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.

Centrifugo-Pneumatic Reciprocating Flowing Coupled with a Spatial Confinement Strategy for an Ultrafast Multiplexed Immunoassay.

Immunoassays serve as powerful diagnostic tools for early disease screening, process monitoring, and precision treatment. However, the current methods are limited by high costs, prolonged processing times (>2 h), and operational complexities that hinder their widespread application in point-of-care testing. Here, we propose a novel centrifugo-pneumatic reciprocating flowing coupled with spatial confinement strategy, termed PRCM, for ultrafast multiplexed immunoassay of pathogens on a centrifugal microfluidic platform. Each chip consists of four replicated units; each unit allows simultaneous detection of three targets, thereby facilitating high-throughput parallel analysis of multiple targets. The PRCM platform enables sequential execution of critical steps such as solution mixing, reaction, and drainage by coordinating inherent parameters, including motor rotation speed, rotation direction, and acceleration/deceleration. By integrating centrifugal-mediated pneumatic reciprocating flow with spatial confinement strategies, we significantly reduce the duration of immune binding from 30 to 5 min, enabling completion of the entire testing process within 20 min. As proof of concept, we conducted a simultaneous comparative test on- and off-the-microfluidics using 12 negative and positive clinical samples. The outcomes yielded 100% accuracy in detecting the presence or absence of the SARS-CoV-2 virus, thus highlighting the potential of our PRCM system for multiplexed point-of-care immunoassays.

Fully Integrated and High-Throughput Microfluidic System for Multiplexed Point-Of-Care Testing.

For every epidemic outbreak, the prevention and treatments in resource-limited areas are always out of reach. Critical to this is that high accuracy, stability, and more comprehensive analytical techniques always rely on expensive and bulky instruments and large laboratories. Here, a fully integrated and high-throughput microfluidic system is proposed for ultra-multiple point-of-care immunoassay, termed Dac system. Specifically, the Dac system only requires a handheld portable device to automatically recycle repetitive multi-step reactions including on-demand liquid releasing, dispensing, metering, collecting, oscillatory mixing, and discharging. The Dac system performs high-precision enzyme-linked immunosorbent assays for up to 17 samples or targets simultaneously on a single chip. Furthermore, reagent consumption is only 2% compared to conventional ELISA, and microbubble-accelerated reactions shorten the assay time by more than half. As a proof of concept, the multiplexed detections are achieved by detecting at least four infection targets for two samples simultaneously on a singular chip. Furthermore, the barcode-based multi-target results can rapidly distinguish between five similar cases, allowing for accurate therapeutic interventions. Compared to bulky clinical instruments, the accuracy of clinical inflammation classification is 92.38% (n = 105), with a quantitative correlation coefficient of R2 = 0.9838, while the clinical specificity is 100% and the sensitivity is 98.93%.

Visible-Light-Promoted Aerobic α-Thiocyanation of Carbonyl Compounds with Ammonium Thiocyanate.

In the present study, we successfully developed an efficient thiocyanation of carbonyl compounds by using low-toxicity and inexpensive ammonium thiocyanate as the thiocyanate source under visible light in air (O2) at room temperature. This unified strategy is very facile for thiocyanation of various carbonyl compound derivatives (ß-keto esters, ß-keto amides, pyrazo-5-ones, isoxazol-5-ones, etc.). More importantly, the reaction proceeded smoothly without the addition of a photocatalyst and strong oxidant, ultimately minimizing the production of chemical waste. Furthermore, this green and sustainable synthetic chemistry can be used in the late-stage functionalization (LSF) of biorelevant compounds, which offers unique opportunities to achieve smooth and clean thiocyanation of drugs under mild reaction conditions.

Research progress of multi-target HDAC inhibitors blocking the BRD4-LIFR-JAK1-STAT3 signaling pathway in the treatment of cancer.

