Development and characterization of polymorphic microsatellite markers in northern fulmar, Fulmarus glacialis (Procellariiformes), and cross-species amplification in eight other seabirds Abstract Background In the North Pacific, northern fulmar (Fulmarus glacialis) forms extensive colonies in few locales, which may lead to limited gene flow and locale-specific population threats. In the Atlantic, there are thousands of colonies of varying sizes and in Europe the species is considered threatened. Prior screens and classical microsatellite development in fulmar failed to provide a suite of markers adequate for population genetics studies. Objectives The objective of this study was to isolate a suite of polymorphic microsatellite loci with sufficient variability to quantify levels of gene flow, population affinity, and identify familial relationships in fulmar. We also performed a cross-species screening of these markers in eight other species. Methods We used shotgun sequencing to isolate 26 novel microsatellite markers in fulmar to screen for variability using individuals from two distinct regions: the Pacific (Chagulak Island, Alaska) and the Atlantic (Hafnarey Island, Iceland). Results Polymorphism was present in 24 loci in Chagulak and 23 in Hafnarey, while one locus failed to amplify in either colony. Polymorphic loci exhibited moderate levels of genetic diversity and this suite of loci uncovered genetic structuring between the regions. Among the other species screened, polymorphism was present in one to seven loci. Conclusion The loci yielded sufficient variability for use in population studies and estimation of familial relationships; as few as five loci provide resolution to determine individual identity. These markers will allow further insight into the global population dynamics and phylogeography of fulmars. We also demonstrated some markers are transferable to other species.

Zebrafish is a central model to dissect the peripheral neuropathy Abstract The peripheral nervous system (PNS) is composed with all nerves extended from the brain and spinal cord, which are the central nervous system to other organs of the body. Dysfunctional peripheral motion resulting from the regressive neuronal axons in the defected PNS leads to several peripheral neuropathies including both inherited and non-inherited disorders. Because of poor understanding of cellular and molecular mechanisms involved in the peripheral neuropathy, there is currently non-targeted treatment of the disorder. Basic researches have paid attention to dissect roles of causative genes, identified from the inherited peripheral neuropathies, in PNS development. However, recent studies focusing on investigation of therapeutic targets have suggested that successful regeneration of the impaired peripheral nerves may be most effective treatment. The regeneration studies have been limited in the rodents system due to some of practical and physiological disadvantages until zebrafish model has emerged as an ideal system. Hence, this review aims to provide a comprehensive overview of the advantages of zebrafish as a model for the peripheral neuropathy researches and to suggest the disease genes-involved potential mechanisms targeting the PNS regeneration that may be demonstrated in zebrafish.

Genome-wide mining of respiratory burst homologs and its expression in response to biotic and abiotic stresses in Triticum aestivum Abstract Background Membrane-bound NADPH oxidases (Nicotinamide adenine ainucleotide phosphate oxidase) also called respiratory burst oxidase homologs (Rboh) play an essential role in ROS production under normal as well as environmental stress conditions in plants. Objective To identify and study respiratory burst homologs (Rboh) from the wheat genome as well as characterize their role in various biological and molecular processes along with expression in response to biotic and abiotic stresses. Methods The Rboh homologs in the wheat genome were predicted based on data processing, alignment of sequences and phylogenetic analysis of sequences in numerous plant species and wheat. The conserved motifs were known followed by domain design study. The 3-D structure prediction and similarity modeling were administered for NADPH enzyme domain. Gene ontology and a functional study were done in addition to expression analysis of Triticum aestivum respiratory burst oxidase (TaRboh) gene family in response to biotic as well as abiotic stress. Results Phylogenetic analysis of Rboh gene family members among seven plant species including wheat, classified the family into four subfamilies. Rboh genes are mainly involved in various biological processes such as Response to oxidative stress, Superoxide anion generation, Hydrogen peroxide biosynthetic process. Among the molecular functions, calcium ion binding, peroxidase activity, oxidoreductase activity, superoxide-generating NADPH oxidase activity are essential. Enzyme annotation of the family and superfamily revealed that it encodes to five structural clusters and coding to enzymes NAD(P)H oxidase (H2O2-forming) (EC:1.6.3.1), Ferric-chelate reductase (NADH) (EC: 1.16.1.7), Peroxidase (EC: 1.11.1.7), Ribose-phosphate diphosphokinase (EC: 2.7.6.1). The enzymes contain six membrane-spanning domains, two hemes, and conserved motifs associated with NADPH, EF-hand and FAD binding. The outcomes additionally reflect a distinct role of this enzyme in different molecular functions which are responsible for the stress signaling. Further, the transcripts of TaRboh found expressed in various plant parts such as stem, leaves, spike, seed, and roots. We also observed expression of these gene family members under drought/combination of drought + heat and important wheat pathogens such as Puccinia striformis, Blumeria graminis f.sp. tritici, Fusarium graminiarum, F. pseudograminiarum, and Zymoseptoria tritici. Conclusions The investigation demonstrated that identified respiratory burst homologs (Rboh) in T. aestivum were involved in pathogen activated ROS production and have regulatory functions in cell death and defense responses.

