• Korean Journal of Agricultural Science
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• v.44 no.4
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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.

• Journal of Korean Society of Forest Science
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• v.112 no.1
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• pp.40-56
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• 2023

Tree species within the Populus genus grow rapidly and have an excellent capacity to absorb carbon, conferring substantial ability to effective purify the environment. Poplar breeding can be achieved rapidly and efficiently if a genetic linkage map is constructed and quantitative trait loci (QTLs) are identified. Here, a high-density genetic linkage map was constructed for the control pollinated progeny using the genotyping-by-sequencing (GBS) technique, which is a next-generation sequencing method. A search was also performed for the genes associated with quantitative traits located in the genetic linkage map by examining the variables of height and diameter at root collar, and resilience to insect damage. The height and diameter at root collar were measured directly, while the ability to recover from insect damage was scored in a 4-year-old breeding population of aspen hybrids (Odae19 × Bonghyeon4 F1) established in the research forest of Seoul National University. After DNA extraction, paternity was confirmed using five microsatellite markers, and only the individuals for which paternity was confirmed were used for the analysis. The DNA was cut using restriction enzymes and the obtained DNA fragments were prepared using a GBS library and sequenced. The analyzed results were sorted using Populus trichocarpa as a reference genome. Overall, 58,040 aligned single-nucleotide polymorphism (SNP) markers were identified, 17,755 of which were used for mapping genetic linkages. The genetic linkage map was divided into 19 linkage groups, with a total length of 2,129.54 cM. The analysis failed to identify any growth-related QTLs, but a gene assumed to be related to recovery from insect damage was identified on linkage group (chromosome) 4 through genome-wide association study.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.17
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• pp.7413-7417
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• 2014

Background: Development of gastric cancer (GC) is a multistep process that requires alterations in the expression of oncogenes and tumor suppressor genes, occurring over several decades. The p53 tumor suppressor protein is involved in cell-cycle control, apoptosis and DNA repair. One of the most important regulators of p53 is MDM2, which acts as a negative regulator in the p53 pathway. Based on the key role of p53 and MDM2 in tumor suppression, polymorphisms that cause change in their function might affect cancer risk. We therefore elevated associations of the polymorphisms of p53 (R72P) and MDM2 (SNP309) with GC in Iran. Materials and Methods: A total of 104 patients with gastric cancer and 100 controls were recruited. Genomic DNA was extracted from fresh gastric samples. Genotyping of the p53 and MDM2 genes was performed using allele specific PCR (AS-PCR). Results: There was no significant difference between the p53 codon 72 polymorphism distribution in control and patient groups (p=0.54), but the G allele of MDM2 was found to be over-represented in patients (p=0. 01, Odds Ratio=2. 08, 95% Confidence Interval= 1.37-4.34). Conclusions: The p53 R72P seems not to be a potential risk factor for development of GC among Iranian patients, but our data suggest that MDM2 SNP309 might modify the risk related to GC.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.13
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• pp.5515-5519
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• 2015

The single nucleotide polymorphism (SNP) ss469415590 in the interferon lambda-4 (IFNL4) gene has recently been reported to have an association with treatment response in chronic hepatitis C. However, any importance of the SNP in association with response to pegylated interferon (PEG-IFN) therapy in patients with chronic hepatitis B (CHB) is unclear. We retrospectively analyzed data for Thai patients with CHB treated with PEG-IFN for 48 weeks. Virological response (VR) for HBeAg-positive CHB was defined as HBeAg seroconversion plus HBV DNA level <2,000 IU/mL at 24 weeks post-treatment. VR for HBeAg-negative CHB was defined as an HBV DNA level <2,000 IU/mL at 48 weeks. The SNP was identified by real time PCR using the TaqMan genotyping assay with MGB probes. A total 254 patients (107 HBeAg-positive and 147 HBeAg-negative) were enrolled in the study. The distribution of TT/TT, ${\Delta}G/TT$ and ${\Delta}G/{\Delta}G$ genotypes was 221 (87.0%), 32 (12.6%) and 1 (0.4%), respectively. Patients with non-TT/TT genotypes had significantly higher baseline HBV DNA levels than patients with the TT/TT genotype. In HBeAg-positive CHB, 41.2% of patients with TT/TT genotype versus 50.0% with non-TT/TT genotype achieved VR (P=0.593). In HBeAg-negative CHB, the corresponding figures were 40.3% and 43.5%, respectively (P=0.777). There was no significant correlation between the SNP genotypes and HBsAg clearance in both groups of patients. In summary, ss469415590 genotypes were not associated with response to PEG-IFN in Thai patients with HBeAg-positive and HBeAg-negative CHB.