Histone deacetylase inhibitors (HDACis) show beneficial effects on different hematological malignancy subtypes. However, their impacts on treating solid tumors are still limited due to diverse resistance mechanisms. Recent studies have found that the feedback activation of BRD4-LIFR-JAK1-STAT3 pathway after HDACi incubation is a vital mechanism inducing resistance of specific solid tumor cells to HDACis. This review summarizes the recent development of multi-target HDACis that can concurrently block BRD4-LIFR-JAK1-STAT3 pathway. Moreover, our findings hope to shed novel lights on developing novel multi-target HDACis with reduced BRD4-LIFR-JAK1-STAT3-mediated drug resistance in some tumors.

Integrating network pharmacology and experimental validation to explore the mitophagy-associated pharmacological mechanism of modified Shisiwei Jianzhong decoction against aplastic anemia.

The aim of this work was to investigate the therapeutic effect of modified Shisiwei Jianzhong Decoction (SJD) on aplastic anemia (AA) and its potential pharmacological mechanism from the perspective of mitophagy. A comprehensive approach combining network pharmacology, mendelian randomization, molecular docking and animal experiments was applied to evaluate the properties of SJD against AA. By integrating multiple databases, it was determined that SJD exerted its therapeutic effect on AA by targeting three key targets [mammalian target of rapamycin (MTOR), poly(ADP-ribose) polymerase 1 (PARP1) and Sirtuin 1 (SIRT1)] through four core compounds (quercetin, resveratrol, genistein and curcumin). Mendelian randomization analysis identified MTOR as a risk factor for AA occurrence while PARP1 was a protective factor. Results of animal experiments showed that SJD improved peripheral blood counts and promoted the proliferation of hematopoietic stem cells. Mechanistically, SJD, especially at high dose, played a therapeutic role in AA by activating mitophagy-related proteins PTEN induced kinase 1 (PINK1)/Parkin and inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase (AKT)/MTOR pathway. This study revealed for the first time the core chemical composition of SJD and its pharmacological effects against AA, which can restore hematopoietic function by activating mitophagy. The results provide inspiration for the clinical application of traditional Chinese medicine in AA treatment.

Effects of probiotic supplementation on 12 min run performance, mood management, body composition and gut microbiota in amateur marathon runners: A double-blind controlled trial.

Background: Probiotic supplementation has a positive effect on endurance exercise performance and body composition in athletes, but the underlying mechanisms remain unclear. Gut microbiota can provide measurable markers of immune function in athletes, and microbial composition analysis may be sensitive enough to detect stress and metabolic disorders caused by exercise. Methods: Nineteen healthy active amateur marathon runners (15 male and 4 female) with a mean age of 29.11 years volunteered to participate in this double-blind controlled study. Based on the performance of the Cooper 12-min running test (CRT), the participants were allocated into two groups to receive either a probiotic formulation comprising lactobacillus acidophilus and bifidobacterium longum (n = 10) or placebo containing maltodextrin (n = 9) for five weeks. Consistency of diet and exercise was ensured throughout the experimental period. Before and after the intervention, all participants were assessed for CRT, emotional stability and gastrointestinal symptoms, gut microbiota composition, body composition and magnetic resonance imaging (MRI) indicators of skeletal muscle microcirculation. Results: Compared to before the intervention, the probiotics group showed an increase in CRT score (2.88 ± 0.57 vs 3.01 ± 0.60 km, P＜0.05), significant improvement in GSRS and GIQLI (9.20 ± 4.64 vs 7.40 ± 3.24, 118.90 ± 12.30 vs 127.50 ± 9.85, P＜0.05), while these indicators remained unchanged in the control group, with a significant time-group interaction effect on gastrointestinal symptoms. Additionally, some MRI metabolic cycling indicators of the thigh skeletal muscle also changed in the probiotics group (P＜0.05). Regarding microbiota abundance, the probiotics group exhibited a significant increase in the abundance of beneficial bacteria and a significant decrease in the abundance of harmful bacteria post-intervention (P＜0.05). Conclusion: As a sports nutritional supplement, probiotics have the potential to improve athletic performance by optimizing the balance of gut microbiota, alleviating gastrointestinal symptoms.

