Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres

Cotton (Gossypium spp.) is the most important natural fiber crop worldwide. The diversity of Gossypium species also provides an ideal model for investigating evolution and domestication of polyploids. However, the huge and complex cotton genome hinders genomic research. Technical advances in high-throughput sequencing and bioinformatics analysis have now largely overcome these obstacles, bringing about a new era of cotton genomics. Here, we review recent progress in Gossypium genomics based on whole genome sequencing, resequencing, and comparative genomics, which have provided insights about the genomic basis of fiber biogenesis and the landscape of cotton functional genomics. We address current challenges and present multidisciplinary genomics-enabled breeding strategies covering the breadth of high fiber yield, quality, and environmental resilience for future cotton breeding programs.

There have been conflicting results reported about the effect on cotton (Gossypium spp.) lint yield of altering planting and irrigation termination (IT) timing. The objectives of this study were to identify a planting window (PW), on a heat unit (HU) basis, and IT timing, as a function of crop growth stage, for optimum yield potential of Upland (G. hirsutum L.) and American Pima (G. babadense L.) cotton. Two PWs of Upland 'Deltapine 90' (DPL 90), Pima 'S-6', and IT treatments were included in field experiments for 11 site-years. Planting windows were defined as PW1 and PW2 for plantings prior to and following 600 HU accumulated after 1 January, respectively. Two IT treatments were imposed for each planting. Irrigation termination in the desert Southwest generally results in cessation of growth (crop termination). The first IT treatment (IT1), was imposed to ensure full development of bolls set up to cutout, and the second (IT2) was after two additional irrigations. From covariate analysis, there was no evidence of interaction between PW and IT, indicating that these treatments responded the same across the different environments for both cotton species. There were, however, differences in lint yields among treatments. For DPL 90, PW1 IT2 yielded 83 and 97 Ib/acre more than PW1 IT1 and PW2 IT2; and for Pima S-6, PW1 IT2 was 118 and 204 Ib/acre more than PW1 IT1 and PW2 IT2, respectively. Early planting is necessary for optimum yield potential of full-season cotton varieties; with the greatest yield coming from early planting and termination after the development of a second fruiting cycle (PW1 IT2). However, if a reduction in input costs and the avoidance of late-season insect pests are important considerations then cotton should be planted early (300 to 600 HU after 1 Jan) and terminated at the end of the first fruiting cycle (approximately 600 HU past cutout) to maintain the lint yield potential of full-season maturity types of Upland and Pima cotton.

Cotton fiber is an excellent model system of cellulose biosynthesis; however, it has not been widely studied due to the lack of information about the cellulose synthase (CESA) family of genes in cotton. In this study, we initially identified six full-length CESA genes designated as GhCESA5-GhCESA10. Phylogenetic analysis and gene co-expression profiling revealed that CESA1, CESA2, CESA7, and CESA8 were the major isoforms for secondary cell wall biosynthesis, whereas CESA3, CESA5, CESA6, CESA9, and CESA10 should involve in primary cell wall formation for cotton fiber initiation and elongation. Using integrative analysis of gene expression patterns, CESA protein levels, and cellulose biosynthesis in vivo, we detected that CESA8 could play an enhancing role for rapid and massive cellulose accumulation in Gossypium hirsutum and Gossypium barbadense. We found that CESA2 displayed a major expression in non-fiber tissues and that CESA1, a housekeeping gene like, was predominantly expressed in all tissues. Further, a dynamic alteration was observed in cell wall composition and a significant discrepancy was observed between the cotton species during fiber elongation, suggesting that pectin accumulation and xyloglucan reduction might contribute to cell wall transition. In addition, we discussed that callose synthesis might be regulated in vivo for massive cellulose production during active secondary cell wall biosynthesis in cotton fibers.

