BMC Genomics

Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis.

Publication Date: 2012 Feb 2 PMID: 22300648
Authors: Couturier, M. - Navarro, D. - Olive, C. - Chevret, D. - Haon, M. - Favel, A. - Lesage-Meessen, L. - Henrissat, B. - Coutinho, P. M. - Berrin, J. G.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Filamentous fungi are potent biomass degraders due to their ability to thrive in ligno(hemi)cellulose-rich environments. During the last decade, fungal genome sequencing initiatives have yielded abundant information on the genes that are putatively involved in lignocellulose degradation. At present, additional experimental studies are essential to provide insights into the fungal secreted enzymatic pools involved in lignocellulose degradation. RESULTS: In this study, we performed a wide analysis of 20 filamentous fungi for which genomic data are available to investigate their biomass-hydrolysis potential. A comparison of fungal genomes and secretomes using enzyme activity profiling revealed discrepancies in carbohydrate active enzymes (CAZymes) sets dedicated to plant cell wall. Investigation of the contribution made by each secretome to the saccharification of wheat straw demonstrated that most of them individually supplemented the industrial Trichoderma reesei CL847 enzymatic cocktail. Unexpectedly, the most striking effect was obtained with the phytopathogen Ustilago maydis that improved the release of total sugars by 57% and of glucose by 22%. Proteomic analyses of the best-performing secretomes indicated a specific enzymatic mechanism of U. maydis that is likely to involve oxido-reductases and hemicellulases. CONCLUSION: This study provides insight into the lignocellulose-degradation mechanisms by filamentous fungi and allows for the identification of a number of enzymes that are potentially useful to further improve the industrial lignocellulose bioconversion process.

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The diversity of cyanobacterial metabolism: genome analysis of multiple phototrophic microorganisms.

Publication Date: 2012 Feb 2 PMID: 22300633
Authors: Beck, C. - Knoop, H. - Axmann, I. M. - Steuer, R.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Cyanobacteria are among the most abundant organisms on Earth and represent one of the oldest and most widespread clades known in modern phylogenetics. As the only known prokaryotes capable of oxygenic photosynthesis, cyanobacteria are considered to be a promising resource for renewable fuels and natural products. Our efforts to harness the sun's energy using cyanobacteria would greatly benefit from an increased understanding of the genomic diversity across multiple cyanobacterial strains. In this respect, the advent of novel sequencing techniques and the availability of several cyanobacterial genomes offers new opportunities for understanding microbial diversity and metabolic organization and evolution in diverse environments. RESULTS: Here, we report a whole genome comparison of multiple phototrophic cyanobacteria. We describe genetic diversity found within cyanobacterial genomes, specifically with respect to metabolic functionality. Our results are based on pair-wise comparison of protein sequences and concomitant construction of clusters of likely ortholog genes. We differentiate between core, shared and unique genes and show that the majority of genes are associated with a single genome. In contrast, genes with metabolic function are strongly overrepresented within the core genome that is common to all considered strains. The analysis of metabolic diversity within core carbon metabolism reveals parts of the metabolic networks that are highly conserved, as well as highly fragmented pathways. CONCLUSIONS: Our results have direct implications for resource allocation and further sequencing projects. It can be extrapolated that the number of newly identified genes still significantly increases with increasing number of new sequenced genomes. Furthermore, genome analysis of multiple phototrophic strains allows us to obtain a detailed picture of metabolic diversity that can serve as a starting point for biotechnological applications and automated metabolic reconstructions.

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Conservation and divergence of chemical defence system in the tunicate Oikopleura dioica revealed by genome wide response to two xenobiotics.

