Decoding the Genetic Basis of Salinity Tolerance at Germination and Seedling Traits in HEB-25 Barley NAM Population

Climate change is intensifying soil salinization, posing a major threat to crop establishment and productivity, particularly in arid and semi-arid regions. Barley (Hordeum vulgare L.), one of the most salt-tolerant cereals, offers valuable genetic resources for improving salinity resilience at early growth stages. This study exploited the genetic diversity of the Nested Association Mapping (NAM) population Halle Exotic Barley-25 (HEB-25) to dissect salinity tolerance during germination and seedling developmental stages. First, the HEB-25 parental lines (25 wild barley genotypes and cv. Barke) were evaluated under salinity treatment to identify contrasting responses. Based on this screening, four HEB families (01, 04, 09, and 22) were selected out of 25 HEB families for detailed phenotypic and genomic analysis. Seeds of the selected HEB families were subjected to 40% seawater salinity stress and control treatments to assess germination percentage and seedling traits, including shoot length, root length, fresh weight (FW), dry weight (DW), DW/FW ratio, root-shoot ratio, and salt tolerance index (STI). Substantial variation was observed among families for all measured traits under salinity stress. STI values enabled clear differentiation among families: Family 01 exhibited the most consistent overall tolerance profile, Family 22 showed the strongest sensitivity in biomass traits, and Family 04 displayed a trait-specific response with sensitivity at the family-mean level but exceptional within-family diversity, harboring some of the highest individual TI values across the population. A genome-wide association study was conducted using 32,995 SNP markers. A total of 27 significant SNPs were identified, corresponding to 20 quantitative trait loci (QTLs). Of these, 12 QTLs were detected under control conditions, 16 under seawater treatment, and 21 based on tolerance indices, indicating both constitutive and stress-responsive genetic effects. Gene annotation within these regions revealed approximately 23 candidate genes associated with abiotic stress tolerance, including genes involved in ion transport, osmotic adjustment, kinases and stress signaling pathways. HEB_22_003, HEB_04_087, and HEB_01_013 represent the most promising genotypes for salinity breeding. These findings highlight the effectiveness of combining precise phenotyping with high-resolution genomic analysis in the HEB-25 population to uncover the genetic architecture of salinity tolerance at early developmental stages. We identified 20 salinity-responsive QTLs, including five major-effect loci on chromosomes 2H, 4H, 5H, and 7H that consistently explained the largest share of phenotypic variation. These loci co-localized with candidate genes linked to ion homeostasis, Ca²⁺-mediated signaling, protein glycosylation, epigenetic regulation, and root system plasticity, revealing key mechanisms underlying early-stage salt adaptation in barley. The strong and contrasting responses of Family 01 and Family 04 provide an excellent genetic framework for functional validation of tolerance alleles. Collectively, these genomic resources establish a robust foundation for QTL pyramiding, marker-assisted breeding, and the development of climate-resilient barley cultivars for saline agroecosystems.