Quantitative genomics-enabled selection for simultaneous improvement of lint yield and seed traits in cotton (Gossypium hirsutum L.).

KEY MESSAGE: A Bayesian linkage disequilibrium-based multiple-locus mixed model identified QTLs for fibre, seed and oil traits and predicted breeding worthiness of test lines, enabling their simultaneous improvement in cotton. Improving cotton seed and oil yields has become increasingly important while continuing to breed for higher lint yield. In this study, a novel Bayesian linkage disequilibrium-based multiple-locus mixed model was developed for QTL identification and genomic prediction (GP). A multi-parent population consisting of 256 recombinant inbred lines, derived from four elite cultivars with distinct combinations of traits, was used in the analysis of QTLs for lint percentage, seed index, lint index and seed oil content and their interrelations. All four traits were moderately heritable and correlated but with no large influence of genotype × environment interactions across multiple seasons. Seven to ten major QTLs were identified for each trait with many being adjacent or overlapping for different trait pairs. A fivefold cross-validation of the model indicated prediction accuracies of 0.46-0.62. GP results based on any two-season phenotypes were strongly correlated with phenotypic means of a pooled analysis of three-season experiments (r = 0.83-0.92). When used for selection of improvement in lint, seed and oil yields, GP captured 40-100% of individuals with comparable lint yields of those selected based on the three-season phenotypic results. Thus, this quantitative genomics-enabled approach can not only decipher the genomic variation underlying lint, seed and seed oil traits and their interrelations, but can provide predictions for their simultaneous improvement. We discuss future breeding strategies in cotton that will enhance the entire value of the crop, not just its fibre.

Insights into the biocontrol and plant growth promotion functions of Bacillus altitudinis strain KRS010 against Verticillium dahliae.

BACKGROUND: Verticillium wilt, caused by the fungus Verticillium dahliae, is a soil-borne vascular fungal disease, which has caused great losses to cotton yield and quality worldwide. The strain KRS010 was isolated from the seed of Verticillium wilt-resistant Gossypium hirsutum cultivar "Zhongzhimian No. 2." RESULTS: The strain KRS010 has a broad-spectrum antifungal activity to various pathogenic fungi as Verticillium dahliae, Botrytis cinerea, Fusarium spp., Colletotrichum spp., and Magnaporthe oryzae, of which the inhibition rate of V. dahliae mycelial growth was 73.97% and 84.39% respectively through confrontation test and volatile organic compounds (VOCs) treatments. The strain was identified as Bacillus altitudinis by phylogenetic analysis based on complete genome sequences, and the strain physio-biochemical characteristics were detected, including growth-promoting ability and active enzymes. Moreover, the control efficiency of KRS010 against Verticillium wilt of cotton was 93.59%. After treatment with KRS010 culture, the biomass of V. dahliae was reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 30.76-folds higher than that in the treatment group (KRS010+Vd991). From a molecular biological aspect, KRS010 could trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Its extracellular metabolites and VOCs inhibited the melanin biosynthesis of V. dahliae. In addition, KRS010 had been characterized as the ability to promote plant growth. CONCLUSIONS: This study indicated that B. altitudinis KRS010 is a beneficial microbe with a potential for controlling Verticillium wilt of cotton, as well as promoting plant growth.

Genome-wide identification and expression-pattern analysis of sulfate transporter (SULTR) gene family in cotton under multiple abiotic stresses and fiber development.

