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Abstract Background Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis. Methods The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes. Result The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361. Conclusion The study identified thirty chromosome-encoded genes specific to B. anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.

A well-developed rhizospheric system is crucial for maize to adapt to environmental stresses, thereby enhancing yield and quality. However, nitrogen (N) stress significantly impedes rhizospheric development and growth in maize. The genetic responses of maize's rhizosphere to N stress under monocropping systems with exogenous inorganic N fertilization and intercropping systems reliant on biological N fixation are not well understood, especially regarding common and specific response genes. Therefore, through transcriptomic analysis, this study systematically investigated the gene expression and molecular responses of maize's rhizosphere under two N supply regimes to N stress. The results showed that N stress generated 196 common and 3350 specific differentially expressed genes across the two systems, with the intercropping system exhibiting a stronger specific response. KEGG analysis revealed that the common genes, though few, are involved in key pathways essential for crop growth. Maize monocropping specific differentially expressed genes (MM) were enriched in pathways related to membrane lipids, cell wall formation, and intracellular signaling, while maize/alfalfa intercropping specific differentially expressed genes (MA) were linked to stress resistance through the glutathione metabolic pathway. WGCNA analysis identified five co-expression modules (CM). MA significantly increased the transcription factor families and structural domains directly targeting rhizospheric growth and development genes, including AP2, GRAS, Cys2His2 Zinc Finger, and LBD in CM blue. Conversely, MM significantly increased the transcription factor families and NAC structural domain targeting the promoters of N transporter protein genes in CM pink. This study emphasizes the importance of both common and specific genes in maintaining maize growth under suboptimal N supply in monocropping and intercropping systems. [ABSTRACT FROM AUTHOR]

Background: Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis. Methods: The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes. Result: The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361. Conclusion: The study identified thirty chromosome-encoded genes specific to B.anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis. [ABSTRACT FROM AUTHOR]

Drought stress, which significantly affects growth and reduces grain yield, is one of the main problems for wheat crops. Producing promising drought-tolerant wheat cultivars with high yields is one of the main targets for wheat breeders. In this study, a total of seven drought-tolerant wheat genotypes were screened for the presence of 19 specific drought tolerance genes. The genotypes were tested under normal and drought conditions for two growing seasons. Four spike traits, namely, spike length (SPL), grain number per spike (GNPS), number of spikelets per spike (NSPS), and grain yield per spike (GYPS), were scored. The results revealed that drought stress decreased the SPL, GNPS, NSPS, and GYPS, with ranges ranging from 2.14 (NSPS) to 13.92% (GNPS) and from 2.40 (NSPS) to 11.09% (GYPS) in the first and second seasons, respectively. ANOVA revealed high genetic variation among the genotypes for each trait under each treatment. According to the drought tolerance indices, Omara 007 presented the highest level of drought tolerance (average of sum ranks = 3), whereas both Giza-36 genotypes presented the lowest level of drought tolerance (average of sum ranks = 4.8) among the genotypes tested. Among the 19 genes tested, 11 were polymorphic among the selected genotypes. Omara 007 and Omara 002 presented the greatest number of specific drought tolerance genes (nine) tested in this study, whereas Sohag-5, Giza-36, and PI469072 presented the lowest number of drought tolerance genes (four). The number of different genes between each pair of genotypes was calculated. Seven different genes were found between Omara 007 and Giza-36, Omara 007 and Sohag-5, and Omara 002 and PI469072. The results of this study may help to identify the best genotypes for crossing candidate genotypes, and not merely to genetically improve drought tolerance in wheat. [ABSTRACT FROM AUTHOR]

