Genome-wide association and functional studies reveal the genetic basis of salt tolerance in naked barley

• Whole-genome data from 158 naked barley genotypes are analysed. • Developed an integrated phenotyping pipeline for high-throughput identification of salt-tolerant naked barley. • GWAS of 158 resequenced genotypes identified 76 candidate genes linked to salt tolerance. • Novel functional genes HvCML16, HvBGL10 and HvWARKL2 affect ROS scavenging and regulate salt tolerance. • The whole-genome data and genetic targets will facilitate future breeding for salt-resilient barley. Developing salt-tolerant crops requires the identification of key genetic factors and regulatory networks to mitigate the threat of soil salinization to global food security. This study aimed to identify genotypes, quantitative trait loci (QTLs), and novel genes associated with salt tolerance during germination in naked barley. We performed whole genome resequencing of 158 naked barley genotypes, combined with salt tolerance evaluation, genome-wide association analysis study (GWAS), and functional assays. Resequencing generated over 131 million high-quality SNPs and 11.76 million InDels, predominantly within intergenic regions. GWAS was conducted using a mixed linear model (MLM), which identified 3 QTLs and 76 candidate genes associated with salt tolerance during germination and early seedling growth. RT-qPCR demonstrated that salt stress induced the upregulation of several genes in XN311 (salt-tolerant) including HvCML16 (calmodulin-like protein 16) and HvBGL10 (glucan endo -1,3-beta-glucosidase 10-like). In contrast, HvWAKL2 (wall-associated receptor kinase 2-like) was downregulated in leaves at 7 days post-germination under salt stress. Functional validation via virus-induced gene silencing demonstrated that knockdown of HvCML16 and HvBGL10 in XN311 significantly impaired plant growth and reduced ROS scavenging activity under salt stress, HvWAKL2 affected POD and CAT activities as well as chlorophyll, MDA and H 2 O 2 contents but had minimal effects on biomass accumulations, confirming their roles in salt tolerance. Our findings reveal coordinated regulatory modules governing salt responses in naked barley, with HvWAKL2, HvCML16 and HvBGL10 emerging as promising genetic targets for enhancing salt resilience, providing valuable genetic insights and resources for breeding salt-tolerant naked barley.