Genome-wide identification and mining elite allele variation of the Monoacylglycerol lipase (MAGL) gene family in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Monoacylglycerol lipase (MAGL) genes belong to the alpha/beta hydrolase superfamily, catalyze the terminal step of triglyceride (TAG) hydrolysis, converting monoacylglycerol (MAG) into free fatty acids and glycerol. RESULTS: In this study, 30 MAGL genes in upland cotton have been identified, which have been classified into eight subgroups. The duplication of GhMAGL genes in upland cotton was predominantly influenced by segmental duplication events, as revealed through synteny analysis. Furthermore, all GhMAGL genes were found to contain light-responsive elements. Through comprehensive association and haplotype analyses using resequencing data from 355 cotton accessions, GhMAGL3 and GhMAGL6 were detected as key genes related to lipid hydrolysis processes, suggesting a negative regulatory effect. CONCLUSIONS: In summary, MAGL has never been studied in upland cotton previously. This study provides the genetic mechanism foundation for the discover of new genes involved in lipid metabolism to improve cottonseed oil content, which will provide a strategic avenue for marker-assisted breeding aimed at incorporating desirable traits into cultivated cotton varieties.

Overexpression and knockdown of cotton GhdadD gene reveals its drought and salt stress tolerance role.

The 5'-deoxyadenosine deaminase (DADD), a member of the amidohydrolase family regulates biological purine metabolism. In this study, bioinformatic analysis, overexpression and knockdown of GhdadD gene were detected to identify its potential role in drought and salt stress tolerance. The results revealed that GhdadD was induced by ABA, Auxin, MBS and light responsive elements. In transgenic Arabidopsis, seed germination rate and root length were increased under drought or salt stress. GhdadD overexpressed seedlings resulted in higher plant height, less leaf damage and lower ion permeability. The expression of osmotic stress and ABA-responsive genes were up regulated. While in GhdadD-silenced cotton seedlings, CAT, SOD activity and soluble sugar content were reduced, MDA content was increased, and the stoma opening was depressed under drought or salt stress. Some osmics stress marker genes were also up regulated. These data indicating that GhdadD enhanced plant resistance to drought and salt stress through ABA pathways.

GhVOZ1-AVP1 module positively regulates salt tolerance in upland cotton (Gossypium hirsutum L.).

Vascular Plant One­zinc Finger (VOZ) transcription factor can respond to a variety of abiotic stresses, however its function in cotton and the molecular mechanisms of response to salt tolerance remained unclear. In this study, we found that GhVOZ1 is highly expressed in stamen and stem of cotton under normal conditions. The expression of GhVOZ1 increased significantly after 3 h of salt treatment in three-leaf staged upland cotton. Overexpressed transgenic lines of GhVOZ1 in Arabidopsis and upland cotton were treated with salt stress and we found that GhVOZ1 could respond positively to salt stress. GhVOZ1 can regulate Arabidopsis Vacuolar Proton Pump Pyrophosphatase (H+-PPase) gene (AVP1) expression through specific binding to GCGTCTAAAGTACGC site on GhAVP1 promoter, which was examined through Dual-luciferase assay and Electrophoretic mobility shift assay (EMSA). AVP1 expression was significantly increased in Arabidopsis with GhVOZ1 overexpression, while GhAVP1 expression was decreased in virus induced gene silenced (VIGS) cotton plants of GhVOZ1. Knockdown of GhAVP1 expression in cotton plants by VIGS showed decreased superoxide dismutase (SOD) and peroxidase (POD) activities, whereas an increased malondialdehyde (MDA) content and ultimately decreased salt tolerance. The GhVOZ1-AVP1 module could maintain sodium ion homeostasis through cell ion transport and positively regulate the salt tolerance in cotton, providing new ideas and insights for the study of salt tolerance.

GhMYB30-GhMUR3 affects fiber elongation and secondary wall thickening in cotton.

