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Improving plant resistance to Verticillium wilt (VW), which causes massive losses in Gossypium hirsutum, is a global challenge. Crop plants need to efficiently allocate their limited energy resources to maintain a balance between growth and defense. However, few transcriptional regulators specifically respond to Verticillium dahliae and the underlying mechanism has not been identified in cotton. In this study, we found that the that expression of most R2R3-MYB members in cotton is significantly changed by V. dahliae infection relative to the other MYB types. One novel R2R3-MYB transcription factor (TF) that specifically responds to V. dahliae, GhMYB3D5, was identified. GhMYB3D5 was not expressed in 15 cotton tissues under normal conditions, but it was dramatically induced by V. dahliae stress. We functionally characterized its positive role and underlying mechanism in VW resistance. Upon V. dahliae infection, the up-regulated GhMYB3D5 bound to the GhADH1 promoter and activated GhADH1 expression. In addition, GhMYB3D5 physically interacted with GhADH1 and further enhanced the transcriptional activation of GhADH1. Consequently, the transcriptional regulatory module GhMYB3D5-GhADH1 then promoted lignin accumulation by improving the transcriptional levels of genes related to lignin biosynthesis (GhPAL, GhC4H, Gh4CL, and GhPOD/GhLAC) in cotton, thereby enhancing cotton VW resistance. Our results demonstrated that the GhMYB3D5 promotes defense-induced lignin accumulation, which can be regarded as an effective way to orchestrate plant immunity and growth.

Alfin-like (AL) is a small plant-specific gene family characterized by a PHD-finger-like structural domain at the C-terminus and a DUF3594 structural domain at the N-terminus, and these genes play prominent roles in plant development and abiotic stress response. In this study, we conducted genome-wide identification and analyzed the AL protein family in Gossypium hirsutum cv. NDM8 to assess their response to various abiotic stresses for the first time. A total of 26 AL genes were identified in NDM8 and classified into four groups based on a phylogenetic tree. Moreover, cis-acting element analysis revealed that multiple phytohormone response and abiotic stress response elements were highly prevalent in AL gene promoters. Further, we discovered that the GhAL19 gene could negatively regulate drought and salt stresses via physiological and biochemical changes, gene expression, and the VIGS assay. The study found there was a significant increase in POD and SOD activity, as well as a significant change in MDA in VIGS-NaCl and VIGS-PEG plants. Transcriptome analysis demonstrated that the expression levels of the ABA biosynthesis gene (GhNCED1), signaling genes (GhABI1, GhABI2, and GhABI5), responsive genes (GhCOR47, GhRD22, and GhERFs), and the stress-related marker gene GhLEA14 were regulated in VIGS lines under drought and NaCl treatment. In summary, GhAL19 as an AL TF may negatively regulate tolerance to drought and salt by regulating the antioxidant capacity and ABA-mediated pathway.

Cotton fiber quality-related traits, such as fiber length, fiber strength, and fiber elongation, are affected by complex mechanisms controlled by multiple genes. Determining the QTN-by-QTN interactions (QQIs) associated with fiber quality-related traits is therefore essential for accelerating the genetic enhancement of cotton breeding. In this study, a natural population of 1,245 upland cotton varieties with 1,122,352 SNPs was used for detecting the main-effect QTNs and QQIs using the 3V multi-locus random-SNP-effect mixed linear model (3VmrMLM) method. A total of 171 significant main-effect QTNs and 42 QQIs were detected, of which 22 were both main-effect QTNs and QQIs. Of the detected 42 QQIs, a total of 13 significant loci and 5 candidate genes were reported in previous studies. Among the three interaction types, the AD interaction type has a preference for the trait of FE. Additionally, the QQIs have a substantial impact on the enhancement predictability for fiber quality-related traits. The study of QQIs is crucial for elucidating the genetic mechanism of cotton fiber quality and enhancing breeding efficiency.

