Genome-Wide Identification of the Alfin-like Gene Family in Cotton (Gossypium hirsutum) and the GhAL19 Gene Negatively Regulated Drought and Salt Tolerance.

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.

Identification of Crucial Modules and Genes Associated with Bt Gene Expression in Cotton.

The expression of Bacillus thuringiensis (Bt) toxins in transgenic cotton confers resistance to insect pests. However, it has been demonstrated that its effectiveness varies among cotton cultivars and different tissues. In this study, we evaluated the expression of Bt protein in 28 cotton cultivars and selected 7 cultivars that differed in Bt protein expression for transcriptome analysis. Based on their Bt protein expression levels, the selected cultivars were categorized into three groups: H (high Bt protein expression), M (moderate expression), and L (low expression). In total, 342, 318, and 965 differentially expressed genes were detected in the H vs. L, M vs. L, and H vs. M comparison groups, respectively. And three modules significantly associated with Bt protein expression were identified by weighted gene co-expression network analysis. Three hub genes were selected to verify their relationships with Bt protein expression using virus-induced gene silencing (VIGS). Silencing GhM_D11G1176, encoding an MYC transcription factor, was confirmed to significantly decrease the expression of Bt protein. The present findings contribute to an improved understanding of the mechanisms that influence Bt protein expression in transgenic cotton.

Deepening genomic sequences of 1081 Gossypium hirsutum accessions reveals novel SNPs and haplotypes relevant for practical breeding utility.

Fiber quality is a major breeding goal in cotton, but phenotypically direct selection is often hindered. In this study, we identified fiber quality and yield related loci using GWAS based on 2.97 million SNPs obtained from 10.65× resequencing data of 1081 accessions. The results showed that 585 novel fiber loci, including two novel stable SNP peaks associated with fiber length on chromosomes At12 and Dt05 and one novel genome regions linked with fiber strength on chromosome Dt12 were identified. Furthermore, by means of gene expression analysis, GhM_A12G0090, GhM_D05G1692, GhM_D12G3135 were identified and GhM_D11G2208 function was identified in Arabidopsis. Additionally, 14 consistent and stable superior haplotypes were identified, and 25 accessions were detected as possessing these 14 superior haplotype in breeding. This study providing fundamental insight relevant to identification of genes associated with fiber quality and yield will enhance future efforts toward improvement of upland cotton.

Identification and Expression Analysis of EPSPS and BAR Families in Cotton.

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.

Genome-wide association study reveals novel SNPs and genes in Gossypium hirsutum underlying Aphis gossypii resistance.

KEY MESSAGE: A. gossypii resistance showed great variability in G. hirsutum varieties. One hundred and seventy-six SNPs associated with A. gossypii resistance were identified using GWAS. Four candidate resistance genes were functionally validated. Aphis gossypii is an economically important sap-feeding pest and is widely distributed in the world's cotton-producing regions. Identification of cotton genotypes and developing cultivars with improved A. gossypii resistance (AGR) is essential and desirable for sustainable agriculture. In the present study, A. gossypii was offered no choice but to propagate on 200 Gossypium hirsutum accessions. A relative aphid reproduction index (RARI) was used to evaluate the AGR, which showed large variability in cotton accessions and was classified into 6 grades. A significantly positive correlation was found between AGR and Verticillium wilt resistance. A total of 176 SNPs significantly associated with the RARI were identified using GWAS. Of these, 21 SNPs could be repeatedly detected in three replicates. Cleaved amplified polymorphic sequence, a restriction digestion-based genotyping assay, was developed using SNP1 with the highest observed -log10(P-value). Four genes within the 650 kb region of SNP1 were further identified, including GhRem (remorin-like), GhLAF1 (long after far-red light 1), GhCFIm25 (pre-mRNA cleavage factor Im 25 kDa subunit) and GhPMEI (plant invertase/pectin methylesterase inhibitor superfamily protein). The aphid infection could induce their expression and showed a significant difference between resistant and susceptible cotton varieties. Silencing of GhRem, GhLAF1 or GhCFIm25 could significantly increase aphid reproduction on cotton seedlings. Silencing of GhRem significantly reduced callose deposition, which is reasonably believed to be the cause for the higher AGR. Our results provide insights into understanding the genetic regulation of AGR in cotton and suggest candidate germplasms, SNPs and genes for developing cultivars with improved AGR.

Lysine 2-Hydroxyisobutyrylation- and Succinylation-Based Pathways Act Inside Chloroplasts to Modulate Plant Photosynthesis and Immunity.

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.

A regional comparative study on the mismatch between population urbanization and land urbanization in China.

By taking 31 provinces (municipalities/autonomous regions) in Mainland China as the object of research, and using the data on urban population and built-up area of each region from 2005 to 2019, this paper measures the dispersion coefficient of population urbanization and land urbanization in each region through models and visually expresses the level and type of imbalance between them to reveal the temporal and spatial characteristics of imbalance. The results of the research show that since China's state-owned land was sold through bidding, auction, and listing, the overall urbanization of the population and land development have become unbalanced. There is obvious regional and category difference in imbalance between population urbanization and land urbanization. The degree of imbalance increases from the central, eastern, northeastern to western regions. The remaining 29 regions are generally lagging in population urbanization except for Beijing and Hebei province. This imbalance is mainly caused by China's dual household registration system, dual land system and the asymmetrical tax distribution system between financial rights and administrative rights.

Transcriptome, Ectopic Expression and Genetic Population Analysis Identify Candidate Genes for Fiber Quality Improvement in Cotton.

