• Title/Summary/Keyword: High-resolution Melting

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Development of specific SNP molecular marker from Thistle using DNA sequences of ITS region (엉겅퀴의 ITS 영역 염기서열 분석을 통한 특이적 SNP 분자마커의 개발)

  • Lee, Shin-Woo;Lee, Soo Jin;Kim, Yun-Hee
    • Journal of Plant Biotechnology
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    • v.45 no.2
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    • pp.102-109
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    • 2018
  • Thistle is a perennial plant that is widely used for medicinal purposes. Information on the genetic diversity of thistle populations are great important for their conservation and germ plasmic utilization. Although thistle is an important medicinal plant species registered in South Korea, no molecular markers are currently available to distinguish them from other similar species from different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from the nuclear ribosomal DNA internal transcribed spacer (ITS) regions of genomic sequences to identify distinct Korean-specific thistle species via an amplification refractory mutation system (ARMS)-PCR and high resolution melting (HRM) curve analyses. We performed molecular authentication of four different kinds of thistle species from different regions using DNA sequences in the ITS intergenic region. We also developed a quantitative PCR assay using species-specific ITS primers, which allowed us to estimate the ratio of Korean-specific thistle species using varying ratios of mixed genomic DNA templates from the two species. The SNP markers developed in this study are useful for rapidly identifying specific thistle species from different countries.

Association between a p73 Gene Polymorphism and Genetic Susceptibility to Non-small Cell Lung Cancer in the South of China

  • Wang, Shuang-Shuang;Guo, Hai-Yan;Dong, Lin-Li;Zhu, Xiang-Qian;Ma, Liang;Li, Wen;Tang, Jian-Xin
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.23
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    • pp.10387-10391
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    • 2015
  • Background: This study aimed to identify any association between the p73 gene G4C14-to-A4T14 polymorphism and risk of non-small cell lung cancer (NSCLC) in the south of China. Materials and Methods: We genotyped the p73 gene polymorphism of peripheral blood DNA from 168 patients with NSCLC and 195 normal controls using HRM (high resolution melting) and PCR-CTPP (polymerase chain reaction with confronting two-pair primers). Results: The results of genotyping by HRM and PCR-CTPP were consistent with direct sequencing, the p73 genotype distribution in 168 lung cancer patients being as follows: GC/GC 101 cases (60.1%), GC/AT 59 cases (35.1%), AT/AT 8 cases (4.8%). The carriers of AT/AT genotype had a significantly reduced risk of NSCLC (OR=0.370; 95%CI: 0.170-0.806; p=0.010) as compared with non-carriers. However, we found no relations between p73 genotypes and histological type (p=0.798, $x^2=0.452$), tumor stage (p=0.806, $x^2=0.806$), or lymph node metastasis (p=0.578, $x^2=1.098$). Conclusions: Our findings suggest that the p73 G4C14-to-A4T14 polymorphism may be a modifier of NSCLC susceptibility in the Chinese population.

Development of a Single-nucleotide Polymorphism Marker for the Sw-5b Gene Conferring Disease Resistance to Tomato spotted wilt virus in Tomato

  • Lee, Hyung Jin;Kim, Boyoung;Bae, Chungyun;Kang, Won-Hee;Kang, Byoung-Cheorl;Yeam, Inhwa;Oh, Chang-Sik
    • Horticultural Science & Technology
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    • v.33 no.5
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    • pp.730-736
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    • 2015
  • Tomato spotted wilt virus (TSWV) causes one of the most destructive viral diseases that threatens global tomato production. Sw-5b was reported as the resistance gene effective against TSWV. The objective of this research was to develop a single-nucleotide polymorphism (SNP) marker to distinguish tomato cultivars resistant to TSWV from susceptible cultivars for marker-assisted breeding. First, we determined genotypes for TSWV resistance in 32 commercial tomato cultivars using the previously reported Sw-5b gene-based marker. Then, DNA sequences of Sw-5b alleles in tomato cultivars showing resistant or susceptible genotypes were analyzed; a single SNP was found to distinguish tomato cultivars resistant to TSWV from susceptible cultivars. Based on the confirmed SNP, a SNP primer pair was designed. Using this new SNP sequence and high-resolution melting analysis, the same 32 tomato cultivars were screened. The results were perfectly correlated with those from screening with the Sw-5b gene-based marker. These results indicate that the SNP maker developed in this study will be useful for better tracking of resistance to TSWV in tomato breeding.

