BMC Genomics

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Identification and analysis of divergent immune gene families within the Tasmanian devil genome

Thu, 11/26/2015 - 07:00
Background: The Tasmanian devil (Sarcophilus harrisii) is being threatened with extinction in the wild by a disease known as devil facial tumour disease (DFTD). In order to prevent the spread of this disease a thorough understanding of the Tasmanian devil immune system and its response to the disease is required. In 2011 and 2012 two genome sequencing projects of the Tasmania devil were released. This has provided us with the raw data required to begin to investigate the Tasmanian devil immunome in depth. In this study we characterise immune gene families of the Tasmanian devil. We focus on immunoglobulins, T cell receptors and cytokine families. Results: We identify and describe 119 cytokines including 40 interleukins, 39 chemokines, 8 interferons, 18 tumour necrosis family cytokines and 14 additional cytokines. Constant regions for immunoglobulins and T cell receptors were also identified. The repertoire of genes in these families was similar to the opossum, however devil specific duplications were seen and orthologs to eutherian genes not previously identified in any marsupial were also identified. Conclusions: By using multiple data sources as well as targeted search methods, highly divergent genes across the Tasmanian devil immune system were identified and characterised. This understanding will allow for the development of devil specific assays and reagents and allow for future studies into the immune response of the Tasmanian devil immune system to DFTD.

Transcriptome profiling reveals that feeding wild zooplankton to larval Atlantic cod (<it>Gadus morhua</it>) influences suites of genes involved in oxidation-reduction, mitosis, and selenium homeostasis

Thu, 11/26/2015 - 07:00
Background: Larval nutrition and growth are key issues for wild and cultured cod. While it was shown previously that larval cod fed wild zooplankton grow faster than those fed only rotifers, the mechanisms involved in this enhanced growth are not completely understood. We used microarrays to identify larval cod transcripts that respond to feeding with small amounts of wild zooplankton (5–10 % of live prey items). The larval transcriptome was compared between 3 treatment groups [fed rotifers (RA), rotifers with protein hydrolysate (RA-PH), or rotifers with zooplankton (RA-Zoo)] at 9–10 mm length [26–30 days post-hatch (dph)] to identify a robust suite of zooplankton-responsive genes (i.e. differentially expressed between RA-Zoo and both other groups). Results: The microarray experiment identified 147 significantly up-regulated and 156 significantly down-regulated features in RA-Zoo compared with both RA and RA-PH. Gene ontology terms overrepresented in the RA-Zoo responsive gene set included “response to selenium ion” and several related to cell division and oxidation-reduction. Ten selenoprotein-encoding genes, and 2 genes involved in thyroid hormone generation, were up-regulated in RA-Zoo compared with both other groups. Hierarchical clustering of RA-Zoo responsive genes involved in oxidation-reduction and selenium homeostasis demonstrated that only the zooplankton treatment had a considerable and consistent impact on the expression of these genes. Fourteen microarray-identified genes were selected for QPCR involving 9–13 mm larvae, and 13 of these were validated as differentially expressed between RA-Zoo and both other groups at ~9 mm. In contrast, in age-matched (34–35 dph; ~11 mm RA and RA-PH, ~13 mm RA-Zoo) and size-matched (~13 mm) older larvae, only 2 and 3 genes, respectively, showed the same direction of RA-Zoo-responsive change as in ~9 mm larvae. Conclusions: The modulation of genes involved in selenium binding, redox homeostasis, and thyroid hormone generation in ~9 mm RA-Zoo larvae in this study may be in response to the relatively high levels of selenium, iodine, and LC-PUFA (potentially causing oxidative stress) in zooplankton. Nonetheless, only a subset of zooplankton-responsive genes in ~9 mm larvae remained so in older larvae, suggesting that the observed transcriptome changes are largely involved in initiating the period of growth enhancement.

