Upland cotton (Gossypium hirsutum), one of the world’s major fiber crops, faces challenges from the genetic homogeneity of modern varieties. Here we present 107 gold-standard genome assemblies spanning the wild-to-domesticated continuum, revealing six large-scale structural variations, including a chromosomal reciprocal translocation and five inversions tracing the evolutionary history of cultivated cotton in the Americas. This history also involved continuous introgression from Gossypium barbadense, shaping the genetic diversity of G. hirsutum landraces and cultivars. Leveraging the graph pan-genome, we capture the sequence and structural diversity of nucleotide-binding site–leucine-rich repeat genes, uncovering pathogen-driven selection signatures and loci associated with disease resistance. A presence–absence variation genome-wide association study (GWAS) identified previously overlooked loci for key fiber traits, complementing single-nucleotide polymorphism–GWAS findings. Additionally, we construct a detailed map of large inversions, offering insights into hybridization dynamics and strategies to mitigate linkage drag. This study enhances our understanding of cotton evolution and domestication while delivering a valuable resource to enhance breeding. Genome assemblies of 100 cultivated and seven semi-wild Gossypium hirsutum accessions provide insights into the evolutionary history of upland cotton and the genetic basis of fiber trait variation.

Cotton fiber length (FL) and strength (FS) are critical determinants of yarn quality and overall textile performance. Although numerous quantitative trait loci (QTLs) associated with these traits have been reported, their practical application in breeding programs is often constrained by broad confidence intervals, limited mapping resolution, and poor reproducibility across studies. In this study, we performed a genome-wide meta-QTL (MQTL) analysis by integrating 677 previously reported QTLs into a high‑density consensus map. This approach pinpointed 49 stable MQTLs with confidence intervals reduced by an average of 2.9‑fold. Among the 49 MQTLs, 28 were supported by colocalization with independent GWAS signals (designated as high-confidence MQTLs), while the remaining 21 lacked GWAS support (putative MQTLs). The high-confidence MQTLs provide robust targets for further investigation. Transcriptomic analysis linked several candidate genes within MQTL regions to key biological processes, including cell wall composition. Notably, we identified a receptor-like kinase gene, LECRK3, in which a missense mutation exerts pleiotropic effects on both FL and FS. Collectively, these findings refine the genetic architecture underlying cotton fiber quality. A functional KASP marker was developed and validated for the pleiotropic candidate gene LECRK3, providing an immediately applicable tool for marker-assisted breeding aimed at improving both fiber length and strength.

Cold stress is a significant challenge to cotton (Gossypium hirsutum L.) production during seed emergence and early seedling establishment, as cotton is native to tropical and subtropical environments. Low temperatures during these sensitive stages impair photosynthetic efficiency, damage cellular structures, and reduce yield. Although cotton responses to cold stress have been extensively investigated at physiological, molecular, and transcriptional levels, increasing evidence suggests that transient gene expression changes alone are insufficient to explain sustained stress performance. This review synthesizes current knowledge on cotton cold-stress responses, emphasizing the regulatory roles of key histone modifications histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) in transcriptional control and within-generation (somatic) epigenetic priming. Although cotton cold epigenome profiling is beginning to emerge, cotton-specific, time-resolved chromatin datasets that span chilling, recovery, and recurrent chilling (stress-recovery-re-stress) remain limited; therefore, several mechanistic inferences necessarily rely on indirect evidence from cotton studies under other conditions and on well-characterized model plant systems. H3K27me3 is implicated in Polycomb-mediated gene silencing and may regulate gene reactivation during cold stress and recovery, whereas H3K4me3 is proposed to support rapid induction of cold-responsive genes. Bivalent chromatin domains containing H3K4me3 and H3K27me3 may maintain stress-related genes in a poised transcriptional state, enabling swift activation while preserving developmental regulation. We highlight key knowledge gaps and experimental priorities for establishing cotton-specific chromatin mechanisms of cold memory and for translating these insights into epigenome-assisted breeding and biotechnological strategies to develop cotton varieties with improved and stable cold resilience.

