Cotton (Gossypium hirsutum) fibers, vital natural textile materials, are single-cell trichomes that differentiate from the ovule epidermis. These fibers are categorized as lint (longer fibers useful for spinning) or fuzz (shorter, less useful fibers). Currently, developing cotton varieties with high lint yield but without fuzz remains challenging due to our limited knowledge of the molecular mechanisms underlying fiber initiation. This study presents the identification and characterization of a naturally occurring dominant negative mutation GhMYB25-like_At^hapT, which results in a reduced lint and fuzzless phenotype. The GhMYB25-like_At^hapT protein exerts its dominant negative effect by suppressing the activity of GhMYB25-like during lint and fuzz initiation. Intriguingly, the negative effect of GhMYB25-like_At^hapT could be alleviated by high expression levels of GhMYB25-like. We also uncovered the role of GhMYB25-like in regulating the expression of key genes such as GhPDF2 (PROTODERMAL FACTOR 2), CYCD3; 1 (CYCLIN D3; 1) and PLD (Phospholipase D), establishing its significance as a pivotal transcription factor in fiber initiation. We identified other genes within this regulatory network, expanding our understanding of the determinants of fiber cell fate. These findings offer valuable insights for cotton breeding and contribute to our fundamental understanding of fiber development.

Fiber strength is a critical determinant of fiber quality, with stronger fibers being highly preferred in the cotton textile industry. However, the genetic basis and the specific regulatory mechanism underlying the formation of cotton fiber strength remain largely unknown. To explore fiber strength-related genes, QTL mapping, map-based cloning, and gene function verification were conducted in a backcross inbred line BS41 derived from interspecific hybridization between upland cotton and sea-island cotton. Upland cotton Emian22 (E22) and an interspecific backcross inbred line (BIL) BS41 were used as parents to construct secondary segregation populations for BSA and QTL mapping of fiber strength. The candidate gene GbNTL9 was identified through map-based cloning and expression analysis. The function of NTL9 was determined through transgenic experiments and cytological observations. The regulatory mechanisms of NTL9 were explored using RNA-seq, RT-qPCR, yeast two-hybrid, bimolecular fluorescence complementation, and yeast one-hybrid. A major QTL for fiber strength, qFS-A11-1, was mapped to a 14.6-kb genomic region using segregating populations from E22 × BS41. GbNTL9, which encodes a NAC transcription factor, was identified as the candidate gene. Overexpression of both upland cotton genotype NTL9^E22 and sea-island genotype NTL9^BS41 in upland cotton enhanced fiber strength by facilitating the dense accumulation and orderly organization of cellulose microfibrils within the cell wall. Transcriptomic analysis revealed that NTL9 inhibited the expression of genes involved in secondary wall synthesis, such as CESA4, CESA7, and CESA8, thereby delaying cell wall cellulose deposition and altering the microfibril deposition pattern. NTL9 interacted with MYB6 and functioned as a downstream gene in the ethylene signaling pathway. Additionally, an effective gene marker NTL9-24 was developed to distinguish haplotypes from G. barbadense and G. hirsutum for fiber quality breeding program. Our findings demonstrate that GbNTL9 positively regulates fiber strength through altering the microfibril deposition pattern, and provide a new insight into the molecular mechanism underlying fiber strength.

Cotton is of great economic value because of its fiber that is used in natural textile commodities and its seeds that contain an edible oil with a high content of unsaturated fatty acids and biodiesel applications. Here, we reported that GhKASI_A05 was associated with the cottonseed oil content (SOC) in a natural population via candidate gene association analysis. An 11-bp Indel located in the GhKASI_A05 promoter was found to contribute to SOC and differential expression in upland cotton inbred accessions. Interaction analysis showed that GhWRI1, an AP2/EREBP family transcription factor, that reportedly functions in plant seed oil and fatty acids (FAs) accumulation, directly bound to AW-box cis-elements in two haplotypes of the GhKASI_A05 promoter and activated the expression of GhKASI_A05 at different levels. The seed-specific overexpression of GhKASI_A05 resulted in increased seed size, weight, and protein content, and C16:0 and C18:1 contents but reduced SOC. Our results provide new insights into the biological function of GhKASI in SOC and effective strategies for cotton breeding in the future.

Cotton fiber initiation determines the fiber yield, yet the genetic basis underlying lint and fuzz initiation has still not been fully uncovered. Here, map-based cloning was carried out to identify the fiberless mutant genes derived from a cross between Gossypium hirsutum acc. WT and a natural fiberless mutant, fbl_SHZ. The 12:3:1 segregation ratio in F₂ populations (including 1848 and 3100 individuals that were developed in 2016 and 2018, respectively) revealed dominant epistasis, with the fuzz gene exerting dominance over the lint gene. Genetic linkage analysis revealed that GhMYB25like_A12 controls fuzz fiber initiation, while both GhMYB25like_A12 and GhMYB25like_D12 regulate lint fiber development. Sequencing analyses showed that the fbl_SHZ mutant exhibited a K104M mutation in the R2R3 domain of GhMYB25like_A12 and a transposable element insertion in GhMYB25like_D12, leading to fiberless seeds. Knockout of GhMYB25like_A12 produced fuzzless seeds, knockout of GhMYB25like_D12 led to no obvious change in seeds, and knockout of both (GhMYB25like_A12&D12) resulted in fiberless seeds. The 12:3:1 ratio reappeared in the F₂ population developed from the GhMYB25like_A12&D12 mutated plants as female and Jin668 as the male, which further confirmed the genetic interaction observed in fbl_SHZ. RNA-seq analysis revealed that GhMYB25like regulates cotton fiber initiation through multiple pathways, especially fatty acid metabolism. This study elucidates the key genes and their genetic interaction mechanisms governing cotton fiber initiation, providing a theoretical foundation for genetic improvement of cotton fiber traits.