Population genetic variations of the matrix metalloproteinases-3 gene revealed hypoxia adaptation in domesticated yaks (Bos grunniens)

  • Ding, Xuezhi;Yang, Chao;Bao, Pengjia;Wu, Xiaoyun;Pei, Jie;Yan, Ping;Guo, Xian
    • Asian-Australasian Journal of Animal Sciences
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    • v.32 no.12
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    • pp.1801-1808
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    • 2019
  • Objective: As an iconic symbol of Qinghai-Tibetan Plateau and of high altitude, yak are subjected to hypoxic conditions that challenge aerobic metabolism. Matrix metalloproteinases-3 (MMP3) is assumed to be a key target gene of hypoxia-inducible factor-$1{\alpha}$ that function as a master regulator of the cellular response to hypoxia. Therefore, the aim of this investigation was to identify the DNA polymorphism of MMP3 gene in domestic yak and to explore its possible association with high-altitude adaptation. Methods: The single-nucleotide polymorphisms (SNPs) genotyping and mutations scanning at the MMP3 locus were conducted in total of 344 individuals from four domestic Chinese yak breeds resident at different altitudes on the Qinghai-Tibetan Plateau, using high-resolution melting analysis and DNA sequencing techniques. Results: The novel of SNPs rs2381 $A{\rightarrow}G$ and rs4331 $C{\rightarrow}G$ were identified in intron V and intron VII of MMP3, respectively. Frequencies of the GG genotype and the G allele of SNP rs2381 $A{\rightarrow}G$ observed in high-altitude Pali yak were significantly higher than that of the other yak breeds resident at middle or low altitude (p<0.01). No significant difference was mapped for SNP rs4331 $C{\rightarrow}G$ in the yak population (p>0.05). Haplotype GC was the dominant among the 4 yak breeds, and Pearson correlation analysis showed that the frequencies of GC was significantly lower in Ganan (GN), Datong (DT), and Tianzhu white yaks (TZ) compared with Pali (PL) yak. The two SNPs were in moderate linkage disequilibrium in high-altitude yaks (PL) but not in middle-altitude (GN, DT) and low-altitude (TZ) yaks. Conclusion: These results indicate that MMP3 may have been subjected to positive selection in yak, especially that the SNP rs2381 $A{\rightarrow}G$ mutation and GC haplotypes might contribute to adaptation for yak in high-altitude environments.

Global DNA Methylation Patterns and Gene Expression Associated with Obesity-Susceptibility in Offspring of Pregnant Sprague-Dawley Rats Exposed to BDE-47 and BDE-209 (임신 중 BDE-47 및 BDE-209에 노출된 어미와 새끼 Sprague-Dawley 랫드의 Global DNA 메틸화 양상과 비만 감수성과 연관된 유전자 발현)

  • Park, Byeong-Min;Yoon, Ok-Jin;Lee, Do-Hoon
    • Korean Journal of Clinical Laboratory Science
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    • v.49 no.1
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    • pp.28-39
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    • 2017
  • Persistent organic pollutants (POPs) can affect epigenetic mechanisms and obesity development. Polybrominated diphenyl ethers (PBDEs)-widely used to make flames-are one of the important POPs. Prenatal exposure to endocrine disrupting chemicals (EDCs), such as POPs, may affect global DNA methylation in long interspersed nuclear elements (LINE-1), increasing the risk of obesity later in life. Therefore, pregnant Sprague-Dawley (SD) rats were used to elucidate whether BDE-47 and BDE-209 transferred through placenta and breast milk cause epigenetic changes in LINE-1 and increase genetic susceptibility to obesity as obesogen during the developmental periods. Global DNA methylation in LINE-1 and gene expression related to obesity were measured in dams and offspring, using a methylation-sensitive high resolution melting analysis (MS-HRM) and direct bisulfite sequencing and quantitative real time polymerase chain reaction (qPCR), respectively. The results of MS-HRM showed global DNA hypomethylation patterns in LINE-1 of exposed offspring (2 of total 4) at PND 4, but bisulfite sequencing showed no difference in both the exposed and non-exposed groups. Gene expression in dams related to ${\beta}$-oxidation pathway and those related to adipokines showed different patterns between the two groups. On the contrary, gene expressions of offspring showed a similar pattern. Gene expressions related to ${\beta}$-oxidation pathway and obesity were significantly increased when compared with 'at birth', but not $PPAR-{\alpha}$. In conclusion, this study demonstrated the possibility that co-exposure to BDE-47 and BDE-209-via the placenta and breast milk-may affect epigenetic changes and modulate gene expression levels related to obesity.