The soil-borne fungal pathogen Verticillium dahliae (V. dahliae) is the causal agent of Verticillium wilt (VW), a vascular disease that severely threatens global cotton production. Although cell wall lignification represents a cornerstone of plant immunity, the precise regulatory circuits that bridge this structural reinforcement with Verticillium dahliae resistance in cotton have yet to be fully elucidated. Here, we demonstrate that the NAC transcription factor GhNAC043 is a key positive regulator of this defense. GhNAC043 expression was rapidly induced upon V. dahliae infection. Silencing GhNAC043 in cotton compromised resistance, reducing lignin accumulation and downregulating lignin biosynthesis genes. Conversely, heterologous overexpression of GhNAC043 in Arabidopsis enhanced VW tolerance. We further identified GhBPM2 as a nuclear interaction partner of GhNAC043. Profiling of the transcriptome demonstrated that the GhNAC043-GhBPM2 module alters the expression profile of genes pivotal for jasmonic acid (JA) and abscisic acid (ABA) signal transduction. Collectively, these results highlight a previously unknown regulatory pathway in which the GhNAC043-GhBPM2 complex drives lignin deposition through the modulation of JA and ABA signaling, thereby fortifying cotton against VW infection.

Auxin homeostasis is hypothesized to play an important role in the growth-defense trade-off in plants; however, the potential mechanisms by which auxin metabolism correlates with cell wall dynamics and immune signaling during vascular pathogen infection in cotton warrant further investigation. In this study, we characterized GhGH3.1, an IAA-conjugating candidate belonging to the GH3 class II lineage, and explored its potential involvement in defense responses against Verticillium dahliae (V. dahliae) infection in Gossypium hirsutum. GhGH3.1 appears evolutionarily conserved across Gossypium species, exhibited a root-predominant expression pattern, and was induced upon V. dahliae challenge. Functional investigations via transient virus-induced gene silencing (VIGS) indicated that the knockdown of GhGH3.1 in cotton was associated with an altered host susceptibility; complementarily, its heterologous expression within Arabidopsis thaliana was associated with relative tolerance. Pairwise interaction assays tentatively showed that the GhGH3.1 protein physically associates with the epidermis development-related protein GhPDF1. Comparative transcriptomic and physiological profiling further indicated that GhGH3.1 deficiency was accompanied by transcriptional co-alterations in jasmonic acid (JA) signaling marker genes, secondary cell wall structural components, and reactive oxygen species (ROS) accumulation. Collectively, these findings tentatively present a preliminary working model where GhGH3.1, potentially through its interaction with GhPDF1, may contribute to V. dahliae defense, likely by modulating auxin-amido conjugation and aligning downstream JA signaling, cell wall reinforcement, and redox balance during the immune response.

Excessive soil salinity poses a significant threat to plant growth and agricultural production. However, the dynamic transcriptional processes underlying plant salt stress response, particularly the transition from transient to steady-state responses and the involvement of rhythmic regulation, remain poorly understood. Here we conducted phenotypic and transcriptomic analyses of upland cotton (Gossypium hirsutum) under salt stress, generating a high-resolution temporal expression profile. Our results identified distinct stress perception and homeostasis responsive phases of gene regulation and revealed a substantial disruption of rhythmic gene expression under stress. A large proportion of rhythmically expressed genes (REGs) lost rhythmicity, with significant alterations in expression phase, period and amplitude. Furthermore, we found that salt stress induced notable shifts in homoeolog expression bias (HEB), suggesting a role for subgenome regulation in environmental plasticity. Collectively, our findings distinguish short-term from long-term adaptive changes and underscore the interplay between stress-induced and rhythmic-regulated transcription. This work provides valuable insights into the temporal and regulatory complexity of salt stress in cotton.

