Fiber neps (FN), composed of entangled fibers, and seed coat neps (SCN), consisting of fiber-entangled seed coat fragments, are critical raw fiber defects that significantly reduce the quality and market value of fibers and downstream textiles. Despite the textile industry's need for solutions, the genetic bases for FN and SCN formation remain poorly understood. This study explored the genetic bases of FN and SCN by analyzing a 550-member multi-parent advanced generation inter-cross (MAGIC) population of upland cotton (Gossypium hirsutum L.), cultivated under three environmental conditions. By employing both Advanced Fiber Information System (AFIS) single-fiber and High Volume Instrument (HVI) bundle fiber phenotyping, the study revealed that FN and SCN exhibit differential environmental responses and have distinct relationships with a standard fiber trait, suggesting independent biological origins. Furthermore, results demonstrated that post-ginning nep-removal processes cause a substantial decline in overall fiber quality, reinforcing the necessity of genetic-based control strategies. Genome-wide association study (GWAS), utilizing 1,481,252 single nucleotide polymorphisms (SNPs), identified stable genomic loci associated with FN on chromosome (Chr.) D13, and SCN on Chr. A03 and A07. Further functional annotation and expression profiling prioritized candidate genes involved in fiber maturation, seed coat integrity, and mechanical strength within these loci. These results clarify the complex genetic basis of cotton nep formation, providing foundational knowledge essential for causal gene discovery and the development of targeted breeding programs aimed at enhancing cotton fiber quality and production efficiency.

Introduction:The naturally brown colored fibers of some cottons exist in varying shades of brown. Linkage analyses have revealed up to six individual loci (Lc1-Lc6) for brown color and suggested a separate genetic basis for each color value of fibers. It was previously reported that the brown color and flame-retardant (FR) properties of Lc1 fibers resulted from an upstream inversion mutation that highly upregulates the GhTT2_A07 gene and, consequently, the entire phenylpropanoid pathway. However, the genes responsible for the coloration and FR of other brown cotton fibers remained undetermined. Methods:In the current study, we used a previously uncharacterized SA-40 germplasm that produces rust-brown cotton. An F2 mapping population derived from the cross of SA-40 and TM-1 was used in an association mapping study to elucidate the genetics of SA-40 rust-brown fiber color and FR. Results and discussion:The rust-brown locus was mapped to GhTT2 on chromosome A07 (GhTT2_A07). Comparison of the upstream sequence of GhTT2_A07 between TM-1 and SA-40 revealed the absence of a Ty3-like LTR retrotransposon in the SA-40 line. No remnants of the LTR retrotransposon in the SA-40 indicated that the retrotransposon insertion did not happen in that line. However, all investigated white cotton lines in this study displayed the presence of the LTR retrotransposon upstream of GhTT2_A07. Transcript analysis showed that the absence of retrotransposon upstream of GhTT2_A07 was associated with higher expression of this gene, but at lower levels than previously reported for the upstream inversion mutation in the MC-BL line, where the retrotransposon has been removed by structural rearrangement. Our finding suggests that the value of brown coloration in Lc1 fibers is directly controlled by the expression level of GhTT2_A07, with higher expression levels resulting in darker fibers.

Breeding valuable traits in crop plants requires identifying diverse alleles in the germplasm that are likely to affect desirable characteristics. The genetic diversity of historic cultivars of cotton is a reservoir of potentially important genes for crop improvement and genetic research. Diversity in the characteristics of harvested cotton fibers affects their suitability for end-use applications. Candidate loci and genes have been identified that affect the length, strength, and maturity of cotton fibers which affect the quality and value of the yarn, thread and textile. Natural genetic mechanisms in the plant may also affect the flammability of the produced textiles. Here we show that a combination of allele mining and transcriptome analysis can identify candidate genes for cotton fiber traits including strength and perhaps flammability. We found novel DNA variants in fiber-expressed gene families in 132 newly sequenced cotton varieties and identified genes with genotype-specific RNA expression. Among these, we identified novel variation in DNA sequence and RNA expression in genes at major QTL qD04-ELO-WLIM (JGI-Gohir.D04G160000), qA13-MIC (Gohir.A13G157500), qA07-STR (Gohir.A07G191600), supported the candidacy of qD11-UHML-KRP6 (Gohir.D11G197900) and qD13-STR (Gohir.D13G17450), and identified an additional A03-WLIM transcription factor gene (Gohir.A03G182100) and several RNA expression variant candidates of potential flammability genes that may be useful for plant biologists and cotton breeders. Candidate genes for traits like flame resistance that are likely due to the combination of many small effect QTL can benefit from this multi-mining approach. We provide an annotated variant call format (vcf) file with variations at 24,996 loci that are predicted to affect 10,418 cotton fiber genes in the historic breeding germplasm.