Xyloglucan, an important hemicellulose, plays a crucial role in maintaining cell wall structure and cell elongation. However, the effects of xyloglucan on cotton fiber development are not well understood. GhMUR3 encodes a xyloglucan galactosyltransferase that is essential for xyloglucan synthesis and is highly expressed during fiber elongation. In this study, we report that GhMUR3 participates in cotton fiber development under the regulation of GhMYB30. Overexpression GhMUR3 affects the fiber elongation and cell wall thickening. Transcriptome showed that the expression of genes involved in secondary cell wall synthesis was prematurely activated in OE-MUR3 lines. In addition, GhMYB30 was identified as a key regulator of GhMUR3 by Y1H, Dual-Luc, and electrophoretic mobility shift assay (EMSA) assays. GhMYB30 directly bound the GhMUR3 promoter and activated GhMUR3 expression. Furthermore, DAP-seq of GhMYB30 was performed to identify its target genes in the whole genome. The results showed that many target genes were associated with fiber development, including cell wall synthesis-related genes, BR-related genes, reactive oxygen species pathway genes, and VLCFA synthesis genes. It was demonstrated that GhMYB30 may regulate fiber development through multiple pathways. Additionally, GhMYB46 was confirmed to be a target gene of GhMYB30 by EMSA, and GhMYB46 was significantly increased in GhMYB30-silenced lines, indicating that GhMYB30 inhibited GhMYB46 expression. Overall, these results revealed that GhMUR3 under the regulation of GhMYB30 and plays an essential role in cotton fiber elongation and secondary wall thickening. Additionally, GhMYB30 plays an important role in the regulation of fiber development and regulates fiber secondary wall synthesis by inhibiting the expression of GhMYB46.

Widespread incomplete lineage sorting and introgression shaped adaptive radiation in the Gossypium genus.

Cotton (Gossypium) stands as a crucial economic crop, serving as the primary source of natural fiber for the textile sector. However, the evolutionary mechanisms driving speciation within the Gossypium genus remain unresolved. In this investigation, we leveraged 25 Gossypium genomes and introduced four novel assemblies-G. harknessii, G. gossypioides, G. trilobum, and G. klotzschianum (Gklo)-to delve into the speciation history of this genus. Notably, we encountered intricate phylogenies potentially stemming from introgression. These complexities are further compounded by incomplete lineage sorting (ILS), a factor likely to have been instrumental in shaping the swift diversification of cotton. Our focus subsequently shifted to the rapid radiation episode during a concise period in Gossypium evolution. For a recently diverged lineage comprising G. davidsonii, Gklo, and G. raimondii, we constructed a finely detailed ILS map. Intriguingly, this analysis revealed the non-random distribution of ILS regions across the reference Gklo genome. Moreover, we identified signs of robust natural selection influencing specific ILS regions. Noteworthy variations pertaining to speciation emerged between the closely related sister species Gklo and G. davidsonii. Approximately 15.74% of speciation structural variation genes and 12.04% of speciation-associated genes were estimated to intersect with ILS signatures. These findings enrich our understanding of the role of ILS in adaptive radiation, shedding fresh light on the intricate speciation history of the Gossypium genus.

Genome-wide analysis of SET domain genes and the function of GhSDG51 during salt stress in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Cotton, being extensively cultivated, holds immense economic significance as one of the most prominent crops globally. The SET (Su(var), E, and Trithorax) domain-containing protein is of significant importance in plant development, growth, and response to abiotic stress by modifying the lysine methylation status of histone. However, the comprehensive identification of SET domain genes (SDG) have not been conducted in upland cotton (Gossypium hirsutum L.). RESULTS: A total of 229 SDGs were identified in four Gossypium species, including G. arboretum, G. raimondii, G. hirsutum, and G. barbadense. These genes could distinctly be divided into eight groups. The analysis of gene structure and protein motif revealed a high degree of conservation among the SDGs within the same group. Collinearity analysis suggested that the SDGs of Gossypium species and most of the other selected plants were mainly expanded by dispersed duplication events and whole genome duplication (WGD) events. The allopolyploidization event also has a significant impact on the expansion of SDGs in tetraploid Gossypium species. Furthermore, the characteristics of these genes have been relatively conserved during the evolution. Cis-element analysis revealed that GhSDGs play a role in resistance to abiotic stresses and growth development. Furthermore, the qRT-PCR results have indicated the ability of GhSDGs to respond to salt stress. Co-expression analysis revealed that GhSDG51 might co-express with genes associated with salt stress. In addition, the silencing of GhSDG51 in cotton by the virus-induced gene silencing (VIGS) method suggested a potential positive regulatory role of GhSDG51 in salt stress. CONCLUSIONS: The results of this study comprehensively analyze the SDGs in cotton and provide a basis for understanding the biological role of SDGs in the stress resistance in upland cotton.