Cotton fiber was first used in 6000 B.C. The two New World cotton species that are most of today’s production include G. hirsutum (Upland) and G. barbadense (Extra Long Staple [ELS]). The first cotton gin existed by the 5th century A.D (single-roller gin). The next development was the churka gin (double-roller gin) which ginned cotton five times faster than the single-roller gin. The churka gin was widely used in North America by 1750 and ginned both Upland and Sea Island (ELS) cotton. The spike-tooth cotton gin was developed by Eli Whitney in 1794. Hodgen Holmes developed a continuous flow gin with toothed saw blades in 1796. These were a different concept than the double-roller gins. Holmes’ saw gin dominated the industry for Upland cotton (and still does), whereas double-roller gin use continued for Sea Island cotton. In 1840, Fones McCarthy developed a reciprocating-knife roller gin. The saw gin had a significantly higher ginning capacity than the McCarthy gin, so it was used with Upland cotton and the McCarthy roller gin was used with Sea Island cotton to preserve the long-staple cotton’s quality. Sea Island production ceased in 1923 because of the boll weevil, but Pima (ELS) cotton had developed by this time in the Southwest, so roller gin use continued. In 1963, a rotary-knife roller gin was developed that ginned at five times the rate of a reciprocating-knife gin. A high-speed roller gin was developed in 2005 with a ginning capacity, on a per-width basis, comparable to modern-day saw gins.

The single-celled cotton (Gossypium hirsutum) fiber provides an excellent model to investigate how human selection affects phenotypic evolution. To gain insight into the evolutionary genomics of cotton domestication, we conducted comparative transcriptome profiling of developing cotton fibers using RNA-Seq. Analysis of single-celled fiber transcriptomes from four wild and five domesticated accessions from two developmental time points revealed that at least one-third and likely one-half of the genes in the genome are expressed at any one stage during cotton fiber development. Among these, ∼5,000 genes are differentially expressed during primary and secondary cell wall synthesis between wild and domesticated cottons, with a biased distribution among chromosomes. Transcriptome data implicate a number of biological processes affected by human selection, and suggest that the domestication process has prolonged the duration of fiber elongation in modern cultivated forms. Functional analysis suggested that wild cottons allocate greater resources to stress response pathways, while domestication led to reprogrammed resource allocation toward increased fiber growth, possibly through modulating stress-response networks. This first global transcriptomic analysis using multiple accessions of wild and domesticated cottons is an important step toward a more comprehensive systems perspective on cotton fiber evolution. The understanding that human selection over the past 5,000+ years has dramatically re-wired the cotton fiber transcriptome sets the stage for a deeper understanding of the genetic architecture underlying cotton fiber synthesis and phenotypic evolution.

The cotton (Gossypium spp.) fiber is a unique elongated cell that is useful for investigating cell differentiation. Previous studies have demonstrated the importance of factors such as sugar metabolism, the cytoskeleton, and hormones, which are commonly known to be involved in plant cell development, while the secondary metabolites have been less regarded. By mining public data and comparing analyses of fiber from two cotton species (Gossypium hirsutum and Gossypium barbadense), we found that the flavonoid metabolism is active in early fiber cell development. Different flavonoids exhibited distinct effects on fiber development during ovule culture; among them, naringenin (NAR) could significantly retard fiber development. NAR is a substrate of flavanone 3-hydroxylase (F3H), and silencing the F3H gene significantly increased the NAR content of fiber cells. Fiber development was suppressed following F3H silencing, but the overexpression of F3H caused no obvious effects. Significant retardation of fiber growth was observed after the introduction of the F3H-RNA interference segment into the high-flavonoid brown fiber G. hirsutum T586 line by cross. A greater accumulation of NAR as well as much shorter fibers were also observed in the BC1 generation plants. These results suggest that NAR is negatively associated with fiber development and that the metabolism mediated by F3H is important in fiber development, thus highlighting that flavonoid metabolism represents a novel pathway with the potential for cotton fiber improvement.

BackgroundPIN proteins are an important class of auxin polar transport proteins that play an important regulatory role in plant growth and development. However, their characteristics and functions have not been identified in Gossypium barbadense.MethodsPIN family genes were identified in the cotton species G. barbadense, Gossypium hirsutum, Gossypium raimondii, and Gossypium arboreum, and detailed bioinformatics analyses were conducted to explore the roles of these genes in G. barbadense using transcriptome data and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) technology. Functional verification of the genes was performed using virus-induced gene silencing (VIGS) technology.ResultsA total of 138 PIN family genes were identified in the four cotton species; the genes were divided into seven subgroups. GbPIN gene family members were widely distributed on 20 different chromosomes, and most had repeated duplication events. Transcriptome analysis showed that some genes had differential expression patterns in different stages of fiber development. According to ‘PimaS-7’ and ‘5917’ transcript component association analysis, the transcription of five genes was directly related to endogenous auxin content in cotton fibers. qRT-PCR analysis showed that the GbPIN7 gene was routinely expressed during fiber development, and there were significant differences among materials. Transient silencing of the GbPIN7 gene by VIGS led to significantly higher cotton plant growth rates and significantly lower endogenous auxin content in leaves and stems. This study provides comprehensive analyses of the roles of PIN family genes in G. barbadense and their expression during cotton fiber development. Our results will form a basis for further PIN auxin transporter research.