Potassium (K+), an essential macronutrient for plant growth and stress adaptation, becomes physiologically stressful when overaccumulated in soil. While K fertilization enhances cotton (Gossypium hirsutum) fiber quality and yield, the consequential KCl-induced ionic stress has emerged as a critical agricultural challenge demanding molecular-level resolution. This study unveils the previously unexplored epigenetic mechanisms mediated by histone H3 lysine 27 trimethylation (H3K27me3) in cotton's adaptation to KCl stress. Through integrated cleavage under targets and tagmentation (CUT&Tag) chromatin profiling and transcriptome sequencing, we demonstrate that KCl stress triggers genome-wide attenuation of H3K27me3 deposition, concomitant with characteristic stress phenotypes in cotton seedlings. Suppression of H3K27me3 using RDS 3434 significantly ameliorated KCl-induced physiological damage, thereby supporting a functional correlation between this epigenetic mark and stress tolerance. Mechanistic analyses revealed 48 genes exhibiting inverse correlation between H3K27me3 enrichment and transcriptional activation, including 2 that encode pivotal salt-tolerance regulators: Glutathione Synthase1 (GhGSH1) and Salt-Related MYB1 (GhSRM1). Virus-induced gene silencing validation confirmed these H3K27me3-associated genes as essential components of cotton's ionic stress response network. Our findings delineate the epigenetic landscape associated with KCl stress adaptation and highlight H3K27me3-mediated chromatin remodeling as a critical regulatory layer in plant abiotic stress responses. This work provides insights into epigenetic engineering strategies for developing stress-resilient cotton cultivars.

Cotton originated in the tropics and is sensitive to low temperatures. Low-temperature stress has always been a significant limiting factor restricting its domestication and spread. The H3K4me3 histone mark regulates plant gene expression and transcriptional memory while influencing several developmental processes and stress responses. However, the potential function of cold stress-induced H3K4 trimethylation of cold-related genes in cotton's response to cold is mostly underinvestigated. Here, we found that low temperatures significantly alter the levels of H3K4me3 modification in cotton, and the levels of H3K4me3 modification significantly correlate with the expression levels of cold-responsive genes. H3K4me3 modification has a role in regulating the expression of positive or negative cold-responsive genes, with positive regulation predominant in upland cotton (71.1%). 5182 core cold-induced genes marked by H3K4me3 (CCRGs), including the crucial cold signaling regulatory pathway ICE-CBF-CORs, have been identified. We revealed that within the ICE-CBF-CORs regulatory pathway, ICE1, CBF1, and most COR genes are positively regulated by H3K4me3. Weighted Gene Co-expression Network Analysis (WGCNA) analysis demonstrated that the expression patterns of CCRG genes are significantly correlated with the elongation of cotton radicles under low temperatures. We conducted preliminary functional validation of cold-responsive gene GhZAT11 modified by H3K4me3 and found that its silencing significantly reduces the cold tolerance of cotton. This supports the application of CCRG genes in cold tolerance research in cotton.

Cotton (Gossypium hirsutum L.) is a key allopolyploid crop with global economic importance. Here we present a telomere-to-telomere assembly of the elite variety Zhongmian 113. Leveraging technologies including PacBio HiFi, Oxford Nanopore Technology (ONT) ultralong-read sequencing and Hi-C, our assembly surpasses previous genomes in contiguity and completeness, resolving 26 centromeric and 52 telomeric regions, 5S rDNA clusters and nucleolar organizer regions. A phylogenetically recent centromere repositioning on chromosome D08 was discovered specific to G. hirsutum, involving deactivation of an ancestral centromere and the formation of a unique, satellite repeat-based centromere. Genomic analyses evaluated favorable allele aggregation for key agronomic traits and uncovered an early-maturing haplotype derived from an 11 Mb pericentric inversion that evolved early during G. hirsutum domestication. Our study sheds light on the genomic origins of short-season adaptation, potentially involving introgression of an inversion from primitively domesticated forms, followed by subsequent haplotype differentiation in modern breeding programs.

