DeepMicroGen: a generative adversarial network-based method for longitudinal microbiome data imputation.

MOTIVATION: The human microbiome, which is linked to various diseases by growing evidence, has a profound impact on human health. Since changes in the composition of the microbiome across time are associated with disease and clinical outcomes, microbiome analysis should be performed in a longitudinal study. However, due to limited sample sizes and differing numbers of timepoints for different subjects, a significant amount of data cannot be utilized, directly affecting the quality of analysis results. Deep generative models have been proposed to address this lack of data issue. Specifically, a generative adversarial network (GAN) has been successfully utilized for data augmentation to improve prediction tasks. Recent studies have also shown improved performance of GAN-based models for missing value imputation in a multivariate time series dataset compared with traditional imputation methods. RESULTS: This work proposes DeepMicroGen, a bidirectional recurrent neural network-based GAN model, trained on the temporal relationship between the observations, to impute the missing microbiome samples in longitudinal studies. DeepMicroGen outperforms standard baseline imputation methods, showing the lowest mean absolute error for both simulated and real datasets. Finally, the proposed model improved the predicted clinical outcome for allergies, by providing imputation for an incomplete longitudinal dataset used to train the classifier. AVAILABILITY AND IMPLEMENTATION: DeepMicroGen is publicly available at https://github.com/joungmin-choi/DeepMicroGen.

Visible-light Excited and Highly Photostable Organic Fluorophore with dual-state Emission.

Organic fluorophores with dual-state emission (DSE) are rare or difficult to observe because most of them display either aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ). Amazing works have been accomplished, yet most of the DSE compounds were excited by UV light which limits their wide application in bioimaging. In this work, we achieved a visible-light excited DSE fluorophore and realized its imaging in SKOV-3 cells and zebrafish. The naphtho[2',3':4,5]imidazo[1,2-a]pyridine (NIP) core ensures its emission in dilute solution. Meanwhile, the twisted phenyl ring blocks fluorescence quenching induced by the π-π stacking and leads to the emission of the solid. The fluorescence intensity is steady even after 6 h of continuous intense sunlight. More importantly, photostability of NIP in cells is much better than commercial dye (mitochondrial green).

The greener the living environment, the better the health? Examining the effects of multiple green exposure metrics on physical activity and health among young students.

The sedentary and less active lifestyle of modern college students has a significant impact on the physical and mental well-being of the college community. Campus Green Spaces (GSs) are crucial in promoting physical activity and improving students' health. However, previous research has focused on evaluating campuses as a whole, without considering the diverse spatial scenarios within the campus environment. Accordingly, this study focused on the young people's residential scenario in university and constructed a framework including a comprehensive set of objective and subjective GSs exposure metrics. A systematic, objective exposure assessment framework ranging from 2D (GSs areas), and 2.5D (GSs visibility) to 3D (GSs volume) was innovatively developed using spatial analysis, deep learning technology, and unmanned aerial vehicle (UAV) measurement technology. Subjective exposure metrics incorporated GSs visiting frequency, GSs visiting duration, and GSs perceived quality. Our cross-sectional study was based on 820 university students in Nanjing, China. Subjective measures of GSs exposure, physical activity, and health status were obtained through self-reported questionnaires. The Generalized Linear Model (GLM) was used to evaluate the associations between GSs exposure, physical activity, and perceived health. Physical activity and social cohesion were considered as mediators, and path analysis based on Structural Equation Modeling (SEM) was used to disentangle the mechanisms linking GSs exposure to the health status of college students. We found that (1) 2D indicator suggested significant associations with health in the 100m buffer, and the potential underlying mechanisms were: GSs area → Physical activity → Social cohesion → Physical health → Mental health; GSs area → Physical activity → Social cohesion → Mental health. (2) Subjective GSs exposure indicators were more relevant in illustrating exposure-response relationships than objective ones. This study can clarify the complex nexus and mechanisms between campus GSs, physical activity, and health, and provide a practical reference for health-oriented campus GSs planning.

The Efficiency of Respiratory Exercises in Rehabilitation of Low Back Pain: A Systematic Review and Meta-Analysis.

