Elevated salinity significantly threatens cotton growth, particularly during the germination and seedling stages. The utilization of primitive species of Gossypium hirsutum, specifically Gossypium purpurascens, has the potential to facilitate the restoration of genetic diversity that has been depleted due to selective breeding in modern cultivars. This investigation evaluated 45 G. purpurascens varieties and a salt-tolerant cotton variety based on 34 morphological, physiological, and biochemical indicators and comprehensive salt tolerance index values. This study effectively identified a total of 19 salt-tolerant and two salt-resistant varieties. Furthermore, transcriptome sequencing of a salt-tolerant genotype (Nayanmian-2; NY2) and a salt-sensitive genotype (Sanshagaopao-2; GP2) revealed 2776, 6680, 4660, and 4174 differentially expressed genes (DEGs) under 0.5, 3, 12, and 24 h of salt stress. Gene ontology enrichment analysis indicated that the DEGs exhibited significant enrichment in biological processes like metabolic (GO:0008152) and cellular (GO:0009987) processes. MAPK signaling, plant-pathogen interaction, starch and sucrose metabolism, plant hormone signaling, photosynthesis, and fatty acid metabolism were identified as key KEGG pathways involved in salinity stress. Among the DEGs, including NAC, MYB, WRKY, ERF, bHLH, and bZIP, transcription factors, receptor-like kinases, and carbohydrate-active enzymes were crucial in salinity tolerance. Weighted gene co-expression network analysis (WGCNA) unveiled associations of salt-tolerant genotypes with flavonoid metabolism, carbon metabolism, and MAPK signaling pathways. Identifying nine hub genes (MYB4, MYB105, MYB36, bZIP19, bZIP43, FRS2 SMARCAL1, BBX21, F-box) across various intervals offered insights into the transcriptional regulation mechanism of salt tolerance in G. purpurascens. This study lays the groundwork for understanding the important pathways and gene networks in response to salt stress, thereby providing a foundation for enhancing salt tolerance in upland cotton.

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