Abstract Crops must efficiently allocate their limited energy resources to survival, growth and reproduction, including balancing growth and defense. Thus, investigating the underlying molecular mechanism of crop under stress is crucial for breeding. Chloroplasts immunity is an important facet involving in plant resistance and growth, however, whether and how crop immunity modulated by chloroplast is influenced by epigenetic regulation remains unclear. Here, the cotton lysine 2‐hydroxyisobutyrylation (Khib) and succinylation (Ksuc) modifications are firstly identified and characterized, and discover that the chloroplast proteins are hit most. Both modifications are strongly associated with plant resistance to Verticillium dahliae, reflected by Khib specifically modulating PR and salicylic acid (SA) signal pathway and the identified GhHDA15 and GhSRT1 negatively regulating Verticillium wilt (VW) resistance via removing Khib and Ksuc. Further investigation uncovers that photosystem repair protein GhPSB27 situates in the core hub of both Khib‐ and Ksuc‐modified proteins network. The acylated GhPSB27 regulated by GhHDA15 and GhSRT1 can raise the D1 protein content, further enhancing plant biomass‐ and seed‐yield and disease resistance via increasing photosynthesis and by‐products of chloroplast‐derived reactive oxygen species (cROS). Therefore, this study reveals a mechanism balancing high disease resistance and high yield through epigenetic regulation of chloroplast protein, providing a novel strategy to crop improvements.

Weeds seriously affect the yield and quality of crops. Because manual weeding is time-consuming and laborious, the use of herbicides becomes an effective way to solve the harm caused by weeds in fields. Both 5-enolpyruvyl shikimate-3-phosphate synthetase (EPSPS) and acetyltransferase genes (bialaphos resistance, BAR) are widely used to improve crop resistance to herbicides. However, cotton, as the most important natural fiber crop, is not tolerant to herbicides in China, and the EPSPS and BAR family genes have not yet been characterized in cotton. Therefore, we explore the genes of these two families to provide candidate genes for the study of herbicide resistance mechanisms. In this study, 8, 8, 4, and 5 EPSPS genes and 6, 6, 5, and 5 BAR genes were identified in allotetraploid Gossypium hirsutum and Gossypium barbadense, diploid Gossypium arboreum and Gossypium raimondii, respectively. Members of the EPSPS and BAR families were classified into three subgroups based on the distribution of phylogenetic trees, conserved motifs, and gene structures. In addition, the promoter sequences of EPSPS and BAR family members included growth and development, stress, and hormone-related cis-elements. Based on the expression analysis, the family members showed tissue-specific expression and differed significantly in response to abiotic stresses. Finally, qRT-PCR analysis revealed that the expression levels of GhEPSPS3, GhEPSPS4, and GhBAR1 were significantly upregulated after exogenous spraying of herbicides. Overall, we characterized the EPSPS and BAR gene families of cotton at the genome-wide level, which will provide a basis for further studying the functions of EPSPS and BAR genes during growth and development and herbicide stress.

Abstract Background The fiber yield and quality of cotton are greatly and periodically affected by water deficit. However, the molecular mechanism of the water deficit response in cotton fiber cells has not been fully elucidated. Results In this study, water deficit caused a significant reduction in fiber length, strength, and elongation rate but a dramatic increase in micronaire value. To explore genome-wide transcriptional changes, fibers from cotton plants subjected to water deficit (WD) and normal irrigation (NI) during fiber development were analyzed by transcriptome sequencing. Analysis showed that 3427 mRNAs and 1021 long noncoding RNAs (lncRNAs) from fibers were differentially expressed between WD and NI plants. The maximum number of differentially expressed genes (DEGs) and lncRNAs (DERs) was identified in fibers at the secondary cell wall biosynthesis stage, suggesting that this is a critical period in response to water deficit. Twelve genes in cotton fiber were differentially and persistently expressed at ≥ five time points, suggesting that these genes are involved in both fiber development and the water-deficit response and could potentially be used in breeding to improve cotton resistance to drought stress. A total of 540 DEGs were predicted to be potentially regulated by DERs by analysis of coexpression and genomic colocation, accounting for approximately 15.76% of all DEGs. Four DERs, potentially acting as target mimics for microRNAs (miRNAs), indirectly regulated their corresponding DEGs in response to water deficit. Conclusions This work provides a comprehensive transcriptome analysis of fiber cells and a set of protein-coding genes and lncRNAs implicated in the cotton response to water deficit, significantly affecting fiber quality during the fiber development stage.

