Tetraploid cultivated cotton (Gossypium spp.) produces cottonseeds rich in protein and oil. Gossypol and related terpenoids, stored in the pigment glands of cottonseeds, are toxic to human beings and monogastric animals. However, a comprehensive understanding of the genetic basis of gossypol and gland formation is still lacking. We performed a comprehensive transcriptome analysis of four glanded versus two glandless tetraploid cultivars distributed in Gossypium hirsutum and Gossypium barbadense. A weighted gene co-expression network analysis (WGCNA) based on 431 common differentially expressed genes (DEGs) uncovered a candidate module that was strongly associated with the reduction in or disappearance of gossypol and pigment glands. Further, the co-expression network helped us to focus on 29 hub genes, which played key roles in the regulation of related genes in the candidate module. The present study contributes to our understanding of the genetic basis of gossypol and gland formation and serves as a rich potential source for breeding cotton cultivars with gossypol-rich plants and gossypol-free cottonseed, which is beneficial for improving food safety, environmental protection, and economic gains of tetraploid cultivated cotton.

Verticillium wilt, caused by Verticillium dahliae, is a major constraint on cotton production. The GASA (Gibberellic Acid-Stimulated in Arabidopsis) proteins are cysteine-rich peptides induced by gibberellin signaling; however, their systemic role in upland cotton (Gossypium hirsutum) resistance to Verticillium wilt remains elusive. Here, we identified 40 GhGASA members from the G. hirsutum genome, which were clustered into three subfamilies. All members possessed a typical gibberellin-regulated domain, and segmental duplication was the primary driver of family expansion. Transcriptomic analyses revealed tissue-specific expression, with high abundance in pistils and roots, and distinct responsive patterns to salt, drought, and cold stresses. Quantitative real-time PCR (qRT-PCR) showed that multiple GhGASA genes were significantly upregulated following V. dahliae inoculation, with transcript levels being much higher in roots and stems than in leaves. Notably, GhGASA17, harboring ethylene-responsive elements, was identified as a key candidate involved in the defense response. Overall, this study provides a comprehensive characterization of the GhGASA gene family, offering a theoretical foundation for understanding its regulatory functions in Verticillium wilt resistance and delivering potential genetic resources for molecular breeding in cotton.

In this study, we performed the first genome-wide identification and characterization of the cinnamoyl-CoA reductase (CCR) gene family in upland cotton (Gossypium hirsutum), focusing on its potential association with stem strength. We identified 76 GhCCR genes and classified them into four subfamilies. We then analyzed their evolutionary relationships, conserved domains, synteny, promoter cis-elements, and expression patterns. All GhCCR proteins possess the NADB_Rossmann superfamily domain, and family expansion appears to have been driven mainly by segmental and tandem duplications. A small number of GhCCR genes showed relatively high expression in leaf, pistil, and torus tissues, while genes such as GhCCR3/9/10 exhibited elevated transcript levels under abiotic stress conditions. RT-qPCR results indicated that three candidate GhCCR genes (GhCCR25, GhCCR52 and GhCCR64) were significantly more highly expressed in multiple tissues of the stiff-stem line JY-25 than in the soft-stem line JR-15. Together, these findings suggest that GhCCR genes may contribute to the regulation of growth, development, and stress adaptation in G. hirsutum. However, direct biochemical or genetic validation is required to confirm their functional roles in lignin biosynthesis and stem rigidity.

The S-locus-specific glycoprotein/EP1 gene family encodes a class of epidermis-specific secretory glycoproteins involved in plant responses to abiotic stress. However, their role in cotton (Gossypium hirsutum) drought response remains unclear. Here, we identified 12 GhEP1 genes from the G. hirsutum genome, clustered into three subfamilies. Structural predictions indicated that most GhEP1 proteins are hydrophilic and localised to the outer side of the plasma membrane or nucleus, with a few containing transmembrane domains. Collinearity analysis revealed that segmental and tandem duplications drove family expansion, with a high degree of collinearity conservation among dicotyledonous plants. Transcriptomic data showed generally low GhEP1 expression across tissues, but GhEP1-2, GhEP1-6, GhEP1-7 and GhEP1-8 responded significantly to NaCl, PEG-induced drought and cold stresses. qRT-PCR further confirmed that the expression of most GhEP1 genes was significantly up-regulated in roots under drought stress, while GhEP1-3 was induced in leaves, indicating tissue-specific regulatory divergence. This study systematically characterises the GhEP1 family and provides potential genetic resources for improving drought tolerance in cotton.

The simultaneous improvement of fiber strength (FS) and lint percentage (LP) is a critical objective for achieving high-quality and high-yield cotton production. Identifying key genes and their regulatory networks that govern the synergistic development of FS and LP is essential for achieving their simultaneous improvement. In our previous study, a stable chromosome segment, Seg-D06-2, was identified for its ability to concurrently enhance both FS and LP with high reliability. In the present study, homozygous individuals harboring the Seg-D06-2 segment within a nearly uniform genetic background were selected to construct a large BC₆F₂ chromosome segment substitution line (CSSL) population comprising 3324 individuals. Extreme individuals characterized by simultaneous improvement in FS and LP, which shared similar genetic and phenotypic backgrounds, were subjected to comparative transcriptomic and weighted gene co-expression network analysis (WGCNA) at 0, 5, 10, 15, 20, and 25 days post-anthesis (DPA). The results highlighted the ’blue’ and ’yellow’ modules as being significantly associated with the simultaneous improvement of FS and LP. Four hub genes (GH_D06G0542, GH_D06G1609, GH_D06G0627 and GH_D06G2689) and two DEGs (GH_D06G0564 and GH_D06G0723) were identified in the ’blue’ module. Three hub genes (GH_D06G0540, GH_D06G0558 and GH_D06G0636) and one DEG (GH_D06G0527) were identified in the ’yellow’ module. These 10 key genes likely play pivotal roles in regulating the synergistic development of FS and LP, warranting further investigation. The reliability of the RNA-seq data was confirmed by qRT-PCR. This study provides a valuable resource for molecular breeding aimed at the simultaneous improvement of FS and LP and offers new insights into the molecular mechanisms governing their synergistic development.