Multimodular type I polyketide synthases in algae evolve by module duplications and displacement of AT domains <it>in trans</it>

Thu, 11/26/2015 - 07:00
Background: Polyketide synthase (PKS) catalyzes the biosynthesis of polyketides, which are structurally and functionally diverse natural products in microorganisms and plants. Here, we have analyzed available full genome sequences of microscopic and macroscopic algae for the presence of type I PKS genes. Results: Type I PKS genes are present in 15 of 32 analyzed algal species. In chlorophytes, large proteins in the MDa range are predicted in most sequenced species, and PKSs with free-standing acyltransferase domains (trans-AT PKSs) predominate. In a phylogenetic tree, PKS sequences from different algal phyla form clades that are distinct from PKSs from other organisms such as non-photosynthetic protists or cyanobacteria. However, intermixing is observed in some cases, for example polyunsaturated fatty acid (PUFA) and glycolipid synthases of various origins. Close relationships between type I PKS modules from different species or between modules within the same multimodular enzyme were identified, suggesting module duplications during evolution of algal PKSs. In contrast to type I PKSs, nonribosomal peptide synthetases (NRPSs) are relatively rare in algae (occurrence in 7 of 32 species). Conclusions: Our phylogenetic analysis of type I PKSs in algae supports an evolutionary scenario whereby integrated AT domains were displaced to yield trans-AT PKSs. Together with module duplications, the displacement of AT domains may constitute a major mechanism of PKS evolution in algae. This study advances our understanding of the diversity of eukaryotic PKSs and their evolutionary trajectories.

Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing

Thu, 11/26/2015 - 07:00
Background: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. Results: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. Conclusions: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.

Collaborative cross mice in a genetic association study reveal new candidate genes for bone microarchitecture

Thu, 11/26/2015 - 07:00
Background: The microstructure of trabecular bone is a composite trait governed by a complex interaction of multiple genetic determinants. Identifying these genetic factors should significantly improve our ability to predict of osteoporosis and its associated risks. Genetic mapping using collaborative cross mice (CC), a genetically diverse recombinant inbred mouse reference panel, offers a powerful tool to identify causal loci at a resolution under one mega base-pairs, with a relatively small cohort size.Here, we utilized 31 CC lines (160 mice of both sexes in total) to perform genome-wide haplotype mapping across 77,808 single-nucleotide polymorphisms (SNPs). Haplotype scans were refined by imputation with the catalogue of sequence variation segregating in the CC to suggest potential candidate genes. Trabecular traits were obtained following microtomographic analysis, performed on 10-μm resolution scans of the femoral distal metaphysis. We measured the trabecular bone volume fraction (BV/TV), number (Tb.N), thickness (Tb.Th), and connectivity density (Conn.D). Results: Heritability of these traits ranged from 0.6 to 0.7. In addition there was a significant (P < 0.01) sex effect in all traits except Tb.Th. Our haplotype scans yielded six quantitative trait loci (QTL) at 1 % false discovery rate; BV/TV and Tb.Th produced two proximal loci each, on chromosome 2 and 7, respectively, and Tb.N and Conn.D yielded one locus on chromosomes 8 and 14, respectively. We identified candidate genes with previously-reported functions in bone biology, and implicated unexpected genes whose function in bone biology has yet to be assigned. Based on the literature, among the genes that ranked particularly high in our analyses (P < 10 -6 ) and which have a validated causal role in skeletal biology, are Avp, Oxt, B2m (associated with BV/TV), Cnot7 (with Tb.N), Pcsk6, Rgma (with Tb.Th), Rb1, and Cpb2 (with Conn.D). Other candidate genes strongly suggested by our analyses are Sgcz, Fgf20 (associated with Tb.N), and Chd2 (with Tb.Th). Conclusion: We have demonstrated for the first time genome-wide significant association between several genetic loci and trabecular microstructural parameters for genes with previously reported experimental observations, as well as proposing a role for new candidate genes with no previously characterized skeletal function.