Background: The present study aimed to identify the module genes and key gene functions and biological pathways of septic shock (SS) through integrated bioinformatics analysis. Methods: In the study, we performed batch correction and principal component analysis on 282 SS samples and 79 normal control samples in three datasets, GSE26440, GSE95233 and GSE57065, to obtain a combined corrected gene expression matrix containing 21,654 transcripts. Patients with SS were then divided into three molecular subtypes according to sample subtyping analysis. Results: By analyzing the demographic characteristics of the different subtypes, we found no statistically significant differences in gender ratio and age composition among the three groups. Then, three subtypes of differentially expressed genes (DEGs) and specific upregulated DEGs (SDEGs) were identified by differential gene expression analysis. We found 7361 DEGs in the type I group, 5594 DEGs in the type II group, and 7159 DEGs in the type III group. There were 1698 SDEGs in the type I group, 2443 in the type II group, and 1831 in the type III group. In addition, we analyzed the correlation between the expression data of 5972 SDEGs in the three subtypes and the gender and age of 227 patients, constructed a weighted gene co‐expression network, and identified 11 gene modules, among which the module with the highest correlation with gender ratio was MEgrey. The modules with the highest correlation with age composition were MEgrey60 and MElightyellow. Then, by analyzing the differences in module genes among different subgroups of SS, we obtained the differential expression of 11 module genes in four groups: type I, type II, type III and the control group. Finally, we analyzed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of all module DEGs, and the GO function and KEGG pathway enrichment of different module genes were different. Conclusions: Our findings aim to identify the specific genes and intrinsic molecular functional pathways of SS subtypes, as well as further explore the genetic and molecular pathophysiological mechanisms of SS. [ABSTRACT FROM AUTHOR]

A rapid and sensitive PCR based strategy in combination with microchip capillary electrophoresis (MCE) was employed to simultaneously detect three foodborne pathogenic bacteria. Three pairs of primers were specially designed for the amplification of target genes from Escherichia coli ( E. coli ), Staphylococcus aureus ( S. aureus ) and Salmonella enterica serovar Typhimurium ( S. Typhimurium). The PCR products along with standard DNA fragments were employed to optimize the separation conditions in MCE. Under optimal conditions, detectable separation of the PCR products (1.6–3.5 ng μL −1 ) from the three foodborne pathogenic bacteria was achieved within 135 s. The limits of detection of the three bacteria were concluded to be as low as 45 CFU mL −1 for E. coli , 62 CFU mL −1 for S. aureus and 42 CFU mL −1 for S. Typhimurium. The RSD of migration time was in the range of 0.5–0.8%. We conclude that MCE along with PCR holds real potential for rapid analysis and detection of nucleic acids from routine foodborne pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Leaf scald (caused by Xanthomonas albilineans) is an important bacterial disease affecting sugarcane in most sugarcane growing countries, including China. High genetic diversity exists among strains of X. albilineans from diverse geographic regions. To highlight the genomic features associated with X. albilineans from China, we sequenced the complete genome of a representative strain (Xa-FJ1) of this pathogen using the PacBio and Illumina platforms. The complete genome of strain Xa-FJ1 consists of a circular chromosome of 3,724,581 bp and a plasmid of 31,536 bp. Average nucleotide identity analysis revealed that Xa-FJ1 was closest to five strains from the French West Indies and the USA, particularly to the strain GPE PC73 from Guadeloupe. Comparative genomic analysis between Xa-FJ1 and GPE PC73 revealed prophage integration, homologous recombination, transposable elements, and a clustered regulatory interspaced short palindromic repeats (CRISPR) system that were linked with 16 insertions/deletions (InDels). Ten and 82 specific genes were found in Xa-FJ1 and GPE PC73, respectively, and some of these genes were subjected to phage-related proteins, zona occludens toxin, and DNA methyltransferases. Our findings highlight intra-species genetic variability of the leaf scald pathogen and provide additional genomic resources to investigate its fitness and virulence.

Red clover (Trifolium pratense) is an important forage plant worldwide. This study was directed to broadening current knowledge of red clover's coding regions and enhancing its utilization in practice by specific reanalysis of previously published assembly. A total of 42,996 genes were characterized using Illumina paired-end sequencing after manual revision of Blast2GO annotation. Genes were classified into metabolic and biosynthetic pathways in response to biological processes, with 7,517 genes being assigned to specific pathways. Moreover, 17,727 enzymatic nodes in all pathways were described. We identified 6,749 potential microsatellite loci in red clover coding sequences, and we characterized 4,005 potential simple sequence repeat (SSR) markers as generating polymerase chain reaction products preferentially within 100-350 bp. Marker density of 1 SSR marker per 12.39 kbp was achieved. Aligning reads against predicted coding sequences resulted in the identification of 343,027 single nucleotide polymorphism(SNP) markers, providing marker density of one SNP marker per 144.6 bp. Altogether, 95 SSRs in coding sequences were analyzed for 50 red clover varieties and a collection of 22 highly polymorphic SSRs with pooled polymorphism information content >0.9 was generated, thus obtaining primer pairs for application to diversity studies in T. pratense. A set of 8,623 genome-wide distributed SNPs was developed and used for polymorphism evaluation in individual plants. The polymorphic information content ranged from 0 to 0.375. Temperature switch PCR was successfully used in single-marker SNP genotyping for targeted coding sequences and for heterozygosity or homozygosity confirmation in validated five loci. Predicted large sets of SSRs and SNPs throughout the genome are key to rapidly implementing genome-based breeding approaches, for identifying genes underlying key traits, and for genome-wide association studies. Detailed knowledge of genetic relationships among breeding material can also be useful for breeders in planning crosses or for plant variety protection. Single-marker assays are useful for diagnostic applications. [ABSTRACT FROM AUTHOR]

