Fusarium wilt resistance of Gossypium barbadense is very important to maintain its yield and quality, and to disease resistance breeding. Although many individual genes, which are quantitative trait loci associated with wilt resistance have been identified, knowledge of genes controlling wilt resistance in G. barbadense is still limited. In order to screen the InDel fragment related to Fusarium wilt resistance in G. barbadense, a genome‐wide association study was conducted using 110 recombinant inbred lines of Xinhai 14 (susceptible cotton) and 06–146 (resistant cotton). In this study, 207,040 high‐quality InDel loci were identified, of which 595 and 632 InDels were significantly associated (p < 1 × 10⁻³) with wilt resistance in G. barbadense in the additive and dominant effect module analyses, respectively. Combined transcriptome expression analysis within the FOV7 stably inherited qFOV7‐D03‐1 interval identified three ≥2 bp InDels for two differentially expressed genes. qPCR analysis was used to further validate that the expression of GB_D03G0204 and GB_D03G0238 was significantly different in the parental, resistant, and high susceptibility varieties. The GB_D03G0238 gene InDel was significant in both additive and dominant effect models, and the GB_D03G0204 gene InDel was significantly associated with wilt resistance in G. barbadense in the dominant effect model. The InDel fragments related to wilt resistance in G. barbadense discovered in this study can help gain insights into the genetic basis of wilt resistance and improve cotton breeding with excellent wilt resistance and high fiber quality traits.

Cotton, a globally vital cash crop, is severely constrained by V. dahliae. Lignin, a core structural component of plant cell walls, plays a crucial role in physical defense, with its biosynthesis regulated by hydroxycinnamoyltransferase (HCT)—a key enzyme in the phenylpropanoid pathway. However, the HCT gene family in upland cotton (Gossypium hirsutum) and its role in resistance to V. dahliae remain poorly understood. In this study, we performed a genome-wide identification of the HCT gene family in G. hirsutum, identifying 74 GhHCT genes that were classified into five evolutionary subfamilies. Bioinformatics analysis revealed that GhHCT proteins exhibit conserved functional domains but diverse gene structures, with promoter regions enriched in hormone-responsive and stress-responsive cis-acting elements. Expression profiling revealed that multiple GhHCT genes were significantly induced in response to V. dahliae infection. Three genes, GhHCT2, GhHCT35, and GhHCT47, showed significantly higher expression levels in resistant cultivars than in susceptible cultivars during early infection stages, suggesting pivotal roles in defense. These three candidate genes, which contain MeJA/SA-responsive elements in their promoters, may enhance resistance by regulating lignin synthesis to strengthen the cell wall barrier. In summary, this study provides the first comprehensive characterization of the HCT gene family in upland cotton. It identifies key candidates for improving resistance to V. dahliae, offering valuable genetic resources for molecular breeding.

DUSPs, a diverse group of protein phosphatases, play a pivotal role in orchestrating cellular growth and development through intricate signaling pathways. Notably, they actively participate in the MAPK pathway, which governs crucial aspects of plant physiology, including growth regulation, disease resistance, pest resistance, and stress response. DUSP is a key enzyme, and it is the enzyme that limits the rate of cell metabolism. At present, complete understanding of the DUSP gene family in cotton and its specific roles in resistance to Verticillium wilt (VW) remains elusive. To address this knowledge gap, we conducted a comprehensive identification and analysis of four key cotton species: Gossypium arboreum, Gossypium barbadense, Gossypium hirsutum, and Gossypium raimondii. The results revealed the identification of a total of 120 DUSP genes in the four cotton varieties, which were categorized into six subgroups and randomly distributed at both ends of 26 chromosomes, predominantly localized within the nucleus. Our analysis demonstrated that closely related DUSP genes exhibited similarities in terms of the conserved motif composition and gene structure. A promoter analysis performed on the GhDUSP gene promoter revealed the presence of several cis-acting elements, which are associated with abiotic and biotic stress responses, as well as hormone signaling. A tissue expression pattern analysis demonstrated significant variations in GhDUSP gene expression under different stress conditions, with roots exhibiting the highest levels, followed by stems and leaves. In terms of tissue-specific detection, petals, leaves, stems, stamens, and receptacles exhibited higher expression levels of the GhDUSP gene. The gene expression analysis results for GhDUSPs under stress suggest that DUSP genes may have a crucial role in the cotton response to stress in cotton. Through Virus-Induced Gene Silencing (VIGS) experiments, the silencing of the target gene significantly reduced the resistance efficiency of disease-resistant varieties against Verticillium wilt (VW). Consequently, we conclude that GH_A11G3500-mediated bispecific phosphorylated genes may serve as key regulators in the resistance of G. hirsutum to Verticillium wilt (VW). This study presents a comprehensive structure designed to provide an in-depth understanding of the potential biological functions of cotton, providing a strong foundation for further research into molecular breeding and resistance to plant pathogens.