Genome-wide expression profiles of <it>Pyropia haitanensis</it> in response to osmotic stress by using deep sequencing technology

Thu, 11/26/2015 - 07:00
Background: Pyropia haitanensis is an economically important marine crop grown in harsh intertidal habitats of southern China; it is also an excellent model system for studying mechanisms of stress tolerance. To understand the molecular mechanisms underlying osmotic tolerance and adaptation to intertidal environments, a comprehensive analysis of genome-wide gene expression profiles in response to dehydration and rehydration in Py. haitanensis was undertaken using digital gene expression profile (DGE) approaches combined with de novo transcriptome sequencing. Results: RNA-sequencing of the pooled RNA samples from different developmental phases and stress treatments was performed, which generated a total of 47.7 million clean reads. These reads were de novo assembled into 28,536 unigenes (≥200 bp), of which 18,217 unigenes (63.83 %) were annotated in at least one reference database. DGE analysis was performed on four treatments (two biological replicates per treatment), which included moderate dehydration, severe dehydration, rehydration, and normal conditions. The number of raw reads per sample ranged from 12.47 to 15.79 million, with an average of 14.69 million reads per sample. After quality filtering, the number of clean reads per sample ranged from 11.83 to 15.04 million. All distinct sequencing reads were annotated using the transcriptome of Py. haitanensis as reference. A total of 1,681 unigenes showed significant differential expression between moderate dehydration and normal conditions, in which 977 genes were upregulated, and 704 genes were downregulated. Between severe dehydration and normal conditions, 1,993 unigenes showed significantly altered expression, which included both upregulated (1,219) and downregulated genes (774). In addition, 1,086 differentially expressed genes were detected between rehydration and normal conditions, of which 720 genes were upregulated and 366 unigenes were downregulated. Most gene expression patterns in response to dehydration differed from that of rehydration, except for the synthesis of unsaturated fatty acids, several transcription factor families, and molecular chaperones that have been collectively implicated in the processes of dehydration and rehydration in Py. haitanensis. Conclusions: Taken together, these data provide a global high-resolution analysis of gene expression changes during osmotic stress that could potentially serve as a key resource for understanding the biology of osmotic acclimation in intertidal red seaweed.

A Bayesian model for detection of high-order interactions among genetic variants in genome-wide association studies

Wed, 11/25/2015 - 07:00
Background: A central question for disease studies and crop improvements is how genetics variants drive phenotypes. Genome Wide Association Study (GWAS) provides a powerful tool for characterizing the genotype-phenotype relationships in complex traits and diseases. Epistasis (gene-gene interaction), including high-order interaction among more than two genes, often plays important roles in complex traits and diseases, but current GWAS analysis usually just focuses on additive effects of single nucleotide polymorphisms (SNPs). The lack of effective computational modelling of high-order functional interactions often leads to significant under-utilization of GWAS data. Results: We have developed a novel Bayesian computational method with a Markov Chain Monte Carlo (MCMC) search, and implemented the method as a Bayesian High-order Interaction Toolkit (BHIT) for detecting epistatic interactions among SNPs. BHIT first builds a Bayesian model on both continuous data and discrete data, which is capable of detecting high-order interactions in SNPs related to case—control or quantitative phenotypes. We also developed a pipeline that enables users to apply BHIT on different species in different use cases. Conclusions: Using both simulation data and soybean nutritional seed composition studies on oil content and protein content, BHIT effectively detected some high-order interactions associated with phenotypes, and it outperformed a number of other available tools. BHIT is freely available for academic users at http://digbio.missouri.edu/BHIT/.