Comparative single-cell transcriptomic map reveals divergence in leaves between two cotton species at cell type resolution

Gossypium barbadense L. (Pima) and Gossypium hirsutum L. (upland) cottons are the two major fibre producing species grown in the south-western United States, where lint yields are adversely affected by high temperatures. In these environments, heat-adapted upland cultivars show higher yields and heat resistance than advanced Pima cultivars. Recent studies with an historical series of commercial Pima cultivars have shown that increases in lint yield and heat resistance are tightly coupled to increases in stomatal conductance and photosynthetic rate, and to decreases in leaf area. In the present study, Pima S-6 and Pima S-7 (advanced Pima cultivars) and Deltapine 90 (advanced upland cultivar) were compared under field and laboratory conditions to determine whether the physiological and morphological gradients found in the Pima historical series extrapolate to upland cotton. In the field, Deltapine 90 showed 25–35% higher stomatal conductance, 35–50% higher photosynthetic rate and 45% smaller leaf area than Pima S-6. The higher photosynthetic rate and stomatal conductance of Deltapine 90 leaves were partially related to their sun-tracking ability. In gas exchange experiments that prevented sun-tracking, the two cultivars had comparable photosynthetic rate as a function of incident radiation, while stomatal conductance was higher in upland cotton. In the 25–35°C range, photosynthetic rate as a function of temperature remained nearly constant in both cultivars, and was higher in upland cotton at all temperatures. Stomatal conductance showed a strong temperature-dependence, and conductance value and the slope of the stomatal response to temperature were higher in Deltapine 90. In progeny from a cross between Deltapine 90 and Pima S-7, the segregation of stomatal conductance in F1 and F2 populations showed a clear genetic component. These results indicate that the differences in photosynthetic rate, stomatal conductance and leaf area associated with increases in lint yield and heat resistance in the Pima historical series are also evident in a comparison between advanced cultivars of upland and Pima cotton. Upland cotton could be used as a source of genetic variation for high stomatal conductance in Pima breeding programs.

Cotton fiber is an important natural textile fiber due to its exceptional length and thickness. These properties arise largely through primary and secondary cell wall synthesis. The cotton fiber of commerce is a cellulosic secondary wall surrounded by a thin cuticulated primary wall, but there were only sparse details available about the polysaccharides in the fiber cell wall of any cotton species. In addition, Gossypium hirsutum (Gh) fiber was known to have an adhesive cotton fiber middle lamella (CFML) that joins adjacent fibers into tissue-like bundles, but it was unknown whether a CFML existed in other commercially important cotton fibers. We compared the cell wall chemistry over the time course of fiber development in Gh and Gossypium barbadense (Gb), the two most important commercial cotton species, when plants were grown in parallel in a highly controlled greenhouse. Under these growing conditions, the rate of early fiber elongation and the time of onset of secondary wall deposition were similar in fibers of the two species, but as expected the Gb fiber had a prolonged elongation period and developed higher quality compared to Gh fiber. The Gb fibers had a CFML, but it was not directly required for fiber elongation because Gb fiber continued to elongate rapidly after CFML hydrolysis. For both species, fiber at seven ages was extracted with four increasingly strong solvents, followed by analysis of cell wall matrix polysaccharide epitopes using antibody-based Glycome Profiling. Together with immunohistochemistry of fiber cross-sections, the data show that the CFML of Gb fiber contained lower levels of xyloglucan compared to Gh fiber. Xyloglucan endo-hydrolase activity was also higher in Gb fiber. In general, the data provide a rich picture of the similarities and differences in the cell wall structure of the two most important commercial cotton species.