On-Line Dual-Active Valves Based Centrifugal Microfluidic Chip for Fully Automated Point-of-Care Immunoassay.

There remains an unmet need for a fully integrated microfluidic platform that can automatically perform multistep and multireagent immunoassays. Here, we proposed a novel online dual-active valve-based centrifugal microfluidic chip, termed DAVM, for fully automatic point-of-care immunoassay. Practically, the puncture valve, one of the dual active valves, is capable of achieving precise, on-demand, sequential release of prestored reagents, while the other valve-reversible active valve enables controlled retention and drainage of the reaction solutions. Thereby, our technology mitigates the challenges of hydrophilic/hydrophobic modifications and unstable valve control performance commonly observed in passive valve controls. As a proof of concept, the indirect enzymatic immunoblotting technique was employed on DAVM for fully automated immunological analysis of eight targets, yielding outcomes within an hour. Furthermore, we conducted a comparative analysis of 28 clinical samples with autoimmune diseases. According to 224 clinical data, the sample testing concordance rate between DAVM and the traditional instrument was 82%, with a target compliance rate of 97%. Therefore, our DAVM system has powerful potential for fully automated immunoassays.

Development of a rapid, sensitive detection method for SARS-CoV-2 and influenza virus based on recombinase polymerase amplification combined with CRISPR-Cas12a assay.

Respiratory tract infections are associated with the most common diseases transmitted among people and remain a huge threat to global public health. Rapid and sensitive diagnosis of causative agents is critical for timely treatment and disease control. Here, we developed a novel method based on recombinase polymerase amplification (RPA) combined with CRISPR-Cas12a to detect three viral pathogens, including SARS-CoV-2, influenza A, and influenza B, which cause similar symptom complexes of flu cold in the respiratory tract. The detection method can be completed within 1 h, which is faster than other standard detection methods, and the limit of detection is approximately 102 copies/µL. Additionally, this detection system is highly specific and there is no cross-reactivity with other common respiratory tract pathogens. Based on this assay, we further developed a more simplified RPA/CRISPR-Cas12a system combined with lateral flow assay on a manual microfluidic chip, which can simultaneously detect these three viruses. This low-cost detection system is rapid and sensitive, which could be applied in the field and resource-limited areas without bulky and expensive instruments, providing powerful tools for the point-of-care diagnostic.

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.

Comparison of haploidentical-allogeneic hematopoietic stem cell transplantation and intensive immunosuppressive therapy for patients with severe aplastic anemia with an absolute neutrophil count of zero: a retrospective study.

A retrospective analysis was conducted based on the clinical data from 60 patients older than 16 years from January 2016 to January 2021. All the patients were newly diagnosed with severe aplastic anemia (SAA) with an absolute neutrophil count (ANC) of zero. We compared the hematological response and survival of haploidentical-allogeneic hematopoietic stem cell transplantation (HID-HSCT) (n = 25) and intensive immunosuppressive therapy (IST) (n = 35) treatments. At six months, the overall response rate and complete response were significantly higher in the HID-HSCT group than those in the IST group (84.0% vs. 40.0%, P = 0.001; 80.0% vs. 17.1%, P = 0.001). With a median follow-up of 18.5 months (4.3~30.8 months), patients in the HID-HSCT group had longer overall survival and event-free survival (80.0% vs. 47.9%, P = 0.0419; 79.2% vs. 33.5%, P = 0.0048). These data suggested that HID-HSCT might be an effective alternative treatment option for adult patients with SAA with an ANC of zero, which requires further validation in an additional prospective study.

Whole-exome sequencing identifies FANC heterozygous germline mutation as an adverse factor for immunosuppressive therapy in Chinese aplastic anemia patients aged 40 or younger: a single-center retrospective study.