Shiga-toxin producing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium are important foodborne pathogens which are threatened to human health seriously. In this study, we firstly investigated the application and accuracy of next-generation sequencing (NGS) technology combining with droplet digital PCR (NGS + ddPCR) for the quantification of pathogenic bacteria through amplification of 16S rRNA genes. Absolute quantification by ddPCR was then further developed to identify E. coli O157:H7 and S. Typhimurium targeting the single copy genes Z3276 and invA , respectively. Apple juice, milk and chicken were selected as corresponding represents of fruit, dairy and meat, to investigate the application of ddPCR in different food. The tested microbial communities were all successfully annotated by NGS at the level of species, and the accuracy of absolute abundance calculated by NGS + ddPCR relied on its initial population for each species. When detecting the genes Z3276 and invA simultaneously by dual channels using ddPCR, the detection limit of E. coli O157:H7 and S. Typhimurium in apple juice and milk was 2–3 log cfu/mL, while it was increased to 3–4 log cfu/g on chicken surface. Exist of background species or homologous strains did not affect the accuracy or detection limit of the targeting strain. • NGS combining with ddPCR for the quantification of pathogenic bacteria. • Characterization and quantification of foodborne pathogens at species level. • Exist of homologous strains did not affect the accuracy of targeting strain by ddPCR. • The detection limit of the targeting pathogens by ddPCR was lower than that of qPCR. • E. coli O157:H7 and S. Typhimurium can be quantified in apple juice, milk and chicken. [ABSTRACT FROM AUTHOR]

Environmental insults can lead to alteration in DNA methylation of specific genes. To address the role of altered DNA methylation in prediction of polycyclic aromatic hydrocarbons (PAHs) exposure-induced genetic damage, we recruited two populations, including diesel engine exhausts (low-level) and coke oven emissions (high-level) exposed subjects. The positive correlation was observed between the internal exposure marker (1-hydroxypyrene) and the extents of DNA damage (P < 0.05). The methylation of representative genes, including TRIM36 , RASSF1a , and MGMT in peripheral blood lymphocytes was quantitatively examined by bisulfite-pyrosequencing assay. The DNA methylation of these three genes in response to PAHs exposure were changed in a CpG-site-specific manner. The identified hot CpG site-specific methylation of three genes exhibited higher predictive power for DNA damage than the respective single genes in both populations. Furthermore, the dose-response relationship analysis revealed a nonlinear U-shape curve of TRIM36 or RASSF1a methylation in combined population, which led to determination of the threshold of health risk. Furthermore, we established a prediction model for genetic damage based on the unidirectional-alteration MGMT methylation levels. In conclusion, this study provides new insight into the application of multiple epi-biomarkers for health risk assessment upon PAHs exposure. [Display omitted] [ABSTRACT FROM AUTHOR]

The molecular tools of genomics have great power to reveal patterns of genetic difference within or among species, but must be complemented by the mechanistic study of the genetic variants found if these variants' evolutionary meaning is to be well understood. Central to this purpose is knowledge of the organisms' genotype-phenotype-environment interactions, which embody biological adaptation and constraint and thus drive natural selection. The history of this approach is briefly reviewed. Strategies embracing the complementarity of genomics and specific-gene studies in evolution are considered. Implementation of these strategies, and examples showing their feasibility and power, are discussed. Initial generalizations emphasize: (1) reproducibility of adaptive mechanisms; (2) evolutionary co-importance of variation in protein sequences and expression; (3) refinement of rudimentary molecular functions as an origin of evolutionary innovations; (4) identification of specific-gene mechanisms as underpinnings of genomic or quantitative genetic variation; and (5) multiple forms of adaptive or constraining epistasis among genes. Progress along these lines will advance understanding of evolution and support its use in addressing urgent medical and environmental applications. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pathovar oryzae ( Xoo) and X. oryzae pv. oryzicola ( Xoc) cause bacterial diseases in rice: leaf blight and leaf streak, respectively. Although both the Asian and the African strains of Xoo induce similar symptoms, they are genetically different, with the African Xoo strains being more closely related to the Asian Xoc. To identify the sequences responsible for differences between African and Asian Xoo strains and their relatedness to Xoc strains, a suppression-subtractive hybridization (SSH) procedure was performed, using the African Xoo MAI1 strain as a tester and the Philippine Xoo PXO86 strain and Xoc BLS256 strain as drivers. A nonredundant set of 134 sequences from MAI1 was generated. Several DNA fragments isolated by SSH were similar to genes of unknown function, hypothetical proteins, genes related to the type III secretion system, and other pathogenicity-related genes. The specificity of various fragments was validated by Southern blot analysis. SSH sequences were compared with several xanthomonad genomes. In silico analysis revealed SSH sequences as specific to strain MAI1, revealing their potential as specific markers for further epidemiological and diagnostic studies. SSH proved to be a useful method for rapidly identifying specific genes among closely related X. oryzae strains. [ABSTRACT FROM AUTHOR]