Within-sample variation in cotton fiber len,gth is a major factor influencing the production and quality of yarns. The textile industry has been searching for approaches of improving the long fiber fraction and minimizing the short fiber fraction within a cotton sample to produce superior fiber and yarn quality. USTER® High Volume Instrument (HVI) has been widely used for a rapid assessment of cotton fiber length traits from a fiber bundle. However, its effectiveness for genetic studies has been questioned due to the indirect estimations of the cotton fiber traits that cannot be measured from a fiber bundle. To overcome the limits of the HVI fiber length traits, we utilized the Advanced Fiber Information System (AFIS) measuring fiber length traits directly from individual fibers based on weight or number. Comparative fiber length analyses showed AFIS provided higher sensitivity in detecting the fiber length variations within and among cotton samples than HVI. The weight-based AFIS length traits were strongly correlated with the corresponding HVI lengths, whereas the number-based AFIS mean length showed a relatively weaker correlation with the HVI lengths. Integrations of the weight based-length traits with genome-wide association studies (GWAS) enabled classifying the QTLs specifically associated with long, mean, or short fiber length traits and identified a false positive associated with the indirectly estimated HVI short fiber trait. Unlike the weight based-AFIS length traits, the number-based AFIS length trait did not show a negative correlation with a weight related-HVI property, and identified a single QTL that was not detected by the corresponding HVI trait. These results suggested that integrating the AFIS method with GWAS helped discoveries of the genome loci involved in the within-sample variation in cotton fiber length and characterizations of the fiber length QTLs.

Naturally-colored brown cotton (NBC) fiber is an environmentally friendly raw source of fiber for textile applications. The fiber of some NBC cultivars exhibits flame-retardant properties, which can be used in textiles that require flame resistance. Proanthocyanidins or their derivatives are responsible for the brown pigment in NBC; however, how flame retardancy is related to pigmentation in NBC is poorly understood. To gain insight into brown pigment biosynthesis, we conducted comparative transcripts and metabolites profiling analysis of developing cotton fibers between the brown (MC-BL) and white (MC-WL) cotton near-isogenic lines (NILs), genetically different only in the Lc1 locus. In this study, mass spectrometry was used to detect metabolites in BL and WL developing fibers at 8, 12, 16, 20, 24, 36, and 40 days post anthesis (DPA) and mature fibers. Transcripts analysis was performed at two critical fiber developmental points, 8 DPA (fiber elongation) and 20 DPA (secondary cell wall deposition). We found 5836 (ESI MS positive mode) and 4541 (ESI MS negative mode) metabolites significantly different accumulated between BL and WL. Among them, 142 were known non-redundant metabolites, including organic acids, amino acids, and derivatives of the phenylpropanoid pathway. Transcript analysis determined 1691 (8 DPA) and 5073 (20 DPA) differentially expressed genes (DEGs) between BL and WL, with the majority of DEGs down-regulated at 20 DPA. Organic acids of the citric acid cycle were induced, while most of the detected amino acids were reduced in the MC-BL line. Both cis- and trans-stereoisomers of flavan-3-ols were detected in developing MC-WL and MC-BL fibers; however, the gallocatechin and catechin accumulated multiple times higher. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acids determined that palmitic acid long-chain alcohols were the main constituents of waxes of mature fibers. Energy-dispersive X-ray spectrometry (EDS) analysis of mature fibers revealed that potassium accumulated three times greater in MC-BL than in MC-WL mature fibers. This study provides novel insights into the biosynthesis of pigments and its association with flame retardancy in NBC fibers.