• Food Science of Animal Resources
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• v.30 no.6
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• pp.918-922
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• 2010

Due to the large amount of beef imported from the USA to Korea, Korean consumers have become increasingly interested in the country of origin since it can affect market prices. Previously, Bos indicus and Bos taurus-specific markers were developed for the purpose of cattle breed identification, specifically discrimination of Australian beef. In this study, six SNP markers derived from Illumina 50K bovine SNP chip data were used for the discrimination between Korean cattle (Hanwoo) and imported beef from USA. PCR-RFLP genotyping methods were also developed, which indicates that these markers can be applied relatively easily compared to other markers. Taking into account a discrimination rate of 55% based on MC1R marker between Hanwoo and imported beef from USA, two additional markers, SNPs 23803 and 34776, were ideal and resulted in probability of identification of 0.942 and probability of misjudgment of 0.03. Therefore, the markers developed in this study can greatly contribute to the correct discrimination between beef from USA and Hanwoo beef.

• Korean Journal of Agricultural Science
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• v.40 no.3
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• pp.215-220
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• 2013

Fatty acids (FAs) were considered in activating nuclear hormone receptors that play significant roles in the cellular lipid metabolism by the regulation of several genes. Previously, fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) genes have been known to regulating the FA metabolism. In this study, associations of FASN and SCD genes with fatty acid (FA) composition in broilers were investigated. Tissue samples from 95 Cobb 500 broilers were used for DNA extraction. The g.1222 A>G SNP located in intron 42 of FASN gene and 2 SNPs in SCD gene, one in exon 2 (g.3728A>G) and the other in exon 4 (g.12903G>A), were subjected for genotyping using PCR-RFLP method. One of the SNPs in SCD gene, SNP g.3728A>G had significant association with myristoleic acid (C14:1; P<0.05), palmitic acid (C16:0; P<0.05), palmitoleic acid (C16:1; P<0.05) and saturated FA (SFA; P<0.05). However, the SNP g.1222A>G in FASN gene had only suggestive association with arachidic acid (C20:0; P=0.08). The findings in this study suggest that the SNP in exon 2 of SCD gene can be used as a molecular marker for selecting birds having desirable FA composition in broilers.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.10a
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• pp.19-19
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• 2019

유전체 전장 연관분석 (GWAS)은 단일염기다형성(SNP)의 유전자형과 표현형 간의 통계적인 연관성을 분석함으로써 품종 선발용 SNP Marker 개발에 응용되고 있다. 본 연구에서는 Tano Red와 Ruby seedless 교배실생 278 계통을 대상으로 여러 과실 특성에 따른 관련 SNP를 동정함으로써 육종 선발에 필요한 DNA marker 개발에 필요한 기초 유전 자료를 얻고자 하였다. 한 계통 당 5~10개의 포도알을 선택하여 과립중, 과육탄성, 과피탄성, 과육경도, 과피경도, 과립당 종자갯수, 과립당 종자무게 및 인장강도를 측정하였다. 각 개체는 Genotyping by sequencing (GBS) 방법으로 Sequencing하여 Reference genome (Vitis vinifera PN40024 12X v2.)과 mapping 하였다. MAF (Minor allele frequency) >5%, Missing Data <30% 의 조건을 가진 SNPs 만 1차 선발하여 TASSEL과 GAPIT 프로그램으로 GWAS 분석을 하였다. Manhattan plot 결과 과립중 형질에서는 33개, 과립당 종자무게 25개와 인장강도에서는 20개의 통계학적으로 유의한 SNPs 가 선발되었고, 특이적으로 이들 모두 18번 염색체에서 발견되었다. 그러나 나머지 형질에서는 유의한 차이를 보이는 SNPs를 선발하지 못하였다. 과실의 인장강도는 수확 후 저장성과 유통과정에 영향을 미치기 때문에 Marker 개발을 통한 품종선별이 중요하다. 향후 이러한 특성과 본 연구를 통해 동정된 SNPs 의 상관관계를 구체적으로 연구하여 Marker 개발에 활용하고자 한다.

