• Genomics & Informatics
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• 제10권3호
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• pp.194-199
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• 2012

In addition to single-nucleotide polymorphisms (SNP), copy number variation (CNV) is a major component of human genetic diversity. Among many whole-genome analysis platforms, SNP arrays have been commonly used for genomewide CNV discovery. Recently, a number of CNV defining algorithms from SNP genotyping data have been developed; however, due to the fundamental limitation of SNP genotyping data for the measurement of signal intensity, there are still concerns regarding the possibility of false discovery or low sensitivity for detecting CNVs. In this study, we aimed to verify the effect of combining multiple CNV calling algorithms and set up the most reliable pipeline for CNV calling with Affymetrix Genomewide SNP 5.0 data. For this purpose, we selected the 3 most commonly used algorithms for CNV segmentation from SNP genotyping data, PennCNV, QuantiSNP; and BirdSuite. After defining the CNV loci using the 3 different algorithms, we assessed how many of them overlapped with each other, and we also validated the CNVs by genomic quantitative PCR. Through this analysis, we proposed that for reliable CNV-based genomewide association study using SNP array data, CNV calls must be performed with at least 3 different algorithms and that the CNVs consistently called from more than 2 algorithms must be used for association analysis, because they are more reliable than the CNVs called from a single algorithm. Our result will be helpful to set up the CNV analysis protocols for Affymetrix Genomewide SNP 5.0 genotyping data.

• BMB Reports
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• 제46권6호
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• pp.305-309
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• 2013

The determination of relatedness between individuals in a family is crucial in analysis of common complex diseases. We present a method to infer close inter-familial relationships based on SNP genotyping data and provide the relationship coefficient of kinship in Korean families. We obtained blood samples from 43 Korean individuals in two families. SNP data was obtained using the Affymetrix Genome-wide Human SNP array 6.0 and the Illumina Human 1M-Duo chip. To measure the kinship coefficient with the SNP genotyping data, we considered all possible pairs of individuals in each family. The genetic distance between two individuals in a pair was determined using the allele sharing distance method. The results show that genetic distance is proportional to the kinship coefficient and that a close degree of kinship can be confirmed with SNP genotyping data. This study represents the first attempt to identify the genetic distance between very closely related individuals.

• 한국양봉학회지
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• 제32권3호
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• pp.147-154
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• 2017

꿀벌은 화분매개 뿐만 아니라, 양봉산물을 생산하는 주요한 산업곤충 중 하나이다. 최근 농촌진흥청과 예천곤충 연구소에서는 국내 최초로 수밀력 우수 꿀벌 품종인 '장원벌'을 선발하여 보급하고 있다. 본 연구에서는 장원벌 계통 특이 분자 마커 개발을 위해 장원벌 부계인 D계통 특이적인 분자 마커를 개발 하고자 Sequence-Based Genotyping (SBG) 분석을 수행하였다. SGB 분석은 농촌진흥청 국립농업과학원에서 보존 육성중인 A, C, D, E, F 5개 기본종 계통에 대해 수행되었으며, 이를 통해 1,029개 SNP를 확보 할 수 있었다. 이후 A, C, D, F, E 기본종 계통 및 $D{\times}F$ 교배계통에 대한 SNP filtering 및 validation을 통해 최종적으로 AmD6 및 AmD9 두 개의 SNP 마커를 선발 하였으며, genotyping 분석을 통해 AmD9 마커가 장원벌 부계인 D 계통을 100% 구분 할 수 있는 것으로 확인되었다. 본 마커를 통해 D 계통 및 장원벌을 보다 정확하게 판별하고 육종에 활용 할 수 있을 것으로 기대하고 있다.

