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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.

Summary Verticillium wilt (VW) is a destructive disease that results in great losses in cotton yield and quality. Identifying genetic variation that enhances crop disease resistance is a primary objective in plant breeding. Here we reported a GWAS of cotton VW resistance in a natural‐variation population, challenged by different pathogenicity stains and different environments, and found 382 SNPs significantly associated with VW resistance. The associated signal repeatedly peaked in chromosome Dt11 (68 798 494‐69 212 808) containing 13 core elite alleles undescribed previously. The core SNPs can make the disease reaction type from susceptible to tolerant or resistant in accessions with alternate genotype compared to reference genotype. Of the genes associated with the Dt11 signal, 25 genes differentially expressed upon Verticillium dahliae stress, with 21 genes verified in VW resistance via gene knockdown and/or overexpression experiments. We firstly discovered that a gene cluster of L‐type lectin‐domain containing receptor kinase (GhLecRKs‐V.9) played an important role in VW resistance. These results proved that the associated Dt11 region was a major genetic locus responsible for VW resistance. The frequency of the core elite alleles (FEA) in modern varieties was significantly higher than the early/middle varieties (12.55% vs 4.29%), indicating that the FEA increased during artificial selection breeding. The current developmental resistant cultivars, JND23 and JND24, had fixed these core elite alleles during breeding without yield penalty. These findings unprecedentedly provided genomic variations and promising alleles for promoting cotton VW resistance improvement.

Cotton produces natural fiber for the textile industry. The genetic effects of genomic structural variations underlying agronomic traits remain unclear. Here, we generate two high-quality genomes of Gossypium hirsutum cv. NDM8 and Gossypium barbadense acc. Pima90, and identify large-scale structural variations in the two species and 1,081 G. hirsutum accessions. The density of structural variations is higher in the D-subgenome than in the A-subgenome, indicating that the D-subgenome undergoes stronger selection during species formation and variety development. Many structural variations in genes and/or regulatory regions potentially influencing agronomic traits were discovered. Of 446 significantly associated structural variations, those for fiber quality and Verticillium wilt resistance are located mainly in the D-subgenome and those for yield mainly in the A-subgenome. Our research provides insight into the role of structural variations in genotype-to-phenotype relationships and their potential utility in crop improvement., High-quality genomes of two cultivated tetraploid cottons Gossypium hirsutum cv. NDM8 and Gossypium barbadense acc. Pima90 and resequencing of 1,081 G. hirsutum accessions provide insights into the role of structural variations.

Cotton (Gossypium hirsutum) is constantly attacked by pathogens and insects. The most efficient control strategy is to develop resistant varieties using broad-spectrum gene resources. Several resistance loci harboured by superior varieties have been identified through genome-wide association studies. However, the key genes and/or loci have not been functionally identified. In this study, we identified a locus significantly associated with Verticillium wilt (VW) resistance, and within a 145.5-kb linkage disequilibrium, two non-specific lipid transfer protein genes (named GhnsLTPsA10) were highly expressed under Verticillium pathogen stress. The expression of GhnsLTPsA10 significantly increased in roots upon Verticillium dahliae stress but significantly decreased in leaves under insect attack. Furthermore, GhnsLTPsA10 played antagonistic roles in positively regulating VW and Fusarium wilt resistance and negatively mediating aphid and bollworm resistance in transgenic Arabidopsis and silenced cotton. By combining transcriptomic, histological and physiological analyses, we determined that GhnsLTPsA10-mediated phenylpropanoid metabolism further affected the balance of the downstream metabolic flux of flavonoid and lignin biosynthesis. The divergent expression of GhnsLTPsA10 in roots and leaves coordinated resistance of cotton against fungal pathogens and insects via the redirection of metabolic flux. In addition, GhnsLTPsA10 contributed to reactive oxygen species accumulation. Therefore, in this study, we elucidated the novel function of GhnsLTP and the molecular association between disease resistance and insect resistance, balanced by GhnsLTPsA10. This broadens our knowledge of the biological function of GhnsLTPsA10 in crops and provides a useful locus for genetic improvement of cotton.