BACKGROUND: Low back pain (LBP) is a common musculoskeletal disorder, and respiratory exercise is considered a nonsurgical management method. Therefore, this systematic review and meta-analysis aims to estimate the results of randomized controlled trials on the effect of respiratory training in reducing LBP and its dose relationship. METHODS: The present study was conducted from January 2020 to January 2022, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (2020). Relevant studies were searched in multiple databases including PubMed, Web of Science, the Cochrane Library, EBSCO, Scopus, ScienceDirect, Wan Fang and China Knowledge Network, ClinicalTrials.gov, and Google Scholar, using a combination of MeSH/Emtree terms and free-text words. The heterogeneity of the studies was assessed using the I2 statistic. RESULTS: A total of 14 publications were included in the meta-analysis, with a total sample size of 698 individuals, aged 60-80 years. Respiratory exercise was effective in relieving LBP (standardized mean difference = -0.87, P < .00001) and improving physical disability (standardized mean difference = -0.79, P < .00001). The type of breathing and the total duration of breathing exercises were found to be the source of heterogeneity in this study by subgroup analysis. Subgroup analysis revealed that the most significant effect sizes of breathing resistance exercise to reduce LBP and the most significant effect sizes of breathing relaxation techniques to alleviate physical disability were performed 3 to 5 times per week and period >4 weeks. Respiratory exercise reducing LBP and improving functional disability was most effective when the total duration of the intervention was >500 minutes. Funnel plots showed that the results of the 2 overall studies were reliable without publication bias. CONCLUSIONS: Respiratory exercise can effectively reduce LBP and improve physical disability. Therefore, these exercises can be regarded as a part of a LBP management plan. We recommend an exercise program with 30 to 50 minutes, 3 to 5 times per week, and >4 weeks of breathing resistance exercise program as the most effective for treating LBP.

mobileOG-db: a Manually Curated Database of Protein Families Mediating the Life Cycle of Bacterial Mobile Genetic Elements.

Bacterial mobile genetic elements (MGEs) encode functional modules that perform both core and accessory functions for the element, the latter of which are often only transiently associated with the element. The presence of these accessory genes, which are often close homologs to primarily immobile genes, incur high rates of false positives and, therefore, limits the usability of these databases for MGE annotation. To overcome this limitation, we analyzed 10,776,849 protein sequences derived from eight MGE databases to compile a comprehensive set of 6,140 manually curated protein families that are linked to the "life cycle" (integration/excision, replication/recombination/repair, transfer, stability/transfer/defense, and phage-specific processes) of plasmids, phages, integrative, transposable, and conjugative elements. We overlay experimental information where available to create a tiered annotation scheme of high-quality annotations and annotations inferred exclusively through bioinformatic evidence. We additionally provide an MGE-class label for each entry (e.g., plasmid or integrative element), and assign to each entry a major and minor category. The resulting database, mobileOG-db (for mobile orthologous groups), comprises over 700,000 deduplicated sequences encompassing five major mobileOG categories and more than 50 minor categories, providing a structured language and interpretable basis for an array of MGE-centered analyses. mobileOG-db can be accessed at mobileogdb.flsi.cloud.vt.edu/, where users can select, refine, and analyze custom subsets of the dynamic mobilome. IMPORTANCE The analysis of bacterial mobile genetic elements (MGEs) in genomic data is a critical step toward profiling the root causes of antibiotic resistance, phenotypic or metabolic diversity, and the evolution of bacterial genera. Existing methods for MGE annotation pose high barriers of biological and computational expertise to properly harness. To bridge this gap, we systematically analyzed 10,776,849 proteins derived from eight databases of MGEs to identify 6,140 MGE protein families that can serve as candidate hallmarks, i.e., proteins that can be used as "signatures" of MGEs to aid annotation. The resulting resource, mobileOG-db, provides a multilevel classification scheme that encompasses plasmid, phage, integrative, and transposable element protein families categorized into five major mobileOG categories and more than 50 minor categories. mobileOG-db thus provides a rich resource for simple and intuitive element annotation that can be integrated seamlessly into existing MGE detection pipelines and colocalization analyses.

Mutual Regulation of Receptor-Like Kinase SIT1 and B'κ-PP2A Shapes the Early Response of Rice to Salt Stress.