DNA methylation and mRNA expression of COL6A3 in antler mesenchyme of female and male reindeer Abstract Backgroud Reindeer is the only deer species that both male and female produce antlers, which provides a particularly interesting case in studying the differences between antlers of the two sexes. Alpha 3(VI) Collagen Gene (COL6A3), forms a microfibrillar network associated with the structural integrity and biomechanical properties, has been found to be one of the differentially expressed genes in antler mesenchyme of female and male reindeer. Objective and Methods The promoter sequence of reindeer COL6A3 gene was obtained using the cloning technology and analyzed by the bioinformatics methods. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the COL6A3 promoter in reindeer antler mesenchyme. Real-time quantitative PCR was used to detect COL6A3 expression in the antler mesenchyme of female and male reindeer. Results Sequence analysis revealed that the reindeer COL6A3 partial promoter sequence was 983 bp including the possible promoter region at + 105 bp to + 155 bp. Homology and phylogenetic analysis indicated that the COL6A3 promoter of reindeer had the closest genetic distance with Bos taurus, Capra hircus and Ovis aries. BSP results indicated that the methylation level of COL6A3 promoter in the female reindeer antler mesenchyme was significantly higher than in the male. Correlating with increased methylation status, we also found that COL6A3 mRNA expression in female reindeer antler mesenchyme was significantly lower than in the male. Conclusion The higher methylation level of the COL6A3 gene in female reindeer antler mesenchyme coincides with decreased COL6A3 mRNA expression, thereby affecting the transposon silencing mechanism and possibly contributing to apparent differences of antlers in female and male reindeer.

Human epididymis protein 4 (HE4) protects against cystic pulmonary fibrosis associated-inflammation through inhibition of NF-κB and MAPK singnaling Abstract Background Cystic pulmonary fibrosis (CF) affects mostly the lung of the newborns. Chronic infection and inflammation become the major causes of morbidity and mortality in CF. However, the underlying molecular mechanisms causing CF still remain unclear. Methods ELISA assay was used to examine the expression of HE4 and pro-inflammatory cytokines in W126VA4 cells supernatant fluid. qRT-PCR was applicable to determine the mRNA level of HE4, α-SMA, collagen 1, MMP2, MMP9 and various interleukins. Immunofluorescent assay was used to test the expression of HE4 in WI-26 VA4 cells. Major elements of MAPK and NF-κB signals pathways were examined by western blot. Results We found higher expression of HE4 in CF patients serum and lung biopsy. Interestingly, HE4 expression was positively correlated with fibrosis markers expression. In addition,HE4 overexpression increased inflammatory cytokines secretion and fibrosis markers expression in WI-26 VA4 cells. And NF-κB pathways were responsible for elevated inflammation. In addition, HE4/MAPK/MMPs signaling cascades destroyed the normal extracellular matrix (ECM) and promoted fibrosis. Conclusions Overall, we first identified that HE4 promoted CF-associated inflammation. Additionally, NF-κB and MAPK signalings were further validated to be responsible for CF-associated inflammation and ECM destruction. Characterization of lumacaftor/ivacaftor in CF-associated inflammation may provide a novel insight into clinical CF treatment.