A Study on the Radiometric Correction of Sentinel-1 HV Data for Arctic Sea Ice Detection (북극해 해빙 탐지를 위한 Sentinel-1 HV자료의 방사보정 연구)

  • Kim, Yunjee;Kim, Duk-jin;Kwon, Ui-Jin;Kim, Hyun-Cheol
    • Korean Journal of Remote Sensing
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    • v.34 no.6_2
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    • pp.1273-1282
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    • 2018
  • Recently, active research on the Arctic Ocean has been conducted due to the influence of global warming and new Arctic ship route. Although previous studies already calculated quantitative extent of sea ice using passive microwave radiometers, melting at the edge of sea ice and surface roughness were hardly considered due to low spatial resolution. Since Sentienl-1A/B data in Extended Wide (EW) mode are being distributed as free of charge and bulk data for Arctic sea can be generated during a short period, the entire Arctic sea ice data can be covered in high spatial resolution by mosaicking bulk data. However, Sentinel-1A/B data in EW mode, especially in HV polarization, needs significant radiometric correction for further classification. Thus, in this study, we developed algorithms that can correct thermal noise and scalloping effects, and confirmed that Arctic sea ice and open-water were well classified using the corrected dual-polarization SAR data.

Quantitative Analysis of Snow Particles Using a Multi-Angle Snowflake Camera in the Yeongdong Region (영동지역에서 눈결정 카메라를 활용한 눈결정의 정량 분석)

  • Kim, Su-Hyun;Ko, Dae-Hong;Seong, Dae-Kyung;Eun, Seung-Hee;Kim, Byung-Gon;Kim, Baek-Jo;Park, Chang-Geun;Cha, Ju-Wan
    • Atmosphere
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    • v.29 no.3
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    • pp.311-324
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    • 2019
  • We employed a Multi-Angle Snowflake Camera (MASC) to quantitatively analyze snow particles at the ground level in the Yeongdong region of Korea. The MASC captures high-resolution photographs of hydrometeors from three angles and simultaneously measures fallspeed. Based on snowflake images of the several episodes in 2017 and 2018, we derived statistics of size, aspect ratio, orientation, complexity, and fallspeed of snow crystals, which generally showed similar characteristics to the previous studies in other regions of the world. Dominant snow crystal habits of January 22, 2018 generated by northerly were melted aggregates when 850 hPa temperature was about $-6{\sim}-8^{\circ}C$. Average fallspeed of snow crystals was $1.0m\;s^{-1}$ though its size gradually increased as temperature decreased. Another snowfall event (March 8, 2018) was driven by the baroclinic instability as accompanied with a deep trough. Snow crystal habits were largely rimed aggregates (complexity ~1.8) and melting particles of dark images. Meanwhile, in the extreme snowfall event whose snow rate was greater than $10cm\;hr^{-1}$ on January 20, 2017, main snow crystals appeared to be heavily rimed particles with relatively smaller size when convective clouds developed vertically up to 9 km in association with tropopause folding. MASC also could successfully measure a decrease in snow crystal size and an increase in riming degree after AgI seeding at Daegwallyeong on March 14, 2017.

Study of Crustal Structure in North Korea Using 3D Velocity Tomography (3차원 속도 토모그래피를 이용한 북한지역의 지각구조 연구)

  • So Gu Kim;Jong Woo Shin
    • The Journal of Engineering Geology
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    • v.13 no.3
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    • pp.293-308
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    • 2003
  • New results about the crustal structure down to a depth of 60 km beneath North Korea were obtained using the seismic tomography method. About 1013 P- and S-wave travel times from local earthquakes recorded by the Korean stations and the vicinity were used in the research. All earthquakes were relocated on the basis of an algorithm proposed in this study. Parameterization of the velocity structure is realized with a set of nodes distributed in the study volume according to the ray density. 120 nodes located at four depth levels were used to obtain the resulting P- and S-wave velocity structures. As a result, it is found that P- and S-wave velocity anomalies of the Rangnim Massif at depth of 8 km are high and low, respectively, whereas those of the Pyongnam Basin are low up to 24 km. It indicates that the Rangnim Massif contains Archean-early Lower Proterozoic Massif foldings with many faults and fractures which may be saturated with underground water and/or hot springs. On the other hand, the Pyongyang-Sariwon in the Pyongnam Basin is an intraplatform depression which was filled with sediments for the motion of the Upper Proterozoic, Silurian and Upper Paleozoic, and Lower Mesozoic origin. In particular, the high P- and S-wave velocity anomalies are observed at depth of 8, 16, and 24 km beneath Mt. Backdu, indicating that they may be the shallow conduits of the solidified magma bodies, while the low P-and S-wave velocity anomalies at depth of 38 km must be related with the magma chamber of low velocity bodies with partial melting. We also found the Moho discontinuities beneath the Origin Basin including Sari won to be about 55 km deep, whereas those of Mt. Backdu is found to be about 38 km. The high ratio of P-wave velocity/S-wave velocity at Moho suggests that there must be a partial melting body near the boundary of the crust and mantle. Consequently we may well consider Mt. Backdu as a dormant volcano which is holding the intermediate magma chamber near the Moho discontinuity. This study also brought interesting and important findings that there exist some materials with very high P- and S-wave velocity annomoalies at depth of about 40 km near Mt. Myohyang area at the edge of the Rangnim Massif shield.