The receptor-like kinase SIT1 acts as a sensor in rice (Oryza sativa) roots, relaying salt stress signals via elevated kinase activity to enhance salt sensitivity. Here, we demonstrate that Protein Phosphatase 2A (PP2A) regulatory subunit B'κ constrains SIT1 activity under salt stress. B'κ-PP2A deactivates SIT1 directly by dephosphorylating the kinase at Thr515/516, a salt-induced phosphorylation site in the activation loop that is essential for SIT1 activity. B'κ overexpression suppresses the salt sensitivity of rice plants expressing high levels of SIT1, thereby contributing to salt tolerance. B'κ functions in a SIT1 kinase-dependent manner. During early salt stress, activated SIT1 phosphorylates B'κ; this not only enhances its binding with SIT1, it also promotes B'κ protein accumulation via Ser502 phosphorylation. Consequently, by blocking SIT1 phosphorylation, B'κ inhibits and fine-tunes SIT1 activity to balance plant growth and stress adaptation.

AgroSeek: a system for computational analysis of environmental metagenomic data and associated metadata.

BACKGROUND: Metagenomics is gaining attention as a powerful tool for identifying how agricultural management practices influence human and animal health, especially in terms of potential to contribute to the spread of antibiotic resistance. However, the ability to compare the distribution and prevalence of antibiotic resistance genes (ARGs) across multiple studies and environments is currently impossible without a complete re-analysis of published datasets. This challenge must be addressed for metagenomics to realize its potential for helping guide effective policy and practice measures relevant to agricultural ecosystems, for example, identifying critical control points for mitigating the spread of antibiotic resistance. RESULTS: Here we introduce AgroSeek, a centralized web-based system that provides computational tools for analysis and comparison of metagenomic data sets tailored specifically to researchers and other users in the agricultural sector interested in tracking and mitigating the spread of ARGs. AgroSeek draws from rich, user-provided metagenomic data and metadata to facilitate analysis, comparison, and prediction in a user-friendly fashion. Further, AgroSeek draws from publicly-contributed data sets to provide a point of comparison and context for data analysis. To incorporate metadata into our analysis and comparison procedures, we provide flexible metadata templates, including user-customized metadata attributes to facilitate data sharing, while maintaining the metadata in a comparable fashion for the broader user community and to support large-scale comparative and predictive analysis. CONCLUSION: AgroSeek provides an easy-to-use tool for environmental metagenomic analysis and comparison, based on both gene annotations and associated metadata, with this initial demonstration focusing on control of antibiotic resistance in agricultural ecosystems. Agroseek creates a space for metagenomic data sharing and collaboration to assist policy makers, stakeholders, and the public in decision-making. AgroSeek is publicly-available at https://agroseek.cs.vt.edu/ .

Data Analytics for Environmental Science and Engineering Research.

The advent of new data acquisition and handling techniques has opened the door to alternative and more comprehensive approaches to environmental monitoring that will improve our capacity to understand and manage environmental systems. Researchers have recently begun using machine learning (ML) techniques to analyze complex environmental systems and their associated data. Herein, we provide an overview of data analytics frameworks suitable for various Environmental Science and Engineering (ESE) research applications. We present current applications of ML algorithms within the ESE domain using three representative case studies: (1) Metagenomic data analysis for characterizing and tracking antimicrobial resistance in the environment; (2) Nontarget analysis for environmental pollutant profiling; and (3) Detection of anomalies in continuous data generated by engineered water systems. We conclude by proposing a path to advance incorporation of data analytics approaches in ESE research and application.

Skin transcriptome profiles associated with black- and white-coated regions in Boer and Macheng black crossbred goats.

To increase the current understanding of the gene-expression profiles in different skin regions associated with different coat colors and identify key genes for the regulation of color patterns in goats, we used the Illumina RNA-Seq method to compare the skin transcriptomes of the black- and white-coated regions containing hair follicles from the Boer and Macheng Black crossbred goat, which has a black head and a white body. Six cDNA libraries derived from skin samples of the white-coated region (n = 3) and black-coated region (n = 3) were constructed from three full-sib goats. On average, we obtained approximately 76.5 and 73.5 million reads for skin samples from black- and white-coated regions, respectively, of which 75.39% and 76.05% were covered in the genome database. A total of 165 differentially expressed genes (DEGs) were detected between these two color regions, among which 110 were upregulated and 55 were downregulated in the skin samples of white- vs. black-coated regions. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that some of these DEGs may play an important role in controlling the pigmentation of skin or hair follicles. We identified three key DEGs, i.e., Agouti, DCT, and TYRP1, in the pathway related to melanogenesis in the different skin regions of the crossbred goat. DCT and TYRP1 were downregulated and Agouti was upregulated in the skin of the white-coated region, suggesting a lack of mature melanocytes in this region and that Agouti might play a key developmental role in color-pattern formation. All data sets (Gene Expression Omnibus) are available via public repositories. In addition, MC1R was genotyped in 200 crossbred goats with a black head and neck. Loss-of-function mutations in MC1R as well as homozygosity for the mutant alleles were widely found in this population. The MC1R gene did not seem to play a major role in determining the black head and neck in our crossbred goats. Our study provides insights into the transcriptional regulation of two distinct coat colors, which might serve as a key resource for understanding coat color pigmentation in goats. The region-specific expression of Agouti may be associated with the distribution of pigments across the body in Boer and Macheng Black crossbred goats.