Genetic transformation in cotton facilitates the integration of stress-resilient traits, contributing to improved abiotic and biotic stress tolerance. Consequently, sustained efforts to optimise transformation and regeneration protocols are critical for advancing cotton genetic improvement and boosting yield potential. This review discusses the recent advances in cotton plant transformation, in cognisance of the prospects and challenges characterising each technique and the variations in the transformation efficiency (TE) of specific explants. Furthermore, the mechanisms by which morphogenesis-related genes and other molecular cascades have been used to improve the TE and regeneration of recalcitrant plant species are also addressed. Factors that affect the cotton transformation efficiency were also discussed. These updates could help cotton breeders design more effective strategies for transforming and regenerating recalcitrant plants.

Cotton fiber development entails complex genome-wide gene regulatory networks (GRNs) that remain insufficiently resolved. Here, we present integrative analyses of fiber GRNs using public RNA-seq datasets, integrated with genomic, transcriptomic, and cistromic data. We detail the fiber co-expression dynamics and regulatory connections, validating findings with external datasets and transcription factor (TF) binding site data. We elucidate previously uncharacterized TFs that regulate genes involved in fiber-related functions and cellulose synthesis, and identify the regulatory role of two homoeologous G2-like TFs on fiber length. Analysis of duplicated gene expression and network relationships in allopolyploid cotton, which has two co-resident genomes (A, D), revealed novel aspects of asymmetric subgenomic developmental contributions. Whereas D-biased homoeolog pairs drive higher overall gene expression from the D subgenome, TFs from the A subgenome play a preferential regulatory role in the fiber GRN. Following allopolyploid formation, it appears that the trans-regulatory roles of TFs diversified more rapidly between homoeologs than did the cis-regulatory elements of their target genes. Our approach underscores the utility of network analysis for detecting master regulators and provides fresh perspectives on fiber development and polyploid functional genomics through the lens of co-expression and GRN dynamics.

Histone methylation is pivotal in regulating the expression of numerous essential functional genes in plants. However, its specific function and mechanism in cotton fiber cell initiation remain poorly understood. The upland cotton (Gossypium hirsutum L.) variety Xuzhou142 (Xu142) and its fuzzless-lintless mutant Xu142 fl are ideal model materials for studying cotton fiber cell development. In vitro ovule culture analysis showed that the histone H3 lysine 27 trimethylation (H3K27me3) inhibitor RDS 3434 could inhibit fiber cell initiation and development. Cleavage under targets and tagmentation (CUT&Tag) and RNA sequencing (RNA-seq) data of-1, 0, and 1 day post-anthesis (DPA) ovules showed that H3K27me3 regulates fiber cell initiation by highly accumulating in Xu142 than in Xu142 fl. It was also found that H3K27me3 was negatively correlated with gene transcription, as 227 expressed genes showed an opposite trend to the H3K27me3 modification level and were identified to participate in multiple signaling pathways. Furthermore, the expression levels often potential genes related to fiber development were confirmed to correlate with H3K27me3 levels. The results demonstrate the critical role of H3K27me3 in fiber development process and present evidence supporting its involvement in cotton fiber cell initiation. The study lays the foundation and provides genetic resources for further investigations into genes related to fiber development.

