A high-resolution melt (HRM) assay to characterize CYP51 haplotypes of the wheat pathogen Mycosphaerella graminicola

• We designed a HRM assay to detect major SNPs in the Mycosphaerella graminicola CYP51 gene.
• SNP detection was performed on a Rotor-Gene instrument using 4 amplicons.
• The optimized assay allowed detection and identification of the CYP51 SNPs.

Mycosphaerella graminicola is a fungal pathogen that causes Septoria leaf blotch in wheat, resulting in substantial crop losses in years conducive to infection. The disease is mainly controlled by the use of sterol 14α-demethylation inhibitor (DMI) fungicides, which target the CYP51 protein involved in sterol biosynthesis in the fungus. The field efficacy of these chemicals has however been declining in the latest years, mainly due to single nucleotide polymorphisms (SNPs) in the target gene, leading to reduced binding of the resulting protein to these fungicides. Therefore, DMIs are nowadays used in mixes and alternate spraying schemes, together with new generation compounds belonging to the succinate dehydrogenase class of chemicals. To allow knowledgeable and sustainable control of the disease, monitoring of the field populations is paramount. Molecular characterization of field strains is currently mostly based on amplification of the CYP51 gene followed by classic Sanger sequencing. In this study, we optimized a simpler and more cost-effective genotyping method based on the high-resolution melting (HRM) technology. We demonstrate that this protocol can be used to detect and genotype the most common and resistance-determining SNPs in the M. graminicola CYP51 gene.