Publication Date: 2012 Feb 2 PMID: 22300585
Authors: Yadetie, F. - Butcher, S. - Forde, H. E. - Campsteijn, C. - Bouquet, J. M. - Karlsen, O. A. - Denoeud, F. - Metpally, R. - Thompson, E. M. - Manak, J. R. - Goksoyr, A. - Chourrout, D.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Animals have developed extensive mechanisms of response to xenobiotic chemical attacks. Although recent genome surveys have suggested a broad conservation of the chemical defensome across metazoans, global gene expression responses to xenobiotics have not been well investigated in most invertebrates. Here, we performed genome survey for key defensome genes in Oikopleura dioica genome, and explored genome-wide gene expression using high density tiling arrays with over 2 million probes, in response to two model xenobiotic chemicals - the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) and the pharmaceutical compound Clofibrate (Clo). RESULTS: Oikopleura genome surveys for key genes of the chemical defensome suggested a reduced repertoire. Not more than 23 cytochrome P450 (CYP) genes could be identified, and neither CYP1 family genes nor their transcriptional activator AhR was detected. These two genes were present in deuterostome ancestors. As in vertebrates, the genotoxic compound BaP induced xenobiotic biotransformation and oxidative stress responsive genes. Notable exceptions were genes of the aryl hydrocarbon receptor (AhR) signaling pathway. Clo also affected the expression of many biotransformation genes and markedly repressed genes involved in energy metabolism and muscle contraction pathways. CONCLUSIONS: Oikopleura has the smallest number of CYP genes among sequenced animal genomes and lacks the AhR signaling pathway. However it appears to have basic xenobiotic inducible biotransformation genes such as a conserved genotoxic stress response gene set. Our genome survey and expression study does not support a role of AhR signaling pathway in the chemical defense of metazoans prior to the emergence of vertebrates.

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Zonation related function and ubiquitination regulation in human hepatocellular carcinoma cells in dynamic vs. static culture conditions.

Publication Date: 2012 Feb 1 PMID: 22296956
Authors: Cheng, S. - Prot, J. M. - Leclerc, E. - Bois, F. Y.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Understanding hepatic zonation is important both for liver physiology and pathology. There is currently no effective systemic chemotherapy for human hepatocellular carcinoma (HCC) and its pathogenesis is of special interest. Genomic and proteomic data of HCC cells in different culture models, coupled to pathway-based analysis, can help identify HCC-related gene and pathway dysfunctions. RESULTS: We identified zonation-related expression profiles contributing to selective phenotypes of HCC, by integrating relevant experimental observations through gene set enrichment analysis (GSEA). Analysis was based on gene and protein expression data measured on a human HCC cell line (HepG2/C3A) in two culture conditions: dynamic microfluidic biochips and static Petri dishes. Metabolic activity (HCC-related cytochromes P450) and genetic information processing were dominant in the dynamic cultures, in contrast to kinase signaling and cancer-specific profiles in static cultures. That, together with analysis of the published literature, leads us to propose that biochips culture conditions induce a periportal-like hepatocyte phenotype while standard plates cultures are more representative of a perivenous-like phenotype. Both proteomic data and GSEA results further reveal distinct ubiquitin-mediated protein regulation in the two culture conditions. CONCLUSIONS: Pathways analysis, using gene and protein expression data from two cell culture models, confirmed specific human HCC phenotypes with regard to CYPs and kinases, and revealed a zonation-related pattern of expression. Ubiquitin-mediated regulation mechanism gives plausible explanations of our findings. Altogether, our results suggest that strategies aimed at inhibiting activated kinases and signaling pathways may lead to enhanced metabolism-mediated drug resistance of treated tumors. If that were the case, mitigating inhibition or targeting inactive forms of kinases would be an alternative.

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Gene alterations at Drosophila inversion breakpoints provide prima facie evidence for natural selection as an explanation for rapid chromosomal evolution.

Publication Date: 2012 Feb 1 PMID: 22296923
Authors: Guillen, Y. - Ruiz, A.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Chromosomal inversions have been pervasive during the evolution of the genus Drosophila, but there is significant variation between lineages in the rate of rearrangement fixation. D. mojavensis, an ecological specialist adapted to a cactophilic niche under extreme desert conditions, is a chromosomally derived species with ten fixed inversions, five of them not present in any other species. RESULTS: In order to explore the causes of D. mojavensis rapid chromosomal evolution, we identified and characterized all breakpoints of seven inversions fixed in chromosome 2, the most dynamic one. One of the inversions presents unequivocal evidence for its generation by ectopic recombination between transposon copies and another two harbor inverted duplications of non-repetitive DNA at the two breakpoints and were likely generated by staggered single-strand breaks and repair by non-homologous end joining. Four out of 14 breakpoints lay in the intergenic region between preexisting duplicated genes, suggesting an adaptive advantage of separating previously tightly linked duplicates. Four out of 14 breakpoints are associated with transposed genes, suggesting these breakpoints are fragile regions. Finally two inversions contain novel genes at their breakpoints and another three show alterations of genes at breakpoints with potential adaptive significance. CONCLUSIONS: D. mojavensis chromosomal inversions were generated by multiple mechanisms, an observation that does not provide support for increased mutation rate as explanation for rapid chromosomal evolution. On the other hand, we have found a number of alterations of genes at the breakpoints with putative adaptive consequences that directly point to natural selection as the cause of D. mojavensis rapid chromosomal evolution.

