Genome-Wide Identification, Characterization and Experimental Expression Analysis of CNGC Gene Family in Gossypium.

Cyclic nucleotide-gated ion channels (CNGCs) are channel proteins for calcium ions, and have been reported to play important roles in regulating survival and environmental response of various plants. However, little is known about how the CNGC family works in Gossypium. In this study, 173 CNGC genes, which were identified from two diploid and five tetraploid Gossypium species, were classified into four groups by phylogenetic analysis. The collinearity results demonstrated that CNGC genes are integrally conservative among Gossypium species, but four gene losses and three simple translocations were detected, which is beneficial to analyzing the evolution of CNGCs in Gossypium. The various cis-acting regulatory elements in the CNGCs' upstream sequences revealed their possible functions in responding to multiple stimuli such as hormonal changes and abiotic stresses. In addition, expression levels of 14 CNGC genes changed significantly after being treated with various hormones. The findings in this study will contribute to understanding the function of the CNGC family in cotton, and lay a foundation for unraveling the molecular mechanism of cotton plants' response to hormonal changes.

Phenomics and transcriptomics analyses reveal deposition of suberin and lignin in the short fiber cell walls produced from a wild cotton species and two mutants.

Fiber length is one of the major properties determining the quality and commercial value of cotton. To understand the mechanisms regulating fiber length, genetic variations of cotton species and mutants producing short fibers have been compared with cultivated cottons generating long and normal fibers. However, their phenomic variation other than fiber length has not been well characterized. Therefore, we compared physical and chemical properties of the short fibers with the long fibers. Fiber characteristics were compared in two sets: 1) wild diploid Gossypium raimondii Ulbrich (short fibers) with cultivated diploid G. arboreum L and tetraploid G. hirsutum L. (long fibers); 2) G. hirsutum short fiber mutants, Ligon-lintless 1 (Li1) and 2 (Li2) with their near isogenic line (NIL), DP-5690 (long fibers). Chemical analyses showed that the short fibers commonly consisted of greater non-cellulosic components, including lignin and suberin, than the long fibers. Transcriptomic analyses also identified up-regulation of the genes related to suberin and lignin biosynthesis in the short fibers. Our results may provide insight on how high levels of suberin and lignin in cell walls can affect cotton fiber length. The approaches combining phenomic and transcriptomic analyses of multiple sets of cotton fibers sharing a common phenotype would facilitate identifying genes and common pathways that significantly influence cotton fiber properties.

Fuzzy clustering for the within-season estimation of cotton phenology.

Crop phenology is crucial information for crop yield estimation and agricultural management. Traditionally, phenology has been observed from the ground; however Earth observation, weather and soil data have been used to capture the physiological growth of crops. In this work, we propose a new approach for the within-season phenology estimation for cotton at the field level. For this, we exploit a variety of Earth observation vegetation indices (derived from Sentinel-2) and numerical simulations of atmospheric and soil parameters. Our method is unsupervised to address the ever-present problem of sparse and scarce ground truth data that makes most supervised alternatives impractical in real-world scenarios. We applied fuzzy c-means clustering to identify the principal phenological stages of cotton and then used the cluster membership weights to further predict the transitional phases between adjacent stages. In order to evaluate our models, we collected 1,285 crop growth ground observations in Orchomenos, Greece. We introduced a new collection protocol, assigning up to two phenology labels that represent the primary and secondary growth stage in the field and thus indicate when stages are transitioning. Our model was tested against a baseline model that allowed to isolate the random agreement and evaluate its true competence. The results showed that our model considerably outperforms the baseline one, which is promising considering the unsupervised nature of the approach. The limitations and the relevant future work are thoroughly discussed. The ground observations are formatted in an ready-to-use dataset and will be available at https://github.com/Agri-Hub/cotton-phenology-dataset upon publication.

Systematic analysis and expression of Gossypium 2ODD superfamily highlight the roles of GhLDOXs responding to alkali and other abiotic stress in cotton.