Mitogen-activated protein kinase kinase kinases (MAPKKKs) are pivotal upstream regulators of MAPK cascades, integrating signals that coordinate plant development and stress responses. However, the specific functions of MAPKKKs, particularly within the MEKK subfamily, in mediating cotton resistance to Verticillium wilt and Fusarium wilt remain poorly characterized. To address this, we conducted a systematic, cross-species analysis of the MAPKKK family in four key cotton species: Gossypium arboreum, Gossypium barbadense, Gossypium hirsutum, and Gossypium raimondii. Genome-wide identification and phylogenetic analysis revealed 660 MAPKKK genes, classifying them into the MEKK, Raf, and ZIK subfamilies. Evolutionary analysis indicated that Whole-Genome Duplication (WGD) events were the primary driver of family expansion. Promoter cis-element and Gene Ontology (GO) enrichment analyses implicated these genes in hormone signaling and stress adaptation. Expression profiling demonstrated functional modularity, with distinct members responding specifically to cold stress or cooperatively to drought and salt stresses. Upon pathogen infection, members diverged into regulatory modules associated with immune homeostasis, tissue-specific defense, and core signaling potentially governing systemic acquired resistance (SAR). The temporal expression patterns of core candidate genes were validated by qRT-PCR. This study provides, for the first time, a comprehensive evolutionary and functional framework for the MEKK subfamily within the cotton MAPKKK family. It reveals the conserved and divergent roles of this subfamily in stress adaptation and identifies key candidate genes for breeding disease-resistant cotton varieties.

Cotton, as an economically important crop, suffers significant yield losses from infection by Fusarium oxysporum f. sp. vasinfectum race 7 (Fov7) infection. To elucidate the molecular mechanisms underlying host responses to Fov7 infection, we conducted a comprehensive transcriptional analysis of Gossypium barbadense using integrated bulk RNA sequencing (RNA-seq) and single-nucleus RNA sequencing (snRNA-seq) approaches. Our time-course study encompassed four critical infection stages (0, 2, 12, and 48 h post-inoculation) to characterize spatiotemporal transcriptomic dynamics. Bulk RNA-seq analysis revealed that the number of differentially expressed gene (DEG) numbers peaked at 2 h post-inoculation (hpi), identifying 9230 DEGs and demonstrating substantial transcriptional activation during early pathogen challenge. K-means clustering classified DEGs into six temporal expression patterns, with cluster C4 showing significant enrichment of defense-related pathways. Weighted gene co-expression network analysis (WGCNA) identified ten distinct co-expression modules, among which the turquoise module exhibited strong correlation with early infection responses. Integration of K-means clustering and WGCNA results pinpointed 1503 key defense-related genes. A single-nucleus atlas of cotton roots during Fov7 infection was constructed, comprising 32,773 nuclei, which revealed the cellular heterogeneity of cotton root tips. Functional enrichment analysis using these key genes emphasized the crucial roles of root cap and epidermal cells in pathogen perception and defense activation. Pseudotime trajectory mapping further delineated transcriptional dynamics in these cell populations during infection progression, demonstrating pathway activation in the root cap and epidermis at later infection stages. This multi-omics study establishes a valuable transcriptional resource for dissecting cotton’s resistance mechanisms against Fov7 and provides theoretical insights for developing disease-resistant cultivars through molecular breeding strategies.