SEP genes are famous for their function in the morphological novelty of bisexual flowers. Although the diverse functions of SEP genes were reported, only the regulatory mechanisms underlying floral organ development have been addressed. In this study, we identified SEP-like genes in Gossypium and found that SEP3 genes were duplicated in diploid cotton varieties. GhSEP4.1 and GhSEP4.2 were abundantly transcribed in the shoot apical meristem (SAM), but only GhSEP4.2 was expressed in the leaf vasculature. The expression pattern of GhSEPs in floral organs was conserved with that of homologs in Arabidopsis, except for GhSEP2 that was preponderantly expressed in ovules and fibers. The overexpression and silencing of each single GhSEP gene suggested their distinct role in promoting flowering via direct binding to GhAP1 and GhLFY genomic regions. The curly leaf and floral defects in overexpression lines with a higher expression of GhSEP genes revealed the concentration-dependent target gene regulation of GhSEP proteins. Moreover, GhSEP proteins were able to dimerize and interact with flowering time regulators. Together, our results suggest the dominant role of GhSEP4.2 in leaves to promote flowering via GhAP1-A04, and differently accumulated GhSEP proteins in the SAM alternately participate in forming the dynamic tetramer complexes to target at the different loci of GhAP1 and GhLFY to maintain reproductive growth. The regulatory roles of cotton SEP genes reveal their conserved and diversified functions.

Comparative transcriptome analysis of fiber tissues between Gossypium barbadense and Gossypium hirsutum could reveal the molecular mechanisms underlying high-quality fiber formation and identify candidate genes for fiber quality improvement. In this study, 759 genes were found to be strongly upregulated at the elongation stage in G. barbadense, which showed four distinct expression patterns (I–IV). Among them, the 346 genes of group IV stood out in terms of the potential to promote fiber elongation, in which we finally identified 42 elongation-related candidate genes by comparative transcriptome analysis between G. barbadense and G. hirsutum. Subsequently, we overexpressed GbAAR3 and GbTWS1, two of the 42 candidate genes, in Arabidopsis plants and validated their roles in promoting cell elongation. At the secondary cell wall (SCW) biosynthesis stage, 2275 genes were upregulated and exhibited five different expression profiles (I–V) in G. barbadense. We highlighted the critical roles of the 647 genes of group IV in SCW biosynthesis and further picked out 48 SCW biosynthesis-related candidate genes by comparative transcriptome analysis. SNP molecular markers were then successfully developed to distinguish the SCW biosynthesis-related candidate genes from their G. hirsutum orthologs, and the genotyping and phenotyping of a BC3F5 population proved their potential in improving fiber strength and micronaire. Our results contribute to the better understanding of the fiber quality differences between G. barbadense and G. hirsutum and provide novel alternative genes for fiber quality improvement.

