• Korean Journal of Plant Resources
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• v.32 no.4
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• pp.303-311
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• 2019

Various colors of fruit skin and flesh are the most popular commercial criteria for peach classification. In order to breed new red-fleshed peach cultivar, many cross seedlings and generations should be maintained. Therefore it is necessary to develop early selection markers to screen seedlings with target traits to increase breeding efficiency. For the comparison of transcription profiles in peach cultivars differing in flesh color expression, two cDNA libraries were constructed. Differences in gene expression between red-fleshed peach cultivar, 'Josanghyeoldo' and white-fleshed peach cultivar, 'Mibaekdo' were analyzed by next-generation sequencing (NGS). Expressed sequence tag (EST) of clones from the two cultivars were selected for nucleotide sequence determination and homology searches. Putative single nucleotide polymorphisms (SNP) were screened from peach EST contigs by high resolution melting (HRM) analysis displayed specific difference between 8 red-fleshed peach cultivars and 24 white-fleshed peach cultivars. All 72 pairs of SNPs were discriminated and the HRM profiles of amplicons were established. In the study reported here, the development of SNP markers for distinguishing between red and white fleshed peach cultivars by HRM analysis offers the opportunity to use DNA markers. This SNP marker could be useful for peach marker assisted breeding and provide a good reference for relevant research on molecular mechanisms of color variation in peach cultivars.

• Parasites, Hosts and Diseases
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• v.51 no.6
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• pp.689-694
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• 2013

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at $82.4{\pm}0.09^{\circ}C$ and $85.9{\pm}0.08^{\circ}C$ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.5
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• pp.1873-1880
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• 2015

Background: A recent genome-wide association study (GWAS) on patients with chronic hepatitis C (CHC) treated with peginterferon and ribavirin (pegIFN-${\alpha}$/RBV) identified a single nucleotide polymorphism (SNP) on chromosome 19 (rs12979860) which was strongly associated with a sustained virological response (SVR). The aim of this study was twofold: to study the relationship between IL28B rs12979860 and sustained virological response (SVR) to pegIFN-${\alpha}$/RVB therapy among CHC patients and to detect the rs12979860 polymorphism by high resolution melting curve (HRM) assay as a simple, fast, sensitive, and inexpensive method. Materials and Methods: The study examined outcomes in 100 patients with chronic hepatitis C in 2 provinces of Iran from December 2011 to June 2013. Two methods were applied to detect IL28B polymorphisms: PCR-sequencing as a gold standard method and HRM as a simple, fast, sensitive, and inexpensive method. Results: The frequencies of IL28B rs12979860 CC, CT, and TT alleles in chronic hepatitis C genotype 1a patients were 10% (10/100), 35% (35/100), and 6% (6/100) and in genotype 3a were 13% (13/100), 31% (31/100), and 5% (5/100), respectively. In genotype 3a infected patients, rs12979860 (CC and CT alleles) and in genotype 1a infected patients (CC allele) were significantly associated with a sustained virological response (SVR). The SVR rates for CC, CT and TT (IL28B rs12979860) were 18%, 34% and 4%, respectively. Multiple logistic regression analysis identified two independent factors that were significantly associated with SVR: IL-28B genotype (rs 12979860 CC vs TT and CT; odds ratio [ORs], 7.86 and 4.084, respectively), and HCV subtype 1a (OR, 7.46). In the present study, an association between SVR rates and IL28B polymorphisms was observed. Conclusions: The HRM assay described herein is rapid, inexpensive, sensitive and accurate for detecting rs12979860 alleles in CHC patients. This method can be readily adopted by any molecular diagnostic laboratory with HRM capability and will be clinically beneficial in predicting treatment response in HCV genotype 1 and 3 infected patients. In addition, it was demonstrated that CC and CT alleles in HCV-3a and the CC allele in HCV-1a were significantly associated with response to pegIFN-${\alpha}$/RBV treatment. The present results may help identify subjects for whom the therapy might be successful.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.5019-5022
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• 2014