Identification of novel candidate loci and genes for seed vigor-related traits in upland cotton (Gossypium hirsutum L.) via GWAS.

Seed vigor (SV) is a crucial trait determining the quality of crop seeds. Currently, over 80% of China's cotton-planting area is in Xinjiang Province, where a fully mechanized planting model is adopted, accounting for more than 90% of the total fiber production. Therefore, identifying SV-related loci and genes is crucial for improving cotton yield in Xinjiang. In this study, three seed vigor-related traits, including germination potential, germination rate, and germination index, were investigated across three environments in a panel of 355 diverse accessions based on 2,261,854 high-quality single-nucleotide polymorphisms (SNPs). A total of 26 significant SNPs were detected and divided into six quantitative trait locus regions, including 121 predicted candidate genes. By combining gene expression, gene annotation, and haplotype analysis, two novel candidate genes (Ghir_A09G002730 and Ghir_D03G009280) within qGR-A09-1 and qGI/GP/GR-D03-3 were associated with vigor-related traits, and Ghir_A09G002730 was found to be involved in artificial selection during cotton breeding by population genetic analysis. Thus, understanding the genetic mechanisms underlying seed vigor-related traits in cotton could help increase the efficiency of direct seeding by molecular marker-assisted selection breeding.

A systematic analysis of the phloem protein 2 (PP2) proteins in Gossypium hirsutum reveals that GhPP2-33 regulates salt tolerance.

BACKGROUND: Phloem protein 2 (PP2) proteins play a vital role in the Phloem-based defense (PBD) and participate in many abiotic and biotic stress. However, research on PP2 proteins in cotton is still lacking. RESULTS: A total of 25, 23, 43, and 47 PP2 genes were comprehensively identified and characterized in G.arboretum, G.raimondii, G.barbadense, and G.hirsutum. The whole genome duplication (WGD) and allopolyploidization events play essential roles in the expansion of PP2 genes. The promoter regions of GhPP2 genes contain many cis-acting elements related to abiotic stress and the weighted gene co-expression network analysis (WGCNA) analysis displayed that GhPP2s could be related to salt stress. The qRT-PCR assays further confirmed that GhPP2-33 could be dramatically upregulated during the salt treatment. And the virus-induced gene silencing (VIGS) experiment proved that the silencing of GhPP2-33 could decrease salt tolerance. CONCLUSIONS: The results in this study not only offer new perspectives for understanding the evolution of PP2 genes in cotton but also further explore their function under salt stress.

GhCDPK60 positively regulates drought stress tolerance in both transgenic Arabidopsis and cotton by regulating proline content and ROS level.

Calcium-Dependent Protein Kinases (CDPKs) involved in regulating downstream components of calcium signaling pathways play a role in tolerance to abiotic stresses and seed development in plants. However, functions of only a few cotton CDPKs have been clarified at present. In this study, 80 conserved CDPKs in Gossypium hirsutum L. were identified and characterized, which was divided into four subgroups. Among them, the transcript level of GhCDPK60 was significantly upregulated under drought and several hormone treatments. And we found that the expression levels of several stress-inducible genes down-regulated in GhCDPK60-silence cotton and up-regulated in GhCDPK60-overexpressing Arabidopsis. In addition, physiological analyses demonstrated that GhCDPK60 improved drought stress tolerance by improving the osmotic adjustment ability and reducing the accumulation of reactive oxygen species (ROS) in plants. These findings broaden our understanding of the biological roles of GhCDPK60 and mechanisms underlying drought stress tolerance in cotton.