BACKGROUND: 2-oxoglutarate-dependent dioxygenase (2ODD) is the second largest family of oxidases involved in various oxygenation/hydroxylation reactions in plants. Many members in the family regulate gene transcription, nucleic acid modification/repair and secondary metabolic synthesis. The 2ODD family genes also function in the formation of abundant flavonoids during anthocyanin synthesis, thereby modulating plant development and response to diverse stresses. RESULTS: Totally, 379, 336, 205, and 204 2ODD genes were identified in G. barbadense (Gb), G. hirsutum (Gh), G. arboreum (Ga), and G. raimondii (Gb), respectively. The 336 2ODDs in G. hirsutum were divided into 15 subfamilies according to their putative functions. The structural features and functions of the 2ODD members in the same subfamily were similar and evolutionarily conserved. Tandem duplications and segmental duplications served essential roles in the large-scale expansion of the cotton 2ODD family. Ka/Ks values for most of the gene pairs were less than 1, indicating that 2ODD genes undergo strong purifying selection during evolution. Gh2ODDs might act in cotton responses to different abiotic stresses. GhLDOX3 and GhLDOX7, two members of the GhLDOX subfamily from Gh2ODDs, were significantly down-regulated in transcription under alkaline stress. Moreover, the expression of GhLDOX3 in leaves was significantly higher than that in other tissues. These results will provide valuable information for further understanding the evolution mechanisms and functions of the cotton 2ODD genes in the future. CONCLUSIONS: Genome-wide identification, structure, and evolution and expression analysis of 2ODD genes in Gossypium were carried out. The 2ODDs were highly conserved during evolutionary. Most Gh2ODDs were involved in the regulation of cotton responses to multiple abiotic stresses including salt, drought, hot, cold and alkali.

Cell-specific clock-controlled gene expression program regulates rhythmic fiber cell growth in cotton.

BACKGROUND: The epidermis of cotton ovule produces fibers, the most important natural cellulose source for the global textile industry. However, the molecular mechanism of fiber cell growth is still poorly understood. RESULTS: Here, we develop an optimized protoplasting method, and integrate single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) to systematically characterize the cells of the outer integument of ovules from wild type and fuzzless/lintless (fl) cotton (Gossypium hirsutum). By jointly analyzing the scRNA-seq data from wildtype and fl, we identify five cell populations including the fiber cell type and construct the development trajectory for fiber lineage cells. Interestingly, by time-course diurnal transcriptomic analysis, we demonstrate that the primary growth of fiber cells is a highly regulated circadian rhythmic process. Moreover, we identify a small peptide GhRALF1 that circadian rhythmically controls fiber growth possibly through oscillating auxin signaling and proton pump activity in the plasma membrane. Combining with scATAC-seq, we further identify two cardinal cis-regulatory elements (CREs, TCP motif, and TCP-like motif) which are bound by the trans factors GhTCP14s to modulate the circadian rhythmic metabolism of mitochondria and protein translation through regulating approximately one third of genes that are highly expressed in fiber cells. CONCLUSIONS: We uncover a fiber-specific circadian clock-controlled gene expression program in regulating fiber growth. This study unprecedentedly reveals a new route to improve fiber traits by engineering the circadian clock of fiber cells.

Fine mapping and candidate gene analysis of qFL-A12-5: a fiber length-related QTL introgressed from Gossypium barbadense into Gossypium hirsutum.

