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Within-sample variation in cotton fiber length 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., Competing Interests: Author DJ was employed by the company Cotton Incorporated. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision. The study was partially supported by Cotton Incorporated. The private funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 Kim, Thyssen, Delhom, Fang, Naoumkina, Florane, Li, Jenkins, McCarty, Zeng, Campbell and Jones.)

Key Message: Significant associations between candidate genes and six major cotton fiber quality traits were identified in a MAGIC population using GWAS and whole genome sequencing. Upland cotton (Gossypium hirsutum L.) is the world's major renewable source of fibers for textiles. To identify causative genetic variants that influence the major agronomic measures of cotton fiber quality, which are used to set discount or premium prices on each bale of cotton in the USA, we measured six fiber phenotypes from twelve environments, across three locations and 7 years. Our 550 recombinant inbred lines were derived from a multi-parent advanced generation intercross population and were whole-genome-sequenced at 3× coverage, along with the eleven parental cultivars at 20× coverage. The segregation of 473,517 single nucleotide polymorphisms (SNPs) in this population, including 7506 non-synonymous mutations, was combined with phenotypic data to identify seven highly significant fiber quality loci. At these loci, we found fourteen genes with non-synonymous SNPs. Among these loci, some had simple additive effects, while others were only important in a subset of the population. We observed additive effects for elongation and micronaire, when the three most significant loci for each trait were examined. In an informative subset where the major multi-trait locus on chromosome A07:72-Mb was fixed, we unmasked the identity of another significant fiber strength locus in gene Gh_D13G1792 on chromosome D13. The micronaire phenotype only revealed one highly significant genetic locus at one environmental location, demonstrating a significant genetic by environment component. These loci and candidate causative variant alleles will be useful to cotton breeders for marker-assisted selection with minimal linkage drag and potential biotechnological applications.

The interactions of nanoparticles with polymer hosts have important implications for directing the macroscopic properties of composite fibers, yet little is known about such interactions with hierarchically ordered natural polymers due to the difficulty of achieving uniform dispersion of nanoparticles within semi-crystalline natural fiber. In this study we have homogeneously dispersed silver nanoparticles throughout an entire volume of cotton fiber. The resulting electrostatic interaction and distinct supramolecular structure of the cotton fiber provided a favorable environment for the controlled formation of nanoparticles (12 ± 3 nm in diameter). With a high surface-to-volume ratio, the extensive interfacial contacts of the nanoparticles efficiently "glued" the structural elements of microfibrils together, producing a unique inorganic-organic hybrid substructure that reinforced the multilayered architecture of the cotton fiber.

Background: Cotton supplies a great majority of natural fiber for the global textile industry. The negative correlation between yield and fiber quality has hindered breeders' ability to improve these traits simultaneously. A multi-parent advanced generation inter-cross (MAGIC) population developed through random-mating of multiple diverse parents has the ability to break this negative correlation. Genotyping-by-sequencing (GBS) is a method that can rapidly identify and genotype a large number of single nucleotide polymorphisms (SNP). Genotyping a MAGIC population using GBS technologies will enable us to identify marker-trait associations with high resolution., Results: An Upland cotton MAGIC population was developed through random-mating of 11 diverse cultivars for five generations. In this study, fiber quality data obtained from four environments and 6071 SNP markers generated via GBS and 223 microsatellite markers of 547 recombinant inbred lines (RILs) of the MAGIC population were used to conduct a genome wide association study (GWAS). By employing a mixed linear model, GWAS enabled us to identify markers significantly associated with fiber quantitative trait loci (QTL). We identified and validated one QTL cluster associated with four fiber quality traits [short fiber content (SFC), strength (STR), length (UHM) and uniformity (UI)] on chromosome A07. We further identified candidate genes related to fiber quality attributes in this region. Gene expression and amino acid substitution analysis suggested that a regeneration of bulb biogenesis 1 (GhRBB1_A07) gene is a candidate for superior fiber quality in Upland cotton. The DNA marker CFBid0004 designed from an 18 bp deletion in the coding sequence of GhRBB1_A07 in Acala Ultima is associated with the improved fiber quality in the MAGIC RILs and 105 additional commercial Upland cotton cultivars., Conclusion: Using GBS and a MAGIC population enabled more precise fiber QTL mapping in Upland cotton. The fiber QTL and associated markers identified in this study can be used to improve fiber quality through marker assisted selection or genomic selection in a cotton breeding program. Target manipulation of the GhRBB1_A07 gene through biotechnology or gene editing may potentially improve cotton fiber quality.

