Document Type : Original Article
Authors
1
PhD Student, Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
2
Associate Professor, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education and Extension Organization, Karaj, Iran.
3
Professor, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education and Extension Organization, Karaj, Iran.
Abstract
Objective
High-resolution melting (HRM) analysis is an affordable, specific, and rapid tool for analyzing various sequences, screening, and genotyping. This method detects small nucleotide changes based on the melting properties of double-stranded DNA. This study aimed to optimize the HRM assay for safflower genotyping concerning oleic acid content. The FAD2-1 gene introduces a double bond into the oleic acid substrate, converting it into linoleic acid. In high-oleic acid varieties, a single nucleotide polymorphism (SNP) in this gene has been reported to introduce a premature stop codon, leading to enzyme inactivation. Therefore, accurately detecting genotypes carrying this SNP in the FAD2-1 gene could be a reliable method for identifying safflower genotypes with high oleic acid content.
Materials and Methods
HRM analysis was performed using two fluorescent dyes (EvaGreen and SYBR Green), three different concentrations of template genomic DNA, and two pairs of primers (HRM1 and HRM2, producing amplicons of 117 bp and 265 bp, respectively) on eight safflower genotypes. The aim was to determine the optimal conditions for distinguishing genotypes based on the presence or absence of the target SNP in the melting curves.
Results
Between the two primer sets, HRM1, which amplified a smaller 117 bp fragment, provided better differentiation among genotypes than HRM2, which amplified a larger 265 bp fragment. In comparing fluorescent dyes, EvaGreen enabled more apparent discrimination of genotypes in a single reaction than SYBR Green. Furthermore, genomic DNA concentration did not significantly affect the melting curve distinction within the tested range.
Conclusion
This study confirms and extends previous findings, demonstrating that smaller amplicons yield better melting curve distinction. EvaGreen fluorescent dye is also a suitable alternative to SYBR Green, providing improved genotyping efficiency.
Keywords
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