Genome-wide association study and functional validation of candidate genes conferring seedling-stage salt stress tolerance in upland cotton (Gossypium hirsutum)

Salt stress is one of the most critical abiotic constraints limiting cotton productivity, with particularly severe effects during germination and seedling establishment. Although cotton is commonly grown on saline–alkali soils and is generally regarded as moderately salt tolerant, the genetic and molecular mechanisms underlying its salt tolerance during the critical seedling stage remain incompletely characterized. In this research, we assessed 419 diverse upland cotton (Gossypium hirsutum L.) accessions from China and Pakistan for five key morphological traits under both control and salt-stress conditions. From these, 15 salt-tolerance indices were derived, including absolute differences and relative change rates. Genome-wide association analysis based on 47,235 high-quality SNP markers revealed 34 significant associations distributed across 10 chromosomes. Two stable signals, designated L2 on Chromosome 3 and L12 on Chromosome 16, were consistently detected across traits and considered priority regions for candidate gene identification. Functional annotation identified eight genes within these loci, including transcription factors, transporters, and enzymes. Expression profiling under salt stress revealed distinct and divergent expression patterns of candidate genes between salt-tolerant and salt-sensitive accessions, and four genes from the L2 locus, three GRAS transcription factors (Gohir.A03G043500, Gohir.A03G043600, Gohir.A03G043700) and one laccase (Gohir.A03G044300), were further validated by virus-induced gene silencing. Silencing each gene significantly reduced seedling salt tolerance, with the laccase gene showing the strongest phenotypic effect. This study provides new insights into the molecular regulation of seedling-stage salt tolerance in cotton. By integrating GWAS with functional validation, this study provides foundational insights and identifies preliminary genetic targets, thereby establishing a basis for the future development of salt-tolerant cotton varieties.