Sulfate transporter (SULTR) proteins are in charge of the transport and absorption on sulfate substances, and have been reported to play vital roles in the biological processes of plant growth and stress response. However, there were few reports of genome-wide identification and expression-pattern analysis of SULTRs in Hibiscus mutabilis. Gossypium genus is a ideal model for studying the allopolyploidy, therefore two diploid species (G. raimondii and G. arboreum) and two tetraploid species (G. hirsutum and G. barbadense) were chosen in this study to perform bioinformatic analyses, identifying 18, 18, 35, and 35 SULTR members, respectively. All the 106 cotton SULTR genes were utilized to construct the phylogenetic tree together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 8 Zea mays ones, which was divided into Group1-Group4. The clustering analyses of gene structures and 10 conserved motifs among the cotton SULTR genes showed the consistent evolutionary relationship with the phylogenetic tree, and the results of gene-duplication identification among the four representative Gossypium species indicated that genome-wide or segment duplication might make main contributions to the expansion of SULTR gene family in cotton. Having conducted the cis-regulatory element analysis in promoter region, we noticed that the existing salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) elements could have influences with expression levels of cotton SULTR genes. The expression patterns of GhSULTR genes were also investigated on the 7 different tissues or organs and the developing ovules and fibers, most of which were highly expressed in root, stem, sepal, receptacel, ovule at 10 DPA, and fiber at 20 and 25 DPA. In addition, more active regulatory were observed in GhSULTR genes responding to multiple abiotic stresses, and 12 highly expressed genes showed the similar expression patterns in the quantitative Real-time PCR experiments under cold, heat, salt, and drought treatments. These findings broaden our insight into the evolutionary relationships and expression patterns of the SULTR gene family in cotton, and provide the valuable information for further screening the vital candidate genes on trait improvement.

Exploring agro-morphological and fiber traits diversity in cotton (G. barbadense L.).

Cotton (Gossypium barbadense L.) is a leading fiber and oilseed crop globally, but genetic diversity among breeding materials is often limited. This study analyzed genetic variability in 14 cotton genotypes from Egypt and other countries, including both cultivated varieties and wild types, using agro-morphological traits and genomic SSR markers. Field experiments were conducted over two seasons to evaluate 12 key traits related to plant growth, yield components, and fiber quality. Molecular diversity analysis utilized 10 SSR primers to generate DNA profiles. The Molecular diversity analysis utilized 10 SSR primers to generate DNA profiles. Data showed wide variation for the morphological traits, with Egyptian genotypes generally exhibiting higher means for vegetative growth and yield parameters. The top-performing genotypes for yield were Giza 96, Giza 94, and Big Black Boll genotypes, while Giza 96, Giza 92, and Giza 70 ranked highest for fiber length, strength, and fineness. In contrast, molecular profiles were highly polymorphic across all genotypes, including 82.5% polymorphic bands out of 212. Polymorphism information content was high for the SSR markers, ranging from 0.76 to 0.86. Genetic similarity coefficients based on the SSR data varied extensively from 0.58 to 0.91, and cluster analysis separated genotypes into two major groups according to geographical origin. The cotton genotypes displayed high diversity in morphology and genetics, indicating sufficient variability in the germplasm. The combined use of physical traits and molecular markers gave a thorough understanding of the genetic diversity and relationships between Egyptian and global cotton varieties. The SSR markers effectively profiled the genotypes and can help select ideal parents for enhancing cotton through hybridization and marker-assisted breeding.

GhCLCc-1, a Chloride Channel Gene from Upland Cotton, Positively Regulates Salt Tolerance by Modulating the Accumulation of Chloride Ions.

The ionic toxicity induced by salinization has adverse effects on the growth and development of crops. However, researches on ionic toxicity and salt tolerance in plants have focused primarily on cations such as sodium ions (Na+), with very limited studies on chloride ions (Cl-). Here, we cloned the homologous genes of Arabidopsis thaliana AtCLCc, GhCLCc-1A/D, from upland cotton (Gossypium hirsutum), which were significantly induced by NaCl or KCl treatments. Subcellular localization showed that GhCLCc-1A/D were both localized to the tonoplast. Complementation of Arabidopsis atclcc mutant with GhCLCc-1 rescued its salt-sensitive phenotype. In addition, the silencing of the GhCLCc-1 gene led to an increased accumulation of Cl- in the roots, stems, and leaves of cotton seedlings under salt treatments, resulting in compromised salt tolerance. And ectopic expression of the GhCLCc-1 gene in Arabidopsis reduced the accumulation of Cl- in transgenic lines under salt treatments, thereby enhancing salt tolerance. These findings elucidate that GhCLCc-1 positively regulates salt tolerance by modulating Cl- accumulation and could be a potential target gene for improving salt tolerance in plants.