Comprehensive characterization of a time-course transcriptional response induced by autotoxins in <it>Panax ginseng</it> using RNA-Seq

Wed, 11/25/2015 - 07:00
Background: As a valuable medicinal plant, the yield of Panax ginseng is seriously affected by autotoxicity, which is a common phenomenon due to continuous cropping. However, the mechanism of autotoxicity in P. ginseng is still unknown. Results: In total, high throughput sequencing of 18 RNA-Seq libraries produced 996,000 000 100-nt reads that were assembled into 72,732 contigs. Compared with control, 3697 and 2828 genes were significantly up- and down-regulated across different tissues and time points, respectively. Gene Ontology enrichment analysis showed that ‘enzyme inhibitor activity’, ‘carboxylesterase activity’, ‘pectinesterase activity’, ‘centrosome cycle and duplication’ and ‘mitotic spindle elongation’ were enriched for the up-regulated genes. Transcription factors including AP2s/ERFs, MYBs, and WRKYs were up-regulated in roots after benzoic acid treatment. Moreover, reactive oxygen species, peroxidases and superoxide dismutase contigs were up-regulated in roots after benzoic acid treatment. Physiological and biochemical indexes showed that the proline and malondialdehyde content were restored to lower levels at a later stage after benzoic acid treatment. Benzoic acid inhibited the root hair development in a dose-dependent manner, and several differential expressed genes potentially involved in hair development were identified. Several key contigs in the flavonoid and ginsenoside biosynthesis pathways were repressed. Finally, 58,518 alternative splicing (AS) events from 12,950 genes were found after benzoic acid treatment. Interestingly, contigs in the ginsenoside biosynthetic pathway underwent AS, providing useful information about post-transcriptional regulation in P. ginseng. Conclusions: This study revealed the stress-response molecular mechanisms in P. ginseng induced by benzoic acid.

Determining multiallelic complex copy number and sequence variation from high coverage exome sequencing data

Mon, 11/02/2015 - 07:00
Background: Copy number variation (CNV) is a major component of genomic variation, yet methods to accurately type genomic CNV lag behind methods that type single nucleotide variation. High-throughput sequencing can contribute to these methods by using sequence read depth, which takes the number of reads that map to a given part of the reference genome as a proxy for copy number of that region, and compares across samples. Furthermore, high-throughput sequencing also provides information on the sequence differences between copies within and between individuals. Methods: In this study we use high-coverage phase 3 exome sequences of the 1000 Genomes project to infer diploid copy number of the beta-defensin genomic region, a well-studied CNV that carries several beta-defensin genes involved in the antimicrobial response, signalling, and fertility. We also use these data to call sequence variants, a particular challenge given the multicopy nature of the region. Results: We confidently call copy number and sequence variation of the beta-defensin genes on 1285 samples from 26 global populations, validate copy number using Nanostring nCounter and triplex paralogue ratio test data. We use the copy number calls to verify the genomic extent of the CNV and validate sequence calls using analysis of cloned PCR products. We identify novel variation, mostly individually rare, predicted to alter amino-acid sequence in the beta-defensin genes. Such novel variants may alter antimicrobial properties or have off-target receptor interactions, and may contribute to individuality in immunological response and fertility. Conclusions: Given that 81 % of identified sequence variants were not previously in dbSNP, we show that sequence variation in multiallelic CNVs represent an unappreciated source of genomic diversity.