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.

The transcription factor GhWRKY70 from gossypium hirsutum enhances resistance to verticillium wilt via the jasmonic acid pathway.

BACKGROUND: The WRKY transcription factors play significant roles in plant growth, development, and defense responses. However, in cotton, the molecular mechanism of most WRKY proteins and their involvement in Verticillium wilt tolerance are not well understood. RESULTS: GhWRKY70 is greatly up-regulated in cotton by Verticillium dahliae. Subcellular localization suggests that GhWRKY70 is only located in the nucleus. Transcriptional activation of GhWRKY70 further demonstrates that GhWRKY70 function as a transcriptional activator. Transgenic Arabidopsis plants overexpressing GhWRKY70 exhibited better growth performance and higher lignin content, antioxidant enzyme activities and jasmonic acid (JA) levels than wild-type plants after infection with V. dahliae. In addition, the transgenic Arabidopsis resulted in an enhanced expression level of AtAOS1, a gene related to JA synthesis, further leading to a higher JA accumulation compared to the wild type. However, the disease index (DI) values of the VIGS-treated cotton plants with TRV:WRKY70 were also significantly higher than those of the VIGS-treated cotton plants with TRV:00. The chlorophyll and lignin contents of TRV:WRKY70 plants were significantly lower than those of TRV:00 plants. GhAOS1 expression and JA abundance in TRV:WRKY70 plants were decreased. The GhWRKY70 protein was confirmed to bind to the W-box element in the promoter region of GhAOS by yeast one-hybrid assay and transient expression. CONCLUSION: These results indicate that the GhWRKY70 transcription factor is a positive regulator in Verticillium wilt tolerance of cotton, and may promote the production of JA via regulation of GhAOS1 expression.

SEP-like genes of Gossypium hirsutum promote flowering via targeting different loci in a concentration-dependent manner.

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.

A Genome Wide Association Study Revealed Key Single Nucleotide Polymorphisms/Genes Associated With Seed Germination in Gossypium hirsutum L.

Fast and uniform seed germination is essential to stabilize crop yields in agricultural production. It is important to understand the genetic basis of seed germination for improving the vigor of crop seeds. However, little is known about the genetic basis of seed vigor in cotton. In this study, we evaluated four seed germination-related traits of a core collection consisting of 419 cotton accessions, and performed a genome-wide association study (GWAS) to explore important loci associated with seed vigor using 3.66 million high-quality single nucleotide polymorphisms (SNPs). The results showed that four traits, including germination potential, germination rate, germination index, and vigor index, exhibited broad variations and high correlations. A total of 92 significantly associated SNPs located within or near 723 genes were identified for these traits, of which 13 SNPs could be detected in multiple traits. Among these candidate genes, 294 genes were expressed at seed germination stage. Further function validation of the two genes of higher expression showed that Gh_A11G0176 encoding Hsp70-Hsp90 organizing protein negatively regulated Arabidopsis seed germination, while Gh_A09G1509 encoding glutathione transferase played a positive role in regulating tobacco seed germination and seedling growth. Furthermore, Gh_A09G1509 might promote seed germination and seedling establishment through regulating glutathione metabolism in the imbibitional seeds. Our findings provide unprecedented information for deciphering the genetic basis of seed germination and performing molecular breeding to improve field emergence through genomic selection in cotton.

Development and Utilization of Functional Kompetitive Allele-Specific PCR Markers for Key Genes Underpinning Fiber Length and Strength in Gossypium hirsutum L.

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.

GhENODL6 Isoforms from the Phytocyanin Gene Family Regulated Verticillium Wilt Resistance in Cotton.

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.

Novel insights into water-deficit-responsive mRNAs and lncRNAs during fiber development in Gossypium hirsutum.

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.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene editing analysis in cotton.

CRIPSR/Cas9 gene editing system is an effective tool for genome modification in plants. Multiple target sites are usually designed and the effective target sites are selected for editing. Upland cotton (Gossypium hirsutum L., hereafter cotton) is allotetraploid and is commonly considered as difficult and inefficient to transform, it is important to select the effective target sites that could result in the ideal transgenic plants with the CRISPR-induced mutations. In this study, Agrobacterium rhizogenes-mediated hairy root method was optimized to detect the feasibility of the target sites designed in cotton phytoene desaturase (GhPDS) gene. A. rhizogenes showed the highest hairy root induction (30%) when the bacteria were cultured until OD600 reached to 0.8. This procedure was successfully applied to induce hairy roots in the other three cultivars (TM-1, Lumian-21, Zhongmian-49) and the mutations were detected in GhPDS induced by CRISPR/Cas9 system. Different degrees of base deletions at two sgRNAs (sgRNA5 and sgRNA10) designed in GhPDS were detected in R15 hairy roots. Furthermore, we obtained an albino transgenic cotton seeding containing CRISPR/Cas9-induced gene editing mutations in sgRNA10. The hairy root transformation system established in this study is sufficient for selecting sgRNAs in cotton, providing a technical basis for functional genomics research of cotton.

High-quality genome assembly and resequencing of modern cotton cultivars provide resources for crop 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.

Cotton GhSSI2 isoforms from the stearoyl acyl carrier protein fatty acid desaturase family regulate Verticillium wilt resistance.

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.

A large-scale genomic association analysis identifies a fragment in Dt11 chromosome conferring cotton Verticillium wilt resistance.

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.

Tissue-specific expression of GhnsLTPs identified via GWAS sophisticatedly coordinates disease and insect resistance by regulating metabolic flux redirection in cotton.

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.