scholarly journals A High Resolution Melting Analysis-Based Genotyping Toolkit for the Peach (Prunus persica) Chilling Requirement

2020 ◽  
Vol 21 (4) ◽  
pp. 1543
Author(s):  
Lin Chou ◽  
Shih-Jie Huang ◽  
Chen Hsieh ◽  
Ming-Te Lu ◽  
Chia-Wei Song ◽  
...  
Keyword(s):  
High Resolution ◽  
Prunus Persica ◽  
High Resolution Melting ◽  
Physical Map ◽  
Low Cost ◽  
Principal Component ◽  
Chilling Requirement ◽  
Nucleotide Polymorphisms ◽  
Genotyping Platform ◽  
Peach Breeding

The chilling requirement (CR) is the main factor controlling the peach floral bud break and subsequent reproductive growth. To date, several peach CR quantitative trait loci (QTLs) have been identified. To improve the accessibility and convenience of this genetic information for peach breeders, the aim of this study was to establish an easy-to-use genotype screening system using peach CR molecular markers as a toolkit for marker-assisted selection. Here, we integrated 22 CR-associated markers from three published QTLs and positioned them on the Prunus persica physical map. Then, we built a PCR-based genotyping platform by using high-resolution melting (HRM) analysis with specific primers and trained this platform with 27 peach cultivars. Due to ambiguous variant calls from a commercial HRM software, we developed an R-based pipeline using principal component analysis (PCA) to accurately differentiate genotypes. Based on the PCA results, this toolkit was able to determine the genotypes at the CR-related single nucleotide polymorphisms (SNPs) in all tested peach cultivars. In this study, we showed that this HRM-PCA pipeline served as a low-cost, high-throughput, and non-gel genotyping solution. This system has great potential to accelerate CR-focused peach breeding.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

scholarly journals Population Structure and Phylogeny of Some U.S. Peach Cultivars

2022 ◽  
Vol 147 (1) ◽  
pp. 1-6
Author(s):  
Chunxian Chen ◽  
William R. Okie
Keyword(s):  
Population Structure ◽  
Prunus Persica ◽  
Principal Component ◽  
Pairwise Distance ◽  
Limiting Factors ◽  
Genetic Admixture ◽  
Department Of Agriculture ◽  
Phylogenetic Relations ◽  
Cultivar Groups ◽  
Peach Breeding

Peach (Prunus persica) cultivars maintained at the U.S. Department of Agriculture program at Byron, GA, have never been characterized with any molecular markers. In this study, 20 microsatellite markers were used to genotype 112 cultivars and the data were analyzed to discern their population structure and phylogenetic relationships. STRUCTURE simulations revealed four K clusters and broad genetic admixture among the cultivars. Principal coordinate analysis (PCoA) showed the cultivar groups from western, northeastern, and southeastern U.S. states were adjacent to each other except cultivars from Michigan (close to most southeastern state groups) and Florida (most distant from the other groups). Principal component analysis (PCA) showed that these cultivars had no obvious PCA partitioning boundaries. The intertwined distribution in both PCoA and PCA partitions suggested many of them were genetically closely related to each other largely because most shared same ancestral parentages. Most pairwise distance means within and between the cultivar groups were relatively low, suggesting close phylogenetic relations among those cultivars, as were demonstrated in the phylogenetic tree. Limiting factors and perspectives relevant to peach breeding are discussed.

scholarly journals The development of unlabeled probes-high resolution melting (UP-HRM) marker on SAD, IAA27 and ACC genes of oil palm

2021 ◽  
Vol 22 (6) ◽  
Author(s):  
TENGKU IMAM SAPUTRA ◽  
ROBERDI ROBERDI ◽  
YOGO ADHI NUGROHO ◽  
WULAN ARTUTININGSIH ◽  
OLIVIA S. PURBA ◽  
...  
Keyword(s):  
High Resolution ◽  
Oil Palm ◽  
High Resolution Melting ◽  
Elaeis Guineensis ◽  
Acyl Carrier Protein ◽  
Nucleotide Polymorphisms ◽  
Snp Discovery ◽  
Single Nucleotide ◽  
Elaeis Oleifera ◽  
Genotype Model

Abstract. Saputra TI, Roberdi, Nugroho YA, Artutiningsih W, Purba OS, Maryanto SD, Yono D, Utomo C, Liwang T. 2021. The development of unlabeled probes-high resolution melting (UP-HRM) marker on SAD, IAA27 and ACC genes of oil palm. Biodiversitas 22: 3356-3362. The unlabeled probes-high resolution melting (UP-HRM) marker is a useful tool for detecting of single nucleotide polymorphisms (SNPs). The objectives of this study were to develop UP-HRM markers to differentiate specific SNPs patterns on oil palm. The marker was developed and tested with Elaeis guineensis (Eg), Elaeis oleifera (Eo), Eo x Eg (hybrid), and was validated with 53 individuals of BC1F1 populations ((Eo x Eg) x Eg). Four UP-HRM markers were developed based on 2 SNPs in the stearoyl-acyl-carrier-protein 9-desaturase (EgSAD), 1 SNP in the auxin-responsive protein IAA27-like (EgIAA27), and 1 SNP in the 1-amino cyclopropane-1-carboxylate oxidase (EgACC) genes. The SNP discovery result showed that Eg was represented a reference homozygote genotype, while Eo was represented as an alternative homozygote genotype and the Eo x Eg hybrid was represented as a heterozygote genotype in all genes. The typical UP-HRM melt curve graph was successfully generated. This result was consistent with each genotype model for all four markers. The UP-HRM markers can distinguish each genotype according to the single-pass sequencing results. Furthermore, dendrogram analysis on validation divided 53 BC1F1 samples into three cluster groups.

scholarly journals High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

2021 ◽  
pp. 1-8
Author(s):  
Oytip Nathalang ◽  
Kamphon Intharanut ◽  
Sarisa Chidtrakoon
Keyword(s):  
High Resolution ◽  
Blood Group ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Blood Group Antigens ◽  
Red Cell ◽  
Nucleotide Polymorphisms ◽  
Hrm Analysis ◽  
Antibody Specificities

<b><i>Background:</i></b> High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. <b><i>Methods:</i></b> Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of <i>KEL</i>*<i>01</i> and <i>KEL</i>*<i>02</i>, <i>JK</i>*<i>01</i> and <i>JK</i>*<i>02</i>, <i>FY</i>*<i>01</i>, <i>FY</i>*<i>02</i>, and <i>FY</i>*<i>02 N.01</i>, <i>DI</i>*<i>01</i> and <i>DI</i>*<i>02</i>, <i>GYPB</i>*<i>03</i> and <i>GYPB</i>*<i>04</i>, <i>RHCE</i>*<i>E</i> and <i>RHCE</i>*<i>e,</i> and <i>DO</i>*<i>01</i> and <i>DO</i>*<i>02.</i> Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. <b><i>Results:</i></b> The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. <b><i>Conclusions:</i></b> We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

scholarly journals High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Lucas B. Harrison ◽  
Nancy D. Hanson
Keyword(s):  
Escherichia Coli ◽  
Principal Component Analysis ◽  
High Resolution ◽  
High Resolution Melting ◽  
Principal Component ◽  
Cost Effective ◽  
Sequence Type ◽  
Content Type ◽  
E Coli ◽  
Sequence Types

ABSTRACT Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification.

Sign in / Sign up

Export Citation Format

Share Document