Plant mitochondrial genomes (mitogenomes) exhibit extensive structural variation yet extremely low nucleotide mutation rates, phenomena that remain only partially understood. The genus Gossypium, a globally important source of cotton, offers a wealth of long-read sequencing resources to explore mitogenome and plastome variation and dynamics accompanying the evolutionary divergence of its approximately 50 diploid and allopolyploid species. Here, we assembled 19 mitogenomes from Gossypium species, representing all genome groups (diploids A through G, K, and the allopolyploids AD) based on a uniformly applied strategy. A graph-based mitogenome assembly method revealed more alternative structural conformations than previously recognized, some of which confirmed the mitogenome structure reported in earlier studies on cotton. Using long-read data, we quantified alternative conformations mediated by recombination events between repeats, and phylogenetically informative structural variants were noted. Nucleotide substitution rate comparisons between coding and non-coding regions revealed low mutation rates across the entire mitogenome. Genome-wide mapping of nuclear organellar DNA transfers (NUOTs) in Gossypium revealed a nonrandom distribution of transfers in the nuclear genome. In cotton, the fate of NUOT events varied, with mitochondrion-to-nucleus transfer (NUMT) predominantly retained as short fragments in the nuclear genome, with more plastid sequences integrated into the nucleus. Phylogenetic relationships inferred using different data sets highlighted distinct evolutionary histories among these cellular compartments, providing ancillary evidence relevant to the evolutionary history of Gossypium. A comprehensive analysis of organellar genome variation demonstrates complex structural variation and low mutation rates across the entire mitogenome and reveals the history of organellar genome transfer among the three genomes throughout the cotton genus. The findings enhance our general understanding of mitogenome evolution, comparative organellar and nuclear evolutionary rates, and the history of inter-compartment genomic integration.

The cytokinin response regulator (ARR) gene is essential for cytokinin signal transduction, which plays a crucial role in plant growth and development. However, the functional mechanism of ARR genes in cotton leaf abscission remains incompletely understood. In this study, a total of 86 ARR genes were identified within the genome of Gossypium hirsutum. These genes were categorized into four distinct groups based on their phylogenetic characteristics, supported by analyses of gene structures and conserved protein motifs. The GhARR genes exhibited an uneven distribution across 25 chromosomes, with three pairs of tandem duplication events observed. Both segmental and tandem duplication events significantly contributed to the expansion of the ARR gene family. Furthermore, numerous putative cis-elements were identified in the promoter regions, with hormone and stress-related elements being common among all 86 GhARRs. Transcriptome expression profiling screening results demonstrated that GhARRs may play a mediating role in cotton’s response to TDZ (thidiazuron). The functional validation of GhARR16, GhARR43, and GhARR85 using virus-induced gene silencing (VIGS) technology demonstrated that the silencing of these genes led to pronounced leaf wilting and chlorosis in plants, accompanied by a substantial decrease in petiole fracture force. Overall, our study represents a comprehensive analysis of the G. hirsutum ARR gene family, revealing their potential roles in leaf abscission regulation.

Cotton fibers are single cells that develop from the epidermal cells in the outer integument of developing seeds. The processes regulating fiber cell development have been extensively studied; however, the spatiotemporal transcriptome and metabolome profiles during the early stages of fiber development remain largely unknown. In this study, we profile the dynamics of transcriptome and metabolome during the early stages of cotton fiber cell development using a combination of spatial transcriptomic, single-cell transcriptomic, and spatial metabolomic analyses. We identify the key genes (e.g., DOX2, KCS19.4, BEE3, and HOS3.7) and metabolites (e.g., linoleic acid, spermine, spermidine, and α-linolenic acid) that may regulate the early development of fiber cells. Finally, knockdown and gain-of-function analyses identify the crucial role of GhBEE3/Gh_A09G062900 in cotton fiber initiation. We also construct a publicly accessible website ( https://cotton.cricaas.com.cn/ovule/ ) for visualization of the spatiotemporal gene expression in cotton, providing a reference dataset for further studies on cotton fiber development. While cotton fiber development has been extensively studied, the spatiotemporal transcriptome and metabolome landscape remain largely unknown. Here, the authors profile early stage cotton fiber cell using a combination of spatial transcriptomic, single-cell transcriptomic, and spatial metabolomic analyses.