Comparison of de-novo assembly tools for plasmid metagenome analysis Abstract Background With the advent of next-generation sequencing techniques, culture-independent metagenome approaches have now made it possible to predict possible presence of genes in the environmental bacteria most of which may be non-cultivable. Short reads obtained from the deep sequencing can be assembled into long contigs some of which include plasmids. Plasmids are the circular double stranded DNA in bacteria and known as one of the major carriers of antibiotic resistance genes. Objective Metagenomic analyses, especially focused on plasmids, could help us predict dissemination mechanisms of antibiotic resistance genes in the environment. However, with the availability of a myriad of metagenomic assemblers, the selection of the most appropriate metagenome assembler for the plasmid metagenome study might be challenging. Therefore, in this study, we compared five open source assemblers to suggest most effective way of plasmid metagenome analysis. Methods IDBA-UD, MEGAHIT, SPAdes, SOAPdenovo2, and Velvet are compared for conducting plasmid metagenome analyses using two water samples. Results Our results clearly showed that abundance and types of antibiotic resistance genes on plasmids varied depending on the selection of assembly tools. IDBA-UD and MEGAHIT demonstrated the overall best assembly statistics with high N50 values with higher portion of longer contigs. Conclusion These two assemblers also detected more diverse plasmids. Among the two, MEGAHIT showed more memory efficient assembly, therefore we suggest that the use of MEGAHIT for plasmid metagenome analysis may offer more diverse plasmids with less computer resource required. Here, we also summarized a fundamental plasmid metagenome work flow, especially for antibiotic resistance gene investigation.

Complete genome sequence and phylogenetic analysis of nosocomial pathogen Acinetobacter nosocomialis strain NCTC 8102 Abstract Background Acinetobacter has emerged recently as one of the most challenging nosocomial pathogens because of its increased rate of antimicrobial resistance. The genetic complexity and genome diversity, as well as the lack of adequate knowledge on the pathogenic determinants of Acinetobacter strains often hinder with pathogenesis studies for the development of better therapeutics to tackle this nosocomial pathogen. Objectives In this study, we comparatively analyzed the whole genome sequence of a virulent Acinetobacternosocomialis strain NCTC 8102. Methods The genomic DNA of A. nosocomialis NCTC 8102 was isolated and sequenced using PacBio RS II platform. The sequenced genome was functionally annotated and gene prediction was carried out using the program, Glimmer 3. The phylogenetic analysis of the genome was performed using Mega 6 program and the comparative genome analysis was carried out by BLAST (Basic Local Alignment Search Tool). Results The complete genome analysis depicted that the genome consists of a circular chromosome with an average G + C content of 38.7%. The genome comprises 3700 protein-coding genes, 96 RNA genes (18 rRNA, 74 tRNA and 4 ncRNA genes), and 91 pseudogenes. In addition, 6 prophage regions comprising 2 intact, 1 incomplete and 3 questionable ones and 18 genomic islands were identified in the genome, suggesting the possible occurrence of horizontal gene transfer in this strain. Comparative genome analysis of A. nosocomialis NCTC 8102 genome with the already sequenced A. nosocomialis strain SSA3 showed an average nucleotide identity of 99.0%. In addition, the number of prophages and genomic islands were higher in the A. nosocomialis NCTC 8102 genome compared to that of the strain SSA3. 14 of the genomic islands were unique to A. nosocomialis NCTC 8102 compared to strain SSA3 and they harbored genes which are involved in virulence, multidrug resistance, biofilm formation and bacterial pathogenesis. Conclusion We sequenced the whole genome of A. nosocomialis strain NCTC 8102 followed by comparatively genome analysis. The study provides valuable information on the genetic features of A. nosocomialis strain and the data from this study would assist in further studies for the development of control measures for this nosocomial pathogen.