A bioinformatic survey of RNA-binding proteins in <it>Plasmodium</it>

Mon, 11/02/2015 - 07:00
Background: The malaria parasites in the genus Plasmodium have a very complicated life cycle involving an invertebrate vector and a vertebrate host. RNA-binding proteins (RBPs) are critical factors involved in every aspect of the development of these parasites. However, very few RBPs have been functionally characterized to date in the human parasite Plasmodium falciparum. Methods: Using different bioinformatic methods and tools we searched P. falciparum genome to list and annotate RBPs. A representative 3D models for each of the RBD domain identified in P. falciparum was created using I-TESSAR and SWISS-MODEL. Microarray and RNAseq data analysis pertaining PfRBPs was performed using MeV software. Finally, Cytoscape was used to create protein-protein interaction network for CITH-Dozi and Caf1-CCR4-Not complexes. Results: We report the identification of 189 putative RBP genes belonging to 13 different families in Plasmodium, which comprise 3.5 % of all annotated genes. Almost 90 % (169/189) of these genes belong to six prominent RBP classes, namely RNA recognition motifs, DEAD/H-box RNA helicases, K homology, Zinc finger, Puf and Alba gene families. Interestingly, almost all of the identified RNA-binding helicases and KH genes have cognate homologs in model species, suggesting their evolutionary conservation. Exploration of the existing P. falciparum blood-stage transcriptomes revealed that most RBPs have peak mRNA expression levels early during the intraerythrocytic development cycle, which taper off in later stages. Nearly 27 % of RBPs have elevated expression in gametocytes, while 47 and 24 % have elevated mRNA expression in ookinete and asexual stages. Comparative interactome analyses using human and Plasmodium protein-protein interaction datasets suggest extensive conservation of the PfCITH/PfDOZI and PfCaf1-CCR4-NOT complexes. Conclusions: The Plasmodium parasites possess a large number of putative RBPs belonging to most of RBP families identified so far, suggesting the presence of extensive post-transcriptional regulation in these parasites. Taken together, in silico identification of these putative RBPs provides a foundation for future functional studies aimed at defining a unique network of post-transcriptional regulation in P. falciparum.

Comparison of <it>Xenorhabdus bovienii</it> bacterial strain genomes reveals diversity in symbiotic functions

Mon, 11/02/2015 - 07:00
Background: Xenorhabdus bacteria engage in a beneficial symbiosis with Steinernema nematodes, in part by providing activities that help kill and degrade insect hosts for nutrition. Xenorhabdus strains (members of a single species) can display wide variation in host-interaction phenotypes and genetic potential indicating that strains may differ in their encoded symbiosis factors, including secreted metabolites. Methods: To discern strain-level variation among symbiosis factors, and facilitate the identification of novel compounds, we performed a comparative analysis of the genomes of 10 Xenorhabdus bovienii bacterial strains. Results: The analyzed X. bovienii draft genomes are broadly similar in structure (e.g. size, GC content, number of coding sequences). Genome content analysis revealed that general classes of putative host-microbe interaction functions, such as secretion systems and toxin classes, were identified in all bacterial strains. In contrast, we observed diversity of individual genes within families (e.g. non-ribosomal peptide synthetase clusters and insecticidal toxin components), indicating the specific molecules secreted by each strain can vary. Additionally, phenotypic analysis indicates that regulation of activities (e.g. enzymes and motility) differs among strains. Conclusions: The analyses presented here demonstrate that while general mechanisms by which X. bovienii bacterial strains interact with their invertebrate hosts are similar, the specific molecules mediating these interactions differ. Our data support that adaptation of individual bacterial strains to distinct hosts or niches has occurred. For example, diverse metabolic profiles among bacterial symbionts may have been selected by dissimilarities in nutritional requirements of their different nematode hosts. Similarly, factors involved in parasitism (e.g. immune suppression and microbial competition factors), likely differ based on evolution in response to naturally encountered organisms, such as insect hosts, competitors, predators or pathogens. This study provides insight into effectors of a symbiotic lifestyle, and also highlights that when mining Xenorhabdus species for novel natural products, including antibiotics and insecticidal toxins, analysis of multiple bacterial strains likely will increase the potential for the discovery of novel molecules

Variation detection based on next-generation sequencing of type Chinese 1 strains of <it>Toxoplasma gondii</it> with different virulence from China

