• Title, Summary, Keyword: Gene Marker

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Utility of the pat gene as a selectable marker gene in production of transgenic Dunaliella salina

  • Jung, Hyo Sun;Kim, Dong Soo
    • Fisheries and aquatic sciences
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    • v.19 no.7
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    • pp.31.1-31.6
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    • 2016
  • Background: The objective of this study was to develop an efficient selectable marker for transgenic Dunaliella salina. Results: Tests of the sensitivity of D. salina to the antibiotic chloramphenicol and the herbicide Basta$^{(R)}$ showed that cells ($1.0{\times}10^6cells/ml$) treated with 1000 or $1500{\mu}g/ml$ chloramphenicol died in 8 or 6 days, respectively, whereas D. salina cells ($1.0{\times}10^6cells/ml$) treated with 5, 10, 20, or $40{\mu}g/ml$ Basta$^{(R)}$ died in 2 days. Therefore, D. salina is more sensitive to Basta$^{(R)}$ than to chloramphenicol. To examine the possibility of using the phosphinothricin N-acetyltransferase (pat) gene as a selectable marker gene, we introduced the pat genes into D. salina with particle bombardment system under the condition of helium pressure of 900 psi from a distance of 3 cm. PCR analysis confirmed that the gene was stably inserted into the cells and that the cells survived in $5{\mu}g/ml$ Basta$^{(R)}$, the medium used to select the transformed cells. Conclusions: The findings of this study suggest that the pat gene can be used as an efficient selectable marker when producing transgenic D. salina.

Identification of chromosomal translocation causing inactivation of the gene encoding anthocyanidin synthase in white pomegranate (Punica granatum L.) and development of a molecular marker for genotypic selection of fruit colors

  • Jeong, Hyeon-ju;Park, Moon-Young;Kim, Sunggil
    • Horticulture, Environment, and Biotechnology : HEB
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    • v.59 no.6
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    • pp.857-864
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    • 2018
  • Previous studies have not detected transcripts of the gene encoding anthocyanidin synthase (ANS) in white pomegranates (Punica granatum L.) and suggest that a large-sized insertion in the coding region of the ANS gene might be the causal mutation. To elucidate the identity of the putative insertion, 3887-bp 5' and 3392-bp 3' partial sequences of the insertion site were obtained by genome walking and a gene coding for an expansin-like protein was identified in these genome-walked sequences. An identical protein (GenBank accession OWM71963) isolated from pomegranate was identified from BLAST search. Based on information of OWM71963, a 5.8-Mb scaffold sequence with genes coding for the expansin-like protein and ANS were identified. The scaffold sequence assembled from a red pomegranate cultivar also contained all genome-walked sequences. Analysis of positions and orientations of these genes and genome-walked sequences revealed that the 27,786-bp region, including the 88-bp 5' partial sequences of the ANS gene, might be translocated into an approximately 22-kb upstream region in an inverted orientation. Borders of the translocated region were confirmed by PCR amplification and sequencing. Based on the translocation mutation, a simple PCR codominant marker was developed for efficient genotyping of the ANS gene. This molecular marker could serve as a useful tool for selecting desirable plants at young seedling stages in pomegranate breeding programs.

Effect of Single Nucleotide Polymorphism of Endothelial Differentiation G-Protein Coupled Receptor 1 (EDG1) Gene on Marbling Score in Hanwoo

  • Shin, Sung-Chul;Chung, Eui-Ryong
    • Food Science of Animal Resources
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    • v.32 no.6
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    • pp.776-782
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    • 2012
  • Marbling (intramuscular fat) is the most economically important meat quality trait in Hanwoo (Korean cattle). The endothelial differentiation G-protein coupled receptor 1 (EDG1) gene, involved in blood vessel formation, is located within the genomic region of a quantitative trait locus (QTL) for marbling on bovine chromosome 3. Thus, the EDG1 gene can be considered as a positional and functional candidate gene for meat quality in beef cattle. This study aimed to identify single nucleotide polymorphisms (SNPs) in the EDG1 gene and to evaluate their associations with carcass traits in Hanwoo population. We have sequenced a fragment of 5'-UTR of the EDG1 gene and identified one SNP. Genotyping of the g.166A>G SNP marker was carried out using PCR-RFLP analysis in 309 Hanwoo steers in order to evaluate their association with carcass traits. The g.166A>G SNP marker showed a significant effect on the marbling score. Animals with the GG genotype had higher marbling score compared with AA and AG genotypes (p<0.05). This SNP marker also showed a significant additive effects for the marbling score (p<0.05). These results suggest that the EDG1 gene can be used as a molecular marker for DNA marker-assisted selection in order to increase the levels of the marbling score in Hanwoo.