Some naturally coloured brown cotton fibres from accessions of Gossypium hirsutum L. can be used to make textiles with enhanced flame retardancy (FR). Several independent brown fibre loci have been identified and mapped to chromosomes, but the underlying genes have not yet been identified, and the mechanism of lint fibre FR is not yet fully understood. In this study, we show that both the brown colour and enhanced FR of the Lc1 lint colour locus are linked to a 1.4Mb inversion on chromosome A07 that is immediately upstream of a gene with similarity to Arabidopsis TRANSPARENT TESTA 2 (TT2). As a result of the alternative upstream sequence, the transcription factor GhTT2_A07 is highly up-regulated in developing fibres. In turn, genes in the phenylpropanoid metabolic pathway are activated, leading to biosynthesis of proanthocyanidins and accumulation of inorganic elements. We show that enhanced FR and anthocyanin precursors appear in developing brown fibres well before the brown colour is detectible, demonstrating for the first time that the polymerized proanthocyanidins that constitute the brown colour are not the source of enhanced FR. Identifying the particular colourless metabolite that provides Lc1 cotton with enhanced FR could help minimize the use of synthetic chemical flame retardant additives in textiles., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Key Message: Three QTL regions controlling three fiber quality traits were validated and further fine-mapped with 27 new single nucleotide polymorphism (SNP) markers. Transcriptome analysis suggests that receptor-like kinases found within the validated QTLs are potential candidate genes responsible for superior fiber strength in cotton line MD52ne. Fiber strength, length, maturity and fineness determine the market value of cotton fibers and the quality of spun yarn. Cotton fiber strength has been recognized as a critical quality attribute in the modern textile industry. Fine mapping along with quantitative trait loci (QTL) validation and candidate gene prediction can uncover the genetic and molecular basis of fiber quality traits. Four previously-identified QTLs (qFBS-c3, qSFI-c14, qUHML-c14 and qUHML-c24) related to fiber bundle strength, short fiber index and fiber length, respectively, were validated using an F3 population that originated from a cross of MD90ne × MD52ne. A group of 27 new SNP markers generated from mapping-by-sequencing (MBS) were placed in QTL regions to improve and validate earlier maps. Our refined QTL regions spanned 4.4, 1.8 and 3.7 Mb of physical distance in the Gossypium raimondii reference genome. We performed RNA sequencing (RNA-seq) of 15 and 20 days post-anthesis fiber cells from MD52ne and MD90ne and aligned reads to the G. raimondii genome. The QTL regions contained 21 significantly differentially expressed genes (DEGs) between the two near-isogenic parental lines. SNPs that result in non-synonymous substitutions to amino acid sequences of annotated genes were identified within these DEGs, and mapped. Taken together, transcriptome and amino acid mutation analysis indicate that receptor-like kinase pathway genes are likely candidates for superior fiber strength and length in MD52ne. MBS along with RNA-seq demonstrated a powerful strategy to elucidate candidate genes for the QTLs that control complex traits in a complex genome like tetraploid upland cotton.

