Combination of high resolution melting (HRM) analysis and SSR molecular markers speeds up plum genotyping: case study genotyping the Greek plum GeneBank collection

2016 ◽  
Vol 15 (4) ◽  
pp. 366-375 ◽  
Author(s):  
Georgios Merkouropoulos ◽  
Ioannis Ganopoulos ◽  
Athanasios Tsaftaris ◽  
Ioannis Papadopoulos ◽  
Pavlina Drogoudi
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Ex Situ ◽  
Hrm Analysis ◽  
Prunus Salicina ◽  
Genebank Collection ◽  
Of Greece

AbstractIn a germplasm bank collection the conservation and characterization of genetic resources is a prerequisite in order to use the material in breeding projects aiming the creation of new cultivars. In the present study, 54 Prunus salicina domestica and Prunus domestica genotypes (including seven Greek cultivars), maintained in the ex situ National Genebank Collection of Greece, were classified using microsatellite (simple sequence repeat, SSR) markers on high resolution melting (HRM) analysis. The SSR primer pairs were chosen from the published literature as originally designed on Prunus species. This combined approach was used to genotype all plum accessions of the collection highlighting the benefits of either method (HRM and SSRs) for cultivar identification. Dendrograms for P. domestica and P. salicina and a combined one with all the genotypes assayed were produced. A total of 15 from the 19 P. domestica accessions analysed, including all the Greek accessions but ‘Avgati Skopelou’, were grouped into the same clade in the combined dendrogram, whereas the remaining four were dispersed into the P. salinica clades. Bayesian structure analysis confirmed that ‘Avgati Skopelou’ differs from the rest of the Greek plum cultivars since it was not grouped into the same cluster. The combination of HRM and SSRs, provided a considerably faster, cost-effective, closed-tube microsatellite genotyping method for molecular characterization of plum cultivars.

scholarly journals Genotyping of GATA4 Gene Variant (G296S) in Malaysian Congenital Heart Disease Subjects by Real-Time PCR High Resolution Melting Analysis

2013 ◽  
Vol 32 (2) ◽  
pp. 152-157
Author(s):  
Nora Fawzi ◽  
Ramachandran Vasudevan ◽  
Patimah Ismail ◽  
Mazeni Alwi ◽  
Ahmad Fazli Abdul Aziz ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
High Resolution ◽  
Congenital Heart ◽  
High Resolution Melting ◽  
New Technology ◽  
Cost Effective ◽  
Study Cohort ◽  
Hrm Analysis ◽  
Melting Analysis

Summary Background: Congenital heart disease (CHD) is the most common birth defect; however, the underlying etiology is unrecognized in the majority of cases. GATA-binding protein 4 (GATA4), a cardiac transcription factor gene, has a crucial role in the cardiogenesis process; hence, a number of heterozygote sequence variations were identified as a cause of CHD. G296S heterozygote variant is the most frequently reported GATA4 gene sequence alteration. This study aims to investigate the role of G296S variant of the GATA4 gene in Malaysian CHD subjects. Methods: We have investigated 86 Malaysian CHD subjects with cardiac septation defects for the presence of the GATA4 gene heterozygote variant (G296S) by the new technology of high resolution melting (HRM) analysis. Results: Genotyping of G296S (c.886G>A) by HRM analysis shows that all the sample genotypes were of the wild GG type genotype and the heterozygote mutant GA genotype was totally absent from this study cohort. Conclusions: The results of our study showed that the G296S variant of the GATA4 gene was not associated with the development of CHD in Malaysian subjects. The use of HRM analysis proved a cost-effective, high-throughput, specific and sensitive genotyping technique which eliminates the need for unnecessary sequencing.

scholarly journals Traceability of PDO Olive Oil “Terra di Bari” Using High Resolution Melting

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Cinzia Montemurro ◽  
Monica Marilena Miazzi ◽  
Antonella Pasqualone ◽  
Valentina Fanelli ◽  
Wilma Sabetta ◽  
...  
Keyword(s):  
High Resolution ◽  
Olive Oil ◽  
High Resolution Melting ◽  
Virgin Olive Oil ◽  
Cost Effective ◽  
Extra Virgin Olive Oil ◽  
Hrm Analysis ◽  
Olive Cultivars ◽  
Pcr Products ◽  
Set Up

The aim of the research was to verify the applicability of microsatellite (SSR) markers in High Resolution Melting (HRM) analysis for the identification of the olive cultivars used in the “Terra di Bari” PDO extra virgin olive oil. A panel of nine cultivars, widespread in Apulia region, was tested with seventeen SSR primer pairs and the PCR products were at first analysed with a Genetic Analyzer automatic sequencer. An identification key was obtained for the nine cultivars, which showed an unambiguous discrimination among the varieties constituting the “Terra di Bari” PDO extra virgin olive oil: Cima di Bitonto, Coratina, and Ogliarola. Subsequently, an SSR based method was set up with the DCA18 marker, coupled with HRM analysis for the distinction of the Terra di Bari olive oil from non-Terra di Bari olive oil using different mixtures. Thus, this analysis enabled the distinction and identification of the PDO mixtures. Hence, this assay provided a flexible, cost-effective, and closed-tube microsatellite genotyping method, well suited to varietal identification and authentication analysis in olive oil.

