Hybrid identification in Nothofagus subgenus using high resolution melting with ITS and trnL approach

The genus Nothofagus is the main component of southern South American temperate forests. The 40 Nothofagus species, evergreen and deciduous, and some natural hybrids are spread among Central and Southern Chile, Argentina, New Zealand, Australia, New Guinea and New Caledonia. Nothofagus nervosa, Nothofagus obliqua and Nothofagus dombeyi are potentially very important timber producers due to their high wood quality and relative fast growth; however, indiscriminate logging has degraded vast areas the Chilean forest causing a serious state of deterioration of their genetic resource. The South of Chile has a large area covered by secondary forests of Nothofagus dombeyi. These forests have a high diversity of species, large amount of biomass and high silvicultural potential. This work shows a case of hybrid identification in Nothofagus subgenus in different secondary forests of Chile, using high resolution melting. Unknown samples of Nothofagus subgenus are genetically distinguishable with the ITS region of Nothofagus antarctica, Nothofagus nitida and N. obliqua species. It was not possible to distinguish between unknown samples of Andean versus coastal origin. Melting curves with ITS approach of unknown material are genetically similar, positioned between N. dombeyi and N. antarctica and distant from N. nitida. The unknown samples are genetically very close to Nothofagus dombeyi. This suggests the presence of hybrid individuality between species (N. dombeyi × N. antarctica) with the possibility of introgression towards the gene pool of N. antarctica, producing the deciduous foliage that is both present. The trnL locus has no distinction between the N. dombeyi and N. antarctica species, since a similar melting curve is present and equal Tm (80.00 °C). The trnL locus cannot be genetically distinguished from one unknown sample of Nothofagus to another, as highlighted in this study.

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A Forensic Detection Method for Hallucinogenic Mushrooms via High-Resolution Melting (HRM) Analysis

Genes ◽

10.3390/genes12020199 ◽

2021 ◽

Vol 12 (2) ◽

pp. 199

Author(s):

Xiaochun Zhang ◽

Huan Yu ◽

Qi Yang ◽

Ziwei Wang ◽

Ruocheng Xia ◽

...

Keyword(s):

High Resolution ◽

Dna Barcoding ◽

High Resolution Melting ◽

Its Region ◽

Its Sequencing ◽

Melting Temperatures ◽

Hrm Analysis ◽

National Drug ◽

Species Specific ◽

Its1 And Its2

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

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Development of a High Resolution Melting Curve Analysis for the Detection of Hemoglobin δ-Chain Variants in Thailand and Identification of Hb A2-Walsgrave [codon 52 (GAT>CAT), Asp→His; HBD:c.157G>C] in a Pregnant Woman from Southern Thailand

Genetic Testing and Molecular Biomarkers ◽

10.1089/gtmb.2020.0291 ◽

2021 ◽

Vol 25 (6) ◽

pp. 426-433

Author(s):

Thanet Prajantasen ◽

Patcharawadee Prayalaw ◽

Sitthichai Panyasai ◽

Sukanya Binlee ◽

Supang Nongnuan

Keyword(s):

Pregnant Woman ◽

High Resolution ◽

High Resolution Melting ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis ◽

High Resolution Melting Curve ◽

Southern Thailand ◽

Δ Chain

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Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01083-17 ◽

2017 ◽

Vol 61 (12) ◽

Cited By ~ 9

Author(s):

L. Bernal-Martínez ◽

H. Gil ◽

O. Rivero-Menéndez ◽

S. Gago ◽

M. Cuenca-Estrella ◽

...

Keyword(s):

High Resolution ◽

Aspergillus Fumigatus ◽

Tandem Repeats ◽

High Resolution Melting ◽

Screening Method ◽

Melting Curve ◽

Health Concern ◽

Azole Resistance ◽

Content Type ◽

Selection Of

ABSTRACT The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A. Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus. PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR34, TR46, and TR53. Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

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