Association between Microsatellite DNA Marker of Leptin Gene and Carcass Traits in Korean Cattle

  • Chung Eui-Ryong;Chung Ku-Young
    • Food Science of Animal Resources
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    • v.25 no.1
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    • pp.26-31
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    • 2005
  • Leptin, the product of the obesity (ob) gene, is synthesized in adipocytes or fat cells and has been implicated in the regulation of food intake, energy balance and body composition in mammals. Therefore, the leptin gene could be a candidate gene controlling fat deposition, meat quality and carcass traits in cattle. In this study the microsatellite genotypes for leptin gene were determined and their effects on carcass traits and meat quality were estimated in Korean cattle. Six different microsatellite alleles within leptin gene were identified and gene frequencies of 173, 177, 184, 186, 190 and 192 bp alleles were 0.012, 0.308, 0.067, 0.260, 0.342 and 0.016, respectively. The microsatellite marker of the leptin gene showed a significant association with the carcass percentage (CP) and marbling score (MS). Animals with genotypes 192/192 and 177/184 had higher CP than animals with other genotypes. Animals with genotypes 184/192 and 177/184 had higher MS compared with animals with other genotypes. Thus, the results suggest that the 177, 184 and 192 bp alleles may be associated with increased carcass percentage and intramuscular fat levels. No associations were found between the microsatellite genotypes of the leptin gene and other carcass traits such as carcass weight (CW), backfat thickness (BF) and M. longissimus dorsi area (LDA). In conclusion, the microsatellite markers of the leptin gene may be useful for marker-assisted selection of carcass traits and meat quality in Korean cattle.

Efficient Gene Targeting using Nuclear Localization Signal (NLS) and Negative Selection Marker Gene in Porcine Somatic Cells

  • Kim, Hye Min;Lee, Sang Mi;Park, Hyo Young;Kang, Man-Jong
    • Reproductive and Developmental Biology
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    • v.38 no.2
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    • pp.71-77
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    • 2014
  • The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

Association of SNP Marker in the Thyroglobulin Gene with Carcass and Meat Quality Traits in Korean Cattle

  • Shin, S.C.;Chung, E.R.
    • Asian-Australasian Journal of Animal Sciences
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    • v.20 no.2
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    • pp.172-177
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    • 2007
  • Thyroid hormones play an important role in regulating metabolism and can affect homeostasis of fat depots. The gene encoding thyroglobulin (TG), producing the precursor for thyroid hormones, has been proposed as a positional and functional candidate gene for a QTL with an effect on fat deposition. The SNP occurs in the 5' promoter region of the TG gene and is widely used in marker assisted selection (MAS) programs to improve the predictability of marbling level and eating quality in beef cattle. In this study, we identified three SNPs at the 5' promoter region of the TG gene in Korean cattle. Of the three SNPs identified in TG gene, the C257T and A335G were previously unreported new SNPs. The sequence data were submitted to GenBank (GenBank accession number: AY615525). The previously reported C422T SNP showed three genotypes, CC, CT and TT, by digestion with the restriction enzyme MflI using the PCR-RFLP method. A new allelic variant corresponding to the C${\rightarrow}$T and A${\rightarrow}$G mutations at positions 257 and 335, respectively, could be detected by the SSCP analysis. The gene-specific SNP marker association analysis indicated that the C422T SNP marker was significantly associated (p<0.05) with marbling score. Animals with the CC and CT genotypes had higher marbling score than those with the TT genotype. Results from this study suggest that TG gene-specific SNP may be a useful marker for meat quality traits in future MAS programs in Korean cattle.