Enhancing cadmium extraction potential of Brassica napus: Effect of rhizosphere interactions.

Brassica napus L. (oilseed rape) was grown with daikon and white lupin in a polyvinyl chloride split pot experiment (with no barrier between the compartments or by a nylon mesh barrier (37 µm) to license partial root interaction, or a solid barrier to stop any root interactions) to examine the effect of rhizosphere interaction on the cadmium uptake. The results showed that shoot and root biomasses of oilseed rape were 40.66% and 26.94% less than that of the monocropped treatment (solid barrier) when intercropping with daikon under the rhizosphere complete interaction. However, the intermingling of roots between oilseed rape and white lupin notably enhanced the dry biomass of oilseed rape by 40.23% and decreased with the reduction of root contact. Oilseed rape intercropping with daikon enhanced the shoot Cd concentration of oilseed rape. The shoot Cd concentration (44.8 mg/kg) of oilseed rape when intercropped white lupin under complete rhizosphere interaction were greater than those of other treatments. Additionally, the intermingling of roots played a positive role in the content of citric and malic acids when intercropping with white lupin. In all systems, the BCF values of oilseed rape >5. Therefore, intercropping with white lupin may contribute to higher biomass and increased uptake Cd by oilseed rape. We can toward sustainable positive effects on phytoremediation that based on a better understanding of rhizosphere processes.

A Pilot Study of Chicago Waterways as Reservoirs of Multidrug-Resistant Enterobacteriaceae (MDR-Ent) in a High-Risk Region for Community-Acquired MDR-Ent Infection in Children.

Community-acquired multidrug resistant Enterobacteriaceae (MDR-Ent) infections continue to increase in the United States. In prior studies, we identified neighboring regions in Chicago, Illinois, where children have 5 to 6 times greater odds of MDR-Ent infections. To prevent community spread of MDR-Ent, we need to identify the MDR-Ent reservoirs. A pilot study of 4 Chicago waterways for MDR-Ent and associated antibiotic resistance genes (ARGs) was conducted. Three waterways (A1 to A3) are labeled safe for "incidental contact recreation" (e.g., kayaking), and A4 is a nonrecreational waterway that carries nondisinfected water. Surface water samples were collected and processed for standard bacterial culture and shotgun metagenomic sequencing. Generally, A3 and A4 (neighboring waterways which are not hydraulically connected) were strikingly similar in bacterial taxa, ARG profiles, and abundances of corresponding clades and genera within the Enterobacteriaceae Additionally, total ARG abundances recovered from the full microbial community were strongly correlated between A3 and A4 (R2 = 0.97). Escherichia coli numbers (per 100 ml water) were highest in A4 (783 most probable number [MPN]) and A3 (200 MPN) relative to A2 (84 MPN) and A1 (32 MPN). We found concerning ARGs in Enterobacteriaceae such as MCR-1 (colistin), Qnr and OqxA/B (quinolones), CTX-M, OXA and ACT/MIR (beta-lactams), and AAC (aminoglycosides). We found significant correlations in microbial community composition between nearby waterways that are not hydraulically connected, suggesting cross-seeding and the potential for mobility of ARGs. Enterobacteriaceae and ARG profiles support the hypothesized concerns that recreational waterways are a potential source of community-acquired MDR-Ent.

Correction to: Functional analysis of GhCHS, GhANR and GhLAR in colored fiber formation of Gossypium hirsutum L.

In the original publication of this article [1], the authors pointed out the Fig. 4b was same with Fig. 4c. The correct Fig. 4b should be below.

vi-HMM: a novel HMM-based method for sequence variant identification in short-read data.

