Abstract
Cold stress severely restricts the winter survival and grain yield of barley (Hordeum vulgare L.) in the Eurasian steppe and continental regions. To unravel the genetic architecture governing seedling cold tolerance, a genome-wide association study (GWAS) was conducted using a diverse panel of 215 Eurasian winter barley accessions. Plants were evaluated under controlled freezing stress (-8 °C) at the three-leaf stage for electrolyte leakage (EL), seedling survival rate (SSR), and maximum quantum efficiency of PSII (Fv/Fm). High-density genotyping using a 50k SNP array yielded 34,120 polymorphic markers for association analysis via a mixed linear model (MLM) accounting for population structure and kinship. A total of 14 significant single nucleotide polymorphisms (SNPs) associated with cold tolerance traits were identified across chromosomes 2H, 4H, 5H, and 7H at a threshold of -log10(p) > 4.5. Notably, major QTL clusters on chromosome 5H overlapped with the well-known Frost Resistance loci (Fr-H1 and Fr-H2), confirming their pivotal role in winter survival. Furthermore, two novel loci on chromosomes 4H (peak marker SNP_4H_321094) and 7H (peak marker SNP_7H_883210) were strongly associated with reduced electrolyte leakage and sustained PSII efficiency. Candidate gene annotation within 100 kb genomic windows revealed genes encoding C-repeat binding factors (CBF), dehydrins, and a RING-type E3 ubiquitin ligase. These findings provide candidate genomic regions and breeder-friendly SNP markers to enhance marker-assisted selection for freezing tolerance in Eurasian barley breeding programs.