Fri, 10/30/2015 - 07:00
Background: Toxoplasma gondii is an intracellular protozoan that affects most species of endothermic animals including humans with a great infection rate. The vertical transmission of T. gondii causes abortion, constituting a serious threat to humans and leading to great losses in livestock production. Distinct from population structure of T. gondii in North America and Europe, Chinese 1 (ToxoDB #9) is a dominant genotype prevalent in China. Among the isolates of Chinese 1, the Wh3 and Wh6 have different virulence and pathogenicity in mice. However, little has been known about their difference at the genomic level. Thus the next-generation sequencing (NGS) approach was used to discover the association of the phenotypical variations with the genome sequencing data and the expression and polymorphisms of the key effectors. Results: We successfully sequenced the genome of Chinese 1 strains of Wh3 and Wh6. The average sequencing depths were 63.91 and 63.61 for Wh3 and Wh6, respectively. The variations of both isolates were identified in comparison with reference genome of type I GT1 strain. There were 505,645 and 505,856 SNPs, 30,658 and 30,004 indels, 4661 and 2320 SVs, and 1942 and 3080 CNVs for Wh3 and Wh6, respectively. In target search variations of particular factors of T. gondii, the dense granule protein 3 (GRA3) and rhoptry neck protein 3 (RON3) were found to have 35 SNPs, 2 indels and 89 SNPs, 6 indels, respectively. GRA3 and RON3 were both found to have higher expression levels in less virulent Wh6 than in virulent Wh3. Both strains of type Chinese 1 share polymorphic GRA15 II and ROP I/III with type I, II, and III strains. Conclusions: Sequencing of the two strains revealed that genome structure of Chinese 1 and type I strains has considerable genomic variations. Sequencing and qRT-PCR analyses of 26 effectors displayed a remarkable variation that may be associated with phenotype and pathogenic differences.

The <it>Leishmania</it> metaphylome: a comprehensive survey of <it>Leishmania</it> protein phylogenetic relationships

Fri, 10/30/2015 - 07:00
Background: Leishmaniasis is a neglected parasitic disease with diverse clinical manifestations and a complex epidemiology. It has been shown that its parasite-related traits vary between species and that they modulate infectivity, pathogenicity, and virulence. However, understanding of the species-specific adaptations responsible for these features and their evolutionary background is limited. To improve our knowledge regarding the parasite biology and adaptation mechanisms of different Leishmania species, we conducted a proteome-wide phylogenomic analysis to gain insights into Leishmania evolution. Results: The analysis of the reconstructed phylomes (totaling 45,918 phylogenies) allowed us to detect genes that are shared in pathogenic Leishmania species, such as calpain-like cysteine peptidases and 3'a2rel-related proteins, or genes that could be associated with visceral or cutaneous development. This analysis also established the phylogenetic relationship of several hypothetical proteins whose roles remain to be characterized. Our findings demonstrated that gene duplication constitutes an important evolutionary force in Leishmania, acting on protein families that mediate host-parasite interactions, such as amastins, GP63 metallopeptidases, cathepsin L-like proteases, and our methods permitted a deeper analysis of their phylogenetic relationships. Conclusions: Our results highlight the importance of proteome wide phylogenetic analyses to detect adaptation and evolutionary processes in different organisms and underscore the need to characterize the role of expanded and species-specific proteins in the context of Leishmania evolution by providing a framework for the phylogenetic relationships of Leishmania proteins.Phylogenomic data are publicly available for use through PhylomeDB (http://www.phylomedb.org).

MicroRNA and mRNA expression profiling analysis revealed the regulation of plant height in <it>Gossypium hirsutum</it>