Lipids are major and essential constituents of plant cells and provide energy for various metabolic processes. However, the function of the lipid signal in defence against Verticillium dahliae, a hemibiotrophic pathogen, remains unknown. Here, we characterized 19 conserved stearoyl‐ACP desaturase family proteins from upland cotton (Gossypium hirsutum). We further confirmed that GhSSI2 isoforms, including GhSSI2‐A, GhSSI2‐B, and GhSSI2‐C located on chromosomes A10, D10, and A12, respectively, played a dominant role to the cotton 18:1 (oleic acid) pool. Suppressing the expression of GhSSI2s reduced the 18:1 level, which autoactivated the hypersensitive response (HR) and enhanced cotton Verticillium wilt and Fusarium wilt resistance. We found that low 18:1 levels induced phenylalanine ammonia‐lyase‐mediated salicylic acid (SA) accumulation and activated a SA‐independent defence response in GhSSI2s‐silenced cotton, whereas suppressing expression of GhSSI2s affected PDF1.2‐dependent jasmonic acid (JA) perception but not the biosynthesis and signalling cascade of JA. Further investigation showed that structurally divergent resistance‐related genes and nitric oxide (NO) signal were activated in GhSSI2s‐silenced cotton. Taken together, these results indicate that SA‐independent defence response, multiple resistance‐related proteins, and elevated NO level play an important role in GhSSI2s‐regulated Verticillium wilt resistance. These findings broaden our knowledge regarding the lipid signal in disease resistance and provide novel insights into the molecular mechanism of cotton fungal disease resistance., GhSACPD gene family isoforms GhSSI2‐A, GhSSI2‐B, and GhSSI2‐C displayed an important role in regulating 18:1 fatty acid level and negatively regulated cotton disease resistance to Verticillium wilt.

Background Verticillium wilt, caused by the soil borne fungus Verticillium dahliae, is a major threat to cotton production worldwide. An increasing number of findings indicate that WAK genes participate in plant−pathogen interactions, but their roles in cotton resistance to V. dahliae remain largely unclear. Results Here, we carried out a genome-wide analysis of WAK gene family in Gossypium hirsutum that resulted in the identification of 81 putative GhWAKs, which were all predicated to be localized on plasma membrane. In which, GhWAK77 as a representative was further located in tobacco epidermal cells using transient expression of fluorescent fusion proteins. All GhWAKs could be classified into seven groups according to their diverse protein domains, indicating that they might sense different outside signals to trigger intracellular signaling pathways that were response to various environmental stresses. A lot of cis-regulatory elements were predicted in the upstream region of GhWAKs and classified into four main groups including hormones, biotic, abiotic and light. As many as 28 GhWAKs, playing a potential role in the interaction between cotton and V. dahliae, were screened out by RNA-seq and qRT-PCR. To further study the function of GhWAKs in cotton resistance to V. dahliae, VIGS technology was used to silence GhWAKs. At 20 dpi, VIGSed plants exhibited more chlorosis and wilting than the control plants. The disease indices of VIGSed plants were also significantly higher than those of the control. Furthermore, silencing of GhWAKs significantly affected the expression of JA- and SA-related marker genes, increased the spread of V. dahliae in the cotton stems, dramatically compromised V. dahliae-induced accumulation of lignin, H2O2 and NO, but enhanced POD activity. Conclusion Our study presents a comprehensive analysis on cotton WAK gene family for the first time. Expression analysis and VIGS assay provided direct evidences on GhWAKs participation in the cotton resistance to V. dahliae.

A high-density linkage map of an intraspecific RIL population was constructed using 6187 bins to identify QTLs for fibre quality- and yield-related traits in upland cotton by whole-genome resequencing. Good fibre quality and high yield are important production goals in cotton (Gossypium hirsutum L.), which is a leading natural fibre crop worldwide. However, a greater understanding of the genetic variants underlying fibre quality- and yield-related traits is still required. In this study, a large-scale population including 588 F7 recombinant inbred lines, derived from an intraspecific cross between the upland cotton cv. Nongdamian13, which exhibits high quality, and Nongda601, which exhibits a high yield, was genotyped by using 232,946 polymorphic single-nucleotide polymorphisms obtained via a whole-genome resequencing strategy with 4.3-fold genome coverage. We constructed a high-density bin linkage map containing 6187 bin markers spanning 4478.98 cM with an average distance of 0.72 cM. We identified 58 individual quantitative trait loci (QTLs) and 25 QTL clusters harbouring 94 QTLs, and 119 previously undescribed QTLs controlling 13 fibre quality and yield traits across eight environments. Importantly, the QTL counts for fibre quality in the Dt subgenome were more than two times that in the At subgenome, and chromosome D02 harboured the greatest number of QTLs and clusters. Furthermore, we discovered 24 stable QTLs for fibre quality and 12 stable QTLs for yield traits. Four novel major stable QTLs related to fibre length, fibre strength and lint percentage, and seven previously unreported candidate genes with significantly differential expression between the two parents were identified and validated by RNA-seq. Our research provides valuable information for improving the fibre quality and yield in cotton breeding.