• 한국작물학회지
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• 제50권5호
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• pp.361-367
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• 2005

Single nucleotide polymorphisms (SNPs) are valuable DNA markers due to their abundance and potential for use in automated high-throughput genotyping. Numerous SNP genotyping assays have been developed. In this report, one of effective and high throughput SNP genotyping assays, which was named the template-directed dye-terminator incorporation with fluorescence polarization detection (FP-TDI) was described. Although the most of this assay succeed, the objective of this work was to deter­mine the reasons for the failures, find ways to improve the assay and reduce the running cost. Ninety $F_2$-derived soybean, Glycine max (L.) Merr., RILs from a cross between 'Pureunkong' and 'Jinpumkong 2' were genotyped at four SNPs. FP measurement was done on $Victot^3$ microplate reader (perkinelmer Inc., Boston, MA, USA). Increasing the number of thermal cycles in the single-base extension step increased the separation of the FP values between the products corresponding to different genotypes. But in some assays, excess of heterozygous genotypes was observed with increase of PCR cycles. We discovered that the excess heterozygous was due to misincorporation of one of the dye­terminators during the primer extension reaction. After pyrophosphatase incubation and thermal cycle control, misincoporation can be effectively prevented. Using long amplicons instead of short amplicons for SNP genotyping and decreasing the amount of dye terminator and Acyclopol Taq polymerase to 1/2 or 1/3 decreased the cost of the assay. With these minor adjustments, the FP-TDI assay can be used more accurately and cost-effectively.

• Journal of Crop Science and Biotechnology
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• 제11권2호
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• pp.91-100
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• 2008

Melting curve analysis of fluorescently labeled DNA fragments is used extensively for genotyping single nucleotide polymorphism(SNP). Here, we evaluated a SNP genotyping method by melting curve analysis with the two probe chemistries in a 384-well plate format on a Roche LightCycler 480. The HybProbe chemistry is based on the fluorescence resonance energy transfer(FRET) and the SimpleProbe chemistry uses a terminal self-quenching fluorophore. We evaluated FRET HybProbes and SimpleProbes for two SNP sites closely linked to two quantitative trait loci(QTL) for southern root-knot nematode resistance. These probes were used to genotype the two parents and 94 $F_2$ plants from the cross of PI 96354$\times$Bossier. The SNP genotypes of all samples determined by the LightCycler software agreed with previously determined SSR genotypes and the SNP genotypes determined on a Luminex 100 flow cytometry instrument. Multiplexed HybProbes for the two SNPs showed a 98.4% success rate and 100% concordance between repeats two of the same 96 DNA samples. Also, we developed a HybProbe assay for the Rcs3 gene conditioning broad resistance to the frogeye leaf spot(FLS) disease. The LightCycler 480 provides rapid PCR on 384-well plate and allows simultaneous amplification and analysis in approximately 2 hours without any additional steps after amplification. This allowed for a reduction of the potential contamination of PCR products, simplicity, and enablement of a streamlined workflow. The melting curve analysis on the LightCycler 480 provided high-throughput and rapid SNP genotyping and appears highly effective for marker-assisted selection in soybean.

• Journal of Ginseng Research
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• 제35권4호
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• pp.504-513
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• 2011

In order to develop a novel system for the discrimination of five ginseng cultivars (Panax ginseng Meyer), single nucleotide polymorphism (SNP) genotyping assays with real-time polymerase chain reaction were conducted. Nucleotide substitution in gDNA library clones of P. ginseng cv. Yunpoong was targeted for the SNP genotyping assay. From these SNP sites, a set of modified SNP specific fluorescence probes (PGP74, PGP110, and PGP130) and novel primer sets have been developed to distinguish among five ginseng cultivars. The combination of the SNP type of the five cultivars, Chungpoong, Yunpoong, Gopoong, Kumpoong, and Sunpoong, was identified as 'ATA', 'GCC', 'GTA', 'GCA', and 'ACC', respectively. This study represents the first report of the identification of ginseng cultivars by fluorescence probes. An SNP genotyping assay using fluorescence probes could prove useful for the identification of ginseng cultivars and ginseng seed management systems and guarantee the purity of ginseng seed.