KEY MESSAGE: The fiber length-related qFL-A12-5 identified in CSSLs introgressed from Gossypium barbadense into Gossypium hirsutum was fine-mapped to an 18.8 kb region on chromosome A12, leading to the identification of the GhTPR gene as a potential regulator of cotton fiber length. Fiber length is a key determinant of fiber quality in cotton, and it is a key target of artificial selection for breeding and domestication. Although many fiber length-related quantitative trait loci have been identified, there are few reports on their fine mapping or candidate gene validation, thus hampering efforts to understand the mechanistic basis of cotton fiber development. Our previous study identified the qFL-A12-5 associated with superior fiber quality on chromosome A12 in the chromosome segment substitution line (CSSL) MBI7747 (BC4F3:5). A single segment substitution line (CSSL-106) screened from BC6F2 was backcrossed to construct a larger segregation population with its recurrent parent CCRI45, thus enabling the fine mapping of 2852 BC7F2 individuals using denser simple sequence repeat markers to narrow the qFL-A12-5 to an 18.8 kb region of the genome, in which six annotated genes were identified in Gossypium hirsutum. Quantitative real-time PCR and comparative analyses led to the identification of GH_A12G2192 (GhTPR) encoding a tetratricopeptide repeat-like superfamily protein as a promising candidate gene for qFL-A12-5. A comparative analysis of the protein-coding regions of GhTPR among Hai1, MBI7747, and CCRI45 revealed two non-synonymous mutations. The overexpression of GhTPR resulted in longer roots in Arabidopsis, suggesting that GhTPR may regulate cotton fiber development. These results provide a foundation for future efforts to improve cotton fiber length.

Genome scale analysis of 1-aminocyclopropane-1-carboxylate oxidase gene family in G. barbadense and its functions in cotton fiber development.

A class of proteins, 1-aminocyclopropane-1-carboxylate oxidase (ACO), is required in the final step of production of ethylene from its immediate precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Despite the crucial and regulatory role of ACO gene family in the fiber development, it has not been thoroughly analyzed and annotated in G. barbadense genome. In the present study, we have identified and characterized all isoforms of ACO gene family from genomes of Gossypium arboreum, G. barbadense, G. hirsutum and G. raimondii. Phylogenetic analysis classified all ACO proteins into six distinct groups on the basis of maximum likelihood. Gene locus analysis and circos plots indicated the distribution and relationship of these genes in cotton genomes. Transcriptional profiling of ACO isoforms in G. arboreum, G. barbadense and G. hirsutum fiber development exhibited the highest expression in G. barbadense during early fiber elongation. Moreover, the accumulation of ACC was found highest in developing fibers of G. barbadense in comparison with other cotton species. ACO expression and ACC accumulation correlated with the fiber length in cotton species. Addition of ACC to the ovule cultures of G. barbadense significantly increased fiber elongation while ethylene inhibitors hindered fiber elongation. These findings will be helpful in dissecting the role of ACOs in cotton fiber development and pave a way towards genetic manipulations for fiber quality improvement.

The CCCH-Type Zinc-Finger Protein GhC3H20 Enhances Salt Stress Tolerance in Arabidopsis thaliana and Cotton through ABA Signal Transduction Pathway.

The CCCH zinc-finger protein contains a typical C3H-type motif widely existing in plants, and it plays an important role in plant growth, development, and stress responses. In this study, a CCCH zinc-finger gene, GhC3H20, was isolated and thoroughly characterized to regulate salt stress in cotton and Arabidopsis. The expression of GhC3H20 was up-regulated under salt, drought, and ABA treatments. GUS activity was detected in the root, stem, leaves, and flowers of ProGhC3H20::GUS transgenic Arabidopsis. Compared with the control, the GUS activity of ProGhC3H20::GUS transgenic Arabidopsis seedlings under NaCl treatment was stronger. Through the genetic transformation of Arabidopsis, three transgenic lines of 35S-GhC3H20 were obtained. Under NaCl and mannitol treatments, the roots of the transgenic lines were significantly longer than those of the wild-type (WT) Arabidopsis. The leaves of the WT turned yellow and wilted under high-concentration salt treatment at the seedling stage, while the leaves of the transgenic Arabidopsis lines did not. Further investigation showed that compared with the WT, the content of catalase (CAT) in the leaves of the transgenic lines was significantly higher. Therefore, compared with the WT, overexpression of GhC3H20 enhanced the salt stress tolerance of transgenic Arabidopsis. A virus-induced gene silencing (VIGS) experiment showed that compared with the control, the leaves of pYL156-GhC3H20 plants were wilted and dehydrated. The content of chlorophyll in pYL156-GhC3H20 leaves was significantly lower than those of the control. Therefore, silencing of GhC3H20 reduced salt stress tolerance in cotton. Two interacting proteins (GhPP2CA and GhHAB1) of GhC3H20 have been identified through a yeast two-hybrid assay. The expression levels of PP2CA and HAB1 in transgenic Arabidopsis were higher than those in the WT, and pYL156-GhC3H20 had expression levels lower than those in the control. GhPP2CA and GhHAB1 are the key genes involved in the ABA signaling pathway. Taken together, our findings demonstrate that GhC3H20 may interact with GhPP2CA and GhHAB1 to participate in the ABA signaling pathway to enhance salt stress tolerance in cotton.

