Natural variation of response to glyphosate in <scp> <i>Sinapis alba</i> </scp> (Brassicaceae): Physiological effects, shikimate accumulation and genetic mechanisms

Abstract Glyphosate is the most widely used herbicide worldwide, and glyphosate‐resistant weeds have increased globally. Resistance mechanisms, including target‐site resistance (TSR) and non‐target‐site resistance (NTSR), are well documented, but their extent and importance are poorly understood among biotypes. This study examines the natural variation in glyphosate sensitivity and its genetic basis in white mustard ( Sinapis alba L.) across 192 accessions in Southern Spain. We conducted a multi‐population screening to evaluate plant performance, shikimate accumulation, survival and yield under glyphosate exposure and analysed the spatial response pattern. We also assessed the expression of two genes linked to glyphosate resistance, EPSPS and ABCC8 , sequenced EPSPS to detect potential mutations and quantified its copy number. Results show glyphosate significantly reduces physiological parameters, with a 90% decrease in photosynthetic rate, a 22% reduction in chlorophyll concentration and a 38% decrease in specific leaf area. However, 32% of accessions survived and produced seeds under glyphosate exposure (1598 g.a.e. ha −1 ) in the vegetative stage. Survivors accumulated 13% less shikimate, indicating a reduced glyphosate effect on the shikimate pathway. Overexpression of the NTS ABCC8 gene was the most common mechanism in 39% of non‐susceptible accessions. Yet, about 20% of survivors were not linked to EPSPS duplication or overexpression of EPSPS or ABCC8 , suggesting other genetic mechanisms may contribute to glyphosate resistance in S. alba . Resistant phenotypes were widely dispersed, not confined to specific areas, highlighting the potential role of gene flow in the evolution of herbicide response in S. alba . Our study suggests that the plant resistance to glyphosate may arise from multiple and non‐exclusive genetic mechanisms.