Identification of cotton PIP5K genes and role of GhPIP5K9a in primary root development.

Phosphatidylinositol 4 phosphate 5-kinase (PIP5K) is crucial for the phosphatidylinositol (PI) signaling pathway. It plays a significant role in plant growth and development, as well as stress response. However, its effects on cotton are unknown. This study identified PIP5K genes from four cotton species and conducted bioinformatic analyses, with a particular emphasis on the functions of GhPIP5K9a in primary roots. The results showed that cotton PIP5Ks were classified into four subgroups. Analysis of gene structure and motif composition showed obvious conservation within each subgroup. Synteny analysis suggested that the PIP5K gene family experienced significant expansion due to both whole-genome duplication (WGD) and segmental duplication. Transcriptomic data analysis revealed that the majority of GhPIP5K genes had the either low or undetectable levels of expression. Moreover, GhPIP5K9a is highly expressed in the root and was located in plasmalemma. Suppression of GhPIP5K9a transcripts resulted in longer primary roots, longer primary root cells and increased auxin polar transport-related genes expression, and decreased abscisic acid (ABA) content, indicating that GhPIP5K9a negatively regulates cotton primary root growth. This study lays the foundation for further exploration of the role of the PIP5K genes in cotton.

GhMYB52 Like: A Key Factor That Enhances Lint Yield by Negatively Regulating the Lignin Biosynthesis Pathway in Fibers of Upland Cotton (Gossypium hirsutum L.).

In the context of sustainable agriculture and biomaterial development, understanding and enhancing plant secondary cell wall formation are crucial for improving crop fiber quality and biomass conversion efficiency. This is especially critical for economically important crops like upland cotton (Gossypium hirsutum L.), for which fiber quality and its processing properties are essential. Through comprehensive genome-wide screening and analysis of expression patterns, we identified a particularly high expression of an R2R3 MYB transcription factor, GhMYB52 Like, in the development of the secondary cell wall in cotton fiber cells. Utilizing gene-editing technology to generate a loss-of-function mutant to clarify the role of GhMYB52 Like, we revealed that GhMYB52 Like does not directly contribute to cellulose synthesis in cotton fibers but instead represses a subset of lignin biosynthesis genes, establishing it as a lignin biosynthesis inhibitor. Concurrently, a substantial decrease in the lint index, a critical measure of cotton yield, was noted in parallel with an elevation in lignin levels. This study not only deepens our understanding of the molecular mechanisms underlying cotton fiber development but also offers new perspectives for the molecular improvement of other economically important crops and the enhancement of biomass energy utilization.

Fourier Transform Infrared (FT-IR) Spectroscopy and Simple Algorithm Analysis for Rapid and Non-Destructive Assessment of Cotton Fiber Maturity and Crystallinity for Plant Mapping.

Information on boll distribution within a cotton plant is critical to evaluate the adaptation and response of cotton plants to environmental and biotic stress in cotton production. Cotton researchers have applied available conventional fiber measurements, such as the high volume instrument (HVI) and advanced fiber information system (AFIS), to map the location and the timing of boll development and distribution within plants and further to determine within-plant variability of cotton fiber properties. Both HVI and AFIS require numerous cotton bolls combined for the measurement. As an alternative approach, attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy was proposed to measure fiber maturity (MIR) and crystallinity (CIIR) of a sample as little as 0.5 mg lint. Extending fiber maturity and crystallinity measurement into a single boll for node-by-node mapping, FT-IR method might be advantageous due to less sampling amount compared with HVI and AFIS methods. Results showed that FT-IR technique enabled the evaluation of fiber MIR and CIIR at a boll level, which resulted in average MIR and CIIR values highly correlated with HVI micronaire (MIC) and AFIS maturity ratio (M). Hence, FT-IR technique possesses a good potential for a rapid and non-destructive node-by-node mapping of cotton boll maturity and crystallinity distribution.