Fasciclin-like arabinogalactan (FLA) protein is a cell-wall-associated protein playing crucial roles in regulating plant growth and development, and it was characterized in different plants including Upland cotton (Gossypium hirsutum L.). In cDNA-AFLP analysis of 25 DPA (days post anthesis) fiber mRNA, two FLA gene-related transcripts exhibit differential expression between Sea Island cotton ( G . barbadense L.) and Upland cotton. Based on the transcript-derived fragment, RACE-PCR and realtime PCR technique, GbFLA5 full-length cDNA was isolated and its expression profiles were characterized in both cotton plant tissues and secondary cell wall (SCW) fibers in this study. The 1154 bp GbFLA5 cDNA contains an ORF of 720 bp, encoding GbFLA5 protein of 239 amino acids residues in length with an estimated molecular mass of 25.41 kDa and isoelectric point of 8.63. The deduced GbFLA5 protein contains an N-terminal signal sequence, two AGP-like domains, a single fasciclin-like domain, and a GPI anchor signal sequence. Phylogenetic analysis shows that GbFLA5 protein is homologous to some known SCW-specific expressed FLAs of plant developing xylem, tension wood and cotton fibers. In the SCW deposition stage from 15 to 45 DPA detected, FLA5 maintains a significantly higher expression level in Sea Island cotton fibers than in Upland cotton fibers. The increasing FLA5 transcript abundance coincided with the SCW deposition process and the expression intensity differences coincided with their fiber strength differences between Sea Island cotton and Upland cotton. These expression profile features of GbFLA5 in cotton fibers revealed its tissue-specific and SCW developmental stage-specific expression characters. Further analysis suggested that GbFLA5 is a crucial SCW-specific protein which may contribute to fiber strength by affecting cellulose synthesis and microfibril deposition orientation.

Gossypium L. generally called cotton plants are used indiscriminately in traditional medicine without cognisance to the fact that there are different cotton species and the likelihood of differences in phytochemical content and ultimately, their medicinal capabilities. This study reports the differences in the phytochemical contents, antioxidant and antimicrobial properties of extracts of leaves of two cotton species, Gossypium hirsutum L. and Gossypium barbadense L. Leaf samples from mature G. hirsutum and G. barbadense plants were collected, shadedried and powdered. Phytochemical contents were quantified while antioxidant activity was tested through 1,1- diphenyl-2-picrylhydrazyl (DPPH) scavenging activity and reducing power. Aqueous, decocted aqueous and ethanol leaf extracts of G. hirsutum and G. barbadense were tested against Esherichia coli (known to cause gastrointestinal infections and urinary tract infections), Staphylococcus aureus (known to cause skin infection), Klebsiella pneumoniea and Pseudomonas aeruginosa (known to cause bronchial infections and pneumonia). The results showed that tannins and phenols were present in leaves of G. hirsutum but absent in G. barbadense. Leaves of G. hirsutum contained significantly higher (at p<0.05) amount of total glycosides (156.44 ± 2.05 mg /100g) and flavonoid (120.85 ± 0.32 mg /100g, twice as much) than G. barbadense. Gossypium hirsutum showed higher DPPH free radical scavenging activity and reducing power than G. barbadense. Gossypium hirsutum showed higher zones of inhibition (mm) in all organisms than G. barbadense. It can therefore be concluded that G. hirsutum leaves have higher phytochemical contents as well as antioxidant and antimicrobial activities than G. barbadense. Gossypium hirsutum is therefore recommended for use as against G. barbadense where the choice is available.Keywords: Antimicrobial, Antioxidant, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Gossypium hirsutum, Gossypium barbadense, Phytochemical