A Comprehensive Gene Co-Expression Network Analysis Reveals a Role of GhWRKY46 in Responding to Drought and Salt Stresses.

Abiotic stress, such as drought and salinity stress, seriously inhibit the growth and development of plants. Therefore, it is vital to understand the drought and salinity resistance mechanisms to enable cotton to provide more production under drought and salt conditions. In this study, we identified 8806 and 9108 differentially expressed genes (DEGs) through a comprehensive analysis of transcriptomic data related to the PEG-induced osmotic and salt stress in cotton. By performing weighted gene co-expression network analysis (WGCNA), we identified four co-expression modules in PEG treatment and five co-expression modules in salinity stress, which included 346 and 324 predicted transcription factors (TFs) in these modules, respectively. Correspondingly, whole genome duplication (WGD) events mainly contribute to the expansion of those TFs. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analyses revealed those different modules were associated with stress resistance, including regulating macromolecule metabolic process, peptidase activity, transporter activity, lipid metabolic process, and responses to stimulus. Quantitative RT-PCR analysis was used to confirm the expression levels of 15 hub TFs in PEG6000 and salinity treatments. We found that the hub gene GhWRKY46 could alter salt and PEG-induced drought resistance in cotton through the virus-induced gene silencing (VIGS) method. Our results provide a preliminary framework for further investigation of the cotton response to salt and drought stress, which is significant to breeding salt- and drought-tolerant cotton varieties.

Insights into the Genomic Regions and Candidate Genes of Senescence-Related Traits in Upland Cotton via GWAS.

Senescence is the last stage of plant development and is controlled by both internal and external factors. Premature senescence significantly affects the yield and quality of cotton. However, the genetic architecture underlying cotton senescence remains unclear. In this study, genome-wide association studies (GWAS) were performed based on 3,015,002 high-quality SNP markers from the resequencing data of 355 upland cotton accessions to detect genomic regions for cotton senescence. A total of 977 candidate genes within 55 senescence-related genomic regions (SGRs), SGR1-SGR55, were predicted. Gene ontology (GO) analysis of candidate genes revealed that a set of biological processes was enriched, such as salt stress, ethylene processes, and leaf senescence. Furthermore, in the leaf senescence GO term, one candidate gene was focused on: Gohir.A12G270900 (GhMKK9), located in SGR36, which encodes a protein of the MAP kinase kinase family. Quantitative real-time PCR (qRT-PCR) analysis showed that GhMKK9 was up-regulated in old cotton leaves. Overexpression of GhMKK9 in Arabidopsis accelerated natural leaf senescence. Virus-induced gene silencing (VIGS) of GhMKK9 in cotton increased drought tolerance. These results suggest that GhMKK9 is a positive regulator and might be involved in drought-induced senescence in cotton. The results provide new insights into the genetic basis of cotton senescence and will be useful for improving cotton breeding in the future.

Genomewide Identification and Characterization of the Genes Involved in the Flowering of Cotton.

Flowering is a prerequisite for flowering plants to complete reproduction, and flowering time has an important effect on the high and stable yields of crops. However, there are limited reports on flowering-related genes at the genomic level in cotton. In this study, genomewide analysis of the evolutionary relationship of flowering-related genes in different cotton species shows that the numbers of flowering-related genes in the genomes of tetraploid cotton species Gossypium hirsutum and Gossypium barbadense were similar, and that these numbers were approximately twice as much as the number in diploid cotton species Gossypium arboretum. The classification of flowering-related genes shows that most of them belong to the photoperiod and circadian clock flowering pathway. The distribution of flowering-related genes on the chromosomes of the At and Dt subgenomes was similar, with no subgenomic preference detected. In addition, most of the flowering-related core genes in Arabidopsis thaliana had homologs in the cotton genome, but the copy numbers and expression patterns were disparate; moreover, flowering-related genes underwent purifying selection throughout the evolutionary and selection processes. Although the differentiation and reorganization of many key genes of the cotton flowering regulatory network occurred throughout the evolutionary and selection processes, most of them, especially those involved in the important flowering regulatory networks, have been relatively conserved and preferentially selected.