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.

Estimating the combining ability and genetic parameters for growth habit, yield, and fiber quality traits in some Egyptian cotton crosses.

It is crucial to understand how targeted traits in a hybrid breeding program are influenced by gene activity and combining ability. During the three growing seasons of 2015, 2016, and 2017, a field study was conducted with twelve cotton genotypes, comprised of four testers and eight lines. Thirty-two F1 crosses were produced in the 2015 breeding season using the line x tester mating design. The twelve genotypes and their thirty-two F1 crosses were then evaluated in 2016 and 2017. The results demonstrated highly significant differences among cotton genotypes for all the studied traits, showing a wide range of genetic diversity in the parent genotypes. Additionally, the line-x-tester interaction was highly significant for all traits, suggesting the impact of both additive and non-additive variations in gene expression. Furthermore, the thirty-two cotton crosses showed high seed cotton output, lint cotton yield, and fiber quality, such as fiber length values exceeding 31 mm and a fiber strength above 10 g/tex. Accordingly, selecting lines and testers with high GCA effects and crosses with high SCA effects would be an effective approach to improve the desired traits in cotton and develop new varieties with excellent yield and fiber quality.

Pediatric chronic graft-versus-host disease-related dry eye disease and the diagnostic association of potential clinical findings.

Pediatric graft-versus-host-disease (GVHD)-related dry eye disease (DED) is often overlooked due to a lack of subjective symptoms and reliable testing, leading to irreversible corneal damage. To study the clinical findings contributing to the accurate detection of pediatric GVHD-related DED, a retrospective study of pediatric patients treated with hematopoietic stem cell transplantation (HSCT) at Keio University Hospital between 2004 and 2017 was conducted. Association and diagnostic values of ophthalmological findings for DED were analyzed. Twenty-six patients who had no ocular complications before HSCT were included in the study. Eleven (42.3%) patients developed new-onset DED. The cotton thread test showed excellent diagnostic accuracy in detecting DED (area under the receiver operating curve, 0.96; sensitivity, 0.95; specificity, 0.85) with a cut-off of 17 mm, which was higher than the conventional threshold of 10 mm. Additionally, the presence of filamentary keratitis (FK) and pseudomembranous conjunctivitis (PC) were significantly associated with the diagnosis of DED (p value, 0.003 and 0.001 for FK and PC, respectively) and displayed good diagnostic performance (sensitivity, 0.46 and 0.54; specificity, 0.97 and 0.97 for FK and PC, respectively). In conclusion, the cotton thread test with a new threshold, the presence of PC and FK, could be helpful for promptly detecting pediatric GVHD-related DED.

NMR-Based Metabolomics: A New Paradigm to Unravel Defense-Related Metabolites in Insect-Resistant Cotton Variety through Different Multivariate Data Analysis Approaches.