• Plant Breeding and Biotechnology
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• 제6권4호
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• pp.434-443
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• 2018

Genotyping-by-sequencing (GBS) has been used as a viable single nucleotide polymorphism (SNP) validation method that provides reduced representation sequencing by using restriction endonucleases. Although GBS makes it possible to perform marker discovery and genotyping simultaneously with reasonable costs and a simple molecular biology workflow, the standard TASSEL-GBS pipeline was designed for homozygous groups, and genotyping of heterozygous groups is more complicated. To addresses this problem, we developed a GBS pipeline for heterozygous groups that called KNU-GBS pipeline, specifically for apple (Malus domestica). Using KNU-GBS pipeline, we constructed a genetic linkage map consisting of 1,053 SNP markers distributed over 17 linkage groups encompassing a total of 1350.1 cM. The novel GBS pipeline for heterozygous groups will be useful for marker-assisted breeding programs, and diverse heterozygous genome analyses.

• 생명과학회지
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• 제33권12호
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• pp.1025-1035
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• 2023

본 연구에서는 한국산과 중국산 새꼬막(Scapharca subcrenata) 사이의 원산지 판별을 위하여 quantitative real-time PCR (qPCR) 분석을 기반으로 하는 Single-nucleotide polymorphism (SNP) 마커의 primer set를 개발 및 검증하였다. 총 180개의 새꼬막 sample을 genotyping by sequencing으로 분석하여 원산지 판별에 유용할 것이라 판단되는 7개의 후보 MS 마커와 15개의 후보 SNP 마커를 선정하였다. 후보 SNP 마커는 PCR과 sanger sequencing, SYBR green-based qPCR을 통해 원산지별 분리 여부를 확인하였다. 이 중 Insertion 1, SNP 21 마커가 qPCR 증폭 양상에서 집단이 확연히 분리되었으며 예상과 실제 증폭 형태가 일치하였다. 추가적으로 새꼬막을 무작위로 섞어서 진행한 blind test에서 Insertion 1은 새꼬막 100개에 대하여 74%의 정확도, 52%의 민감도, 96%의 특이도를 보였고, SNP 21은 새꼬막 137개에 대하여 86%의 정확도, 79%의 민감도, 93%의 특이도를 보였다. 따라서 개발된 두 개의 SNP 마커는 독립적 또는 복합적으로 사용하면 새꼬막 원산지 판별의 진위 여부를 검증하는 데 유용할 것으로 기대된다.

• Asian-Australasian Journal of Animal Sciences
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• 제23권7호
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• pp.833-847
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• 2010

In the past decade, there have been many advances in whole-genome sequencing in domestic animals, as well as the development of "next-generation" sequencing technologies and high-throughput genotyping platforms. Consequently, these advances have led to the creation of the high-density SNP array as a state-of-the-art tool for genetics and genomics analyses of domestic animals. The emergence and utilization of SNP arrays will have significant impacts not only on the scale, speed, and expense of SNP genotyping, but also on theoretical and applied studies of quantitative genetics, population genetics and molecular evolution. The most promising applications in agriculture could be genome-wide association studies (GWAS) and genomic selection for the improvement of economically important traits. However, some challenges still face these applications, such as incorporating linkage disequilibrium (LD) information from HapMap projects, data storage, and especially appropriate statistical analyses on the high-dimensional, structured genomics data. More efforts are still needed to make better use of the high-density SNP arrays in both academic studies and industrial applications.

• Parasites, Hosts and Diseases
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• 제47권2호
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• pp.83-91
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• 2009

The completion of many malaria parasite genomes provides great opportunities for genomewide characterization of gene expression and high-throughput genotyping. Substantial progress in malaria genomics and genotyping has been made recently, particularly the development of various microarray platforms for large-scale characterization of the Plasmodium falciparum genome. Microarray has been used for gene expression analysis, detection of single nucleotide polymorphism (SNP) and copy number variation (CNV), characterization of chromatin modifications, and other applications. Here we discuss some recent advances in genetic mapping and genomic studies of malaria parasites, focusing on the use of high-throughput arrays for the detection of SNP and CNV in the P. falciparum genome. Strategies for genetic mapping of malaria traits are also discussed.