Characterization and Functional Analysis of GhNAC82, A NAM Domain Gene, Coordinates the Leaf Senescence in Upland Cotton (Gossypium hirsutum L.).

In the process of growth and development, cotton exhibits premature senescence under various abiotic stresses, impairing yield and fiber quality. NAC (NAM, ATAF1,2, and CUC2) protein widely distributed in land plants, play the critical role in responding to abiotic stress and regulating leaf senescence. We have identified and functional analyzed a NAM domain gene GhNAC82 in upland cotton, it was located on the A11 chromosome 4,921,702 to 4,922,748 bp, only containing one exon. The spatio-temporal expression pattern analysis revealed that it was highly expressed in root, torus, ovule and fiber development stage. The results of qRT-PCR validated that GhNAC82 negatively regulated by salt stress, drought stress, H2O2 stress, IAA treatment, and ethylene treatment, positively regulated by the ABA and MeJA treatment. Moreover, heterologous overexpression of GhNAC82 results in leaf premature senescence and delays root system development in Arabidopsis thaliana. The phenotype of delayed-senescence was performed after silencing GhNAC82 by VIGS in premature cotton. Taken together, GhNAC82 was involved in different abiotic stress pathways and play important roles in negatively regulating leaf premature senescence.

Detection of Stable Elite Haplotypes and Potential Candidate Genes of Boll Weight Across Multiple Environments via GWAS in Upland Cotton.

Boll weight (BW) is a key determinant of yield component traits in cotton, and understanding the genetic mechanism of BW could contribute to the progress of cotton fiber yield. Although many yield-related quantitative trait loci (QTLs) responsible for BW have been determined, knowledge of the genes controlling cotton yield remains limited. Here, association mapping based on 25,169 single-nucleotide polymorphisms (SNPs) and 2,315 insertions/deletions (InDels) was conducted to identify high-quality QTLs responsible for BW in a global collection of 290 diverse accessions, and BW was measured in nine different environments. A total of 19 significant markers were detected, and 225 candidate genes within a 400 kb region (± 200 kb surrounding each locus) were predicted. Of them, two major QTLs with highly phenotypic variation explanation on chromosomes A08 and D13 were identified among multiple environments. Furthermore, we found that two novel candidate genes (Ghir_A08G009110 and Ghir_D13G023010) were associated with BW and that Ghir_D13G023010 was involved in artificial selection during cotton breeding by population genetic analysis. The transcription level analyses showed that these two genes were significantly differentially expressed between high-BW accession and low-BW accession during the ovule development stage. Thus, these results reveal valuable information for clarifying the genetic basics of the control of BW, which are useful for increasing yield by molecular marker-assisted selection (MAS) breeding in cotton.

A Comprehensive Identification and Function Analysis of Serine/Arginine-Rich (SR) Proteins in Cotton (Gossypium spp.).

As one of the most important factors in alternative splicing (AS) events, serine/arginine-rich (SR) proteins not only participate in the growth and development of plants but also play pivotal roles in abiotic stresses. However, the research about SR proteins in cotton is still lacking. In this study, we performed an extensive comparative analysis of SR proteins and determined their phylogeny in the plant lineage. A total of 169 SR family members were identified from four Gossypium species, and these genes could be divided into eight distinct subfamilies. The domain, motif distribution and gene structure of cotton SR proteins are conserved within each subfamily. The expansion of SR genes is mainly contributed by WGD and allopolyploidization events in cotton. The selection pressure analysis showed that all the paralogous gene pairs were under purifying selection pressure. Many cis-elements responding to abiotic stress and phytohormones were identified in the upstream sequences of the GhSR genes. Expression profiling suggested that some GhSR genes may involve in the pathways of plant resistance to abiotic stresses. The WGCNA analysis showed that GhSCL-8 co-expressed with many abiotic responding related genes in a salt-responding network. The Y2H assays showed that GhSCL-8 could interact with GhSRs in other subfamilies. The subcellular location analysis showed that GhSCL-8 is expressed in the nucleus. The further VIGS assays showed that the silencing of GhSCL-8 could decrease salt tolerance in cotton. These results expand our knowledge of the evolution of the SR gene family in plants, and they will also contribute to the elucidation of the biological functions of SR genes in the future.