Cotton (Gossypium hirsutum) is an economically important crop and is widely cultivated around the globe. However, the major problem of cotton is its high vulnerability to biotic and abiotic stresses. It has been around three decades since the cotton plant was genetically engineered with genes encoding insecticidal proteins (mainly Cry proteins) with an aim to protect it against insect attack. Several studies have been reported on the impact of these genes on cotton production and fiber quality. However, the metabolites responsible for conferring resistance in genetically modified cotton need to be explored. The current work aims to unveil the key metabolites responsible for insect resistance in Bt cotton and also compare the conventional multivariate analysis methods with deep learning approaches to perform clustering analysis. We aim to unveil the marker compounds which are responsible for inducing insect resistance in cotton plants. For this purpose, we employed 1H-NMR spectroscopy to perform metabolite profiling of Bt and non-Bt cotton varieties, and a total of 42 different metabolites were identified in cotton plants. In cluster analysis, deep learning approaches (linear discriminant analysis (LDA) and neural networks) showed better separation among cotton varieties compared to conventional methods (principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLSDA)). The key metabolites responsible for inter-class separation were terpinolene, α-ketoglutaric acid, aspartic acid, stigmasterol, fructose, maltose, arabinose, xylulose, cinnamic acid, malic acid, valine, nonanoic acid, citrulline, and shikimic acid. The metabolites which regulated differently with the level of significance p < 0.001 amongst different cotton varieties belonged to the tricarboxylic acid cycle (TCA), Shikimic acid, and phenylpropanoid pathways. Our analyses underscore a biosignature of metabolites that might involve in inducing insect resistance in Bt cotton. Moreover, novel evidence from our study could be used in the metabolic engineering of these biological pathways to improve the resilience of Bt cotton against insect/pest attacks. Lastly, our findings are also in complete support of employing deep machine learning algorithms as a useful tool in metabolomics studies.

Genome-Wide Identification and Characterization of the PPO Gene Family in Cotton (Gossypium) and Their Expression Variations Responding to Verticillium Wilt Infection.

Polyphenol oxidases (PPOs) are copper-binding metalloproteinases encoded by nuclear genes, ubiquitously existing in the plastids of microorganisms, plants, and animals. As one of the important defense enzymes, PPOs have been reported to participate in the resistant processes that respond to diseases and insect pests in multiple plant species. However, PPO gene identification and characterization in cotton and their expression patterns under Verticillium wilt (VW) treatment have not been clearly studied. In this study, 7, 8, 14, and 16 PPO genes were separately identified from Gossypium arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively, which were distributed within 23 chromosomes, though mainly gathered in chromosome 6. The phylogenetic tree manifested that all the PPOs from four cotton species and 14 other plants were divided into seven groups, and the analyses of the conserved motifs and nucleotide sequences showed highly similar characteristics of the gene structure and domains in the cotton PPO genes. The dramatically expressed differences were observed among the different organs at various stages of growth and development or under the diverse stresses referred to in the published RNA-seq data. Quantitative real-time PCR (qRT-PCR) experiments were also performed on the GhPPO genes in the roots, stems, and leaves of VW-resistant MBI8255 and VW-susceptible CCRI36 infected with Verticillium dahliae V991, proving the strong correlation between PPO activity and VW resistance. A comprehensive analysis conducted on cotton PPO genes contributes to the screening of the candidate genes for subsequent biological function studies, which is also of great significance for the in-depth understanding of the molecular genetic basis of cotton resistance to VW.

[Revealing the Effect of Saline Water Drip Irrigation on Soil Microorganisms in Cotton Fields Based on Metagenomics].