Cotton seed trichomes are the most important source of natural fibers globally. The major fiber thickness properties influence the price of the raw material, and the quality of the finished product. The recessive immature fiber (im) gene reduces the degree of fiber cell wall thickening by a process that was previously shown to involve mitochondrial function in allotetraploid Gossypium hirsutum Here, we present the fine genetic mapping of the im locus, gene expression analysis of annotated proteins near the locus, and association analysis of the linked markers. Mapping-by-sequencing identified a 22-bp deletion in a pentatricopeptide repeat (PPR) gene that is completely linked to the immature fiber phenotype in 2837 F2 plants, and is absent from all 163 cultivated varieties tested, although other closely linked marker polymorphisms are prevalent in the diversity panel. This frame-shift mutation results in a transcript with two long open reading frames: one containing the N-terminal transit peptide that targets mitochondria, the other containing only the RNA-binding PPR domains, suggesting that a functional PPR protein cannot be targeted to mitochondria in the im mutant. Taken together, these results suggest that PPR gene Gh_A03G0489 is involved in the cotton fiber wall thickening process, and is a promising candidate gene at the im locus. Our findings expand our understanding of the molecular mechanisms that modulate cotton fiber fineness and maturity, and may facilitate the development of cotton varieties with superior fiber attributes., (Copyright © 2016 Thyssen et al.)

Background: Individual fiber strength is an important quality attribute that greatly influences the strength of the yarn spun from cotton fibers. Fiber strength is usually measured from bundles of fibers due to the difficulty of reliably measuring strength from individual cotton fibers. However, bundle fiber strength (BFS) is not always correlated with yarn strength since it is affected by multiple fiber properties involved in fiber-to-fiber interactions within a bundle in addition to the individual fiber strength. Molecular mechanisms responsible for regulating individual fiber strength remain unknown. Gossypium hirsutum near isogenic lines (NILs), MD52ne and MD90ne showing variations in BFS provide an opportunity for dissecting the regulatory mechanisms involved in individual fiber strength., Results: Comprehensive fiber property analyses of the NILs revealed that the superior bundle strength of MD52ne fibers resulted from high individual fiber strength with minor contributions from greater fiber length. Comparative transcriptome analyses of the NILs showed that the superior bundle strength of MD52ne fibers was potentially related to two signaling pathways: one is ethylene and the interconnected phytohormonal pathways that are involved in cotton fiber elongation, and the other is receptor-like kinases (RLKs) signaling pathways that are involved in maintaining cell wall integrity. Multiple RLKs were differentially expressed in MD52ne fibers and localized in genomic regions encompassing the strength quantitative trait loci (QTLs). Several candidate genes involved in crystalline cellulose assembly were also up-regulated in MD52ne fibers while the secondary cell wall was produced., Conclusion: Comparative phenotypic and transcriptomic analyses revealed differential expressions of the genes involved in crystalline cellulose assembly, ethylene and RLK signaling pathways between the MD52ne and MD90ne developing fibers. Ethylene and its phytohormonal network might promote the elongation of MD52ne fibers and indirectly contribute to the bundle strength by potentially improving fiber-to-fiber interactions. RLKs that were suggested to mediate a coordination of cell elongation and SCW biosynthesis in other plants might be candidate genes for regulating cotton fiber cell wall assembly and strength.

Background: Cotton fiber maturity is an important factor for determining the commercial value of cotton. How fiber cell wall development affects fiber maturity is not well understood. A comparison of fiber cross-sections showed that an immature fiber (im) mutant had lower fiber maturity than its near isogenic wild type, Texas marker-1 (TM-1). The availability of the im mutant and TM-1 provides a unique way to determine molecular mechanisms regulating cotton fiber maturity., Results: Transcriptome analysis showed that the differentially expressed genes (DEGs) in the im mutant fibers grown under normal stress conditions were similar to those in wild type cotton fibers grown under severe stress conditions. The majority of these DEGs in the im mutant were related to stress responses and cellular respiration. Stress is known to reduce the activity of a classical respiration pathway responsible for energy production and reactive oxygen species (ROS) accumulation. Both energy productions and ROS levels in the im mutant fibers are expected to be reduced if the im mutant is associated with stress responses. In accord with the prediction, the transcriptome profiles of the im mutant showed the same alteration of transcriptional regulation that happened in energy deprived plants in which expressions of genes associated with cell growth processes were reduced whereas expressions of genes associated with recycling and transporting processes were elevated. We confirmed that ROS production in developing fibers from the im mutant was lower than that from the wild type. The lower production of ROS in the im mutant fibers might result from the elevated levels of alternative respiration induced by stress., Conclusion: The low degree of fiber cell wall thickness of the im mutant fibers is associated with deregulation of the genes involved in stress responses and cellular respiration. The reduction of ROS levels and up-regulation of the genes involved in alternative respirations suggest that energy deprivation may occur in the im mutant fibers.