Overexpression of a Cotton Aquaporin Gene GhTIP1;1-like Confers Cold Tolerance in Transgenic Arabidopsis.

Cold stress can significantly affect the development, yield, and quality of crops and restrict the geographical distribution and growing seasons of plants. Aquaporins are the main channels for water transport in plant cells. Abiotic stresses such as cold and drought dehydrate cells by changing the water potential. In this study, we cloned a gene GhTIP1;1-like encodes tonoplast aquaporin from the transcriptome database of cotton seedlings after cold stress. Expression analysis showed that GhTIP1;1-like not only responds to cold stress but was also induced by heat, drought and salt stress. Subcellular localization showed that the protein was anchored to the vacuole membrane. Promoter deletion analysis revealed that a MYC motif within the promoter region of GhTIP1;1-like were the core cis-elements in response to low temperature. Virus-induced gene silencing (VIGS) and histochemical staining indicate that GhTIP1;1-like plays a positive role in plant cold tolerance. Overexpression of GhTIP1;1-like in Arabidopsis delayed the senescence process and enhanced the cold tolerance of transgenic plants. Compared with the wild type, the soluble protein concentration and peroxidase activity of the transgenic lines under cold stress were higher, while the malondialdehyde content was lower. In addition, the expression levels of cold-responsive genes were significantly increased in transgenic plants under cold stress. Our results indicate that GhTIP1;1-like could respond to different abiotic stresses and be positively involved in regulating the cold tolerance of cotton.

The MADS transcription factor GhFYF is involved in abiotic stress responses in upland cotton (Gossypium hirsutum L.).

Cotton is an important textile industry raw material crops, which plays a critical role in the development of society. MADS transcription factors (TFs) play a key role about the flowering time, flower development, and abiotic stress responses in plants, but little is known about their functions on abiotic stress in cotton. In this study, a MIKCC subfamily gene from cotton, GhFYF (FOREVER YOUNG FLOWER), was isolated and characterized. Our data showed that GhFYF localized to the nucleus. A ß-glucuronidase (GUS) activity assay revealed that the promoter of GhFYF was mainly expressed in the flower and seed of ProGhFYF::GUS transgenic A. thaliana plants. The GUS staining of flowers and seeds was deepened after drought, salt treatment, and the expression level of the GUS gene and corresponding stress genes AtERD10, AtAnnexin1 are up-regulated in the inflorescence. Overexpression GhFYF in A. thaliana could promote the seed germination and growth under different salt concentrations, and determin the proline content. Yeast two-hybrid (Y2H) assays showed that GhFYF interacted with the HAD-like protein GhGPP2, which has responds to abiotic stress. Our findings indicate that GhFYF is involved in abiotic stress responses, especially for salt stress. This work establishes a solid foundation for further functional analysis of the GhFYF gene in cotton.

Phylogenetic Analysis of the Membrane Attack Complex/Perforin Domain-Containing Proteins in Gossypium and the Role of GhMACPF26 in Cotton Under Cold Stress.

