Fuzzless Gossypium hirsutum mutants are ideal materials for investigating cotton fiber initiation and development. In this study, we used the fuzzless G. hirsutum mutant Xinluzao 50 FLM as the research material and combined it with other fuzzless materials for verification by RNA sequencing to explore the gene expression patterns and differences between genes in upland cotton during the fuzz period. A gene ontology (GO) enrichment analysis showed that differentially expressed genes (DEGs) were mainly enriched in the metabolic process, microtubule binding, and other pathways. A weighted gene co-expression network analysis (WGCNA) showed that two modules of Xinluzao 50 and Xinluzao 50 FLM and four modules of CSS386 and Sicala V-2 were highly correlated with fuzz. We selected the hub gene with the highest KME value among the six modules and constructed an interaction network. In addition, we selected some genes with high KME values from the six modules that were highly associated with fuzz in the four materials and found 19 common differential genes produced by the four materials. These 19 genes are likely involved in the formation of fuzz in upland cotton. Several hub genes belong to the arabinogalactan protein and GDSL lipase, which play important roles in fiber development. According to the differences in expression level, 4 genes were selected from the 19 genes and tested for their expression level in some fuzzless materials. The modules, hub genes, and common genes identified in this study can provide new insights into the formation of fiber and fuzz, and provide a reference for molecular design breeding for the genetic improvement of cotton fiber.

Sulfotransferases (SOTs) (EC 2.8.2.-) are sulfate regulatory proteins in a variety of organisms that have been previously shown to be involved in regulating a variety of physiological and biological processes, such as growth, development, adaptation to land, stomatal closure, drought tolerance, and response to pathogen infection. However, there is a lack of comprehensive identification and systematic analysis of SOT in cotton, especially in G. barbadense. In this study, we used bioinformatics methods to analyze the structural characteristics, phylogenetic relationships, gene structure, expression patterns, evolutionary relationships, selection pressure and stress response of SOT gene family members in G. barbadense. In this study, a total of 241 SOT genes were identified in four cotton species, among which 74 SOT gene members were found in G. barbadense. According to the phylogenetic tree, 241 SOT protein sequences were divided into five distinct subfamilies. We also mapped the physical locations of these genes on chromosomes and visualized the structural information of SOT genes in G. barbadense. We also predicted the cis-acting elements of the SOT gene in G. barbadense, and we identified the repetitive types and collinearity analysis of SOT genes in four cotton species. We calculated the Ka/Ks ratio between homologous gene pairs to elucidate the selective pressure between SOT genes. Transcriptome data were used to explore the expression patterns of SOT genes, and then qRT-PCR was used to detect the expression patterns of GBSOT4, GBSOT17 and GBSOT33 under FOV stress. WGCNA (weighted gene co-expression network analysis) showed that GB_A01G0479 (GBSOT4) belonged to the MEblue module, which may regulate the resistance mechanism of G. barbadense to FOV through plant hormones, signal transduction and glutathione metabolism. In addition, we conducted a VIGS (virus-induced gene silencing) experiment on GBSOT4, and the results showed that after FOV inoculation, the plants with a silenced target gene had more serious leaf wilting, drying and cracking than the control group, and the disease index of the plants with the silenced target gene was significantly higher than that of the control group. This suggests that GBSOT4 may be involved in protecting the production of G. barbadense from FOV infection. Subsequent metabolomics analysis showed that some flavonoid metabolites, such as Eupatorin-5-methylether (3′-hydroxy-5,6,7,4′-tetramethoxyflavone, were accumulated in cotton plants in response to FOV infection.

