• Genomics & Informatics
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• v.17 no.4
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• pp.40.1-40.9
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• 2019

While studies aimed at detecting and analyzing indels or single nucleotide polymorphisms within human genomic sequences have been actively conducted, studies on detecting long insertions/deletions are not easy to orchestrate. For the last 10 years, the availability of long read data of human genomes from PacBio or Nanopore platforms has increased, which makes it easier to detect long insertions/deletions. However, because long read data have a critical disadvantage due to their relatively high cost, many next generation sequencing data are produced mainly by short read sequencing machines. Here, we constructed programs to detect so-called unmapped regions (UMRs, where no reads are mapped on the reference genome), scanned 40 Korean genomes to select UMR long deletion candidates, and compared the candidates with the long deletion break points within the genomes available from the 1000 Genomes Project (1KGP). An average of about 36,000 UMRs were found in the 40 Korean genomes tested, 284 UMRs were common across the 40 genomes, and a total of 37,943 UMRs were found. Compared with the 74,045 break points provided by the 1KGP, 30,698 UMRs overlapped. As the number of compared samples increased from 1 to 40, the number of UMRs that overlapped with the break points also increased. This eventually reached a peak of 80.9% of the total UMRs found in this study. As the total number of overlapped UMRs could probably grow to encompass 74,045 break points with the inclusion of more Korean genomes, this approach could be practically useful for studies on long deletions utilizing short read data.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.3
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• pp.315-320
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• 2016

Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5'-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.

• Korean Journal of Poultry Science
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• v.36 no.1
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• pp.85-88
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• 2009

One of the mitochondrial genes, called cytochrome c oxidase I (COI), has been widely used for the species identification (called bio-barcode) in birds. In this study, the bio-barcode has been applied to chicken breeds in Korea whether it also can be used as a molecular marker for breed identification. Data indicated that Korean native chicken has the mixed SNP (single nucleotide polymorphism) patterns between White Leghorn (Layer) and Cornish (Broiler) and ultimately, it can not be used as the marker for breed identification. However, this result indicates the mixed use of the Korean native chicken, since it has been used for dual purpose for producing meat and egg for a long time. In order to use as a marker for species identification, more reliable mitochondrial and/or nuclear DNA markers need to be developed.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.77.1-77
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• 2003

We have isolated a full-length cDNA clone, CaCDPK4 encoding a typical calcium-dependent protein kinase (CDPK) from hot pepper cDNA library. Genomic southern blot analysis showed that it belongs to a multigene family, but represents a single copy gone in hot pepper genome. RNA expression pattern of this gene revealed that it is induced by infiltration of Xanthomonas axonopodis pv. glycines Bra into hot pepper leaves but not by water deficit stress. However, high salt treatment of NaCl (0.4 M) solution to hot pepper plants strongly induced CaCDPK4 gene. In addition, this gene is weakly responsive to the exogenous application of salicylic acid or ethephon. Biochemical study of the GST-CaCDPK4 recominant protein showed that it autophosphorylates in vitro and the presence of EGTA, a calcium chelater, eliminates the kinase activity of the recombinant protein. As a way to identify the in vivo function of CaCDPK4 in plants, VIGS (Virus-Induced Gene Silencing) was employed. Agrobacterium-mediated TRV silencing construct containing the kinase and calmodulin domain of CaCDPK4 resulted in cell death of Nicotiana benthamiana plants. A highly homologous H benthamiana CDPK gene, NbCDPK5, to CaCDPK4 was cloned from N. benthamiana cDNA library. VIGS of NbCDPK5 also resulted in cell death. The molecular characterization of this cell death phenotype is being under investigation.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.382-386
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• 2006

A total number of 20 cat samples including 8 parentage testing and 12 individual identification were genotyped. Genomic DNA was extracted from buccal swab, and genotyped by using 10 microsatellite markers (FCA005, FCA26, FCA224, FCA240, FCA453, FCA293, FCA075, FCA105, FCA229, and FCA651). This method consisted of single PCR procedure and showed reasonable amplification of all PCR products. Genotypes were determined by genetic analyzer. The number of alleles per locus of cats varied from 3 to 8 with a mean value of 5.5. Expected heterozygosity was ranged from 0.390 to 0.827 (mean 0.639) and the total exclusion probability of 10 microsatellite loci was 0.9441. Of the 10 markers, FCA240 marker has relatively high PIC value (>0.7). Of the 8 cats, 7 cats were qualified by compatibility according to the Mendelism. These results can give basic information for developing parentage verification and individual identification system in cat.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.5
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• pp.712-721
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• 2020

