High-throughput qPCR phenotyping of Verticillium stripe resistance in Brassica napus under natural field infection

Verticillium stripe, caused by Verticillium longisporum, is an emerging threat to Brassica napus production in North America, with increasing field incidence and yield loss reported in affected regions. Although visual ratings are useful for rapid field assessment, resistance breeding is constrained by the lack of scalable, objective phenotyping methods that can quantify pathogen colonization across large genotype panels under field conditions. Here, we adapted and validated an established OLG70/OLG71-based SYBR Green qPCR assay within a high-throughput workflow combining freeze-dried hypocotyl sampling and magnetic bead-based DNA extraction to quantify V. longisporum DNA in field-grown B. napus. A panel of 215 B. napus genotypes was evaluated at two naturally infested field sites in Manitoba, Canada. The assay produced robust standard curves across plates, with R² values >0.99, amplification efficiencies of 95–105%, and low intra-plate variability, supporting reliable quantification over a broad dynamic range. Log₁₀-transformed fungal DNA quantities showed strong cross-environment consistency, with genotype rankings largely conserved between sites (Spearman ρ = 0.82). At the Glenlea site, qPCR-derived fungal load was strongly associated with visual Verticillium stripe severity (Spearman ρ = 0.68; R² ≈ 0.44; Kruskal-Wallis P = 1.59 × 10⁻¹⁷), indicating that hypocotyl colonization measured by qPCR captures biologically meaningful variation in disease expression. This scalable qPCR-based platform provides a repeatable, high-resolution phenotype for large-scale germplasm screening and future genetic analyses aimed at improving Verticillium stripe resistance in canola.