A Genome-Wide Analysis of the CEP Gene Family in Cotton and a Functional Study of GhCEP46-D05 in Plant Development.

C-TERMINALLY ENCODED PEPTIDEs (CEPs) are a class of peptide hormones that have been shown in previous studies to play an important role in regulating the development and response to abiotic stress in model plants. However, their role in cotton is not well understood. In this study, we identified 54, 59, 34, and 35 CEP genes from Gossypium hirsutum (2n = 4x = 52, AD1), G. barbadense (AD2), G. arboreum (2n = 2X = 26, A2), and G. raimondii (2n = 2X = 26, D5), respectively. Sequence alignment and phylogenetic analyses indicate that cotton CEP proteins can be categorized into two subgroups based on the differentiation of their CEP domain. Chromosomal distribution and collinearity analyses show that most of the cotton CEP genes are situated in gene clusters, suggesting that segmental duplication may be a critical factor in CEP gene expansion. Expression pattern analyses showed that cotton CEP genes are widely expressed throughout the plant, with some genes exhibiting specific expression patterns. Ectopic expression of GhCEP46-D05 in Arabidopsis led to a significant reduction in both root length and seed size, resulting in a dwarf phenotype. Similarly, overexpression of GhCEP46-D05 in cotton resulted in reduced internode length and plant height. These findings provide a foundation for further investigation into the function of cotton CEP genes and their potential role in cotton breeding.

Machine learning ensembles, neural network, hybrid and sparse regression approaches for weather based rainfed cotton yield forecast.

Cotton is a major economic crop predominantly cultivated under rainfed situations. The accurate prediction of cotton yield invariably helps farmers, industries, and policy makers. The final cotton yield is mostly determined by the weather patterns that prevail during the crop growing phase. Crop yield prediction with greater accuracy is possible due to the development of innovative technologies which analyses the bigdata with its high-performance computing abilities. Machine learning technologies can make yield prediction reasonable and faster and with greater flexibility than process based complex crop simulation models. The present study demonstrates the usability of ML algorithms for yield forecasting and facilitates the comparison of different models. The cotton yield was simulated by employing the weekly weather indices as inputs and the model performance was assessed by nRMSE, MAPE and EF values. Results show that stacked generalised ensemble model and artificial neural networks predicted the cotton yield with lower nRMSE, MAPE and higher efficiency compared to other models. Variable importance studies in LASSO and ENET model found minimum temperature and relative humidity as the main determinates of cotton yield in all districts. The models were ranked based these performance metrics in the order of Stacked generalised ensemble > ANN > PCA ANN > SMLR ANN > LASSO> ENET > SVM > PCA SMLR > SMLR SVM > SMLR. This study shows that stacked generalised ensembling and ANN method can be used for reliable yield forecasting at district or county level and helps stakeholders in timely decision-making.

Changes in carbohydrate distribution in cotton photosynthetic organs and increase in boll weight reduce yield loss under high temperature.

Yield of cotton (Gossypium hirsutum) does not always fall with high temperature (HT) even though this induces significant reductions in fruit retention. To investigate the underlying mechanisms, a greenhouse experiment was conducted with two temperature regimes [control treatment, 28 °C; high temperature (HT), 34 °C] for 7 d. Results showed HT did not significantly influence cotton yield, but reduced boll number and increased boll weight. The 13C distribution ratio of the leaf subtending the cotton boll (LSCB) decreased while that of the cotton boll increased under HT. Transcriptomic and proteomic analyses of the LSCB revealed up-regulated genes involved in cytokinin and jasmonic acid synthesis, as well as SWEET15 (GH_D01G0218), which positively regulated photosynthesis and transport photosynthate, ultimately leading to increased boll weight. After 7 d recovery from HT, the 13C distribution ratio of the LSCB increased while that of the cotton boll decreased. However, boll weight still increased, which was related to increased amylase and sucrose phosphate synthase activities and up-regulated sucrose transport genes in the main-stem leaf and capsule wall. Thus, both accelerated sucrose synthesis and transport in the LSCB under HT and increased sucrose supply ability of the main-stem leaf and capsule wall after recovery from HT contributed to an increased boll weight, which finally maintained cotton yield.

