Abstract
An innovative quantitative PCR-based method derived from the Kompetitive Allele Specific PCR Assay Reagent (KASPar) system was developed to quantify the genomic DNA from two coexisting genotypes on the same tissues of a host-plant. For this purpose, the classical end-point KASPar method was evolved to a real-time method thanks to the addition of an adapted measurement step after each PCR cycle. It was applied to the quantification of the two genotypes G1 and G2 of the Gaeumannomyces graminis var. tritici (Ggt) soilborne fungus, pathogenic on wheat roots. Specific primers targeting a single nucleotide polymorphism from the ITS sequence were used allowing simultaneous quantification of both genotypes in the same reaction. The assays were applied to quantify fungal DNA of each genotype, aside or mixed together, after DNA extraction from fungal pure cultures and from single or co-inoculated roots in artificial medium or in soil. The detection and quantification lower limits for the two genotypes were 1.25 pg and 5 pg for DNA from fungal pure cultures, and 1.8 pg and 7 pg for DNA from fungal inoculated roots. The advantages of this cost-effective method are the high levels of specificity, sensitivity and reproducibility. Moreover, the accuracy of the method is independent of the copy numbers of the target sequences. The method is the first one to adapt the non-quantitative genotyping KASPar system to a quantitative application of two known genotypes of a species simultaneously and is suitable for simultaneous genotype-specific quantification of any other organisms (fungi, bacteria, plants).
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Acknowledgments
This work was supported by grants from INRA (“Institut National de la Recherche Agonomique”), Plant Health and Environment division. We thank J. Wilson, a native English speaker and a professional translator, for her English revisions of the manuscript.
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Kévin Gazengel and Lionel Lebreton equal contributors
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Gazengel, K., Lebreton, L., Guillerm-Erckelboudt, AY. et al. Simultaneous monitoring of two fungal genotypes on plant roots by single nucleotide polymorphism quantification with an innovative KASPar quantitative PCR. Mycol Progress 12, 657–666 (2013). https://doi.org/10.1007/s11557-012-0872-4
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DOI: https://doi.org/10.1007/s11557-012-0872-4