Saline water irrigation has become an important means to alleviate the shortage of freshwater in arid areas. However, long-term saline water irrigation can cause soil salinity accumulation, affect soil microbial community structure, and then affect soil nutrient transformation. In this study, we used metagenomics to investigate the effects of long-term saline water drip irrigation on soil microbial community structure in a cotton field. In the experiment, the salinity of irrigation water (ECw) was set to two treatments:0.35 dS·m-1 and 8.04 dS·m-1 (denoted as FW and SW, respectively), and the nitrogen application rates were 0 kg·hm-2and 360 kg·hm-2 (denoted as N0 and N360, respectively). The results showed that saline water irrigation increased soil water content, salinity, organic carbon, and total nitrogen content and decreased soil pH and available potassium content. Nitrogen fertilizer application increased soil organic carbon, salinity, and total nitrogen content and decreased soil water content, pH, and available potassium content. The dominant bacterial phyla in each treatment were:Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, and Gemmatimonadetes. Saline water irrigation significantly increased the relative abundances of Actinobacteria, Chloroflexi, Gemmatimonadetes, and Firmicutes but significantly decreased the relative abundances of Proteobacteria, Acidobacteria, Cyanobacteria, and Nitrospira. Nitrogen fertilizer application significantly increased the relative abundances of Chloroflexi and Nitrospira but significantly decreased the relative abundances of Acidobacteria, Gemmatimonadetes, Planctomycetes, Cyanobacteria, and Verrucomicrobia. LEfSe analysis showed that saline water irrigation had no significant effect on the number of potential biomarkers, and nitrogen fertilizer application decreased the number of potential biomarkers in soil microbial communities. The correlation network diagram showed that the 20 genera had different degrees of correlation, including 44 positive correlations and 48 negative correlations. The core species in the network diagram were Nocardioides, Streptomyces, Pyrinomonas, Candidatus_Solibacter, and Bradyrhizobium spp. Saline water irrigation increased the relative abundances of the denitrification genes nirK, nirS, nasB, and norC and decreased the relative abundances of the nitrification genes amoB, amoC, and nxrA, whereas nitrogen fertilizer application increased the relative abundances of the nitrification genes amoA, amoB, amoC, hao, and nxrA and decreased the relative abundances of the denitrifying genes narB, napA, nasA, and nosZ. Saline water irrigation could adversely affect soil physicochemical properties; SWC, EC1:5, and BD were the main driving factors affecting soil microbial community structure and function genes; and soil microorganisms adapted to soil salt stress by regulating species composition.

Genome-wide identification and expression analysis of the cotton patatin-related phospholipase A genes and response to stress tolerance.

MAIN CONCLUSION: Patatin-related phospholipase A genes were involved in the response of Gossypium hirsutum to drought and salt tolerance. pPLA (patatin-related phospholipase A) is a key enzyme that catalyzes the initial step of lipid hydrolysis, which is involved in biological processes, such as drought, salt stress, and freezing injury. However, a comprehensive analysis of the pPLA gene family in cotton, especially the role of pPLA in the response to drought and salt tolerance, has not been reported so far. A total of 33 pPLA genes were identified in this study using a genome-wide search approach, and phylogenetic analysis classified these genes into three groups. These genes are unevenly distributed on the 26 chromosomes of cotton, and most of them contain a few introns. The results of the collinear analysis showed that G. hirsutum contained 1-5 copies of each pPLA gene found in G. arboreum and G. raimondii. The subcellular localization analysis of Gh_D08G061200 showed that the protein was localized in the nucleus. In addition, analysis of published upland cotton transcriptome data revealed that six GhPLA genes were expressed in various tissues and organs. Two genes (Gh_A04G142100.1 and Gh_D04G181000.1) were highly expressed in all tissues under normal conditions, showing the expression characteristics of housekeeping genes. Under different drought and salt tolerance stresses, we detected four genes with different expression levels. This study helps to clarify the role of pPLA in the response to drought and salt tolerance.

The effects and interrelationships of intercropping on Cotton Verticillium wilt and soil microbial communities.