Duplication events occur very frequently during plant evolution. The genes in the duplicated pathway or network can evolve new functions through neofunctionalization and subfunctionalization. Flavonoids are secondary metabolites involved in plant development and defense. Our previous transcriptomic analysis of F6 recombinant inbred lines (RILs) and the parent lines after Fusarium oxysporum f. sp. vasinfectum (Fov) infection showed that CHI genes have important functions in cotton. However, there are few reports on the possible neofunctionalization differences of CHI family paralogous genes involved in Fusarium wilt resistance in cotton. In this study, the resistance to Fusarium wilt, expression of metabolic pathway-related genes, metabolite content, endogenous hormone content, reactive oxygen species (ROS) content and subcellular localization of four paralogous CHI family genes in cotton were investigated. The results show that the four paralogous CHI family genes may play a synergistic role in Fusarium wilt resistance. These results revealed a genetic channelization mechanism that can regulate the metabolic flux homeostasis of flavonoids under the mediation of endogenous salicylic acid (SA) and methyl jasmonate (MeJA) via the four paralogous CHI genes, thereby achieving disease resistance. Our study provides a theoretical basis for studying the evolutionary patterns of homologous plant genes and using homologous genes for molecular breeding.

In this study, GH19 chitinase (Chi) gene family was systematically identified and characterized using genomic assemblies from four cotton species: Gossypium barbadense, G. hirsutum, G. arboreum, and G. raimondii. A suite of analyses was performed, including genome-wide gene identification, physicochemical property characterization of the encoded proteins, subcellular localization prediction, phylogenetic reconstruction, chromosomal mapping, promoter cis-element analysis, and comprehensive expression profiling using transcriptomic data and qRT-PCR (including tissue-specific expression, hormone treatments, and Fusarium oxysporum infection assays). A total of 107 GH19 genes were identified across the four species (35 in G. barbadense, 37 in G. hirsutum, 19 in G. arboreum, and 16 in G. raimondii). The molecular weights of GH19 proteins ranged from 9.9 to 97.3 kDa, and they were predominantly predicted to localize to the extracellular space. Phylogenetic analysis revealed three well-conserved clades within this family. In tetraploid cotton, GH19 genes were unevenly distributed across 12 chromosomes, often clustering in certain regions, whereas in diploid species, they were confined to five chromosomes. Promoter analysis indicated that GH19 gene promoters contain numerous stress- and hormone-responsive motifs, including those for abscisic acid (ABA), ethylene (ET), and gibberellin (GA), as well as abundant light-responsive elements. The expression patterns of GH19 genes were largely tissue-specific; for instance, GbChi23 was predominantly expressed in the calyx, whereas GbChi19/21/22 were primarily expressed in the roots and stems. Overall, this study provides the first comprehensive genomic and functional characterization of the GH19 family in G. barbadense, laying a foundation for understanding its role in disease resistance mechanisms and aiding in the identification of candidate genes to enhance plant defense against biotic stress.

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.

High-temperature (HT) stress has been recognized as one of the main factors restricting the normal growth and development of cotton and severely affects fiber quality and yield. To elucidate the regulatory mechanism of male sterility-related hormones in Gossypium barbadense under HT stress, we explored candidate genes closely related to male sterility in G. barbadense. We studied the expression profiles of hormones and genes in the anthers of G. barbadense GB150 under HT stress by combining transcriptomic and metabolomic analyses. Through a combined analysis of the transcriptional metabolism of the anthers of G. barbadense GB150, we determined the contents of ABA, JA, SA, IAA, tZR, and GA₂₀ and the expression of genes related to biosynthetic pathways and signal transduction pathways. The results revealed that the ABA and JA contents significantly increased after HT; the IAA, tZR, and GA₂₀ contents significantly decreased; and the SA content did not significantly change after HT. We then used weighted gene coexpression network analysis (WGCNA) to further analyze the interactions among hormones, transcription factors, and core genes and constructed hormone coexpression networks and genome-wide coexpression networks. Through these network analyses, we ultimately identified 10 candidate genes closely related to male sterility in G. barbadense. Using qRT-PCR, resequencing data from 221 G. barbadense materials revealed that ALA4 (Arabidopsis thaliana has been proven to be associated with male fertility) and SBP1 (two stop gains in the gene structure) may play important roles in the process of male sterility in G. barbadense. The results of this study provide a theoretical basis for the molecular mechanism of male sterility in G. barbadense.