Background: Cotton fiber length is very important to the quality of textiles. Understanding the genetics and physiology of cotton fiber elongation can provide valuable tools to the cotton industry by targeting genes or other molecules responsible for fiber elongation. Ligon Lintless-1 (Li1) is a monogenic mutant in Upland cotton (Gossypium hirsutum) which exhibits an early cessation of fiber elongation resulting in very short fibers (< 6 mm) at maturity. This presents an excellent model system for studying the underlying molecular and cellular processes involved with cotton fiber elongation. Previous reports have characterized Li1 at early cell wall elongation and during later secondary cell wall synthesis, however there has been very limited analysis of the transition period between these developmental time points., Results: Physical and morphological measurements of the Li1 mutant fibers were conducted, including measurement of the cellulose content during development. Affymetrix microarrays were used to analyze transcript profiles at the critical developmental time points of 3 days post anthesis (DPA), the late elongation stage of 12 DPA and the early secondary cell wall synthesis stage of 16 DPA. The results indicated severe disruption to key hormonal and other pathways related to fiber development, especially pertaining to the transition stage from elongation to secondary cell wall synthesis. Gene Ontology enrichment analysis identified several key pathways at the transition stage that exhibited altered regulation. Genes involved in ethylene biosynthesis and primary cell wall rearrangement were affected, and a primary cell wall-related cellulose synthase was transcriptionally repressed. Linkage mapping using a population of 2,553 F2 individuals identified SSR markers associated with the Li1 genetic locus on chromosome 22. Linkage mapping in combination with utilizing the diploid G. raimondii genome sequences permitted additional analysis of the region containing the Li1 gene., Conclusions: The early termination of fiber elongation in the Li1 mutant is likely controlled by an early upstream regulatory factor resulting in the altered regulation of hundreds of downstream genes. Several elongation-related genes that exhibited altered expression profiles in the Li1 mutant were identified. Molecular markers closely associated with the Li1 locus were developed. Results presented here will lay the foundation for further investigation of the genetic and molecular mechanisms of fiber elongation.

Cotton fiber fineness and maturity measured indirectly as micronaire (MIC) are important properties of determining fiber grades in the textile market. To understand the genetic control and molecular mechanisms of fiber fineness and maturity, we studied two near isogenic lines, Gossypium hirsutum, Texas Marker-1 wild type (TM-1) and immature fiber (im) mutant showing a significant difference in MIC values. The fibers from im mutant plants were finer and less mature with lower MIC values than those from the recurrent parent, TM-1. A comprehensive fiber property analysis of TM-1 and im mutant showed that the lower MIC of fibers in im mutant was due to the lower degree of fiber cell wall thickening as compared to the TM-1 fibers. Using an F(2) population comprising 366 progenies derived from a cross between TM-1 and im mutant, we confirmed that the immature fiber phenotype present in a mutant plant was controlled by one single recessive gene im. Furthermore, we identified 13 simple sequence repeat markers that were closely linked to the im gene located on chromosome 3. Molecular markers associated with the im gene will lay the foundation to further investigate genetic information required for improving cotton fiber fineness and maturity.