Recently, mutations in the genes involved in cell cycle control, including CHEK2, are being considered as etiological factors in different kinds of cancers. The CHEK2 protein plays an important role in protecting damaged DNA from entering mitosis. In this study the potential effects of two common mutations $IVS2+1G{\rightarrow}A$ and Ile157Thr of CHEK2 gene in differentiated thyroid carcinoma (DTC) were evaluated. A total of 100 patients admitted to the Research Institute for Nuclear Medicine were diagnosed with DTC based on pathology reports of surgery samples. An additional 100 people were selected as a control group with no cancer history. PCR-HRM (high resolution melting) analysis was performed to deal with each of mutations in all case and control samples separately. During the analysis of $IVS2+1G{\rightarrow}A$ and Ile157Thr mutations of CHEK2 gene in the case and control groups, all the samples were identified as wild homozygote type. The finding suggests that $IVS2+1G{\rightarrow}A$ and Ile157Thr mutations of CHEK2 gene do not constitute a risk factor for DTC in the Iranian population. However, further studies with larger population are required to confirm the outcome.

• 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.

• Korean Journal of Plant Resources
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• v.34 no.5
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• pp.443-450
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• 2021

Peach flesh color is commercially important criteria for classification and has implications for nutritional quality. To breed new yellow-fleshed peach cultivar many cross seedlings and generations should be maintained. Therefore it is necessary to develop early selection molecular markers for screening cross seedlings and germplasm with economically important traits to increase breeding efficiency. For the comparison of transcription profiles in peach varieties with a different flesh color expression, two cDNA libraries were constructed. Differences in gene expression between yellow-fleshed peach cultivar, 'Changhowon Hwangdo' and white-fleshed peach cultivar, 'Mibaekdo' were analyzed by next-generation sequencing (NGS). Expressed sequence tag (EST) of clones from the two varieties was selected for nucleotide sequence determination and homology searches. Putative single nucleotide polymorphisms (SNPs) were screened from peach EST contigs by high resolution melting (HRM) analysis, SNP ID ppa002847m:cds and ppa002540m:cds displayed specific difference between 17 yellow-fleshed and 21 white-fleshed peach varieties. The SNP markers for distinguishing yellow and white fleshed peach varieties by HRM analysis offers the opportunity to use early selection. This SNP markers could be useful for marker assisted breeding and provide a good reference for relevant research on molecular mechanisms of color variation in peach varieties.

• Journal of Plant Biotechnology
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• v.44 no.3
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• pp.235-242
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• 2017

Cudrania tricuspidata Bureau is a widely-used, medicinal, perennial and woody plant. Obtaining information about the genetic diversity of plant populations is highly important with regard toconservation and germplasm utilization. Although C. tricuspidata is an important medicinal plant species registered in South Korea, no molecular markers are currently available to distinguish Korean-specific ecotypes from other ecotypes from different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from the chloroplast and nuclear genomic sequences, which serve to to identify distinct Korean-specific ecotypes of C. tricuspidata via amplification refractory mutation system (ARMS)-PCR and high resolution melting (HRM) curve analyses. We performed molecular authentication of twelve C. tricuspidata ecotypes from different regions using DNA sequences in the maturaseK (MatK) chloroplast intergenic region and nuclear ribosomal DNA internal transcribed spacer (ITS) regions. The SNP markers developed in this study are useful for rapidly identifying specific C. tricuspidata ecotypes from different regions.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.1
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• pp.219-223
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• 2016

Background: Promoter hypermethylation is a frequent epigenetic mechanism for gene transcription repression in cancer and is one of the hallmarks of the disease. Cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) contributes to cell contact-mediated communication. Dysregulation of promoter methylation has been reported in various cancers. Objectives: The objectives of this study were to investigate the CELSR3 hypermethylation level in oral squamous cell carcinomas (OSCCs) using methylation-sensitive high-resolution melting analysis (MS-HRM) and to correlate CELSR3 methylation with patient demographic and clinicopathological parameters. Materials and Methods: Frozen tissue samples of healthy subjects' normal mucosa and OSCCs were examined with regard to their methylation levels of the CELSR3 gene using MS-HRM. Results: MS-HRM analysis revealed a high methylation level of CELSR3 in 86% of OSCC cases. Significant correlations were found between CELSR3 quantitative methylation levels with patient ethnicity (P=0.005), age (P=0.024) and pathological stages (P=0.004). A moderate positive correlation between CELSR3 and patient age was also evident (R=0.444, P=0.001). Conclusions: CELSR3 promoter hypermethylation may be an important mechanism involved in oral carcinogenesis. It may thus be used as a biomarker in OSCC prognostication.

• 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.

• 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.