Fri, 10/30/2015 - 07:00
Background: Dwarf cottons are more resistant to damage from wind and rain and associated with stable, increased yields, and also desirable source for breeding the machine harvest varieties. In an effort to uncover the transcripts and miRNA networks involved in plant height, the transcriptome and small RNA sequencing were performed based on dwarf mutant Ari1327 (A1), tall-culm mutant Ari3697 (A3) and wild type Ari971 (A9) in Gossypium hirsutum. Methods: The stem apexes of wild-type upland cotton (Ari971) and its dwarf mutant (Ari1327) and tall-culm mutant (Ari3697) at the fifth true leaf stage were extracted for RNA, respectively. Transcriptome and small RNA libraries were constructed and subjected to next generation sequencing. Results: The transcriptome sequencing analysis showed that the enriched pathways of top 3 differentially expressed genes (DEGs) were categorized as carotenoid biosynthesis, plant-pathogen interaction and plant hormone signal transduction in both A1–A9 and A3–A9. The ABA and IAA related factors were differentially expressed in the mutants. Importantly, we found the lower expressed SAUR and elevated expressed GH3, and ABA related genes such as NCED and PP2C maybe relate to reduced growth of the plant height in Ari1327 which was consistent with the higher auxin and ABA content in this mutant. Furthermore, miRNA160 targeted to the auxin response factor (ARF) and miRNA166 (gma-miR166u and gma-miR166h-3p) targeted to ABA responsive element binding factor were related to the mutation in cotton. We have noticed that the cell growth related factors (smg7 targeted by gra-miR482 and 6 novel miRNAs and pectate-lyases targeted by osa-miR159f), the redox reactions related factors (Cytochrome P450 targeted by miR172) and MYB genes targeted by miR828, miR858 and miR159 were also involved in plant height of the cotton mutants. A total of 226 conserved miRNAs representing 32 known miRNA families were obtained, and 38 novel miRNAs corresponding to 23 unique RNA sequences were identified. Total 531 targets for 211 conserved miRNAs were obtained. Using PAREsnip, 27 and 29 miRNA/target conserved interactions were validated in A1–A9 and A3–A9, respectively. Furthermore, miRNA160, miRNA858 and miRNA172 were validated to be up-regulated in A1–A9 but down-regulated in A3–A9, whereas miRNA159 showed the opposite regulation. Conclusions: This comprehensive interaction of the transcriptome and miRNA at tall-culm and dwarf mutant led to the discovery of regulatory mechanisms in plant height. It also provides the basis for in depth analyses of dwarf mutant genes for further breeding of dwarf cotton.

The epigenetic processes of meiosis in male mice are broadly affected by the widely used herbicide atrazine

Fri, 10/30/2015 - 07:00
Background: Environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. We studied the effects of the widely used herbicide atrazine (ATZ) on meiosis, a key step of gametogenesis, in male mice. Methods: Gene expression pattern was analysed by Gene–Chip array. Genome-wide mapping of H3K4me3 marks distribution was done by ChIP-sequencing of testis tissue using Illumina technologies. RT-qPCR was used to validate differentially expressed genes or differential peaks. Results: We demonstrate that exposure to ATZ reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. Using Gene-Chip and ChIP-Seq analysis of H3K4me3 marks, we found that a broad range of cellular functions, including GTPase activity, mitochondrial function and steroid-hormone metabolism, are affected by ATZ. Furthermore, treated mice display enriched histone H3K4me3 marks in regions of strong recombination (double-strand break sites), within very large genes and reduced marks in the pseudoautosomal region of X chromosome. Conclusions: Our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.

Deep sequencing shows microRNA involvement in bovine mammary gland adaptation to diets supplemented with linseed oil or safflower oil

Fri, 10/30/2015 - 07:00
Background: Bovine milk fat composition is responsive to dietary manipulation providing an avenue to modify the content of fatty acids and especially some specific unsaturated fatty acid (USFA) isomers of benefit to human health. MicroRNAs (miRNAs) regulate gene expression but their specific roles in bovine mammary gland lipogenesis are unclear. The objective of this study was to determine the expression pattern of miRNAs following mammary gland adaptation to dietary supplementation with 5 % linseed or safflower oil using next generation RNA-sequencing. Methods: Twenty-four Canadian Holstein dairy cows (twelve per treatment) in mid lactation were fed a control diet (total mixed ration of corn:grass silages) for 28 days followed by a treatment period (control diet supplemented with 5 % linseed or safflower oil) of 28 days. Milk samples were collected weekly for fat and individual fatty acid determination. RNA from mammary gland biopsies harvested on day-14 (control period) and on days +7 and +28 (treatment period) from six randomly selected cows per treatment was subjected to small RNA sequencing. Results: Milk fat percentage decreased significantly (P < 0.001) during treatment with the two diets as compared to the control period. The individual saturated fatty acids C4:0, C6:0, C8:0, C14:0 and C16:0 decreased significantly (P < 0.05) while five USFAs (C14:1, C18:1n11t, C20:3n3, C20:5n3 and CLA:t10c12) increased remarkably (P < 0.05) in response to both treatments. Analysis of 361 million sequence reads generated 321 known bovine miRNAs and 176 novel miRNAs. The expression of fourteen and twenty-two miRNAs was affected (P < 0.05) by linseed and safflower oil treatments, respectively. Seven miRNAs including six up-regulated (bta-miR-199c, miR-199a-3p, miR-98, miR-378, miR-148b and miR-21-5p) and one down-regulated (bta-miR-200a) were found to be regulated (P < 0.05) by both treatments, and thus considered core differentially expressed (DE) miRNAs. The gene targets of core DE miRNAs have functions related to gene expression and general cellular metabolism (P < 0.05) and are enriched in four pathways of lipid metabolism (3-phosphoinositide biosynthesis, 3-phosphoinositide degradation, D-myo-inisitol-5-phosphate metabolism and the superpathway of inositol phosphate compounds). Conclusion: Our results suggest that DE miRNAs in this study might be important regulators of bovine mammary lipogenesis and metabolism. The novel miRNAs identified in this study will further enrich the bovine miRNome repertoire and contribute to understanding mammary gland biology.