BACKGROUND: Cotton Verticillium wilt, causing by Verticillium dahliae, has seriously affected the yield and quality of cotton. The incidence of Verticillium wilt in cotton fields has been on the rise for many years, especially after straw has been returned to the fields. Intercropping can reduce the incidence of soil borne diseases and is often used to control crop diseases, but the relationship between the effects of intercropping on microbial communities and the occurrence of plant diseases is unclear. This research explored the relationship between soil microbial community structure and Cotton Verticillium wilt in interplanting of cotton-onion, cotton-garlic, cotton-wheat and cotton monocultures. Amplicon sequencing applied to the profile of bacterial and fungal communities. RESULTS: The results showed that the disease index of Cotton Verticillium wilt was significantly reduced after intercropping with cotton-garlic and cotton-onion. Chao1 and Sobs indices were not significantly different in the rhizosphere soil and pre-plant soils of the four planting patterns, but the pre-plant fungal shannon index was significantly lower in the cotton-onion intercropping plot than in the other three plots. PCoA analysis showed that the soil microbial communities changed to a certain extent after intercropping, with large differences in the microbial communities under different cropping patterns. The abundance of Chaetomium was highest in the cotton-garlic intercropping before planting; the abundance of Penicillium was significantly higher in the cotton-wheat intercropping than in the other three systems. CONCLUSION: Cotton-garlic and cotton-onion interplanting can control Cotton Verticillium wilt by affecting the soil microbial community. Fungi of the genera Chaetomium and Penicillium may be associated with plant disease resistance.

First study on the root endophytic fungus Trichoderma hamatum as an entomopathogen: Development of a fungal bioinsecticide against cotton leafworm (Spodoptera littoralis).

Cotton leaf worm (Spodoptera littoralis) is a pest that produces important losses in horticultural and ornamental crops in greenhouse, being classified as quarantine pest A2 by EPPO. One of the strategies proposed to control agricultural pests in a health and environmentally friendly way is biological control with entomopathogenic fungi. The genus of filamentous fungi Trichoderma includes different species with direct (infection, antibiosis, anti-feeding, etc.) and indirect (systemic activation of plant defenses) insecticidal capacity, however, the species T. hamatum has never been described previously as entomopathogenic. In this work, the entomopathogenic capacity of T. hamatum on S. littoralis L3 larvae was analyzed by applying spores and fungal filtrates (topically and orally). Infection by spores was compared with the commercial entomopathogenic fungus Beauveria bassiana, obtaining similar results with respect to the production of larval mortality. Oral application of spores reported high mortality and fungal colonization of larvae, however, T. hamatum did not show chitinase activity when grown in the presence of S. littoralis tissues. Therefore, infection of S. littoralis larvae by T. hamatum is through natural openings such as mouth, anus or spiracles. With respect to the application of filtrates, only those obtained from the liquid culture of T. hamatum in contact with S. littoralis tissues reported a significant reduction in larval growth. Metabolomic analysis of the filtrates determined that the filtrate with insecticidal capacity presented the siderophore rhizoferrin in large quantities, which could be responsible for this activity. However, the production of this siderophore had never been previously described in Trichoderma and its insecticidal capacity was unknown. In conclusion, T. hamatum presents entomopathogenic capacity against S. littoralis larvae through the application of spores and filtrates, and both ways could be the basis for the development of efficient bioinsecticides against the pest.

Genome-wide association study identifies GhSAL1 affects cold tolerance at the seedling emergence stage in upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Genomic analysis of upland cotton revealed that cold tolerance was associated with ecological distribution. GhSAL1 on chromosome D09 negatively regulated cold tolerance of upland cotton. Cotton can undergo low-temperature stress at the seedling emergence stage, which adversely affects growth and yield; however, the regulatory mechanism underlying cold tolerance remains nebulous. Here, we analyze the phenotypic and physiological parameters in 200 accessions from 5 ecological distributions under constant chilling (CC) and diurnal variation of chilling (DVC) stresses at the seedling emergence stage. All accessions were clustered into four groups, of which Group IV, with most germplasms from the northwest inland region (NIR), had better phenotypes than Groups I-III under the two kinds of chilling stresses. A total of 575 significantly associated single-nucleotide polymorphism (SNP) were identified, and 35 stable genetic quantitative trait loci (QTL) were obtained, of which 5 were associated with traits under CC and DVC stress, respectively, while the remaining 25 were co-associated. The accumulation of dry weight (DW) of seedling was associated with the flavonoid biosynthesis process regulated by Gh_A10G0500. The emergence rate (ER), DW, and total length of seedling (TL) under CC stress were associated with the SNPs variation of Gh_D09G0189 (GhSAL1). GhSAL1HapB was the elite haplotype, which increased ER, DW, and TL by 19.04%, 11.26%, and 7.69%, respectively, compared with that of GhSAL1HapA. The results of virus-induced gene silencing (VIGS) experiment and determination of metabolic substrate content preliminarily illustrated that GhSAL1 negatively regulated cotton cold tolerance through IP3-Ca2+ signaling pathway. The elite haplotypes and candidate genes identified in this study could be used to improve cold tolerance at the seedling emergence stage in future upland cotton breeding.