Additional file 3: Table S3. The correlation (R2) of gene expression (log10 (FPKM+ 1)) between two biological replicates.

Additional file 2: Table S2. G. hirsutum fiber transcriptomes assembly statistics.

Additional file 1: Table S1. Primers used for qPCR.

Background Verticillium wilt is a widespread and destructive disease, which causes serious loss of cotton yield and quality. Long non-coding RNA (lncRNA) is involved in many biological processes, such as plant disease resistance response, through a variety of regulatory mechanisms, but their possible roles in cotton against Verticillium dahliae infection remain largely unclear. Results Here, we measured the transcriptome of resistant G. hirsutum following infection by V. dahliae and 4277 differentially expressed lncRNAs (delncRNAs) were identified. Localization and abundance analysis revealed that delncRNAs were biased distribution on chromosomes. We explored the dynamic characteristics of disease resistance related lncRNAs in chromosome distribution, induced expression profiles, biological function, and these lncRNAs were divided into three categories according to their induced expression profiles. For the delncRNAs, 687 cis-acting pairs and 14,600 trans-acting pairs of lncRNA-mRNA were identified, which indicated that trans-acting was the main way of Verticillium wilt resistance-associated lncRNAs regulating target mRNAs in cotton. Analyzing the regulation pattern of delncRNAs revealed that cis-acting and trans-acting lncRNAs had different ways to influence target genes. Gene Ontology (GO) enrichment analysis revealed that the regulatory function of delncRNAs participated significantly in stimulus response process, kinase activity and plasma membrane components. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that delncRNAs participated in some important disease resistance pathways, such as plant-pathogen interaction, alpha-linolenic acid metabolism and plant hormone signal transduction. Additionally, 21 delncRNAs and 10 target genes were identified as being involved in alpha-linolenic acid metabolism associated with the biosynthesis of jasmonic acid (JA). Subsequently, we found that GhlncLOX3 might regulate resistance to V. dahliae through modulating the expression of GhLOX3 implicated in JA biosynthesis. Further functional analysis showed that GhlncLOX3-silenced seedlings displayed a reduced resistance to V. dahliae, with down-regulated expression of GhLOX3 and decreased content of JA. Conclusion This study shows the dynamic characteristics of delncRNAs in multiaspect, and suggests that GhlncLOX3-GhLOX3-JA network participates in response to V. dahliae invasion. Our results provide novel insights for genetic improvement of Verticillium wilt resistance in cotton using lncRNAs.

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.

As the largest cultivated fiber crop in the world, cotton (Gossypium hirsutum) is often exposed to various biotic stresses during its growth periods. Verticillium wilt caused by Verticillium dahliae is a severe disease in cotton, and the molecular mechanism of cotton resistance for Verticillium wilt needs to be further investigated. Here, we revealed that the cotton genome contains nine types of GST genes. An evolutionary analysis showed that a newly identified cluster (including Gh_A09G1508, Gh_A09G1509 and Gh_A09G1510) located on chromosome 09 of the A-subgenome was under positive selection pressure during the formation of an allotetraploid. Transcriptome analysis showed that this cluster participates in Verticillium wilt resistance. Because the Gh_A09G1509 gene showed the greatest differential expression in the resistant cultivar under V. dahliae stress, we overexpressed this gene in tobacco and found that its overexpression resulted in enhanced Verticillium wilt resistance. Suppression of the gene cluster via virus-induced gene silencing made cotton plants of the resistant cultivar Nongda601 significantly susceptible. These results demonstrated that the GST cluster played an important role in Verticillium wilt resistance. Further investigation showed that the encoded enzymes of the cluster were essential for the delicate equilibrium between the production and scavenging of H2 O2 during V. dahliae stress.