The membrane attack complex/perforin (MACPF) domain-containing proteins are involved in the various developmental processes and in responding to diverse abiotic stress. The function and regulatory network of the MACPF genes are rarely reported in Gossypium spp. We study the detailed identification and partial functional verification of the members of the MACPF family. Totally, 100 putative MACPF proteins containing complete MACPF domain were identified from the four cotton species. They were classified into three phylogenetic groups and underwent multifold pressure indicating that selection produced new functional differentiation. Cotton MACPF gene family members expanded mainly through the whole-genome duplication (WGD)/segmental followed by the dispersed. Expression and cis-acting elements analysis revealed that MACPFs play a role in resistance to abiotic stresses, and some selected GhMACPFs were able to respond to the PEG and cold stresses. Co-expression analysis showed that GhMACPFs might interact with valine-glutamine (VQ), WRKY, and Apetala 2 (AP2)/ethylene responsive factor (ERF) domain-containing genes under cold stress. In addition, silencing endogenous GhMACPF26 in cotton by the virus-induced gene silencing (VIGS) method indicated that GhMACPF26 negatively regulates cold tolerance. Our data provided a comprehensive phylogenetic evolutionary view of Gossypium MACPFs. The MACPFs may work together with multiple transcriptional factors and play roles in acclimation to abiotic stress, especially cold stress in cotton.

QTL and candidate gene identification of the node of the first fruiting branch (NFFB) by QTL-seq in upland cotton (Gossypium hirsutum L.).

BACKGROUND: The node of the first fruiting branch (NFFB) is an important precocious trait in cotton. Many studies have been conducted on the localization of quantitative trait loci (QTLs) and genes related to fiber quality and yield, but there has been little attention to traits related to early maturity, especially the NFFB, in cotton. RESULTS: To identify the QTL associated with the NFFB in cotton, a BC4F2 population comprising 278 individual plants was constructed. The parents and two DNA bulks for high and low NFFB were whole genome sequenced, and 243.8 Gb of clean nucleotide data were generated. A total of 449,302 polymorphic SNPs and 135,353 Indels between two bulks were identified for QTL-seq. Seventeen QTLs were detected and localized on 11 chromosomes in the cotton genome, among which two QTLs (qNFFB-Dt2-1 and qNFFB-Dt3-3) were located in hotspots. Two candidate genes (GhAPL and GhHDA5) related to the NFFB were identified using quantitative real-time PCR (qRT-PCR) and virus-induced gene silencing (VIGS) experiments in this study. Both genes exhibited higher expression levels in the early-maturing cotton material RIL182 during flower bud differentiation, and the silencing of GhAPL and GhHDA5 delayed the flowering time and increased the NFFB compared to those of VA plants in cotton. CONCLUSIONS: Our study preliminarily found that GhAPL and GhHDA5 are related to the early maturity in cotton. The findings provide a basis for the further functional verification of candidate genes related to the NFFB and contribute to the study of early maturity in cotton.

Silencing of GhKEA4 and GhKEA12 Revealed Their Potential Functions Under Salt and Potassium Stresses in Upland Cotton.

The K+ efflux antiporter (KEA) mediates intracellular K+ and H+ homeostasis to improve salt tolerance in plants. However, the knowledge of KEA gene family in cotton is largely absent. In the present study, 8, 8, 15, and 16 putative KEA genes were identified in Gossypium arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively. These KEA genes were classified into three subfamilies, and members from the same subfamilies showed similar motif compositions and gene structure characteristics. Some hormone response elements and stress response elements were identified in the upstream 2000 bp sequence of GhKEAs. Transcriptome data showed that most of the GhKEAs were highly expressed in roots and stems. The quantificational real-time polymerase chain reaction (qRT-PCR) results showed that most of the GhKEAs responded to low potassium, salt and drought stresses. Virus-induced gene silencing (VIGS) experiments demonstrated that under salt stress, after silencing genes GhKEA4 and GhKEA12, the chlorophyll content, proline content, soluble sugar content, peroxidase (POD) activity and catalase (CAT) activity were significantly decreased, and the Na+/K+ ratio was extremely significantly increased in leaves, leading to greater salt sensitivity. Under high potassium stress, cotton plants silenced for the GhKEA4 could still maintain a more stable Na+ and K+ balance, and the activity of transporting potassium ions from roots into leaves was reduced silenced for GhKEA12. Under low potassium stress, silencing the GhKEA4 increased the activity of transporting potassium ions to shoots, and silencing the GhKEA12 increased the ability of absorbing potassium ions, but accumulated more Na+ in leaves. These results provided a basis for further studies on the biological roles of KEA genes in cotton development and adaptation to stress conditions.