Naturally colored cotton (NCC) offers an environmentally friendly fiber for textile applications. Processing white cotton fiber into textiles requires extensive energy, water, and chemicals, whereas processing of NCC skips the most polluting activity, scouring-bleaching and dyeing; therefore, NCC provides an avenue to minimize the harmful impacts of textile production. NCC varieties are suitable for organic agriculture since they are naturally insect and disease-resistant, salt and drought-tolerant. Various fiber shades, ranging from light green to tan and brown, are available in the cultivated NCC (Gossypium hirsutum L.) species. The pigments responsible for the color of brown cotton fiber are proanthocyanidins or their derivatives synthesized by the flavonoid pathway. Due to pigments, the NCC has excellent ultraviolet protection properties. Some brown cotton varieties exhibited superior thermal resistance of fiber that can be used to make fabrics with enhanced flame retardancy. Here, we review molecular mechanisms involved in the pigment production of brown cotton and challenges in breeding NCC varieties with a wide range of colors but without penalty in fiber quality. Also, we discuss opportunities for NCC with flame-retarding properties in textile applications.

Upland cotton (Gossypium hirsutum L.) is the major crop producing renewable fiber for textiles. Cotton fiber length is an economically important trait that affects the quality of the yarn. The Upland cotton multi-parent advanced generation intercross (MAGIC) population was developed for genetics research to understand how fiber traits can be improved without reducing fiber yield. A genome-wide association study of the MAGIC population identified a significant fiber length QTL on Chromosome D11 (qFL-D11-1). To understand how the qFL-D11-1 regulates fiber length, we developed F5 lines by crossing the longest and the shortest fiber recombinant inbred lines (RILs) of the MAGIC population to establish lines with parental haplotypes at the qFL-D11-1 but segregating for other loci. The F5 lines and parental RILs were sequenced to detect exchanges from parents' genomic regions in the progeny. One segregating genomic region in the F5 lines coincided with qFL-D12-1, which additively affected fiber length. Correlation analysis of the expression patterns of genes from the two QTLs with the fiber lengths in the MAGIC population showed the highest correlation of the RING-type ubiquitin E3 ligase (GhUbE3) from qFL-D12-1. Kip-related protein-6 (KRP6) is the candidate gene from the qFL-D11-1. A yeast twohybrid and bimolecular fluorescence complementation assays demonstrated a physical interaction between GhKRP6 (Ghir_D11G020340) and GhUbE3 (Ghir_D12G006080). RNAseq showed that the GhUbE3 expression is reciprocal to the interacting GhKRP6 during fiber development. Virus-induced gene silencing (VIGS) of GhKRP6 increased fiber length. The data indicates that GhUbE3 controls the dose of GhKRP6's inhibitory activity and, therefore, the duration of fiber elongation.

Cotton fibers are the world's most important renewable source of fiber for textile industry. The cell wall of cotton fiber determines fiber quality parameters for textile industry. The thickness of secondary cell wall and the fiber perimeter define the fineness and micronaire (MIC) of cotton fiber. The fineness of cotton fiber plays an important role in affecting the yarn quality. Actin cytoskeleton impacts the cell extension and shape, however how it affects secondary cell wall is unclear. We overexpressed the actin isovariant Gh_D04G0865 (previously shown mutation in this gene impaired fiber length) under fiber-specific promoter in cotton. We obtained two independent transgenic lines. Fiber characteristics of their first-generation progenies were evaluated by high volume instrument (HVI), advanced fiber information system (AFIS), Cottonscope, and Fourier-transform infrared (FT-IR) spectroscopy. Fiber of transgenic lines with excessive expression of actin showed significant reduction in MIC and fineness, while increase in strength. Seed index was increased in transgenic lines overexpressing actin. FT-IR analysis detected changes in vibrations associated with crystalline cellulose. Taken together, our findings revealed that actin cytoskeleton may play roles in reducing fiber wall thickening process during secondary cell wall biosynthesis.

