Genetic dissection of inherited cold-stress reveals resilience hotspots in barley

Cold episodes during early crop development threaten cereal establishment and yield, but it remains unclear whether ancestral cold exposure can generate measurable inherited resilience in barley. We tested this question in a three-generation field experiment using 138 barley accessions and eight third-generation lineage-treatment combinations that separated no-memory, transgenerational, intergenerational and repeated cold-memory classes under control and recurrent cold-stress conditions. Cold stress was imposed by delayed sowing, and establishment, physiological, biochemical, growth, reproductive and yield traits were integrated with standardized cold-memory indices and genome-wide association mapping. Under third-generation cold stress, memory-conditioned lineages outperformed the no-memory cold-stressed lineage for several key traits: germination increased from 79.0% to 83.5-86.6%, seedling survival from 68.0% to 77.7-85.5%, electrolyte leakage declined from 51.5% to 42.1-34.3%, and grain yield per plant increased from 4.27 g to 5.04-5.75 g. The integrated cold resilience index also increased from 38.9 in the no-memory cold-stressed lineage to 49.8, 54.5 and 58.9 in the transgenerational, intergenerational and repeated-memory lineages, respectively. Trait-specific indices confirmed a consistent response hierarchy, with grain-yield memory reaching 26.3%, 36.7% and 44.0% in the same three memory classes. Correlation analysis resolved a stress-protection module centred on membrane stability, oxidative-damage protection and survival, and a productivity module centred on grain-yield memory, reproductive yield and benefit-cost ratio. GWAS using 17,894 quality-filtered SNPs detected 137 significant marker-index-memory associations, including major loci for germination memory on 5H (- log₁₀(P) = 7.58) and benefit-cost ratio on 3H and 6H (- log₁₀(P) = 7.37 and 6.89). Candidate-gene and expression analyses prioritized genes involved in trehalose and sucrose metabolism, receptor-like kinase signalling, ERF-mediated transcriptional regulation, auxin transport, lipid protection, solute transport and genome surveillance. We interpret these results as inherited cold-memory-associated phenotypes rather than direct proof of epigenetic inheritance. The study establishes cold memory as a quantifiable barley resilience trait and provides loci and candidate genes for future functional validation and marker-assisted breeding.