Background: DNA methylation is an important part of epigenetic regulation and plays an important role in the response of plants to adverse stress. Methods: In this study, whole-genome bisulfite sequencing (WGBS) was performed on the high-temperature-resistant material Xinluzao 36 and the high-temperature-sensitive material Che 61–72 at 0 h and 12 h under high-temperature stress conditions. Results: The results revealed that the Gossypium hirsutum methylation levels of CG and CHG (H = A, C, or T) decreased after the high-temperature stress treatment, and the methylation level of the A subgenome was significantly greater than that of the D subgenome. The methylation level of CHH increased, and the methylation level of CHH in the D subgenome was significantly greater than that in the A subgenome after high-temperature stress treatment. The methylation density of CG is lower than that of CHG and CHH, and the methylation density of the middle region of chromosomes is greater than that of both ends, which is opposite to the distribution density of genes. There were 124 common differentially methylated genes in the CG, CHG, and CHH groups, and 5130 common DEGs and differentially methylated genes were found via joint analysis with RNA-seq; these genes were significantly enriched in the biosynthesis of plant hormones, thiamine metabolism, glutathione metabolism, and tyrosine metabolism pathways. DNA methylation did not affect the expression of many genes (accounting for 85.68% of the differentially methylated genes), DNA methylation-promoted gene expression was located mainly in the downstream region of the gene or gene body, and the expression of inhibitory genes was located mainly in the upstream region of the gene. Conclusions: This study provides a theoretical basis for further exploration of the gene expression and functional regulatory mechanism of G. hirsutum DNA methylation under high-temperature stress conditions.

The occurrence and spread of Verticillium dahliae (V. dahliae) in cotton depends on the combined effects of pathogens, host plants, and the environment, among which temperature is one of the most important environmental factors. Studying how temperature impacts the occurrence of V. dahliae in cotton and the mechanisms governing host defense responses is crucial for disease prevention and control. Understanding the dual effects of temperature on both pathogens and hosts can provide valuable insights for developing effective strategies to manage this destructive fungal infection in cotton. This study was based on the deciduous V. dahliae Vd-3. Through cultivation at different temperatures, Vd-3 formed the most microsclerotia and had the largest colony diameter at 25 °C. Endospore toxins were extracted, and 48 h was determined to be the best pathogenic time point for endotoxins to infect cotton leaves through a chlorophyll fluorescence imaging system and phenotypic evaluation. Transcriptome sequencing was performed on cotton leaves infected with Vd-3 endotoxins for 48 h at different culture temperatures. A total of 34,955 differentially expressed genes (DEGs) were identified between each temperature and CK (no pathogen inoculation), including 17,422 common DEGs. The results of the enrichment analysis revealed that all the DEGs were involved mainly in photosynthesis and sugar metabolism. Among the 34,955 DEGs, genes in the biosynthesis and signal transduction pathways of jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) were identified, and their expression patterns were determined. A total of 5652 unique DEGs were clustered into six clusters using the k-means clustering algorithm, and the functions and main transcription factors (TFs) of each cluster were subsequently annotated. In addition, we constructed a gene regulatory network via weighted correlation network analysis (WGCNA) and identified twelve key genes related to cotton defense against V. dahliae at different temperatures, including four genes encoding transcription factors. These findings provide a theoretical foundation for investigating temperature regulation in V. dahliae infecting cotton and introduce novel genetic resources for enhancing resistance to this disease in cotton plants.

Background: In recent years, changes in climate conditions and long-term continuous cropping have led to the increased occurrence of Verticillium wilt in various cotton-growing regions, causing significant economic losses in cotton production. Research has shown that volatile substances are closely linked to plant disease resistance; however, studies on their roles in the response of cotton to Verticillium wilt, including their relationship with gene regulation, are limited. Methods: In this study, the transcriptomes and metabolomes of Xinluzao 57 (a highly susceptible Verticillium wilt variety) and 192,868 (a highly resistant Verticillium wilt variety) were sequenced at different time points after inoculation with Verticillium wilt. Results: A total of 21,911 commonly differentially expressed genes (DEGs) were identified within and between the materials, and they were clustered into eight groups. Significant annotations were made in pathways related to amino acids and anthocyanins. Metabolomics identified and annotated 26,200 volatile metabolites across nine categories. A total of 158 differentially accumulated metabolites (DAMs) were found within and between the materials; three clusters were identified, and the 10 metabolites with the most significant fold changes were highlighted. Weighted gene coexpression network analysis (WGCNA) revealed that 13 genes were significantly correlated with guanosine, 6 genes were correlated with 2-deoxyerythritol, and 32 genes were correlated with raffinose. Conclusions: Our results provide a foundation for understanding the role of volatile substances in the response of cotton to Verticillium wilt and offer new gene resources for future research on Verticillium wilt resistance.

Background: High-temperature stress is one of the major abiotic stresses limiting cotton production. Identifying genetic loci and genes for heat tolerance is crucial for breeding heat-tolerant varieties. Methods: Given the complexity of heat tolerance phenotypes in cotton, this study, which focused on resource materials, identified an A/C SNP mutation at position 5486185 on chromosome D06 within the heat tolerance interval through genome-wide association studies (GWAS) of natural Gossypium hirsutum populations. Results: A total of 308 resource materials were identified and evaluated for their heat tolerance phenotypes over two years of field research. Kompetitive allele-specific PCR (KASP) molecular markers were developed on the basis of the D06-5486185 SNP to characterize the heat tolerance phenotypes of these 308 resource materials. Genotyping for heat tolerance-related traits and agronomic traits was also performed. Materials with the C/C haplotype at position D06-5486185 presented increased heat tolerance (higher pollen viability (PV), leaf area (LA), chlorophyll (Chl) and number of bolls on the third fruit branch (FB3) and a lower number of dry buds (DBs) and drop rate (DR)) without negatively impacting key yield traits. This locus is located in the intergenic region of two adjacent bZIP transcription factor genes (GH_D06G0408 and GH_D06G0409). Expression analysis revealed that the expression levels of these two genes were significantly greater in heat-tolerant accessions (C/C type) than in sensitive accessions and that their expression levels were significantly correlated with multiple heat-tolerant phenotypes. Conclusions: In summary, this study developed a Kompetitive Allele Specific PCR (KASP) marker associated with heat tolerance in G. hirsutum and identified two key heat tolerance candidate genes. These results provide an efficient marker selection tool and important genetic resources for the molecular breeding of heat-tolerant G. hirsutum, laying an important foundation for further establishing a molecular marker-assisted breeding system for heat tolerance in G. hirsutum.

The root phenotype of cotton seedlings serves as a criterion for selecting high-vigour varieties, yet systematic evaluations of Upland Cotton germplasm resources remain limited. We measured nine root- and shoot-related traits in 204 Gossypium hirsutum accessions from diverse ecological regions. Correlation analysis revealed that root surface area, volume, and fresh weight were closely interrelated indicators. Principal component analysis extracted two biologically meaningful factors—‘absorptive capacity’ and ‘morphological strategy’—with a cumulative contribution of 55.72%. Cluster analysis classified the accessions into three functional groups: high-yield potential (n = 15), balanced adaptation (n = 135), and fine-root dominance (n = 54). These clusters corresponded with ecological origins and breeding periods, reflecting local adaptation. We propose root surface area, volume, and fresh weight as cost-effective screening indicators and identified 15 elite accessions as potential parents for root-centred breeding. These findings are particularly relevant for the arid production environments of Northwestern China.