Abstract: O-antigens are highly polymorphic. The genes specifically involved in O-antigen synthesis are generally grouped together on the chromosome as a gene cluster. In Escherichia coli, the O-antigen gene clusters are characteristically located between the housekeeping genes galF and gnd. In this study, the O-antigen gene clusters of E. coli O59 and E. coli O155 were sequenced. The former was found to contain genes for GDP-mannose synthesis, glycosyltransferase genes and the O-antigen polymerase gene (wzy), while the latter contained only glycosyltransferase genes and wzy. O unit flippase genes (wzx) were found immediately downstream of the gnd gene, in the region between the gnd and hisI genes in these two strains. This atypical location of wzx has not been reported before, and furthermore these two genes complemented in trans despite the fact that different O-antigen structures are present in E. coli O59 and O155. A putative acetyltransferase gene was found downstream of wzx in both strains. Comparison of the region between gnd and hisI revealed that the wzx and acetyltransferase genes are closely related between E. coli O59 and O155, indicating that the two gene clusters arose recently from a common ancestor. This work provides further evidence for the O-antigen gene cluster having formed gradually, and selection pressure will eventually bring O-antigen genes into a single cluster. Genes specific for E. coli O59 and O155, respectively, were also identified. [Copyright &y& Elsevier]

h. guo, l. feng, j. tao, c. zhang and l. wang. 2004. To characterize the locus for O-antigen biosynthesis from Escherichia coli O172 type strain and to develop a rapid, specific and sensitive PCR-based method for identification and detection of E. coli O172. DNA of O-antigen gene cluster of E. coli O172 was amplified by long-range PCR method using primers based on housekeeping genes galF and gnd Shot gun bank was constructed and high quality sequencing was performed. The putative genes for synthesis of UDP-FucNAc, O-unit flippase, O-antigen polymerase and glycosyltransferases were assigned by the homology search. The evolutionary relationship between O-antigen gene clusters of E. coli O172 and E. coli O26 is shown by sequence comparison. Genes specific to E. coli O172 strains were identified by PCR assays using primers based on genes for O-unit flippase, O-antigen polymerase and glycosyltransferases. The specificity of PCR assays was tested using all E. coli and Shigella O-antigen type strains, as well as 24 clinical E. coli isolates. The sensitivity of PCR assays was determined, and the detection limits were 1 pg μl−1 chromosomal DNA, 0·2 CFU g−1 pork and 0·2 CFU ml−1 water. The total time required from beginning to end of the procedure was within 16 h. The O-antigen gene cluster of E. coli O172 was identified and PCR assays based on O-antigen specific genes showed high specificity and sensitivity. An O-antigen gene cluster was identified by sequencing. The specific genes were determined for E. coli O172. The sensitivity of O-antigen specific PCR assay was tested. Although Shiga toxin-producing O172 strains were not yet isolated from clinical specimens, they may emerge as pathogens. [ABSTRACT FROM AUTHOR]