Fiber cell initiation and development affect cotton fiber yield and quality. Cotton fiber develops from the ovular epidermis of a seed, and approximately 25-30% of protodermal cells in each cotton ovule develop into fiber. However, the molecular basis for fiber cell development remains elusive. Here, we analyzed single-cell RNA-seq (scRNA-seq) data from over 40,000 cells during early stages of fiber cell development in Upland and Pima cotton and in a naked seed mutant. We found concerted expression changes of 900-1,700 genes in fiber-cell clusters involving gene expression, translation, and peptide biosynthesis, which were substantially delayed or absent in the mutant. Expression of ∼500 and ∼300 genes in Upland and Pima cotton, respectively, was distinguishably different and consistent with overrepresentation of the genes in transcriptional and translational regulation, implying their roles in fiber yield and quality traits. Gene co-expression network analysis (GCNA) of scRNA-seq and scATAC-seq data revealed two modules of fiber gene co-expression networks. One module of the fiber co-expressed genes was associated with elevated chromatin accessibility for transcriptional regulation, whereas the other module of the genes was related to translational regulation and ribosome biogenesis. Indeed, expression of cotton putative translation factor genes was elevated in fiber cell clusters in both Upland and Pima cotton. Finally, cotton transgenic plants expressing promoter::GFP confirmed expression patterns of fiber-expressed GhRDL2_D5 during fiber cell initiation. These single-cell genomic resources provide insights into fiber cell development for breeding and biotechnological improvement of fiber yield and quality in Upland and Pima cotton.

The fiber length-related qFL-A12-5 identified in CSSLs introgressed from Gossypium barbadense into Gossypium hirsutum was fine-mapped to an 18.8 kb region on chromosome A12, leading to the identification of the GhTPR gene as a potential regulator of cotton fiber length. Fiber length is a key determinant of fiber quality in cotton, and it is a key target of artificial selection for breeding and domestication. Although many fiber length-related quantitative trait loci have been identified, there are few reports on their fine mapping or candidate gene validation, thus hampering efforts to understand the mechanistic basis of cotton fiber development. Our previous study identified the qFL-A12-5 associated with superior fiber quality on chromosome A12 in the chromosome segment substitution line (CSSL) MBI7747 (BC4F3:5). A single segment substitution line (CSSL-106) screened from BC6F2 was backcrossed to construct a larger segregation population with its recurrent parent CCRI45, thus enabling the fine mapping of 2852 BC7F2 individuals using denser simple sequence repeat markers to narrow the qFL-A12-5 to an 18.8 kb region of the genome, in which six annotated genes were identified in Gossypium hirsutum. Quantitative real-time PCR and comparative analyses led to the identification of GH_A12G2192 (GhTPR) encoding a tetratricopeptide repeat-like superfamily protein as a promising candidate gene for qFL-A12-5. A comparative analysis of the protein-coding regions of GhTPR among Hai1, MBI7747, and CCRI45 revealed two non-synonymous mutations. The overexpression of GhTPR resulted in longer roots in Arabidopsis, suggesting that GhTPR may regulate cotton fiber development. These results provide a foundation for future efforts to improve cotton fiber length.

Interest in short‐season cotton production is increasing because of late‐season insect problems and, in the West, because of the expense of irrigation. Diverse cultivars of Upland cotton (Gossypium hirsutum L.) and American Pima cotton (G. barbadense L.) were compared at Phoenix, Arizona to evaluate short‐season production using several planting dates in 3 years and three dates of final irrigation in one of the years. The soil was a fine‐loamy, mixed (calcareous), hyperthermic, Anthropic Torrifluvents. Lint yield of Upland cotton was not reduced when planted as late as 7 May, except when irrigation was terminated in mid‐August. In contrast, lint yields of Pima cottons were reduced with each later planting date, except for the earliest planting and latest irrigation and harvest. Plant populations in 2 years had no interaction with planting dates for lint yield. The dates of seedling emergence and dates of early flowering occurred about the same time for Upland and Pima cotton in most plantings and therefore could not account for the differences between species in yield response to dates of planting and final irrigation. The rate of flowering, once started, was much slower for Pima cotton than Upland cotton. ‘Pima S‐5’ reached peak flowering an average of 18 days later than ‘Deltapine 61’ (DPL 61). Weekly lint production far DPL 61 was 4.5 times that for Pima S‐5 in August, twice that of Pima S‐5 in September, but only three‐fifths that of Pima S‐5 in October. Pima strain ‘79–106’ was intermediate between DPL 61 and Pima S‐5 in earliness of lint production. We conclude that Pima cotton requires early planting and nearly full‐season production for highest yield. Early planting is not as critical for Upland cotton, unless early crop termination is planned.