Abstract Background Dirigent (DIR) proteins mediate regioselectivity and stereoselectivity during lignan biosynthesis and are also involved in lignin, gossypol and pterocarpan biosynthesis. This gene family plays a vital role in enhancing stress resistance and in secondary cell-wall development, but systematical understanding is lacking in cotton. Results In this study, 107 GbDIRs and 107 GhDIRs were identified in Gossypium barbadense and Gossypium hirsutum, respectively. Most of these genes have a classical gene structure without intron and encode proteins containing a signal peptide. Phylogenetic analysis showed that cotton DIR genes were classified into four distinct subfamilies (a, b/d, e, and f). Of these groups, DIR-a and DIR-e were evolutionarily conserved, and segmental and tandem duplications contributed equally to their formation. In contrast, DIR-b/d mainly expanded by recent tandem duplications, accompanying with a number of gene clusters. With the rapid evolution, DIR-b/d-III was a Gossypium-specific clade involved in atropselective synthesis of gossypol. RNA-seq data highlighted GhDIRs in response to Verticillium dahliae infection and suggested that DIR gene family could confer Verticillium wilt resistance. We also identified candidate DIR genes related to fiber development in G. barbadense and G. hirsutum and revealed their differential expression. To further determine the involvement of DIR genes in fiber development, we overexpressed a fiber length-related gene GbDIR78 in Arabidopsis and validated its function in trichomes and hypocotyls. Conclusions These findings contribute novel insights towards the evolution of DIR gene family and provide valuable information for further understanding the roles of DIR genes in cotton fiber development as well as in stress responses.

Fiber length (FL) and fiber strength (FS) are the important indicators of fiber quality in cotton. Longer and stronger fibers are preferred for manufacturing finer yarns in the textile industry. Functional markers (FMs) designed from polymorphic sites within gene sequences attributing to phenotypic variation are highly efficient when used for marker-assisted selection (MAS) in breeding superior varieties with longer FL and higher FS. The aims of this study were to develop FMs via kompetitive allele-specific PCR (KASP) assays and to validate the efficacy of the FMs for allele discrimination and the potential value in practice application. We used four single-nucleotide polymorphism markers and 360 cotton accessions and found that two FMs, namely, D11_24030087 and A07_72204443, could effectively differentiate accessions of different genotypes with higher consistency to phenotype. The appeared frequencies of varieties harbored Hap2 (elite alleles G and T) with longer FL (> the mean of accessions with non-elite allele, 28.50 mm) and higher FS (> the mean of accessions with non-elite allele, 29.06 cN•tex–1) were 100 and 72.7%, respectively, which was higher than that of varieties harbored only on a single elite allele (G or T, 77.9 or 61.9%), suggesting a favorable haplotype for selecting varieties with superior FL and FS. These FMs could be valuable for the high-throughput selection of superior materials by providing genotypic information in cotton breeding programs.

Abstract Background Evaluating phenotypic traits is very important for the selection of elite lines in Gossypium hirsutum L. Cotton breeders are interested in using diverse genotypes in hybridization that can segregate for traits of interested with the possibility of selection and genetic gain. Information on phenotypic and molecular diversity helps the breeders for parental selection. Methods In this study, 719 global collections of G. hirsutum L. were evaluated for five fibre-related traits during two consecutive years in eight different environments. A series of phenotypic data for fibre quality traits were obtained and the elite accessions were further screened using principal component analysis and phylogenetic tree construction based on single nucleotide polymorphism markers. Results We found that fibre quality traits showed a wide range of variation among the G. hirsutum accessions over 2 years. In general, accessions from outside China tended to have higher fibre length (FL) and fibre strength (FS) than did Chinese accessions. Among different regional accessions in China, North/Northwest accessions tended to have the highest FL, FS and best fibre macronaire. By assessing five fibre quality traits over 2 years with genotypic data, 31 elite germplasms reaching double-thirty quality values (FL ≥ 30 mm and FS ≥ 30 cN·tex− 1) were selected. Conclusions This study provided a detailed phenotypic diversity description of a population representing a wide range of upland cotton germplasm. Our findings provide useful information about possible elite fibre quality parents for cotton breeding programs.