Development of Gene Based STS Markers in Wheat

  • Lee, Sang-Kyu;Heo, Hwa-Young;Kwon, Young-Up;Lee, Byung-Moo
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.57 no.1
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    • pp.71-77
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    • 2012
  • The objective of this study is to develop the gene based sequence tagged site (STS) markers in wheat. The euchromatin enriched genomic library was constructed and the STS primer sets were designed using gene based DNA sequence. The euchromatin enriched genomic (EEG) DNA library in wheat was constructed using the $Mcr$A and $Mcr$BC system in $DH5{\alpha}$ cell. The 2,166 EEG colonies have been constructed by methylated DNA exclusion. Among the colonies, 606 colonies with the size between 400 and 1200 bp of PCR products were selected for sequencing. In order to develop the gene based STS primers, blast analysis comparing between wheat genetic information and rice genome sequence was employed. The 227 STS primers mainly matched on $Triticum$ $aestivum$ (hexaploid), $Triticum$ $turgidum$ (tetraploid), $Aegilops$ (diploid), and other plants. The polymorphisms were detected in PCR products after digestion with restriction enzymes. The eight STS markers that showed 32 polymorphisms in twelve wheat genotypes were developed using 227 STS primers. The STS primers analysis will be useful for generation of informative molecular markers in wheat. Development of gene based STS marker is to identify the genetic function through cloning of target gene and find the new allele of target trait.

System for Repeated Integration of Various Gene Expression Cassettes in the Yeast Chromosome (효모염색체내에 다양한 유전자발현 cassette의 반복적 integration을 위한 system 구축)

  • Kim, Yeon-Hee
    • Journal of Life Science
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    • v.28 no.11
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    • pp.1277-1284
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    • 2018
  • In this study, a repeated yeast integrative plasmid (R-YIp) harboring Cre/loxP system was constructed to integrate various gene expression cassettes into the yeast chromosome. The R-YIp system contains a reusable selective marker (CgTRP1), loxP sequence, and target sequence for integration. Therefore, many gene expression cassettes can be integrated into the same position of the same yeast chromosome. In the present study, several model enzymes involving xylan/xylose metabolism were examined, including endoxylanase (XYLP), ${\beta}$-xylosidase (XYLB), xylose reductase (GRE3) and xylitol dehydrogenase (XYL2). Efficient expression of these genes was obtained using two promoters (GAL10p and ADH1p) and various plasmids (pGMF-GENE and pAMF-GENE plasmids) were constructed. The XYLP, XYLB, GRE3, and XYL2 genes were efficiently expressed under the control of the GAL10 promoter. Subsequently, R-YIps containing the GAL10p-GENE-GAL7t cassette were constructed, resulting in pRS-XylP, pRS-XylB, pRS-Gre3, and pRS-Xyl2 plasmids. These plasmids were sequentially integrated into chromosome VII of a Saccharomyces cerevisiae strain by repeated gene integration and selective marker rescue. These genes were integrated by the R-YIp system and were stably expressed in the yeast transformants to produce active recombinant enzymes. Therefore, we expect that the R-YIp system will be able to overcome current limitations of the host cells and allow selective marker selection for the integration of various genes into the yeast chromosome.

Development and Validation of a Perfect KASP Marker for Fusarium Head Blight Resistance Gene Fhb1 in Wheat

  • Singh, Lovepreet;Anderson, James A;Chen, Jianli;Gill, Bikram S;Tiwari, Vijay K;Rawat, Nidhi
    • The Plant Pathology Journal
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    • v.35 no.3
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    • pp.200-207
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    • 2019
  • Fusarium head blight (FHB) is a devastating wheat disease with a significant economic impact. Fhb1 is the most important large effect and stable QTL for FHB resistance. A pore-forming toxin-like (PFT) gene was recently identified as an underlying gene for Fhb1 resistance. In this study, we developed and validated a PFT-based Kompetitive allele specific PCR (KASP) marker for Fhb1. The KASP marker, PFT_KASP, was used to screen 298 diverse wheat breeding lines and cultivars. The KASP clustering results were compared with gelbased gene specific markers and the widely used linked STS marker, UMN10. Eight disagreements were found between PFT_KASP and UMN10 assays among the tested lines. Based on the genotyping and sequencing of genes in the Fhb1 region, these genotypes were found to be common with a previously characterized susceptible haplotype. Therefore, our results indicate that PFT_KASP is a perfect diagnostic marker for Fhb1 and would be a valuable tool for introgression and pyramiding of FHB resistance in wheat cultivars.