BACKGROUND: Accurate and reliable identification of sequence variants, including single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (INDELs), plays a fundamental role in next-generation sequencing (NGS) applications. Existing methods for calling these variants often make simplified assumptions of positional independence and fail to leverage the dependence between genotypes at nearby loci that is caused by linkage disequilibrium (LD). RESULTS AND CONCLUSION: We propose vi-HMM, a hidden Markov model (HMM)-based method for calling SNPs and INDELs in mapped short-read data. This method allows transitions between hidden states (defined as "SNP," "Ins," "Del," and "Match") of adjacent genomic bases and determines an optimal hidden state path by using the Viterbi algorithm. The inferred hidden state path provides a direct solution to the identification of SNPs and INDELs. Simulation studies show that, under various sequencing depths, vi-HMM outperforms commonly used variant calling methods in terms of sensitivity and F1 score. When applied to the real data, vi-HMM demonstrates higher accuracy in calling SNPs and INDELs.

Preoperative platelet-lymphocyte ratio predicts recurrence of laryngeal squamous cell carcinoma.

Aim: To evaluate the impact of preoperative platelet-lymphocyte ratio (PLR), neutrophil-lymphocyte ratio (NLR), derived NLR (dNLR) and lymphocyte-monocyte ratio (LMR) on the prognosis of laryngeal squamous cell carcinoma. Materials & methods: Overall survival and recurrence-free survival (RFS) were analyzed using Kaplan-Meier estimates. Multivariable Cox regression model was used to evaluate the independent prognostic significance of variables. Results: High PLR (>103.96), NLR (>1.96) and dNLR (>1.70) predicted lower RFS according to Kaplan-Meier method. In COX regression model, patients with high PLR had poor RFS estimates compared with those with lower PLR (p < 0.001). Conclusion: Preoperative PLR was a more valuable prognostic factor than NLR, dNLR and LMR for the recurrence of laryngeal squamous cell carcinoma.

Cadmium accumulation in oilseed rape is promoted by intercropping with faba bean and ryegrass.

The mechanisms of intercropping increasing plant biomass, cadmium accumulation, and organic acids secreted in rhizosphere soil are still unclear. Oilseed rape and intercrops were grown in boxes separated either with no barrier between the compartments or by a nylon mesh barrier (37 µm) to license partial root interaction, or a solid barrier to stop any root interactions. Two intercropping systems (oilseed rape-faba bean and oilseed rape-ryegrass) were carried out in soil with Cd content of 5 mg/kg. The intermingling of roots between oilseed rape and faba bean enhanced the biomass of oilseed rape. However, the biomass was negatively affected implying the higher nutrient apportionment to the ryegrass than oilseed rape. Oilseed rape intercropping with both faba bean and ryegrass played a positive role in the shoot Cd concentration of oilseed rape. The intermingling of roots played a positive role in the citric and malic acids when intercropping with faba bean. A remarkable increase in water-soluble Cd and DTPA-Cd content was observed during oilseed rape-faba bean complete root interaction treatment, up to 175.00% and 46.65%, respectively, which compare with the monoculture treatment. In both systems, the translocation factor values were higher for oilseed rape (O-F system) than for the other test plants and were always >1. Thus the Cd removal potential of oilseed rape can be further improved in the future by optimizing agronomic practices and intercropping with faba bean.

Characterization and Fungicide Sensitivity of Colletotrichum Species Causing Strawberry Anthracnose in Eastern China.

Strawberry anthracnose caused by Colletotrichum spp. is one of the most serious diseases in the strawberry fields of China. In total, 196 isolates of Colletotrichum were obtained from leaves, stolons, and crowns of strawberry plants with anthracnose symptoms in eastern China and were characterized based on morphology, internal transcribed spacer (ITS), and ß-tubulin (TUB2) gene sequences. All 196 isolates were identified as the Colletotrichum gloeosporioides species complex. In total, 62 strains were further identified at the species level by phylogenetic analyses of multilocus sequences of ITS, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat), calmodulin (CAL), and TUB2. Three species from the C. gloeosporioides species complex were identified: Colletotrichum siamense, C. fructicola, and C. aenigma. Isolates of C. siamense were tolerant to high temperatures, with a significantly larger colony diameter than the other two species when grown above 36°C. The inoculation of strawberry plants confirmed the pathogenicity of all three species. C. siamense isolates resulted in the highest disease severity. The in vitro sensitivities of C. siamense and C. fructicola isolates to azoxystrobin and three demethylation-inhibitor (DMI) fungicides (difenoconazole, tebuconazole, and prochloraz) were determined. Both species were sensitive to DMI fungicides but not to azoxystrobin. C. siamense isolates were more sensitive to prochloraz, while C. fructicola isolates were more sensitive to difenoconazole and tebuconazole. The present study provides valuable information for the effective management of strawberry anthracnose.