Textiles made from cotton fibers are flammable and thus often include flame retardant additives for consumer safety. Transgressive segregation in multi-parent populations facilitates new combinations of alleles of genes and can result in traits that are superior to those of any of the parents. A screen of 257 recombinant inbred lines from a multi-parent advanced generation intercross (MAGIC) population for naturally enhance flame retardance (FR) was conducted. All eleven parents, like all conventional white fiber cotton cultivars produce flammable fabric. MAGIC recombinant inbred lines (RILs) that produced fibers with significantly lower heat release capacities (HRC) as measured by microscale combustion calorimetry (MCC) were identified and the stability of the phenotypes of the outliers were confirmed when the RILs were grown at an additional location. Of the textiles fabricated from the five superior RILs, four exhibited the novel characteristic of inherent flame resistance. When exposed to open flame by standard 45° incline flammability testing, these four fabrics self-extinguished. To determine the genetic architecture of this novel trait, linkage, epistatic and multi-locus genome wide association studies (GWAS) were conducted with 473k SNPs identified by whole genome sequencing (WGS). Transcriptomes of developing fiber cells from select RILs were sequenced (RNAseq). Together, these data provide insight into the genetic mechanism of the unexpected emergence of flame-resistant cotton by transgressive segregation in a breeding program. The incorporation of this trait into global cotton germplasm by breeding has the potential to greatly reduce the costs and impacts of flame-retardant chemicals.

Upland cotton (Gossypium hirsutum L.) is the main source of natural fiber for the textile industry. Cotton fibers are unicellular trichomes that emerge from the epidermal cells of the seed. In cultivated cotton species, seed trichomes differentiate into two distinct types, spinnable lint, and short fuzz. The main priority for cotton growers is fiber yield, whereas the textile industry also demands better fiber quality characteristics, such as length, uniformity, strength, maturity, and optimal fineness. However, it is a major challenge for breeders to improve fiber quality while maintaining yield, because of the observed negative correlation between yield and fiber quality traits. Recent technical advances in sequencing and bioinformatics have facilitated the assembly of reference quality genomes of multiple cotton species, bringing a new era for cotton genomics. Available genomic resources will help genetically dissect the agronomic and fiber quality traits of cotton and identify gene variants that can be used for cotton improvement through breeding or biotechnology. Here, we review recent progress in the sequencing of genomes of cotton species and approaches in molecular genetics and genomics for the improvement of cotton fiber quality traits. We discuss progress in the understanding of each stage of cotton fiber development and the remaining challenges.

Fiber length is one of the major properties determining the quality and commercial value of cotton. To understand the mechanisms regulating fiber length, genetic variations of cotton species and mutants producing short fibers have been compared with cultivated cottons generating long and normal fibers. However, their phenomic variation other than fiber length has not been well characterized. Therefore, we compared physical and chemical properties of the short fibers with the long fibers. Fiber characteristics were compared in two sets: 1) wild diploid Gossypium raimondii Ulbrich (short fibers) with cultivated diploid G . arboreum L and tetraploid G . hirsutum L. (long fibers); 2) G . hirsutum short fiber mutants, Ligon-lintless 1 ( Li 1 ) and 2 (Li 2 ) with their near isogenic line (NIL), DP-5690 (long fibers). Chemical analyses showed that the short fibers commonly consisted of greater non-cellulosic components, including lignin and suberin, than the long fibers. Transcriptomic analyses also identified up-regulation of the genes related to suberin and lignin biosynthesis in the short fibers. Our results may provide insight on how high levels of suberin and lignin in cell walls can affect cotton fiber length. The approaches combining phenomic and transcriptomic analyses of multiple sets of cotton fibers sharing a common phenotype would facilitate identifying genes and common pathways that significantly influence cotton fiber properties.

Most cultivated cotton (Gossypium hirsutum L.) varieties have two types of seed fibers: short fuzz fiber strongly adhered to the seed coat, and long lint fiber used in the textile industry. The Ligon lintless-2 (Li2) cotton mutant has a normal vegetative phenotype but produces very short lint fiber on the seeds. The Li2 mutation is controlled by a single dominant gene. We discovered a large structural rearrangement at the end of chromosome D13 in the Li2 mutant based on whole-genome sequencing and genetic mapping of segregating populations. The rearrangement contains a 177-kb deletion and a 221-kb duplication positioned as a tandem inverted repeat. The gene Gh_D13G2437 is located at the junction of the inverted repeat in the duplicated region. During transcription such structure spontaneously forms self-complementary hairpin RNA of Gh_D13G2437 followed by production of small interfering RNA (siRNA). Gh_D13G2437 encodes a Ran-Binding Protein 1 (RanBP1) that preferentially expresses during cotton fiber elongation. The abundance of siRNA produced from Gh_D13G2437 reciprocally corresponds with the abundance of highly homologous (68%-98% amino acid sequence identity) RanBP1 family transcripts during fiber elongation, resulting in a shorter fiber phenotype in the Li2. Overexpression of Gh_D13G2437 in the Li2 mutant recovered the long lint fiber phenotype. Taken together, our findings revealed that siRNA-induced silencing of a family of RanBP1s inhibit elongation of cotton fiber cells in the Li2 mutant.

