Erratic cold spells and soil water deficits increasingly threaten barley productivity. Although model systems suggest that prior stress can confer heritable “stress memory,” its mechanistic basis in cereals remains unresolved. We tracked three successive generations of 138 global accessions under factorial cold × drought versus well-watered controls. Antioxidant enzymes, redox metabolites, and six yield components were integrated into generation-specific stress tolerance indices (STIs). Genome-wide association revealed robust transgenerational priming: lines repeatedly exposed to combined stress mounted 70–90% stronger antioxidant responses. Thirty-three significant SNPs formed five linkage disequilibrium blocks on chromosomes 2H, 3H, 4H, and 5H, explaining 58% of antioxidant STI and 46% of grain number variation. Candidate genes comprise a NAC transcription factor, an AUX/LAX auxin influx carrier, a β-glucosidase, and an F-box/WD40 protein, highlighting a conserved network linking redox homeostasis, hormone signaling, and yield. At the transcript level, combined drought–cold elicited persistent shifts: HvPRX40 and HvBGLU12 were induced, peaking at 12–24 h and remaining elevated to 48 h; the SCF/ubiquitin component HvFBXWD40a was upregulated near 12 h; the auxin carrier HvAUX1 was down-regulated at 6–12 h, with partial restoration by 24–48 h. Together, these patterns support a model in which early reactive oxygen species detoxification and ABA-coupled metabolic mobilization are amplified and prolonged under combined stress, whereas auxin import is briefly curtailed before homeostatic recovery. Our findings show that durable cold–drought tolerance in barley is driven by multigenerational antioxidant priming and delineate marker-rich genomic regions to accelerate climate-resilient breeding via marker-assisted selection or gene editing. • Multi-generational cold×drought exposure induces durable antioxidant priming across three generations. • Five linkage disequilibrium (LD) blocks on 2H, 3H, 4H and 5H integrate redox homeostasis with spike development and seed-setting. • Candidate genes ( HvPRX40, HvFBXWD40a, HvAUX1, HvBGLU12 ) implicate hormone–ROS crosstalk in stress memory. • Marker-rich hotspots provide targets for marker-assisted selection and precision gene editing for climate resilience.
Safhi et al. (Wed,) studied this question.