Genome-wide identification and characterization of polycomb repressive complex 2 core components in upland cotton (Gossypium hirsutum L.).

BACKGROUND: The evolutionarily conserved Polycomb Repressive Complex 2 (PRC2) plays a vital role in epigenetic gene repression by depositing tri-methylation on lysine residue K27 of histone H3 (H3K27me3) at the target loci, thus participating in diverse biological processes. However, few reports about PRC2 are available in plant species with large and complicated genomes, like cotton. RESULTS: Here, we performed a genome-wide identification and comprehensive analysis of cotton PRC2 core components, especially in upland cotton (Gossypium hirsutum). Firstly, a total of 8 and 16 PRC2 core components were identified in diploid and tetraploid cotton species, respectively. These components were classified into four groups, E(z), Su(z)12, ESC and p55, and the members in the same group displayed good collinearity, similar gene structure and domain organization. Next, we cloned G. hirsutum PRC2 (GhPRC2) core components, and found that most of GhPRC2 proteins were localized in the nucleus, and interacted with each other to form multi-subunit complexes. Moreover, we analyzed the expression profile of GhPRC2 genes. The transcriptome data and quantitative real-time PCR (qRT-PCR) assays indicated that GhPRC2 genes were ubiquitously but differentially expressed in various tissues, with high expression levels in reproductive organs like petals, stamens and pistils. And the expressions of several GhPRC2 genes, especially E(z) group genes, were responsive to various abiotic and biotic stresses, including drought, salinity, extreme temperature, and Verticillium dahliae (Vd) infection. CONCLUSION: We identified PRC2 core components in upland cotton, and systematically investigated their classifications, phylogenetic and synteny relationships, gene structures, domain organizations, subcellular localizations, protein interactions, tissue-specific and stresses-responsive expression patterns. Our results will provide insights into the evolution and composition of cotton PRC2, and lay the foundation for further investigation of their biological functions and regulatory mechanisms.

A method for detecting the quality of cotton seeds based on an improved ResNet50 model.

The accurate and rapid detection of cotton seed quality is crucial for safeguarding cotton cultivation. To increase the accuracy and efficiency of cotton seed detection, a deep learning model, which was called the improved ResNet50 (Impro-ResNet50), was used to detect cotton seed quality. First, the convolutional block attention module (CBAM) was embedded into the ResNet50 model to allow the model to learn both the vital channel information and spatial location information of the image, thereby enhancing the model's feature extraction capability and robustness. The model's fully connected layer was then modified to accommodate the cotton seed quality detection task. An improved LRelu-Softplus activation function was implemented to facilitate the rapid and straightforward quantification of the model training procedure. Transfer learning and the Adam optimization algorithm were used to train the model to reduce the number of parameters and accelerate the model's convergence. Finally, 4419 images of cotton seeds were collected for training models under controlled conditions. Experimental results demonstrated that the Impro-ResNet50 model could achieve an average detection accuracy of 97.23% and process a single image in 0.11s. Compared with Squeeze-and-Excitation Networks (SE) and Coordination Attention (CA), the model's feature extraction capability was superior. At the same time, compared with classical models such as AlexNet, VGG16, GoogLeNet, EfficientNet, and ResNet18, this model had superior detection accuracy and complexity balances. The results indicate that the Impro-ResNet50 model has a high detection accuracy and a short recognition time, which meet the requirements for accurate and rapid detection of cotton seed quality.