Abstract Background Cyclophilins (CYPs), belonging to the peptidyl prolyl cis/trans isomerase (PPIase) superfamily, play important roles during plant responses to biotic and abiotic stresses. Results Here, a total of 79 CYPs were identified in the genome of Gossypium hirsutum. Of which, 65 GhCYPs only contained one cyclophilin type PPIase domain, others 14 GhCYPs contain additional domains. A number of cis-acting elements related to phytohormone signaling were predicated in the upstream of GhCYPs ORF. The expression analysis revealed that GhCYPs were induced in response to cold, hot, salt, PEG and Verticillium dahliae infection. In addition, the functional importance of GhCYP-3 in Verticillium wilt resistance was also presented in this study. GhCYP-3 showed both cytoplasmic and nuclear localization. Overexpression of GhCYP-3 in Arabidopsis significantly improved Verticillium wilt resistance of the plants. Recombinant GhCYP-3 displayed PPIase activity and evident inhibitory effects on V. dahliae in vitro. Moreover, the extracts from GhCYP-3 transgenic Arabidopsis displayed significantly inhibit activity to conidia germinating and hyphal growth of V. dahliae. Conclusions Our study identified the family members of cotton CYP genes using bioinformatics tools. Differential expression patterns of GhCYPs under various abiotic stress and V. dahliae infection conditions provide a comprehensive understanding of the biological functions of candidate genes. Moreover, GhCYP-3 involved in the resistance of cotton to V. dahliae infection presumably through antifungal activity.

Verticillium wilt (VW), a fungal disease caused by Verticillium dahliae, currently devastates cotton fiber yield and quality seriously, yet few resistance germplasm resources have been discovered in Gossypium hirsutum. The cotton variety Nongda601 with suitable VW resistance and high yield was developed in our lab, which supplied elite resources for discovering resistant genes. Early nodulin-like protein (ENODL) is mainly related to nodule formation, and its role in regulating defense response has been seldom studied. Here, 41 conserved ENODLs in G. hirsutum were identified and characterized, which could divide into four subgroups. We found that GhENODL6 was upregulated under V. dahliae stress and hormonal signal and displayed higher transcript levels in resistant cottons than the susceptible. The GhENODL6 was proved to positively regulate VW resistance via overexpression and gene silencing experiments. Overexpression of GhENODL6 significantly enhanced the expressions of salicylic acid (SA) hormone-related transcription factors and pathogenicity-related (PR) protein genes, as well as hydrogen peroxide (H2O2) and SA contents, resulting in improved VW resistance in transgenic Arabidopsis. Correspondingly, in the GhENODL6 silenced cotton, the expression levels of both phenylalanine ammonia lyase (PAL) and 4-coumarate-CoA ligase (4CL) genes significantly decreased, leading to the reduced SA content mediating by the phenylalanine ammonia lyase pathway. Taken together, GhENODL6 played a crucial role in VW resistance by inducing SA signaling pathway and regulating the production of reactive oxygen species (ROS). These findings broaden our understanding of the biological roles of GhENODL and the molecular mechanisms underlying cotton disease resistance.

Salt tolerance in cotton is highly imperative for improvement in the response to decreasing farmland and soil salinization. However, little is known about the genetic basis underlying salt tolerance in cotton, especially the seedling stage. In this study, we evaluated two salt-tolerance-related traits of a natural population comprising 713 upland cotton (Gossypium hirsutum L.) accessions worldwide at the seedling stage and performed a genome-wide association study (GWAS) to identify marker-trait associations under salt stress using the Illumina Infinium CottonSNP63K array. A total of 23 single nucleotide polymorphisms (SNPs) that represented seven genomic regions on chromosomes A01, A10, D02, D08, D09, D10, and D11 were significantly associated with the two salt-tolerance-related traits, relative survival rate (RSR) and salt tolerance level (STL). Of these, the two SNPs i46598Gh and i47388Gh on D09 were simultaneously associated with the two traits. Based on all loci, we screened 280 possible candidate genes showing different expression levels under salt stress. Most of these genes were involved in transcription factors, transporters and enzymes and were previously reported as being involved in plant salt tolerance, such as NAC, MYB, NXH, WD40, CDPK, LEA, and CIPK. We further validated six putative candidate genes by qRT-PCR and found a differential expression level between salt-tolerant and salt-sensitive varieties. Our findings provide valuable information for enhancing the understanding of complicated mechanisms of salt tolerance in G. hirsutum seedlings and cotton salt tolerance breeding by molecular marker-assisted selection.