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.

Currently, developing cotton cultivation in saline–alkali soils is a vital focus for restructuring the cotton industry in China. The seedling stage, specifically the three-leaf stage, is a crucial period for assessing the salt tolerance of cotton. This research examined 430 natural populations of upland cotton, including 45 semi-wild germlines of Gossypium purpurascens. We measured the phenotypic responses of salt stress injury on seedlings as well as potassium (K), calcium (Ca), sodium (Na), and magnesium (Mg) concentrations in the roots, stems, and leaves following a 72 h exposure. The comprehensive salt tolerance index (CSTI) was determined using a membership function, principal component analysis, and cluster analysis based on 48 phenotypic traits related to salt tolerance. The results revealed significant variations in the phenotypic traits of the ion group under salt stress. Salt stress greatly affected the relative contents of Mg, K, and Ca ions in the aboveground parts of cotton, and correlations were observed among the 48 indices. The CSTI was calculated using seven principal component indexes, identifying 30 salt-tolerant, 114 weakly salt-tolerant, 39 salt-sensitive, and 4 highly sensitive materials based on cluster analysis. Among the 45 G. purpurascens cotton resources, 28 were weakly salt-tolerant, while 17 were salt-sensitive. Through TMT (Tandem Mass Tag)-based quantitative analysis, we identified 3107 unique peptides among 28,642 detected peptides, resulting in 203,869 secondary mass spectra, with 50,039 spectra successfully matched to peptides. Additionally, we identified several salt tolerance-related pathways (carbon metabolism; glutathione metabolism; the biosynthesis of amino acids, etc.) and proteins classified within the CAZy (Carbohydrate-Active EnZYme) family and expansin proteins. The results of this study concerning salt-tolerant materials provide a crucial theoretical foundation for the identification and evaluation of salt-tolerant breeding parents in cultivated cotton.