Comparison of Whole-Genome Sequences of Legionella pneumophila in Tap Water and in Clinical Strains, Flint, Michigan, USA, 2016.

During the water crisis in Flint, Michigan, USA (2014-2015), 2 outbreaks of Legionnaires' disease occurred in Genesee County, Michigan. We compared whole-genome sequences of 10 clinical Legionella pneumophila isolates submitted to a laboratory in Genesee County during the second outbreak with 103 water isolates collected the following year. We documented a genetically diverse range of L. pneumophila strains across clinical and water isolates. Isolates belonging to 1 clade (3 clinical isolates, 3 water isolates from a Flint hospital, 1 water isolate from a Flint residence, and the reference Paris strain) had a high degree of similarity (2-1,062 single-nucleotide polymorphisms), all L. pneumophila sequence type 1, serogroup 1. Serogroup 6 isolates belonging to sequence type 2518 were widespread in Flint hospital water samples but bore no resemblance to available clinical isolates. L. pneumophila strains in Flint tap water after the outbreaks were diverse and similar to some disease-causing strains.

Enhanced GII.4 human norovirus infection in gnotobiotic pigs transplanted with a human gut microbiota.

The role of commensal microbiota in enteric viral infections has been explored extensively, but the interaction between human gut microbiota (HGM) and human norovirus (HuNoV) is poorly understood. In this study, we established an HGM-Transplanted gnotobiotic (Gn) pig model of HuNoV infection and disease, using an infant stool as HGM transplant and a HuNoV GII.4/2006b strain for virus inoculation. Compared to germ-free Gn pigs, HuNoV inoculation in HGMT Gn pigs resulted in increased HuNoV shedding, characterized by significantly higher shedding titres on post inoculation day (PID) 3, 4, 6, 8 and 9, and significantly longer mean duration of virus shedding. In addition, virus titres were significantly higher in duodenum and distal ileum of HGMT Gn pigs on PID10, while comparable and transient HuNoV viremia was detected in both groups. 16S rRNA gene sequencing demonstrated that HuNoV infection dramatically altered intestinal microbiota in HGMT Gn pigs at the phylum (Proteobacteria, Firmicutes and Bacteroidetes) and genus (Enterococcus, Bifidobacterium, Clostridium, Ruminococcus, Anaerococcus, Bacteroides and Lactobacillus) levels. In summary, enhanced GII.4 HuNoV infection was observed in the presence of HGM, and host microbiota was susceptible to disruption upon HuNoV infection.

Functional analysis of GhCHS, GhANR and GhLAR in colored fiber formation of Gossypium hirsutum L.

BACKGROUND: The formation of natural colored fibers mainly results from the accumulation of different anthocyanidins and their derivatives in the fibers of Gossypium hirsutum L. Chalcone synthase (CHS) is the first committed enzyme of flavonoid biosynthesis, and anthocyanidins are transported into fiber cells after biosynthesis mainly by Anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR) to present diverse colors with distinct stability. The biochemical and molecular mechanism of pigment formation in natural colored cotton fiber is not clear. RESULTS: The three key genes of GhCHS, GhANR and GhLAR were predominantly expressed in the developing fibers of colored cotton. In the GhCHSi, GhANRi and GhLARi transgenic cottons, the expression levels of GhCHS, GhANR and GhLAR significantly decreased in the developing cotton fiber, negatively correlated with the content of anthocyanidins and the color depth of cotton fiber. In colored cotton Zongxu1 (ZX1) and the GhCHSi, GhANRi and GhLARi transgenic lines of ZX1, HZ and ZH, the anthocyanidin contents of the leaves, cotton kernels, the mixture of fiber and seedcoat were all changed and positively correlated with the fiber color. CONCLUSION: The three genes of GhCHS, GhANR and GhLAR were predominantly expressed early in developing colored cotton fibers and identified to be a key genes of cotton fiber color formation. The expression levels of the three genes affected the anthocyanidin contents and fiber color depth. So the three genes played a crucial part in cotton fiber color formation and has important significant to improve natural colored cotton quality and create new colored cotton germplasm resources by genetic engineering.