Background Salinity is a major abiotic stress seriously hindering crop yield. Development and utilization of tolerant varieties is the most economical way to address soil salinity. Upland cotton is a major fiber crop and pioneer plant on saline soil and thus its genetic architecture underlying salt tolerance should be extensively explored. Results In this study, genome-wide association analysis and RNA sequencing were employed to detect salt-tolerant qualitative-trait loci (QTLs) and candidate genes in 196 upland cotton genotypes at the germination stage. Using comprehensive evaluation values of salt tolerance in four environments, we identified 33 significant single-nucleotide polymorphisms (SNPs), including 17 and 7 SNPs under at least two and four environments, respectively. The 17 stable SNPs were located within or near 98 candidate genes in 13 QTLs, including 35 genes that were functionally annotated to be involved in salt stress responses. RNA-seq analysis indicated that among the 98 candidate genes, 13 were stably differentially expressed. Furthermore, 12 of the 13 candidate genes were verified by qRT-PCR. RNA-seq analysis detected 6640, 3878, and 6462 differentially expressed genes at three sampling time points, of which 869 were shared. Conclusions These results, including the elite cotton accessions with accurate salt tolerance evaluation, the significant SNP markers, the candidate genes, and the salt-tolerant pathways, could improve our understanding of the molecular regulatory mechanisms under salt stress tolerance and genetic manipulation for cotton improvement.

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