Single-cell transcriptome atlas reveals somatic cell embryogenic differentiation features during regeneration.

Understanding somatic cell totipotency remains a challenge facing scientific inquiry today. Plants display remarkable cell totipotency expression, illustrated by single-cell differentiation during somatic embryogenesis (SE) for plant regeneration. Determining cell identity and exploring gene regulation in such complex heterogeneous somatic cell differentiation have been major challenges. Here, we performed high-throughput single-cell sequencing assays to define the precise cellular landscape and revealed the modulation mode of marker genes during embryogenic differentiation in cotton (Gossypium hirsutum L.) as the crop for biotechnology application. We demonstrated that nonembryogenic calli (NEC) and primary embryogenic calli (PEC) tissues were composed of heterogeneous cells that could be partitioned into four broad populations with six distinct cell clusters. Enriched cell clusters and cell states were identified in NEC and PEC samples, respectively. Moreover, a broad repertoire of new cluster-specific genes and associated expression modules were identified. The energy metabolism, signal transduction, environmental adaptation, membrane transport pathways, and a series of transcription factors were preferentially enriched in cell embryogenic totipotency expression. Notably, the SE-ASSOCIATED LIPID TRANSFER PROTEIN (SELTP) gene dose-dependently marked cell types with distinct embryogenic states and exhibited a parabolic curve pattern along the somatic cell embryogenic differentiation trajectory, suggesting that SELTP could serve as a favorable quantitative cellular marker for detecting embryogenic expression at the single-cell level. In addition, RNA velocity and Scissor analysis confirmed the pseudo-temporal model and validated the accuracy of the scRNA-seq data, respectively. This work provides valuable marker-genes resources and defines precise cellular taxonomy and trajectory atlases for somatic cell embryogenic differentiation in plant regeneration.

Cotton (Gossypium hirsutum) VIRMA as an N6-Methyladenosine RNA Methylation Regulator Participates in Controlling Chloroplast-Dependent and Independent Leaf Development.

N6-methyladenosine (m6A) is one of the most abundant internal modifications of mRNA, which plays important roles in gene expression regulation, and plant growth and development. Vir-like m6A methyltransferase associated (VIRMA) serves as a scaffold for bridging the catalytic core components of the m6A methyltransferase complex. The role of VIRMA in regulating leaf development and its related mechanisms have not been reported. Here, we identified and characterized two upland cotton (Gossypium hirsutum) VIRMA genes, named as GhVIR-A and GhVIR-D, which share 98.5% identity with each other. GhVIR-A and GhVIR-D were ubiquitously expressed in different tissues and relatively higher expressed in leaves and main stem apexes (MSA). Knocking down the expression of GhVIR genes by the virus-induced gene silencing (VIGS) system influences leaf cell size, cell shape, and total cell numbers, thereby determining cotton leaf morphogenesis. The dot-blot assay and colorimetric experiment showed the ratio of m6A to A in mRNA is lower in leaves of GhVIR-VIGS plants compared with control plants. Messenger RNA (mRNA) high-throughput sequencing (RNA-seq) and a qRT-PCR experiment showed that GhVIRs regulate leaf development through influencing expression of some transcription factor genes, tubulin genes, and chloroplast genes including photosystem, carbon fixation, and ribosome assembly. Chloroplast structure, chlorophyll content, and photosynthetic efficiency were changed and unsuitable for leaf growth and development in GhVIR-VIGS plants compared with control plants. Taken together, our results demonstrate GhVIRs function in cotton leaf development by chloroplast dependent and independent pathways.