Upland cotton is the most important natural-fiber crop. The genomic variation of diverse germplasms and alleles underpinning fiber quality and yield should be extensively explored. Here, we resequenced a core collection comprising 419 accessions with 6.55-fold coverage depth and identified approximately 3.66 million SNPs for evaluating the genomic variation. We performed phenotyping across 12 environments and conducted genome-wide association study of 13 fiber-related traits. 7,383 unique SNPs were significantly associated with these traits and were located within or near 4,820 genes; more associated loci were detected for fiber quality than fiber yield, and more fiber genes were detected in the D than the A subgenome. Several previously undescribed causal genes for days to flowering, fiber length, and fiber strength were identified. Phenotypic selection for these traits increased the frequency of elite alleles during domestication and breeding. These results provide targets for molecular selection and genetic manipulation in cotton improvement.

Verticillium dahliae seriously affects the yield of cotton. Here, V. dahliae infection induced the significant reduction of protein concentration in cotton xylem sap (CXS), suggesting that the protein composition have changed. Thus, the proteomics in CXS from resistant Gossypium hirsutum cv. ND601 and susceptible CCRI8 infected by V. dahliae were analyzed using the label-free method. A total of 3047 proteins were identified across all four CXS sample groups. 1717 and 1476 proteins were differentially accumulated in ND601 and CCRI8 after infection with V. dahliae, respectively. The majority of up-accumulated and induced proteins belongs to pathogenesis-related proteins and associates with cell wall (CWRPs). Down-accumulated and disappeared proteins were principally related to plant growth and development. Differentially accumulated CWRPs from ND601 and CCRI8 in type and quantity were not entirely consistent with each other, leading to different cell wall dynamics and strength, which were partly proved by the measurement of stem mechanical strength. Most of proteins related to growth and development were down-accumulated in ND601 compared to CCRI8, suggesting that the resistant variety may transfer more energy for defense responses or reduce nutrient acquisition of V. dahliae for colonization more effectively than the susceptible. SIGNIFICANCE: Verticillium wilt, mainly caused by V. dahliae, is one of the most destructive diseases in cotton. V. dahliae usually penetrates the root epidermis, reaches vascular tissues, and eventually extends to the above-ground tissues along the xylem vessels. Obviously, xylem is an important battlefront for plant defense to V. dahliae. Therefore, we analyzed the proteome profiles of xylem saps from resistant and susceptible cotton cultivars. Our findings provide valuable insights into the molecular mechanism underlying the interaction between V. dahliae and cotton.

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. Electronic supplementary material The online version of this article (10.1186/s12870-019-1848-1) contains supplementary material, which is available to authorized users.

Background Developing tolerant cultivars by incorporating resistant genes is regarded as a potential strategy for controlling Verticillium wilt that causes severe losses in the yield and fiber quality of cotton. Results Here, we identified the gene GbHyPRP1 in Gossypium barbadense, which encodes a protein containing both proline-rich repetitive and Pollen Ole e I domains. GbHyPRP1 is located in the cell wall. The transcription of this gene mainly occurs in cotton roots and stems, and is drastically down-regulated upon infection with Verticillium dahliae. Silencing HyPRP1 dramatically enhanced cotton resistance to V. dahliae. Over-expression of HyPRP1 significantly compromised the resistance of transgenic Arabidopsis plants to V. dahliae. The GbHyPRP1 promoter region contained several putative phytohormone-responsive elements, of which SA was associated with gene down-regulation. We compared the mRNA expression patterns of HyPRP1-silenced plants and the control at the global level by RNA-Seq. A total of 1735 unique genes exhibited significant differential expression. Of these, 79 DEGs involved in cell wall biogenesis and 43 DEGs associated with the production of ROS were identified. Further, we observed a dramatic thickening of interfascicular fibers and vessel walls and an increase in lignin in the HyPRP1-silenced cotton plants compared with the control after inoculation with V. dahliae. Additionally, silencing of HyPRP1 markedly enhanced ROS accumulation in the root tips of cotton inoculated with V. dahliae. Conclusions Taken together, our results suggest that HyPRP1 performs a role in the negative regulation of cotton resistance to V. dahliae via the thickening of cell walls and ROS accumulation. Electronic supplementary material The online version of this article (10.1186/s12870-018-1565-1) contains supplementary material, which is available to authorized users.