Objective: The Wannan Black pig is a typical Chinese indigenous, disease-resistant pig breed with high fertility, and a crude-feed tolerance that has been bred by artificial selection in the south of Anhui province for a long time. However, genome variation, genetic relationships with other pig breeds, and domestication, remain poorly understood. Here, we focus on elucidating the genetic characteristics of the Wannan Black pig and identifying selection signatures during domestication and breeding. Methods: We identified the whole-genome variation in the Wannan Black pig and performed population admixture analyses to determine genetic relationships with other domesticated pig breeds and wild boars. Then, we identified the selection signatures between the Wannan Black pig and Asian wild boars in 100-kb windows sliding in 10 kb steps by using two approaches: the fixation index (F_ST) and π ratios. Results: Resequencing the Wannan Black pig genome yielded 501.52 G of raw data. After calling single-nucleotide variants (SNVs) and insertions/deletions (InDels), we identified 21,316,754 SNVs and 5,067,206 InDels (2,898,582 inserts and 2,168,624 deletions). Additionally, we found genes associated with growth, immunity, and digestive functions. Conclusion: Our findings help in explaining the unique genetic and phenotypic characteristics of Wannan Black pigs, which in turn can be informative for future breeding programs of Wannan Black pigs.

• Development and Reproduction
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• v.4 no.1
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• pp.61-66
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• 2000

This study was investigated to analyze the inactivating point mutation and expression level of follicle-stimulating hormone(FSH) receptor mRNA. In first experiment, we analyzed the point mutation. Peripheral blood was collected from each patient. To screen individuals for the C566T mutation, PCR was performed for exon 7 of the FSH receptor gene in 10 patients. No inactivating point mutation of FSH receptor gene was identified in women with premature ovarian failure. To analyze the expression level of FSH receptor, mRNA expressions were examined by RT-PCR method using specific primers for the FSH receptor. The amount of FSH receptor mRNA expressed in POF patients was lower than that in the control group. But it was not significantly different. These finding suggests that lower expression of FSH receptor in premature ovarian failure patients might be the cause of the low response to the gonadotropin during the hyperstimulation in IVF-ET cycles.

• Development and Reproduction
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• v.17 no.3
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• pp.199-205
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• 2013

Genomic DNAs were extracted from the turtle leg (Pollicipes mitella, 1798) population of Tongyeong, Yeosu and Manjaedo located in the southern sea of Korea. The turtle leg population from Tongyeong (0.929) exhibited higher bandsharing values than did turtle leg from Manjaedo (0.852). The higher fragment sizes (>1,200 bp) are much more observed in the Yeosu population. The number of unique loci to each population and number of shared loci by the three populations, generated by PCR using 7 primers in the turtle leg (P. mitella) population of Tongyeong, Yeosu and Manjaedo. Genetic distances among different individuals of the Tongyeong population of the turtle leg (lane 1-07), Yeosu population of the turtle leg (lane 08-14) and Manjaedo population of the turtle leg (lane 15-21), respectively, were generated using the CLASSIFICATION option in Systat version 10 according to the bandsharing values and similarity matrix. The dendrogram, obtained by the seven decamer primers, indicated three genetic clusters: cluster 1 (TONGYEONG 01 TONGYEONG 07), cluster 2 (YEOSU 08 YEOSU 14), and cluster 3 (MANJEDO 15 MANJEDO 21). Tongyeong population could be evidently discriminated with the other two Yeosu and Manjaedo populations among three populations. The longest genetic distance (0.305) was found to exist between individuals' no. 02 of the Tongyeong population and no. 13 of the Yeosu population. It seems to the authors that this is a result of a high degree of inbreeding in narrow region for a long while.

• Journal of Animal Science and Technology
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• v.47 no.3
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• pp.317-324
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• 2005

A method for sex determination of pigs was examined using polymerase chain reaction(PCR). Sex determining region Y(SRY) gene encoded on Y chromosome plays a key role for primary male development. Zinc finger X-Y(ZFX-ZFY) gene, one of the X-V homology gene group was found on the X and Y chromosomes, respectively, We tested for molecular sexing by amplification patterns of SRY and ZF genes. Genomic DNAs from various resources including porcine hairs and semen collected from domestic pig breeds and native pigs was used for PCR assay of each gene. The amplified products for porcine SRY gene were yielded only in males but not in females. On the other hand, two differential patterns were observed in amplification of ZF gene reflecting the chromosomal dimorphism by a length polymorphism between X and Y chromosomes. Of both, a common band was detected in all individuals tested so that this band might be amplified from ZFX gene as a PCR template, but another is specific for males indicated that from ZFY. The result of PCR assay provides identical information to that from investigation of phenotypic genders of the pigs tested. We suggest that this PCR strategy to determine porcine sexes using comparison of the amplification patterns of the SRY gene specific for Y chromosome and the dimorphic ZF gene between X and Y chromosomes may be a rapid and precise method for discrimination of two sexes and applied to DNA analysis of small samples such as embryonic blastomere, semen, and hairs.