GhTCE1-GhTCEE1 dimers regulate transcriptional reprogramming during wound-induced callus formation in cotton.

Wounded plant cells can form callus to seal the wound site. Alternatively, wounding can cause adventitious organogenesis or somatic embryogenesis. These distinct developmental pathways require specific cell fate decisions. Here, we identify GhTCE1, a basic helix-loop-helix family transcription factor, and its interacting partners as a central regulatory module of early cell fate transition during in vitro dedifferentiation of cotton (Gossypium hirsutum). RNAi- or CRISPR/Cas9-mediated loss of GhTCE1 function resulted in excessive accumulation of reactive oxygen species (ROS), arrested callus cell elongation, and increased adventitious organogenesis. In contrast, GhTCE1-overexpressing tissues underwent callus cell growth, but organogenesis was repressed. Transcriptome analysis revealed that several pathways depend on proper regulation of GhTCE1 expression, including lipid transfer pathway components, ROS homeostasis, and cell expansion. GhTCE1 bound to the promoters of the target genes GhLTP2 and GhLTP3, activating their expression synergistically, and the heterodimer TCE1-TCEE1 enhances this activity. GhLTP2- and GhLTP3-deficient tissues accumulated ROS and had arrested callus cell elongation, which was restored by ROS scavengers. These results reveal a unique regulatory network involving ROS and lipid transfer proteins, which act as potential ROS scavengers. This network acts as a switch between unorganized callus growth and organized development during in vitro dedifferentiation of cotton cells.

Germination and seed persistence of Amaranthus retroflexus and Amaranthus viridis: Two emerging weeds in Australian cotton and other summer crops.

Redroot pigweed (Amaranthus retroflexus L.) and slender amaranth (Amaranthus viridis L.) are becoming problematic weeds in summer crops, including cotton in Australia. A series of laboratory and field experiments were performed to examine the germination ecology, and seed persistence of two populations of A. retroflexus and A. viridis collected from the Goondiwindi and Gatton regions of Australia. Both populations of A. retroflexus and A. viridis behaved similarly to different environmental conditions. Initial dormancy was observed in fresh seeds of both species; however, germination reached maximum after an after-ripening period of two months at room temperature. Light was not a mandatory prerequisite for germination of both species as they could germinate under complete darkness. Although both species showed very low germination at the alternating day/night temperature of 15/5 C, these species germinated more than 40% between ranges of 25/15 C to 35/25 C. Maximum germination of A. retroflexus (93%) and A. viridis (86%) was observed at 35/25 C and 30/20, respectively. Germination of A. retroflexus and A. viridis was completely inhibited at osmotic potentials of -1.0 and -0.6 MPa, respectively. No germination was observed in both species at the sodium chloride concentration of 200 mM. A. retroflexus seedling emergence (87%) was maximum from the seeds buried at 1 cm while the maximum germination of A. viridis (72%) was observed at the soil surface. No seedling emergence was observed from a burial depth of 8 cm for both species. In both species, seed persistence increased with increasing burial depth. At 24 months after seed placement, seed depletion ranged from 75% (10 cm depth) to 94% (soil surface) for A. retroflexus, and ranged from 79% to 94% for A. viridis, respectively. Information gained from this study will contribute to an integrated control programs for A. retroflexus and A. viridis.

Founder transformants of cotton (Gossypium hirsutum L.) obtained through the introduction of DS-Red, Rec, Rep and CRISPR/Cas9 expressing constructs for developing base lines of recombinase mediated gene stacking.

