genotype (Duroc, Landrace, Berkshire and Yorkshire). Statistical association between genotypes and fatty acid composition traits were tested in the Korean native pig and Yorkshire crossed F2 pigs. The CYP2A6 SchI polymorphism was associated with only fatty acid composition C20:3n3 level (cis11,14,17-Eicosatrienoic acid, p=0.0252). The 'T' allele was associated with lower C20:3n3 level. Further study is required to validate the genotypic association and biological consequence of the CYP2A6 gene polymorphism in pigs.

• Journal of Life Science
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• 제20권5호
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• pp.789-793
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• 2010

Artificial chromosome manipulation and amplification of single-copy yeast artificial chromosome (YAC) are usually required in order to use YACs for applications such as physical mapping and functional analysis in eukaryotes. We designed and implemented a Simultaneous YAC Manipulation-Amplification (SYMA) system that combines the copy number amplification system of YAC with a convenient YAC manipulation system. To achieve the desired split and to amplify a YAC clone-harboring plant chromosome, a pBGTK plasmid containing a conditional centromere and thymidine kinase (TK) gene was constructed as a template to amplify the splitting fragment via PCR. By splitting, new 490-kb and 100-kb split YACs containing the elements for copy number amplification were simultaneously generated from a 590-kb YAC clone. The 100-kb split YAC was then successfully amplified 14.4-fold by adding 3 mg/ml sulfanilamide and $50\;{\mu}g/ml$ methotrexate (S3/M50) as inducing substances.

• Horticulture, Environment, and Biotechnology : HEB
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• 제59권6호
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• pp.865-873
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• 2018

Allium ochotense and Allium microdictyon are commonly known as 'Mountain garlic' and are popular, economically important species in many countries such as Korea, China, and Mongolia. Their leaves are used as culinary side dishes and in traditional medicines. In Korea, these two species are at risk of extinction due to damage to their natural habitat and thus, conservation and breeding programs are needed. However, their identification relies mostly on morphological data, which is limited and until recently, led to classifying these two species under A. victorialis. In the present study, a simple and reliable method of molecular identification was developed to distinguish A. ochotense from A. microdictyon that targets four barcoding regions: the internal transcribed spacer (ITS), the maturase K gene (matK), the chloroplast psbA-trnH intergenic region, and the ribulose-bisphosphate carboxylase large subunit gene (rbcL). Single nucleotide polymorphisms (SNPs) were found in ITS and matK regions, and species-specific primers were designed based solely on the SNP at position 680 of the ITS region that could differentiate A. ochotense from A. microdictyon. Using these primers in amplification refractory mutation system (ARMS)-PCR, A. ochotense, and A. microdictyon could be simultaneously and efficiently distinguished. This study is the first to report a simple, rapid, and efficient method for discriminating A. ochotense and A. microdictyon, indicating the utility of species-specific markers in the development of conservation and breeding programs.