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.

G. barbadense represents an indispensable germplasm resource for high-quality textile fiber and disease resistance; nevertheless, systematic information regarding its ZRT/IRT-like protein (ZIP) gene family remains limited. Here, a total of 46 GbZIP genes were identified across the G. barbadense genome. Comprehensive bioinformatic investigations revealed uneven chromosomal distribution and confirmed that segmental/whole-genome duplications, supplemented by localized tandem duplications, drove family expansion. Members clustered within the same phylogenetic clades shared conserved motif organization and gene architecture, while promoter regions harbored abundant cis-acting elements associated with phytohormone and stress signaling. Transcriptome profiling indicated distinct expression patterns across vegetative/reproductive tissues, fiber and ovule developmental stages, and diverse abiotic stress conditions (cold, hot, drought, and salt). Quantitative Real-Time PCR (qRT-PCR) further validated that several GbZIP candidates exhibited temporal expression variations upon exposure to cadmium toxicity, V. dahliae infection, and combined Cd-V. dahliae stress. Specifically, GbZIP13, GbZIP18, GbZIP27, and GbZIP36 displayed prominent broad-spectrum responses to all three stress conditions, whereas GbZIP16, GbZIP29, and GbZIP30 showed stress-specific regulatory divergence. Overall, this study aims to systematically analyze the evolutionary characteristics and expression patterns of the GbZIP family, and to specifically evaluate the response differences under Cd stress, V. dahliae stress, and combined stress, in order to identify potential key candidate genes.

The translocase of the inner membrane (TIM) family plays an essential role in mediating the transport of nuclear-encoded precursor proteins across the inner mitochondrial membrane and regulating cellular energy metabolism. Sea-island cotton (Gossypium barbadense) is valued for its superior fiber quality, yet the composition, evolution, and expression patterns of its TIM genes remain unclear. To address this, we performed a genome-wide analysis of the TIM family in G. barbadense, using the diploid cotton G. arboreum, and G. raimondii and the allotetraploid G. hirsutum as comparative references. A total of 44 GbTIM genes were identified in G. barbadense and characterized through phylogenetic, structural, collinearity, promoter, and expression analyses. The TIM family expanded during cotton polyploidization, with tetraploid species containing approximately twice as many genes as diploids. Phylogenetic analysis categorized TIM proteins into seven subfamilies, with Group VI representing the largest clade. Promoter regions were enriched in light- and phytohormone-responsive elements. Interspecific synteny analysis demonstrated extensive collinearity between G. barbadense and its diploid ancestors but limited collinearity with G. hirsutum, indicating divergence among tetraploids. Transcriptomic profiling revealed highly spatiotemporal expression patterns. Gbar_A11G001280 was abundantly expressed during fiber initiation and upregulated in low-lint-percentage germplasm, whereas Gbar_D07G010770 was continuously expressed throughout fiber development. This study clarifies the evolutionary lineage of the TIM family across four cotton species, with a primary focus on G. barbadense, and provides candidate genes for molecular breeding of high-quality fiber. Functional validation of these candidate genes is recommended in future studies.

Understanding the commonalities and regional divergence of lint yield-contributing traits is vital for cotton (Gossypium hirsutum L.) breeding. Using 1954 cultivar records (1773 unique cultivar names) approved in China between 2001 and 2025 across three macro-regions, this study applied a multi-method consensus framework combining false discovery rate (FDR)-adjusted association tests, Random Forest (RF), permutation importance, and Boruta feature selection, supported by dual-track resampling stability analysis; SHapley Additive exPlanations (SHAP) was used to describe effect direction, response shapes, and trait interactions. Under cultivar-grouped cross-validation, RF explained more variance than a Ridge baseline (R² = 0.52–0.64). Lint percentage, boll weight, and plant height were Tier 1 drivers in all three regions, although plant height was the least stable in the Yangtze River Basin. Regional divergence was evident: lint percentage dominated in the Northwest Inland region, boll number per plant in the Yangtze River Basin, and a balanced multi-trait structure, including growth duration as a negative factor, in the Yellow River Basin. Formal comparison of response shapes classified 15 of 18 trait–region responses as smooth transition zones rather than sharp thresholds (lint percentage reference range 40.5–42%; boll weight 5.7–6.1 g). These exploratory associations derive from observational registration-trial records. Because the consensus criteria are not statistically independent and no independent or temporal validation was performed, tier assignments are descriptive and the reference values provide hypotheses for regional ideotype breeding that require validation in independent trials.