• Animal Bioscience
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• v.36 no.1
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• pp.19-28
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• 2023

Objective: The objective of this study was to perform genome (genome wide association studies [GWAS]) and chromosome (CWAS) wide association analyses to identify single nucleotide polymorphisms (SNPs) associated with growth traits in registered Simmental and Simbrah cattle. Methods: The phenotypes were deregressed BLUP EBVs for birth weight, weaning weight direct, weaning weight maternal, and yearling weight. The genotyping was performed with the GGP Bovine 150k chip. After the quality control analysis, 105,129 autosomal SNP from 967 animals (473 Simmental and 494 Simbrah) were used to carry out genotype association tests. The two association analyses were performed per breed and using combined information of the two breeds. The SNP associated with growth traits were mapped to their corresponding genes at 100 kb on either side. Results: A difference in magnitude of posterior probabilities was found across breeds between genome and chromosome wide association analyses. A total of 110, 143, and 302 SNP were associated with GWAS and CWAS for growth traits in the Simmental-, Simbrah- and joint -data analyses, respectively. It stands out from the enrichment analysis of the pathways for RNA polymerase (POLR2G, POLR3E) and GABAergic synapse (GABRR1, GABRR3) for Simmental cattle and p53 signaling pathway (BID, SERPINB5) for Simbrah cattle. Conclusion: Only 6,265% of the markers associated with growth traits were found using CWAS and GWAS. The associated markers using the CWAS analysis, which were not associated using the GWAS, represents information that due to the model and priors was not associated with the traits.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.12
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• pp.1689-1695
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• 2008

Leptin, the hormone product of the obese gene, is secreted predominately from white adipose tissue and regulates feed intake, energy metabolism and body composition. It has been considered a candidate gene for performance, carcass and meat quality traits in beef cattle. The objective of this study was to identify SNPs in the promoter region of the leptin gene and to evaluate the possible association of the SNP genotypes with carcass and meat quality traits in Korean cattle. We identified a total of 25 SNPs in the promoter region (1,208-3,049 bp upstream from the transcription start site) of the leptin gene, eleven (g.1508C>G, g.1540G>A, g.1545G>A, g.1551C>T, g.1746T>G, g.1798ins(G), g.1932del(T), g.1933del(T), g.1934del(T), g.1993C>T and g.2033C>T) of which have not been reported previously. Their sequences were deposited in GenBank database with accession number DQ202319. Genotyping of the SNPs located at positions g.2418C>G and g.2423G>A within the promoter region was performed by direct sequencing and PCR-SSCP method to investigate the effects of SNP genotypes on carcass and meat quality traits in Korean cattle. The SNP and SSCP genotypes from the two mutations of the leptin promoter were shown to be associated with the BF trait. The average BF value of animals with heterozygous SNP genotype was significantly greater than that of animals with the homozygous SNP genotypes for the g.2418C>G and g.2423G>A SNPs (p<0.05). Analysis of the combined genotype effect in both SNPs showed that animals with the AC SSCP genotype had higher BF value than animals with BB or AA SSCP genotypes (p<0.05). These results suggest that SNP of the leptin promoter region may be useful markers for selection of economic traits in Korean cattle.

• Journal of Plant Biotechnology
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• v.46 no.3
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• pp.165-171
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• 2019

Marker-assisted backcrossing (MABC) is useful for selecting an offspring with a highly recovered genetic background for a recurrent parent at early generation to various crops. Moreover, marker-assisted backcrossing (MABC) along with marker-assisted selection (MAS) contributes immensely to overcome the main limitation of the conventional breeding and it accelerates recurrent parent genome (RPG) recovery. In this study, we were employed to incorporate rin gene(s) from the donor parent T13-1084, into the genetic background of HK13-1151, a popular high-yielding tomato elite inbred line that is a pink color fruit, in order to develop a rin HK13-1084 improved line. The recurrent parent genome recovery was analyzed in early generations of backcrossing using SNP markers obtained from genotyping-by-sequencing analysis. From the $BC_1F_1$ and $BC_2F_1$ plants, 3,086 and 4868 polymorphic SNP markers were obtained via GBS analysis, respectively. These markers were present in all twelve chromosomes. The background analysis revealed that the extent of RPG recovery ranged from 56.7% to 84.5% and from 87.8% to 97.8% in $BC_1F_1$ and $BC_2F_1$ generations, respectively. In this study, No 5-1 with 97.8% RPG recovery rate among $BC_2F_1$ plants was similar to HK13-1151 strain in the fruit shape. Therefore, the selected plants were fixed in $BC_2F_2$ generation through selfing. MAS allowed identification of the plants that are more similar to the recurrent parent for the loci evaluated in the backcross generations. MABC can greatly reduce breeding time as compared to the conventional backcross breeding. For instance, MABC approach greatly shortened breeding time in tomato.