Genotypic and phenotypic analyses of a <it>Pseudomonas aeruginosa</it> chronic bronchiectasis isolate reveal differences from cystic fibrosis and laboratory strains

Fri, 10/30/2015 - 07:00
Background: Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized. Results: This strain displays phenotypes that have been associated with chronic respiratory infections in CF including alginate over-production, rough lipopolysaccharide, quorum-sensing deficiency, loss of motility, decreased protease secretion, and hypermutation. Hypermutation is a key adaptation of this bacterium during the course of chronic respiratory infections and analysis indicates that PAHM4 encodes a mutated mutS gene responsible for a ~1,000-fold increase in mutation rate compared to wild-type laboratory strain P. aeruginosa PAO1. Antibiotic resistance profiles and sequence data indicate that this strain acquired numerous mutations associated with increased resistance levels to β-lactams, aminoglycosides, and fluoroquinolones when compared to PAO1. Sequencing of PAHM4 revealed a 6.38 Mbp genome, 5.9 % of which were unrecognized in previously reported P. aeruginosa genome sequences. Transcriptome analysis suggests a general down-regulation of virulence factors, while metabolism of amino acids and lipids is up-regulated when compared to PAO1 and metabolic modeling identified further potential differences between PAO1 and PAHM4. Conclusions: This work provides insights into the potential differential adaptation of this bacterium to the lung of patients with bronchiectasis compared to other clinical settings such as cystic fibrosis, findings that should aid the development of disease-appropriate treatment strategies for P. aeruginosa infections.

Functionally conserved enhancers with divergent sequences in distant vertebrates

Fri, 10/30/2015 - 07:00
Background: To examine the contributions of sequence and function conservation in the evolution of enhancers, we systematically identified enhancers whose sequences are not conserved among distant groups of vertebrate species, but have homologous function and are likely to be derived from a common ancestral sequence. Our approach combined comparative genomics and epigenomics to identify potential enhancer sequences in the genomes of three groups of distantly related vertebrate species. Results: We searched for sequences that were conserved within groups of closely related species but not between groups of more distant species, and were associated with an epigenetic mark of enhancer activity. To facilitate inferring orthology between non-conserved sequences, we limited our search to introns whose orthology could be unambiguously established by mapping the bracketing exons. We show that a subset of these non-conserved but syntenic sequences from the mouse and zebrafish genomes have homologous functions in a zebrafish transgenic enhancer assay. The conserved expression patterns driven by these enhancers are probably associated with short transcription factor-binding motifs present in the divergent sequences. Conclusions: We have identified numerous potential enhancers with divergent sequences but a conserved function. These results indicate that selection on function, rather than sequence, may be a common mode of enhancer evolution; evidence for selection at the sequence level is not a necessary criterion to define a gene regulatory element.