Integrated physiological, ionic, and transcriptomic screening over four years identifies highly salt tolerant barley genotypes for arid agroecosystems using field lysimeters

• Sixty-four barley genotypes were evaluated under four salinity levels (0, 8, 12, and 16 dS/m). • Eight promising lines were identified, with Lines 2 and 5 showing the highest salinity tolerance. • GGE biplot and PCA consistently highlighted Lines 2 and 5 as stable high-yielders under salinity stress. • Lines 2 and 5 maintained superior yield traits, a favorable K + /Na + ratio, and reduced ROS accumulation. • Transcriptomic analysis with SSR markers confirmed high genetic diversity, distinguishing tolerant lines. Soil salinization is accelerating across the world’s drylands, jeopardizing both crop production and the ecological services delivered by agroecosystems. This study aimed to identify the elite salt-tolerant barley genotypes and provide insights for breeding new barley varieties with high salt tolerance. Initially, 10 genotypes were selected from a pool of 64 based on their superior performance under multiple salinity stress conditions (0, 8, 12, and 16 dS/m) during the 2020/2021 and 2021/2022 growing seasons. These genotypes were further evaluated in soil pot experiments, incorporating physiological, molecular, and elemental analyses. Of them, Lines 2 and 5 demonstrated superior salt tolerance, exhibiting the highest geometric mean productivity (GMP), salt tolerance index (STI), and low-stress susceptibility index (SSI). These lines also exhibited lower reactive oxygen species (ROS) accumulation and better ion balance than sensitive varieties, such as Giza 129 and Giza 135, which showed a 29- and 33-fold increase in shoot Na + content, respectively. Genetic screening with 20 SSR markers identified 62 alleles, 61 of which were polymorphic. Transcriptomic analysis revealed that tolerant genotypes (Lines 2 and 5) exhibited broader stress-response mechanisms, with differential gene expression in stress-related, oxidative stress, and metabolic pathways. This study highlights the complexity of salt tolerance in barley and demonstrates the importance of integrating phenotypic, physiological, and molecular analyses to identify salt-tolerant genotypes. Lines 2 and 5 were the most salt-tolerant, making them valuable candidates for targeted breeding programs in saline environments. Deploying such genotypes can reduce the freshwater footprint of barley cultivation and help stabilize yields on marginal lands, thereby enhancing the resilience of arid agro-ecosystems threatened by ongoing salinity expansion.