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Microgravity simulation by diamagnetic levitation: effects of a strong gradient magnetic field on the transcriptional profile of Drosophila melanogaster.

Publication Date: 2012 Feb 1 PMID: 22296880
Authors: Herranz, R. - Larkin, O. J. - Dijkstra, C. E. - Hill, R. J. - Anthony, P. - Davey, M. R. - Eaves, L. - van Loon, J. J. - Medina, F. J. - Marco, R.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Many biological systems respond to the presence or absence of gravity. Since experiments performed in space are expensive and can only be undertaken infrequently, Earth-based simulation techniques are used to investigate the biological response to weightlessness. A high gradient magnetic field can be used to levitate a biological organism so that its net weight is zero. RESULTS: We have used a superconducting magnet to assess the effect of diamagnetic levitation on the fruit fly D. melanogaster in levitation experiments that proceeded for up to 22 consecutive days. We have compared the results with those of similar experiments performed in another paradigm for microgravity simulation, the Random Positioning Machine (RPM). We observed a delay in the development of the fruit flies from embryo to adult. Microarray analysis indicated changes in overall gene expression of imagoes that developed from larvae under diamagnetic levitation, and also under simulated hypergravity conditions. Significant changes were observed in the expression of immune-, stress-, and temperature-response genes. For example, several heat shock proteins were affected. We also found that a strong magnetic field, of 16.5 Tesla, had a significant effect on the expression of these genes, independent of the effects associated with magnetically-induced levitation and hypergravity. CONCLUSIONS: Diamagnetic levitation can be used to simulate an altered effective gravity environment in which gene expression is tuned differentially in diverse Drosophila melanogaster populations including those of different age and gender. Exposure to the magnetic field per se induced similar, but weaker, changes in gene expression.

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Genome-wide analysis of hepatic LRH-1 reveals a promoter binding preference and suggests a role in regulating genes of lipid metabolism in concert with FXR.

Publication Date: 2012 Feb 1 PMID: 22296850
Authors: Kim Chong, H. - Biesinger, J. - Seo, Y. K. - Xie, X. - Osborne, T. F.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: In a previous genome-wide analysis of FXR binding to hepatic chromatin, we noticed that an extra nuclear receptor (NR) half-site was co-enriched close to the FXR binding IR-1 elements and we provided limited support that the monomeric LRH-1 receptor that binds to NR half-sites might function together with FXR to activate gene expression. RESULTS: To analyze the global pattern for LRH-1 binding and to determine whether it might associate with FXR on a whole genome-wide scale, we analyzed LRH-1 binding to the entire hepatic genome using a non-biased genome-wide ChIP-seq approach. We identified over 10,600 LRH-1 binding sites in hepatic chromatin and over 20% were located within 2 kb of the 5' end of a known mouse gene. Additionally, the results demonstrate that a significant fraction of the genome sites occupied by LRH-1 are located close to FXR binding sites revealed in our earlier study. A Gene ontology analysis revealed that genes preferentially enriched in the LRH-1/FXR overlapping gene set are related to lipid metabolism. These results demonstrate that LRH-1 recruits FXR to lipid metabolic genes. A significant fraction of FXR binding peaks also contain a nuclear receptor half-site that does not bind LRH-1 suggesting that additional monomeric nuclear receptors such as RORs and NR4As family members may also target FXR to other pathway selective genes related to other areas of metabolism such as glucose metabolism where FXR has also been shown to play an important role. CONCLUSION: These results document an important role for LRH-1 in hepatic metabolism through acting predominantly at proximal promoter sites and working in concert with additional nuclear receptors that bind to neighboring sites.