Cotton (Gossypium hirsutum L.) fiber is the most important resource of natural and renewable fiber for the textile industry. However, the understanding of genetic components and their genome-wide interactions controlling fiber quality remains fragmentary. Here, we sequenced a multiple-parent advanced-generation inter-cross (MAGIC) population, consisting of 550 individuals created by inter-crossing 11 founders, and established a mosaic genome map through tracing the origin of haplotypes that share identity-by-descent (IBD). We performed two complementary GWAS methods-SNP-based GWAS (sGWAS) and IBD-based haplotype GWAS (hGWAS). A total of 25 sQTLs and 14 hQTLs related to cotton fiber quality were identified, of which 26 were novel QTLs. Two major QTLs detected by both GWAS methods were responsible for fiber strength and length. The gene Ghir_D11G020400 (GhZF14) encoding the MATE efflux family protein was identified as a novel candidate gene for fiber length. Beyond the additive QTLs, we detected prevalent epistatic interactions that contributed to the genetics of fiber quality, pinpointing another layer for trait variance. This study provides new targets for future molecular design breeding of superior fiber quality.

Cotton fiber mutants are valuable resources for studying functions of altered genes and their roles in fiber development. The n4t is a recessive tufted-fuzzless seed mutant created through chemical mutagenesis with ethyl methanesulfonate. Genetic analysis indicated that the tufted-fuzzless phenotype is controlled by a single recessive locus. In this study, we developed an F2 population of 602 progeny plants and sequenced the genomes of the parents and two DNA bulks from F2 progenies showing the mutant phenotype. We identified DNA sequence variants between the tufted-fuzzless mutant and wild type by aligning the sequence reads to the reference TM-1 genome and designed subgenome-specific SNP markers. We mapped the n4t locus on chromosome D04 within a genomic interval of about 411 kb. In this region, seven genes showed significant differential expression between the tufted-fuzzless mutant and wild type. Possible candidate genes are discussed in this study. The utilization of the n4t mutant along with other fiber mutants will facilitate our understanding of the molecular mechanisms of cotton fiber cell growth and development.

Most commercially produced cotton cultivars have two types of fibers on the seed coat, short fuzz and long lint. Lint fiber is used in the textile industry, while fuzz is considered an undesirable trait. Both types of fibers are believed to be controlled by the same regulators; however, their mechanisms of actions are still obscure. Cotton fiber mutants provide an excellent system to study the genes that regulate fiber development. Here we described four uncharacterized and three previously reported cotton mutants with fuzzless seed phenotypes. To evaluate whether or not the genes previously associated with fuzzless seed phenotypes have mutations we sequenced whole genomic DNA of seven mutants and wild type varieties. We identified multiple polymorphic changes among the tested genes. Non-synonymous SNPs in the coding region of the MML3-A gene was common in the six mutant lines tested in this study, showing both dominant and recessive fuzzless phenotypes. We have mapped the locus of the causative mutation for one of the uncharacterized fuzzless lines using an F2 population that originated from a cross between the dominant fuzzless mutant and a wild type. Further, we have clarified the current knowledge about the causative n2 mutations by analyzing the sequence data and previously reported mapping data. The key genes and possible mechanisms of fiber differentiation are discussed in this study.