Acquired aplastic anemia (AA) is a bone marrow failure disorder characterized by pancytopenia, and immunosuppressive therapy (IST) is the optional first-line management. Several studies identified the influencing factors on IST response; however, there are still a considerable number of patients suffering from poor prognoses. In this study, we enrolled 61 AA patients aged ≤ 40 years old, and whole-exome sequencing (WES) found unexpected high FANC heterozygous germline mutations (28/61, 45.9%). Patients with FANC mutations have a significantly lower absolute reticulocyte count and CD34+ % in the bone marrow and also lower 3-, 6-, and 9-month IST response than that without mutation, which were 0% vs. 25% (P = 0.017), 26.3% vs. 42.1% (P = 0.495), and 29.4% vs. 72.2% (P = 0.011), especially in anti-thymocyte globulin combined with the cyclosporin A (ATG + CsA) group, which were 0% vs.33.4% (P = 0.143), 25% vs.83.3% (P = 0.103), and 25% vs. 100% (P = 0.003), respectively. The event-free survival in the FANCwt group was also better than that in the FANCmut group (P = 0.016) and also showed in patients who received ATG + CsA treatment (P = 0.045). In addition, all the adverse effects of FANC germline mutation were not significant in stem cell-transplanted group. Our result indicated that the WES-based detection of FANC heterozygous germline mutations may have a great meaning in predicting IST response of acquired AA. This study was registered at chictr.org.cn (# ChiCTR2100054992).

Design, synthesis, and biological evaluation of quinolinedione-linked sulfonylpiperazine derivatives as NQO1-directed antitumor agents.

In the current study, a series of novel quinolinedione-linked sulfonylpiperazine derivatives have been reported as NQO1-directed antitumor agents. A majority of compounds in this study were found to be more effective in resisting the proliferation of cancer cells than that of the positive control 5-Fu and TSA. Among the tested compounds, the derivative 22r exhibited considerable effect (IC50, 3.29-5.19 µM) against the proliferation of three NQO1-rich cancer cells (HepG2, MCF-7, and A549), and was recognized to be an excellent NQO1 substrate as revealed by in vitro enzyme reduction assay and molecular docking study with NQO1. In studies on the mechanisms involved, 22r induced reactive oxygen species (ROS) production, caused DNA damage, and induced apoptosis in HepG2 cells. Remarkably, compound 22r exhibited excellent anticancer activity against HepG2 xenograft models in vivo. The study demonstrated that compound 22r provided a promising strategy for the management of malignant tumors.

Design, synthesis, and biological evaluation of novel napabucasin-melatonin hybrids as potent STAT3 inhibitors.

The current work developed diverse novel napabucasin-melatonin hybrids as potent STAT3 inhibitors. Several biological studies have suggested many compounds demonstrating potent inhibition against different tumor cells. Among these, compound 7e depicted enhanced inhibition against HepG2, MDA-MB-231, and A549 cells than napabucasin, with IC50 values of 1.06, 1.38, and 1.3 µM, respectively. Based on fluorescence polarization analysis, compound 7e was bound to the SH2 domain in STAT3, with an IC50 value of 12.95 µM. Molecular docking further confirmed the 7e binding mode inside the SH2 domain of STAT3. Further mechanistic studies indicated that 7e inhibited the activation of STAT3 (Y705), and thus reduced the expression of STAT3 downstream genes (CyclinD1, Bcl-2 and c-Myc) instead of affecting p-STAT1 expression. Meanwhile, the phosphorylation levels of its upstream kinases JAK2 and bypass kinase Erk1/2 remain unaffected. Simultaneously, 7e induced cancer cell apoptosis in a concentration-dependent manner. Significantly, 20 mg/kg (i.p.) compound 7e suppressed the mouse HepG2 xenograft development in vivo without body weight loss, suggesting that it could be an effective antitumor agent.

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.

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.

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.

Evolution, expression and functional analysis of cultivated allotetraploid cotton DIR genes.

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.

Dynamic characteristics and functional analysis provide new insights into long non-coding RNA responsive to Verticillium dahliae infection in Gossypium hirsutum.

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.

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.