Background: The B3-domain transcription factor ABI3 (ABSCISIC ACID INSENSITIVE 3) is a critical regulator of seed maturation, stress adaptation, and hormonal signaling in plants. However, its evolutionary dynamics and functional roles in cotton (Gossypium spp.) remain poorly characterized. Methods: We conducted a comprehensive genome-wide investigation of the ABI3 gene family across 26 plant species, with a focus on 8 Gossypium species. Analyses included phylogenetics, chromosomal localization, synteny assessment, gene duplication patterns, protein domain characterization, promoter cis-regulatory element identification, and tissue-specific/spatiotemporal expression profiling under different organizations of Gossypium hirsutum. Results: Phylogenetic and chromosomal analyses revealed conserved ABI3 evolutionary patterns between monocots and dicots, alongside lineage-specific expansion events within Gossypium spp. Syntenic relationships and duplication analysis in G. hirsutum (upland cotton) indicated retention of ancestral synteny blocks and functional diversification driven predominantly by segmental duplication. Structural characterization confirmed the presence of conserved B3 domains in all G. hirsutum ABI3 homologs. Promoter analysis identified key stress-responsive cis-elements, including ABA-responsive (ABRE), drought-responsive (MYB), and low-temperature-responsive (LTRE) motifs, suggesting a role in abiotic stress regulation. Expression profiling demonstrated significant tissue-specific transcriptional activity across roots, stems, leaves, and fiber developmental stages. Conclusions: This study addresses a significant knowledge gap by elucidating the evolution, structure, and stress-responsive expression profiles of the ABI3 gene family in cotton. It establishes a foundational framework for future functional validation and targeted genetic engineering strategies aimed at developing stress-resilient cotton cultivars with enhanced fiber quality.

Cotton fiber quality improvement remains a fundamental challenge in breeding programs due to the complex genetic architecture underlying fiber development. The narrow genetic base of upland cotton (Gossypium hirsutum L.) and the quantitative nature of fiber quality traits necessitate innovative approaches for identifying and incorporating superior alleles from related species. We developed a BC₆F₂ population by introgressing chromosome segments from the sea island cotton variety Xinhai 36 (G. barbadense) into the upland cotton variety Xinluzhong 60 (G. hirsutum). Based on fiber strength phenotyping, we constructed two DNA bulks representing extreme phenotypes (20 superior and 12 inferior individuals) for bulked segregant analysis sequencing (BSA-Seq). High-throughput sequencing generated 225.13 Gb of raw data with average depths of 20× for parents and 30× for bulks. SNP calling and annotation were performed using GATK and ANNOVAR against the upland cotton reference genome (TM-1). BSA-Seq analysis identified 13 QTLs primarily clustered within a 1.6 Mb region (20.6–22.2 Mb) on chromosome A10. Within this region, we detected nonsynonymous mutation genes involving a total of six genes. GO and KEGG enrichment analyses revealed significant enrichment for carbohydrate metabolic processes, protein modification, and secondary metabolite biosynthesis pathways. Integration with transcriptome data prioritized GH_A10G1043, encoding a β-amylase family protein, as the key candidate gene. Functional validation through overexpression and RNAi knockdown in Arabidopsis thaliana demonstrated that GH_A10G1043 significantly regulates starch content and β-amylase activity, though without visible morphological alterations. This study successfully identified potential genomic regions and candidate genes associated with cotton fiber strength using chromosome segment substitution lines combined with BSA-Seq. The key candidate gene GH_A10G1043 provides a valuable target for marker-assisted selection in cotton breeding programs. Our findings establish a foundation for understanding the molecular mechanisms of fiber quality formation and offer genetic resources for developing superior cotton varieties with enhanced fiber strength.

Drought is one of the most severe and frequently occurring abiotic stresses that significantly threaten cotton production. In this study, to identify key genes associated with drought resistance in upland cotton, BSA-seq was integrated with RNA-seq (R) and proteomic (P) approaches conducted on an F₂ population and two parental accessions exhibiting extreme drought resistance: the drought-sensitive accession “2406211” (A) and the drought-resistant accession “2403739” (B). After crossing the two parent plants, the F₂ generation from these accessions was used to construct pools representing the two extreme drought resistance traits. BSA-seq analysis identified five QTL regions on chromosomes A02 (9.10 Mb–11.10 Mb), A07 (18.20 Mb–20.70 Mb), A09 (39.80 Mb–42.40 Mb), D03 (8.90 Mb–13.80 Mb), and D04 (10.80 Mb–13.40 Mb). RNA-Seq analysis revealed a total of 5844 differentially expressed genes (DEGs) in RAW1 (W1, control condition) versus RAW2 (W2, drought stress), comprising 2499 up-regulated and 3345 down-regulated genes. In RBW1 versus RBW2, 6586 DEGs were detected, with 3286 up-regulated and 3300 down-regulated genes. Proteomic analysis identified 1352 differentially expressed proteins (DEPs) in PAW1 versus PAW2, including 453 up-regulated and 899 down-regulated proteins, and 3440 DEPs in PBW1 versus PBW2, with 1773 up-regulated and 1667 down-regulated proteins. By integrating transcriptomic and proteomic data to analyze genes located within the BSA target regions, we identified 5 DEGs/DEPs, including GH_D04G0687, GH_A02G0703, GH_A07G1211, GH_A07G1202, and GH_D04G0702, as potential candidate genes within the drought-resistance target intervals. This study expands transcriptomic and proteomic resources and identifies novel gene targets for the genetic improvement and breeding of cotton cultivars.