Microsatellite high-resolution melting (SSR-HRM) analysis for genotyping and molecular characterization of an Olea europaea germplasm collection

2014 ◽  
Vol 12 (3) ◽  
pp. 273-277 ◽  
Author(s):  
Aliki Xanthopoulou ◽  
Ioannis Ganopoulos ◽  
Georgios Koubouris ◽  
Athanasios Tsaftaris ◽  
Chrysa Sergendani ◽  
...  
Keyword(s):  
High Resolution ◽  
Microsatellite Markers ◽  
Molecular Characterization ◽  
Olea Europaea ◽  
High Resolution Melting ◽  
Germplasm Collection ◽  
Hrm Analysis ◽  
Olive Cultivars ◽  
Olea Europaéa

Olea europaea L. has been cultivated in the Mediterranean region for thousands of years and is of major economic importance. The origin of olive cultivars remains as complex to trace as their identification. Thus, their molecular characterization and discrimination will enable olive germplasm management. In addition, it would be a useful tool for authentication of olive products. High-resolution melting (HRM) analysis, coupled with five microsatellite markers, was integrated to facilitate molecular identification and characterization of main O. europaea cultivars collected from the National Olive Tree Germplasm Collection established in Chania, Greece. The five microsatellite loci used were highly informative and generated a unique melting curve profile for each of the 47 cultivars and for each microsatellite tested. In particular, three microsatellite markers (DCA03, DCA09 and DCA17), which generated 29 HRM profiles, were sufficient to genotype all the olive cultivars studied, highlighting their potential use for cultivar identification. Furthermore, this assay provided a flexible, cost-effective and closed-tube microsatellite genotyping method well suited for molecular characterization of olive cultivars.

scholarly journals Differentiation of Mitragyna speciosa, a narcotic plant, from allied Mitragyna species using DNA barcoding-high-resolution melting (Bar-HRM) analysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chayapol Tungphatthong ◽  
Santhosh Kumar J. Urumarudappa ◽  
Supita Awachai ◽  
Thongchai Sooksawate ◽  
Suchada Sukrong
Keyword(s):  
High Resolution ◽  
Dna Barcoding ◽  
High Resolution Melting ◽  
Dna Barcode ◽  
Herbal Medicines ◽  
Efficient Tool ◽  
Hrm Analysis ◽  
Mitragyna Speciosa ◽  
Leaf Powder

AbstractMitragyna speciosa (Korth.) Havil. [MS], or “kratom” in Thai, is the only narcotic species among the four species of Mitragyna in Thailand, which also include Mitragyna diversifolia (Wall. ex G. Don) Havil. [MD], Mitragyna hirsuta Havil. [MH], and Mitragyna rotundifolia (Roxb.) O. Kuntze [MR]. M. speciosa is a tropical tree belonging to the Rubiaceae family and has been prohibited by law in Thailand. However, it has been extensively covered in national and international news, as its abuse has become more popular. M. speciosa is a narcotic plant and has been used as an opium substitute and traditionally used for the treatment of chronic pain and various illnesses. Due to morphological disparities in the genus, the identification of plants in various forms, including fresh leaves, dried leaf powder, and finished products, is difficult. In this study, DNA barcoding combined with high-resolution melting (Bar-HRM) analysis was performed to differentiate M. speciosa from allied Mitragyna and to assess the capability of Bar-HRM assays to identify M. speciosa in suspected kratom or M. speciosa-containing samples. Bar-HRM analysis of PCR amplicons was based on the ITS2, rbcL, trnH-psbA, and matK DNA barcode regions. The melting profiles of ITS2 amplicons were clearly distinct, which enabled the authentication and differentiation of Mitragyna species from allied species. This study reveals that DNA barcoding coupled with HRM is an efficient tool with which to identify M. speciosa and M. speciosa-containing samples and ensure the safety and quality of traditional Thai herbal medicines.

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

Genes ◽  
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.

scholarly journals The Case Study of the Medieval Town Walls of Gubbio in Italy: First Results on the Characterization of Mortars and Binders

Heritage ◽  
2018 ◽  
Vol 1 (2) ◽  
pp. 468-478 ◽  
Author(s):  
Fernanda Carvalho ◽  
Andreia Lopes ◽  
Antonella Curulli ◽  
Teresa da Silva ◽  
Maria Lima ◽  
...  
Keyword(s):  
Polarized Light ◽  
Ex Situ ◽  
X Ray Diffraction ◽  
X Ray ◽  
Heritage Sites ◽  
First Results ◽  
Restoration Practices ◽  
Climate Events

Good conservation and restoration practices of cultural heritage assets rely on the knowledge of original materials. In the framework of the HERACLES Project (HERACLES—HEritage Resilience Against CLimate Events on Site, H2020 Grant Agreement 700395), dealing with the effects of climatic actions and natural hazards on built heritage, a set of important heritage sites are currently under study to improve their resilience against climate events. Among these are the medieval Gubbio Town Walls in Italy. The present work focuses on the mortars and binders of this monument and collected samples related to different parts of the Walls, corresponding to various historical periods of construction and interventions. They were characterized to determine their minerochemical composition, thermal behavior, and morphology. For that purpose, ex-situ laboratory techniques, such as X-ray diffraction (XRD), wavelength dispersive X-ray fluorescence (WDXRF), optical microscopy (OM), polarized light microscopy (PLM), scanning electron microscopy (SEM), and simultaneous differential thermal analysis and thermogravimetry (TG-DTA) were used to discern trends in different sampling areas due to construction/reconstruction periods and building techniques.

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