Unlabeled probe melting analysis for SNP genotyping of Puccinia horiana isolates

Kris Van Poucke, Grace O'Keefe, Mathias De Backer, Don Davis, Martine Maes, Kurt Heungens

    Onderzoeksoutput: Hoofdstuk in Boek/Rapport/CongresprocedureC3: Congres abstract


    Chrysanthemum White Rust is a fungal disease caused by Puccinia horiana. Although this microcyclic rust is a regulated pathogen, it was able to spread to most of the chrysanthemum-producing areas in the world. Based on CRoPS analysis, De Backer et al. (2013) identified 32 single nucleotide polymorphisms (SNPs) and one single sequence repeat (SSR) in a worldwide collection of 45 isolates. These polymorphisms were analyzed via sequencing and used to determine the genetic diversity of this P. horiana population and help reconstruct international migration pathways. These markers are ideal tools to analyse larger collections of isolates and address questions regarding migration and local survival. For genotyping large populations, sequencing these markers would be expensive and time-consuming. High resolution melting (HRM) provides a cost-effective and faster alternative. During HRM analysis, a PCR amplicon that includes the SNP is melted in the presence of a dye that exclusively binds to double stranded DNA. The melting temperature (Tm) of the amplicon depends largely on its base pair composition, allowing for easy and fast SNP calling. However, in this case amplicon melting was not sufficiently reliable for genotyping because in some cases the difference in Tm between the two SNP variants was limited (< 1°C) and the amount of target DNA of this obligate parasite in DNA extracts was variable. Adding an unlabeled probe to the reaction mixture in combination with asymmetric PCR amplification of the genomic target sequence, followed by melting analysis of the probe, was a successful alternative method. When the probe matches the sequence of the amplicon, the probe-Tm is on average 5°C higher compared to the Tm of the mismatched probe. This allows for a reliable SNP calling and assignment of the isolate to a certain genotype. This approach was validated using DNA that was extracted from pustules from fresh leaves as well as from herbarium samples, and on DNA that was whole-genome amplified. The technique was subsequently applied to a collection of American P. horiana isolates.
    Oorspronkelijke taalEngels
    Titelabstract book 66th International Symposium on Crop Protection
    PublicatiestatusGepubliceerd - 2014
    Evenement66th International Symposium on Crop Protection - Gent, België
    Duur: 20-mei-201420-mei-2014


    • B390-gewasbescherming
    • B390-fytopathologie

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