A set of 12,779 expressed sequence tags (ESTs), both the 5′-most and 3′-most ends, derived from Ciona intestinalis tadpole larvae was categorized into 3521 independent clusters, from which 1013 clusters corresponding to 9424 clones were randomly selected to analyze genetic information and gene expression profiles. When compared with sequences in databases, 545 of the clusters showed significant matches (P < E-15) with reported proteins, while 153 showed matches with putative proteins for which there is not enough information to categorize their function, and 315 had no significant sequence similarities to known proteins. Sequence-similarity analyses of the 545 clusters in relation to the biological functions demonstrated that 407 of them have functions that many kinds of cells use, 104 are associated with cell–cell communication, and 34 are transcription factors or other gene-regulatory proteins. Sequence prevalence distribution analysis demonstrated that more than one-half of the mRNAs are rare mRNAs. All of the 1013 clusters were subjected to whole-mount in situ hybridization to analyze the gene expression profile in the tadpole larva. A total of 361 clusters showed expression specific to a certain tissue or organ: 96 showed epidermis-specific expression, 60 were specific to the nervous system, 108 to endoderm, 34 to mesenchyme, 5 to trunk lateral cells, 4 to trunk ventral cells, 23 to notochord, 28 to muscle, and 3 to siphon rudiments. In addition, 190 clusters showed expression in multiple tissues. Moreover, nervous system-specific genes showed intriguing expression patterns dependent on the cluster. The present study highlights a broad spectrum of genes that are used in the formation of one of the most primitive chordate body plans as well as for the function of various types of tissue and organ and also provides molecular markers for individual tissues and organs constituting the Ciona larva. [Copyright &y& Elsevier]

The occurrence of tadpole-type larvae is one of the key events used to understand the origin and evolution of chordates, and the tail bud of chordate embryos is thought to be associated with formation of the tip of the tail. Although some transcriptional factor genes including Brachyury are expressed in the tail bud, no structural genes have been reported to be expressed there. We report here that an ascidian gene HrTT-1 is expressed exclusively in the tip of elongating tail of the tail-bud embryo. This gene encodes a possible secreted protein of 415 amino acids with the SCP/TPX family consensus sequences. [ABSTRACT FROM AUTHOR]

The Lactobacillus plantarum group is composed of four closely related species or subspecies. To clarify the L. plantarum group classification and accurately provide health benefits of probiotics, it is important to distinguish them correctly. In this study, species- or subspecies-specific genes were identified to distinguish the L. plantarum group, and a real-time polymerase chain reaction (PCR) assay was developed to accurately detect them. Specific genes, such as protein-coding genes present in all strains of target species or subspecies but not in other bacterial strains, were explored from 70 genome sequences using comparative genomics. As a result, genes encoding a transporter, major facilitator family protein, and hypothetical proteins were confirmed as specific genes. Specificity of primers was evaluated with 55 lactic acid bacteria, and all primers showed 100% specificity. Probiotic products and fermented food samples were used to validate the developed real-time PCR assay. Of the probiotic products, one product was found to contain L. pentosus , even though it was labeled to contain L. plantarum. Using our in-house developed real-time PCR assay, the products labeled as L. plantarum were identified at the subspecies level, and the L. plantarum group species or subspecies in fermented food samples were successfully qualified and quantified. • Comparative genomics revealed novel specific genes to L. plantarum subspecies. • Specific primers for L. plantarum group species showed 100% specificity. • Real-time PCR quantitatively detected L. plantarum group species in food samples. • This real-time PCR is an accurate and simple method to detect L. plantarum group. [ABSTRACT FROM AUTHOR]

Leaf scald (caused by Xanthomonas albilineans) is an important bacterial disease affecting sugarcane in most sugarcane growing countries, including China. High genetic diversity exists among strains of X. albilineans from diverse geographic regions. To highlight the genomic features associated with X. albilineans from China, we sequenced the complete genome of a representative strain (Xa-FJ1) of this pathogen using the PacBio and Illumina platforms. The complete genome of strain Xa-FJ1 consists of a circular chromosome of 3,724,581 bp and a plasmid of 31,536 bp. Average nucleotide identity analysis revealed that Xa-FJ1 was closest to five strains from the French West Indies and the USA, particularly to the strain GPE PC73 from Guadeloupe. Comparative genomic analysis between Xa-FJ1 and GPE PC73 revealed prophage integration, homologous recombination, transposable elements, and a clustered regulatory interspaced short palindromic repeats (CRISPR) system that were linked with 16 insertions/deletions (InDels). Ten and 82 specific genes were found in Xa-FJ1 and GPE PC73, respectively, and some of these genes were subjected to phage-related proteins, zona occludens toxin, and DNA methyltransferases. Our findings highlight intra-species genetic variability of the leaf scald pathogen and provide additional genomic resources to investigate its fitness and virulence. [ABSTRACT FROM AUTHOR]