Allopolyploidization, a process involving interspecific hybridization and whole-genome duplication, has both immediate and long-term evolutionary effects on plants. However, the immediate impact of genome doubling in cotton (Gossypium spp.) remains understudied due to the lack of resynthesized allopolyploids. In this study, we used haploid lines from the allotetraploid species Gossypium hirsutum (AD1) and G. barbadense (AD2) to evaluate the effects of immediate changes in ploidy. We compared the transcriptomes of diploid cotton species G. arboreum (A2), G. raimondii (D5), their F1 hybrid (A2 × D5), natural allotetraploids (AD1 and AD2), and their derived haploids (hAD1 via the GhDMP knockout system and hAD2 via semigamy). We determined that haploidization has a minor effect on changes in gene expression compared to hybridization and allopolyploidization, highlighting the large effect of species-specific long-term evolution. Specifically, ploidy changes in AD1 were linked to chromosome separation and nucleosome dynamics. Moreover, haploidization appeared to obscure homologous expression divergence due to changes in the trans environment, leading to a decreased amount of homoeolog expression bias (HEB). While significant associations were detected between nonadditive patterns of total homoeolog expression, HEB, and cis-trans regulatory categories, no apparent differences in these association patterns were observed among the F1 hybrid, tetraploids, and haploids. These findings provide a more detailed view of the transcriptomic consequences of allopolyploidization in cotton, offering insight into the temporal dynamics of gene expression evolution and the underlying regulatory mechanisms.

Cotton (Gossypium hirsutum L.) is a critical source of natural fiber and cottonseed oil for humans, yielding substantial economic benefits globally. However, the susceptibility of cotton cultivation to compound drought and heat events (CDHEs) brings significant threats to cotton productivity. Despite this, a comprehensive assessment of global CDHE occurrence over cotton-growing areas and its potential impacts on cotton yields remains unresolved, hindering efforts to implement adaptive strategies to ensure global cotton productivity. To address this gap, we analyzed changes in mean temperature and soil moisture within global cotton-growing areas during their respective growing seasons, estimated the probability of CDHEs across multiple spatial scales via copula theorems, and examined the relationship between cotton yield anomalies and CDHEs in major cotton countries. Our results indicate increasing but divergent trends in mean temperature and soil moisture. Specifically, while most regions exhibit drying trends, India and Pakistan show significant wetting trends, with soil moisture increasing during the cotton growing season. The global average probability of CDHEs between 1961-1990 and 1991-2020 showed a more than threefold increase in severity, with such an increase occurring in approximately 61 % of cotton-growing areas due to comparable contributions from drying and warming trends. Furthermore, major cotton-producing countries exhibited similar CDHE trends, leading to a heightened probability of synchronous CDHE occurrences, except in countries connected to India and Pakistan. Such occurrences of CDHEs are significantly related to cotton yield failures in major cotton-producing countries. Our findings emphasize the growing exposures of cotton-growing areas to CDHEs and highlight the urgent need for adaptive strategies to enhance the resilience of cotton production systems under changing climatic conditions.

Salt stress and salt-alkali stress significantly inhibit the normal growth and development of plants. Understanding the molecular mechanisms of cotton responses to these stresses is crucial for improve yield and fiber quality. In this study, we conducted a comprehensive analysis of the transcriptome dynamics under salt and salt-alkali stress conditions, utilizing 234 RNA-seq datasets compiled from 11 previous studies. After systematic evaluation and correction for batch effects, we observed that root transcriptomes clustered more consistently than leaf transcriptomes across stress treatment and time points. Weighted gene co-expression network analysis (WGCNA) on 123 root transcriptomes identified three key modules, with their hub genes significantly associated with salt and salt-alkali tolerance. Virus-induced gene silencing assay and RNA-seq analysis indicated that GhGDH2_D03 (Gohir.D03G104800), a module hub gene encoding Glutamate Dehydrogenase 2, positively regulates salt and salt-alkali tolerance in cotton by modulating multiple signaling pathways and metabolic processes, including the ethylene signaling pathway. This study underscores the pivotal role of GhGDH2_D03 in conferring tolerance to salt and salt-alkali stress, in addition to its previous reported involvement in biotic stress defense, providing valuable insights and genetic resources for cotton breeding.