Cotton being the major fiber crop across the world is exposed to numerous biotic and abiotic stresses. Genetic transformation of cotton is vital to meet the world's food, feed and fiber demands. Genetic manipulation by randomly transferring the genes emanate variable gene expression. Targeted gene insertion by latest genome editing tools results in predictable expression of genes at a specified location. Gene stacking technology emerged as an adaptive strategy to combat biotic and abiotic stresses by integrating 2-3 genes simultaneously and at a specific site to avoid variable gene expression at diverse locations. This study explains the development of cotton's founder transformants to be used as a base line for multiple gene stacking projects. We introduced Cre and PhiC31 mediated recombination sites to specify the locus of incoming genes. CRISPR-Cas9 gene was integrated for developing CRISPR based founder lines of cotton. Cas9 gene along with gRNA was integrated to target Rep (replication) region of cotton leaf curl virus. Replication region of virus was specifically targeted to diminish further proliferation and preventing the virus to develop new strains. To successfully develop these primary transformants, a model transformation system has been optimized with the red color visualization (DS-Red). Following red color transformation system, three baselines with recombination specified site (Rec), targeted replication region (Rep) and Cas9 founder lines have been developed. These founder transformants are useful for developing recombinase mediated and CRISPR/Cas9 based originator lines of cotton. Moreover, these transformants will set up a base system for all the recombinase mediated gene stacking projects.

Identification and expression analysis of arabinogalactan protein genes in cotton reveal the function of GhAGP15 in Verticillium dahliae resistance.

Verticillium wilt, primarily caused by the fungal pathogen Verticillium dahliae, is a serious disease in cotton. Arabinogalactan proteins (AGPs), a class of hydroxyproline-rich glycoproteins, have been widely implicated in plant growth and environmental adaptation. The purpose of this study is to identify and characterize AGP members in cotton plants and explore their roles in responding to environmental stressors. In total, 65 GhAGP members were identified in upland cotton (Gossypium hirsutum), along with 43, 35, and 37 AGP members that were also identified in G. barbadense, G. arboreum, and G. raimondii, respectively. According to gene structure and protein domains analysis, GhAGP genes in upland cotton are highly conserved. Meanwhile, tandem duplication events have occurred frequently throughout cotton's evolutionary history. Expression analysis showed that GhAGP genes were widely expressed during growth and development and in response to abiotic stressors. Many cis-elements related to hormonal responses and environmental stressors were detected in GhAGP promoter regions. GhAGP genes participate in responding to cold, drought, and salt stress, and were sensitive to ET signaling. Furthermore, the expression level of GhAGP15 was elevated during V. dahliae infection and resistance against V. dahliae in upland cotton was significantly weakened by silencing GhAGP15 using a virus-induced gene silencing (VIGS) approach. Our results further suggest that the function of GhAGP15 in V. dahliae resistance might be involved in regulation of the JA, SA, and reactive oxygen species (ROS) pathways. The comprehensive analysis of AGP genes in cotton performed in this study provides a basic framework for further functional research of these genes.

RNA-Seq with a novel glabrous-ZM24fl reveals some key lncRNAs and the associated targets in fiber initiation of cotton.