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Genome-wide landscape of liver X receptor chromatin binding and gene regulation in human macrophages.

Publication Date: 2012 Jan 31 PMID: 22292898
Authors: Pehkonen, P. - Welter-Stahl, L. - Diwo, J. - Ryynanen, J. - Wienecke-Baldacchino, A. - Heikkinen, S. - Treuter, E. - Steffensen, K. R. - Carlberg, C.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The liver X receptors (LXRs) are oxysterol sensing nuclear receptors with multiple effects on metabolism and immune cells. However, the complete genome-wide cistrome of LXR in cells of human origin has not yet been provided. RESULTS: We performed ChIP-seq in phorbol myristate acetate-differentiated THP-1 cells (macrophage-type) after stimulation with the potent synthetic LXR ligand T0901317 (T09). Microarray gene expression analysis was performed in the same cellular model. We identified 1357 genome-wide LXR locations (FDR<1%), of which 526 were observed after T09 treatment. De novo analysis of LXR binding sequences identified a DR4-type element as the major motif. On mRNA level T09 up-regulated 1258 genes and repressed 455 genes. Our results show that LXR actions are focused on 112 genomic regions that contain up to 11 T09 target genes per region under the control of highly stringent LXR binding sites with individual constellations for each region. We could confirm that LXR controls lipid metabolism and transport and observed a strong association with apoptosis-related functions. CONCLUSIONS: This first report on genome-wide binding of LXR in a human cell line provides new insights into the transcriptional network of LXR and its target genes with their link to physiological processes, such as apoptosis. The gene expression microarray and sequence data have been submitted collectively to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE28319.

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Xanthomonas oryzae pv oryzae triggers immediate transcriptomic modulations in rice.

Publication Date: 2012 Jan 31 PMID: 22289642
Authors: Grewal, R. K. - Gupta, S. - Das, S.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Xanthomonas oryzae pv oryzae is a devastating pathogen of rice and has been extensively studied as a model pathogen of monocotyledons. Expressional studies in both the contenders have been undertaken in past to understand the molecular mechanism underlying the compatible and incompatible interactions in the pathosystem. Continuous update on database and gene annotations necessitates constant updating on the roles of the new entities as well as reinterpretation of regulations of the previous ones. Moreover the past endeavors have addressed the middle or late defense responses of the rice plant whereas in the present study an attempt has been made to investigate the early defense responses taking place immediately after inoculation. RESULTS: Microarray was used to study the transcriptional modulations in eighteen days old rice seedling leaves of both susceptible and resistant genotypes one hour after inoculation. In resistant plants as compared to susceptible ones 274 genes were found to be differentially expressed. Annotations could be assigned to 112 up- and 73 down-regulated transcripts and gene interaction maps were generated for 86 transcripts. Expressional data and interaction maps were used to develop a hypothetical scheme of the molecular events taking place during early defense response. Network analysis with the differential transcripts showed up-regulation of major clusters of cell signaling proteins and transcription factors while growth and basal metabolic components were largely found to be down-regulated. CONCLUSIONS: This study provides an understanding of the early defense signaling in rice cells. Components of the calcium and lipid signaling as well as MAPK cascade were modulated, by signals from surface receptors and cytosolic R-proteins, to arouse jasmonic acid and ethylene signaling and suppress auxin signaling through various transcription factors. Abscisic acid modulation was also evident through the expression regulation of transcription factors involved with its functions. Moreover adjustments in expression levels of components of primary as well as secondary metabolism, protein trafficking and turnout were apparent, highlighting the complexity of defense response.

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Combining evidence of selection with association analysis increases power to detect regions influencing complex traits in dairy cattle.

