Functional synergy and genomic linkage of glyphosate resistance traits in Canada fleabane

BACKGROUND Glyphosate resistance in Conyza canadensis (Canada fleabane) has been primarily attributed to non‐target‐site resistance (NTSR) mechanisms such as vacuolar sequestration, though these have not been formally elucidated. While a target‐site mutation at EPSPS2 (P106S) was recently identified, it failed to account for many resistant cases. These findings underscore the need to re‐evaluate the genetic basis of glyphosate resistance in this species. RESULTS Using an F 2 population derived from glyphosate‐resistant and susceptible biotypes, we disentangled the individual and combined effects of target‐site resistance (TSR) and NTSR. Dose–response phenotyping and genotyping revealed that NTSR conferred broad protection across a wide range of glyphosate doses, while TSR provided a more limited, dose‐dependent benefit. When both mechanisms were present, median lethal dose (LD 50 ) values greatly exceeded additive expectations, indicating a synergistic interaction. Quantitative trait locus (QTL) mapping identified a major‐effect locus associated with NTSR on chromosome 4, with candidate genes linked to membrane transport and subcellular compartmentalization processes. Segregation distortion and recombination frequency estimates suggest moderate genetic linkage between TSR and NTSR loci, facilitating co‐inheritance of resistance alleles. CONCLUSION This study provides the first explicit quantitative analysis of gene × gene interactions underlying herbicide resistance in C. canadensis . By disentangling TSR and NTSR, we show that single copies of the TSR and NTSR alleles confer approximately nine‐fold and seven‐fold glyphosate resistance, respectively. When combined, these mechanisms exhibit synergism, resulting in resistance levels that exceed additive LD 50 expectations by more than two‐fold. Both TSR and NTSR loci have been mapped to chromosome 4, and moderate genomic linkage (~27% recombination) between them will likely contribute to the persistence and spread of high‐level resistance, even under low selection pressure.