BACKGROUND: Cotton fiber is an important natural resource for textile industry and an excellent model for cell biology study. Application of glabrous mutant cotton and high-throughput sequencing facilitates the identification of key genes and pathways for fiber development and cell differentiation and elongation. LncRNA is a type of ncRNA with more than 200 nt in length and functions in the ways of chromatin modification, transcriptional and post-transcriptional modification, and so on. However, the detailed lncRNA and associated mechanisms for fiber initiation are still unclear in cotton. RESULTS: In this study, we used a novel glabrous mutant ZM24fl, which is endowed with higher somatic embryogenesis, and functions as an ideal receptor for cotton genetic transformation. Combined with the high-throughput sequencing, fatty acid pathway and some transcription factors such as MYB, ERF and bHLH families were identified the important roles in fiber initiation; furthermore, 3,288 lncRNAs were identified, and some differentially expressed lncRNAs were also analyzed. From the comparisons of ZM24_0 DPA vs ZM24_-2 DPA and fl_0 DPA vs ZM24_0 DPA, one common lncRNA MSTRG 2723.1 was found that function upstream of fatty acid metabolism, MBY25-mediating pathway, and pectin metabolism to regulate fiber initiation. In addition, other lncRNAs MSTRG 3390.1, MSTRG 48719.1, and MSTRG 31176.1 were also showed potential important roles in fiber development; and the co-expression analysis between lncRNAs and targets showed the distinct models of different lncRNAs and complicated interaction between lncRNAs in fiber development of cotton. CONCLUSIONS: From the above results, a key lncRNA MSTRG 2723.1 was identified that might mediate some key genes transcription of fatty acid metabolism, MYB25-mediating pathway, and pectin metabolism to regulate fiber initiation of ZM24 cultivar. Co-expression analysis implied that some other important lncRNAs (e.g., MSTRG 3390.1, MSTRG 48719.1, and MSTRG 31176.1) were also showed the different regulatory model and interaction between them, which proposes some valuable clues for the lncRNAs associated mechanisms in fiber development.

An Unexpected Regulatory Sequence from Rho-Related GTPase6 Confers Fiber-Specific Expression in Upland Cotton.

Cotton fibers, single seed trichomes derived from ovule epidermal cells, are the major source of global textile fibers. Fiber-specific promoters are desirable to study gene function and to modify fiber properties during fiber development. Here, we revealed that Rho-related GTPase6 (GhROP6) was expressed preferentially in developing fibers. A 1240 bp regulatory region of GhROP6, which contains a short upstream regulatory sequence, the first exon, and the partial first intron, was unexpectedly isolated and introduced into transgenic cotton for analyzing promoter activity. The promoter of GhROP6 (proChROP6) conferred a specific expression in ovule surface, but not in the other floral organs and vegetative tissues. Reverse transcription PCR analysis indicated that proGhROP6 directed full-length transcription of the fused ß-glucuronidase (GUS) gene. Further investigation of GUS staining showed that proChROP6 regulated gene expression in fibers and ovule epidermis from fiber initiation to cell elongation stages. The preferential activity was enriched in fiber cells after anthesis and reached to peak on flowering days. By comparison, proGhROP6 was a mild promoter with approximately one-twenty-fifth of the strength of the constitutive promoter CaMV35S. The promoter responded to high-dosage treatments of auxin, gibberellin and salicylic acid and slightly reduced GUS activity under the in vitro treatment. Collectively, our data suggest that the GhROP6 promoter has excellent activity in initiating fibers and has potential for bioengineering of cotton fibers.

Biochemical and Expression Analyses Revealed the Involvement of Proanthocyanidins and/or Their Derivatives in Fiber Pigmentation of Gossypium stocksii.

The wild cotton species Gossypium stocksii produces a brown fiber that provides a valuable resource for the color improvement of naturally colored cotton (NCC) fiber. However, the biochemical basis and molecular mechanism of its fiber pigmentation remain unclear. Herein, we analyzed the dynamics of proanthocyanidins (PAs) accumulation in developing the fiber of G. stocksii, which suggested a similar role of PAs and/or their derivatives in the fiber coloration of G. stocksii. In addition, comparative transcriptomics analyses revealed that the PA biosynthetic genes were expressed at higher levels and for a longer period in developing fibers of G. stocksii than G. arboreum (white fiber), and the transcription factors, such as TT8, possibly played crucial regulatory roles in regulating the PA branch genes. Moreover, we found that the anthocyanidin reductase (ANR) was expressed at a higher level than the leucoanthocyanidin reductases (LARs) and significantly upregulated during fiber elongation, suggesting a major role of ANR in PA synthesis in G. stocksii fiber. In summary, this work revealed the accumulation of PAs and the expression enhancement of PA biosynthetic genes in developing fibers of G. stocksii. We believe this work will help our understanding of the molecular mechanisms of cotton fiber coloration and further promote the future breeding of novel NCCs.