Integrated metatranscriptome and transcriptome reveals the microbial community composition and physiological function of xylem sap on grapevine during bleeding period Abstract Background The xylem sap of fruit trees ensures the survival during the dormant period, and its flow during the bleeding period is correlated with the start of a new life cycle. Though the simple exploration on ingredients in the sap was carried out in the early years, the specific life activities and physiology functions of the sap during bleeding period have not been reported yet and the bleeding period is still a fruit tree development period worthy of attention. Objectives In this study, the microbial community composition during bleeding period were revealed by metatranscriptome and transcriptomic data. For the first time, the microorganism genome and grape genome in xylem sap were analyzed on transcriptional level, based on which the main physiological functions of the sap were also determined. Methods The genomic RNA in the sap was isolated and sequenced. Kyoto Encyclopedia of Gene and Genome (KEGG), Evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) and Carbohydrate-Active enzymes Database (CAZy) functional annotation were used to analysis the function of micro-organisms in xylem sap. DEGs were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The genes responsive to biotic and abiotic stresses were finally screened by transcriptome screening, stress data analysis and vitro validation experiments. Results The analysis exhibited 36,144,564 micro-related clean reads and 244,213 unigene. KEGG, eggNOG and CAZy functional annotation analysis indicated that signal transduction and material metabolism were the most important function of xylem sap. DEGs analysis were mainly about disease resistance, carbon source metabolism and hormone signal transduction, especially in P3 vs P1, enriched in the plant-pathogen interaction pathway. Analysis on grape genome information revealed xylem sap had little RNA with weak life activity. Metabolic pathways, biosynthesis of secondary metabolites, plant hormone signal transduction and plant-pathogen interaction were the four pathways with the largest number of enriched genes. Moreover, 16 genes responsive to biotic and abiotic stresses were screened out. Conclusion Promoting plant growth and resisting pathogens were the most important function of xylem sap during the bleeding period, and the function of microbial community were closely related to microorganisms growth and disease resistance. The 16 stress-related genes might be used for the future grape resistance research.

Genome-wide association study of vitamin E using genotyping by sequencing in sesame ( Sesamum indicum ) Abstract Background At least eight structurally related forms of vitamin E occur in nature, four tocopherols and four tocotrienols, all of which are potent membrane-soluble antioxidants. In this study, we detected two major isoforms in sesame (Sesamum indicum L.) seed: γ-tocopherol and β-tocotrienol. The objective of this study is to investigate the genetic basis of these vitamin E isoforms. Methods We  conducted a genome-wide association study (GWAS) using 5962 genome-wide markers, acquired from 96 core sesame accessions. The GWAS was performed using generalized linear (GLM) and mixed linear (MLM) models. Results LG08_6621957, on chromosome 8, was detected as having a significant association with γ-tocopherol in both models. It explained 20.9% of γ-tocopherol variation in sesame. For β-tocotrienol, no significant loci were detected according to the two models, but one locus, SLG03_13104062, explained 17.8% of the phenotypic variation. Based on structure and phylogenetic studies, the 96 accessions were clearly clustered into two subpopulations. Conclusion This study on sesame demonstrates and provides an evidence that genotyping by sequencing (GBS) based GWAS can be used to identifying important loci for small growing crops. The significant SNPs or genes could be useful for improving the vitamin E content in sesame breeding programs.

In silico mining and FISH mapping of a chromosome-specific satellite DNA in Capsicum annuum L. Abstract Background A large proportion of eukaryote nuclear genomes is composed of repetitive DNA. Tracing the dynamics of repetitive elements in the genomes of related taxa can reveal important information about their phylogenic relationships as well as traits that have become distinct to a lineage. Objective Study the genomic abundance and chromosomal location of repetitive DNA in Capsicum annuum L. to understand the repeat dynamics. Method We quantified repeated DNA content in the C. annuum genome using the RepeatExplorer pipeline. Results About 42% of the C. annuum genome dataset comprised repetitive elements. Of these, 0.011, 0.98, 3.09, and 0.024% represented high and low confidence satellite repeats, putative long-terminal repeats (LTRs), and rDNA sequences, respectively. One novel high confidence 167-bp satellite repeat with a genomic proportion of 0.011%, Ca167TR, was identified. Furthermore, FISH with Ca167TR on metaphase chromosomes of C. annuum revealed signals in the subtelomeric regions of the short and long arms of chromosome 3 and 4, respectively. Conclusion Further understanding of the origin and associated functions of Ca167TR and other repeats in Capsicum will give us insights into the genomic relationships and functions of the genome.