Development of HRM Markers Based on SNPs Identified from Next Generation Resequencing of Susceptible and Resistant Parents to Gummy Stem Blight in Watermelon (수박에서 덩굴마름병 감수성 및 저항성 양친에 대한 차세대 염기서열 재분석으로 탐색된 SNP 기반 HRM 분자표지 개발)

  • Lee, Eun Su;Kim, Jinhee;Hong, Jong Pil;Kim, Do-Sun;Kim, Minkyong;Huh, Yun-Chan;Back, Chang-Gi;Lee, Jundae;Lee, Hye-Eun
    • Korean Journal of Breeding Science
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    • v.50 no.4
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    • pp.424-433
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    • 2018
  • Watermelon (Citrullus lanatus) is an economically important vegetable crop all over the world, which has functional compounds such as lycopene and citrulline. Gummy stem blight caused by Didymella bryoniae is one of the most devastative diseases in watermelon. Single nucleotide polymorphisms (SNPs), which are genetic variations occurring between individuals with respect to a single base, were often used to construct genetic linkage maps and develop molecular markers linked to a variety of horticultural traits and resistance to several diseases. In this study, we developed high-resolution melting (HRM) markers based on SNPs generated from NGS resequencing of two parents in watermelon. Plant materials were C. lanatus '920533' (female and susceptible parent), C. amarus 'PI 189225' (male and resistant parent), and their $F_1$ and $F_2$ progenies. A total of 13.6 Gbp ('920533') and 13.1 Gbp ('PI 189225') of genomic sequences were obtained using NGS analysis. A total of 6.09 million SNPs between '920533' and 'PI 189225' were detected, and 354,860 SNPs were identified as potential HRM primer sets. From these, a total of 330 primer sets for HRM analysis were designed. As a result, a total of 61 HRM markers that have polymorphic melting curves were developed. These HRM markers can be used for the construction of SNP-based linkage maps and for the analysis of quantitative trait loci (QTLs) related to gummy stem blight resistance.

Detection of PIK3CA Gene Mutations with HRM Analysis and Association with IGFBP-5 Expression Levels in Breast Cancer

  • Dirican, Ebubekir;Kaya, Zehra;Gullu, Gokce;Peker, Irem;Ozmen, Tolga;Gulluoglu, Bahadir M.;Kaya, Handan;Ozer, Ayse;Akkiprik, Mustafa
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.21
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    • pp.9327-9333
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    • 2014
  • Breast cancer is the second most common cancer and second leading cause of cancer deaths in women. Phosphatidylinositol-3-kinase (PI3K)/AKT pathway mutations are associated with cancer and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutations have been observed in 25-45% of breast cancer samples. Insulin growth factor binding protein-5 (IGFBP-5) can show different effects on apoptosis, cell motility and survival in breast cancer. We here aimed to determine the association between PIK3CA gene mutations and IGFBP-5 expressions for the first time in breast cancer patients. Frozen tumor samples from 101 Turkish breast cancer patients were analyzed with high resolution melting (HRM) for PIK3CA mutations (exon 9 and exon 20) and 37 HRM positive tumor samples were analyzed by DNA sequencing, mutations being found in 31. PIK3CA exon 9 mutations (Q546R, E542Q, E545K, E542K and E545D) were found in 10 tumor samples, exon 20 mutations (H1047L, H1047R, T1025T and G1049R) in 21, where only 1 tumor sample had two exon 20 mutations (T1025T and H1047R). Moreover, we detected one sample with both exon 9 (E542Q) and exon 20 (H1047R) mutations. 35% of the tumor samples with high IGFBP-5 mRNA expression and 29.4% of the tumor samples with low IGFBP-5 mRNA expression had PIK3CA mutations (p=0.9924). This is the first study of PIK3CA mutation screening results in Turkish breast cancer population using HRM analysis. This approach appears to be a very effective and reliable screening method for the PIK3CA exon 9 and 20 mutation detection. Further analysis with a greater number of samples is needed to clarify association between PIK3CA gene mutations and IGFBP-5 mRNA expression, and also clinical outcome in breast cancer patients.