The analysis of association between genotypic markers and phenotypic traits allows identification of quantitative trait loci (QTLs), which can be applied through marker-assisted selection in breeding programs to improve the quality of crops. However, success in these applications depends primarily on the stability and dominance of the QTL. We previously identified a significant fiber length (FL) QTL on chromosome (Chr.) D11 based on the genome-wide association study (GWAS) of a multi-parent advanced generation inter-cross (MAGIC) population in upland cotton. In this report, we conducted mapping studies with two bi-parental populations to confirm the stability of the FL QTL on Chr. D11 and determine the magnitude of its effect on the fiber length phenotype. One of the F2 populations was developed from a cross between the longest fiber and the shortest fiber recombinant inbred lines (RILs) of the MAGIC population originally used for GWAS, whereas the second F2 population was created from a cross between two cotton lines Acala 1517–80 and JJ1145ne, which were not among the eleven MAGIC parental lines. The populations were grown in different environmental conditions. Genetic mapping of these populations confirmed the stability of the FL QTL on Chr. D11. The highest LOD scores of association with fiber length in both populations showed three SNP markers that resided within 360 kb of the QTL region on Chr. D11. Ten genes possessing non synonymous SNPs (nsSNPs) in their protein coding regions were identified in this region. RNAseq analysis detected activity in developing fiber tissue for seven of these candidate genes.

Cotton fiber mutants are useful tools for understanding the genetics and physiology of cotton fiber development. Currently, there are two naturally occurring, dominant lintless mutant lines, Ligon-lintless-1 (Li1) and Ligon-lintless-2 (Li2), and one man-made mutant line, Ligon-lintless-x (Lix), that exhibit extremely short lint fibers. Here we report a new lintless mutant that is the result of artificial chemical mutagenesis. In 2008, the cotton line MD 15 (PI 642769) was mutagenized with 3.2% v/v ethyl methane sulfonate (EMS). In 2010, a single Ligon-lintless-type plant was identified among the 2,000 M2 mutant progeny plants and was designated liy. This plant was crossed with the wild-type MD 15 with the objective of determining the genetic control of the lintless trait. Unlike Li1, Li2, and Lix, which are controlled by a single dominant gene, this new lintless trait is controlled by a monogenic recessive gene designated as liy. The liy plant is short and stunted and has an okra-leaf phenotype. The liy gene is not allelic to either Li1 or Li2. The genetic loci controlling these four Ligon-lintless mutations are located on four different chromosomes. This new lintless mutant will be useful in further investigating fiber elongation in cotton.

Background: Improving cotton fiber length without reducing yield is one of the major goals of cotton breeding. However, genetic improvement of cotton fiber length by breeding has been a challenge due to the narrow genetic diversity of modern cotton cultivars and negative correlations between fiber quality and yield traits. A multi-parent advanced generation inter-cross (MAGIC) population developed through random mating provides an excellent genetic resource that allows quantitative trait loci (QTL) and causal genes to be identified. Results: An Upland cotton MAGIC population, consisting of 550 recombinant inbred lines (RILs) derived from eleven different cultivars, was used to identify fiber length QTLs and potential genes that contribute to longer fibers. A genome wide association study (GWAS) identified a cluster of single nucleotide polymorphisms (SNPs) on chromosome (Chr.) D11 that is significantly associated with fiber length. Further evaluation of the Chr. D11 genomic region among lines of the MAGIC population detected that 90% of RILs have a D11 haplotype similar to the reference TM-1 genome (D11-ref), whereas 10% of RILs inherited an alternative haplotype from one of the parents (D11-alt). The average length of fibers of D11-alt RILs was significantly shorter compared to D11-ref RILs, suggesting that alleles in the D11-alt haplotype contributed to the inferior fiber quality. RNAseq analysis of the longest and shortest fiber length RILs from D11-ref and D11-alt populations identified 949 significantly differentially expressed genes (DEGs). Gene set enrichment analysis revealed that different functional categories of genes were over-represented during fiber elongation between the four selected RILs. We found 12 genes possessing non-synonymous SNPs (nsSNPs) significantly associated with the fiber length, and three that were highly significant and were clustered at D11:24-Mb, including D11G1928, D11G1929 and D11G1931. Conclusion: The results of this study provide insights into molecular aspects of genetic variation in fiber length and suggests candidate genes for genetic manipulation for cotton improvement.

The results of ANOVA analysis. (XLSX 1088 kb)