Publication Date: 2012 Jan 30 PMID: 22289501
Authors: Schwarzenbacher, H. - Dolezal, M. - Flisikowski, K. - Seefried, F. - Wurmser, C. - Schlotterer, C. - Fries, R.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Hitchhiking mapping and association studies are two popular approaches to map genotypes to phenotypes. In this study we combine both approaches to complement their specific strengths and weaknesses, resulting in a method with higher statistical power and fewer false positive signals. We applied our approach to dairy cattle as they underwent extremely successful selection for milk production traits and since an excellent phenotypic record is available. We performed whole genome association tests with a new mixed model approach to account for stratification, which we validated via Monte Carlo simulations. Selection signatures were inferred with the integrated haplotype score and a locus specific permutation based integrated haplotype score that works with a folded frequency spectrum and provides a formal test of signifance to identify selection signatures. RESULTS: About 1,600 out of 34,851 SNPs showed signatures of selection and the locus specific permutation based integrated haplotype score showed overall good accordance with the whole genome association study. Each approach provides distinct information about the genomic regions that influence complex traits. Combining whole genome association with hitchhiking mapping yielded two significant loci for the trait protein yield. These regions agree well with previous results from other selection signature scans and whole genome association studies in cattle. CONCLUSION: We show that the combination of whole genome association and selection signature mapping based on the same SNPs increases the power to detect loci influencing complex traits. The locus specific permutation based integrated haplotype score provides a formal test of significance in selection signature mapping. Importantly it does not rely on knowledge of ancestral and derived allele states. KEY WORDS selection signature, whole genome association, cattle, complex trait.

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Whole Genome Profiling provides a robust framework for physical mapping and sequencing in the highly complex and repetitive wheat genome.

Publication Date: 2012 Jan 30 PMID: 22289472
Authors: Philippe, R. - Choulet, F. - Paux, E. - van Oeveren, J. - Tang, J. - Wittenberg, A. H. - Janssen, A. - van Eijk, M. J. - Stormo, K. - Alberti, A. - Wincker, P. - Akhunov, E. - van der Vossen, E. - Feuillet, C.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Sequencing projects using a clone-by-clone approach require the availability of a robust physical map. The SNaPshot technology, based on pair-wise comparisons of restriction fragments sizes, has been used recently to build the first physical map of a wheat chromosome and to complete the maize physical map. However, restriction fragments sizes shared randomly between two non-overlapping BACs often lead to chimerical contigs and mis-assembled BACs in such large and repetitive genomes. Whole Genome Profiling (WGPTM) was developed recently as a new sequence-based physical mapping technology and has the potential to limit this problem. RESULTS: A subset of the wheat 3B chromosome BAC library covering 230 Mb was used to establish a WGP physical map and to compare it to a map obtained with the SNaPshot technology. We first adapted the WGP-based assembly methodology to cope with the complexity of the wheat genome. Then, the results showed that the WGP map covers the same length than the SNaPshot map but with 30% less contigs and, more importantly with 3.5 times less mis-assembled BACs. Finally, we evaluated the benefit of integrating WGP tags in different sequence assemblies obtained after Roche/454 sequencing of BAC pools. We showed that while WGP tag integration improves assemblies performed with unpaired reads and with paired-end reads at low coverage, it does not significantly improve sequence assemblies performed at high coverage (25x) with paired-end reads. CONCLUSIONS: Our results demonstrate that, with a suitable assembly methodology, WGP builds more robust physical maps than the SNaPshot technology in wheat and that WGP can be adapted to any genome. Moreover, WGP tag integration in sequence assemblies improves low quality assembly. However, to achieve a high quality draft sequence assembly, a sequencing depth of 25x paired-end reads is required, at which point WGP tag integration does not provide additional scaffolding value. Finally, we suggest that WGP tags can support the efficient sequencing of BAC pools by enabling reliable assignment of sequence scaffolds to their BAC of origin, a feature that is of great interest when using BAC pooling strategies to reduce the cost of sequencing large genomes.

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Identification of differentially expressed genes in SHSY5Y cells exposed to okadaic acid by suppression subtractive hybridization.

Publication Date: 2012 Jan 27 PMID: 22284234
Authors: Valdiglesias, V. - Fernandez-Tajes, J. - Pasaro, E. - Mendez, J. - Laffon, B.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Okadaic acid (OA), a toxin produced by several dinoflagellate species is responsible for frequent food poisonings associated to shellfish consumption. Although several studies have documented the OA effects on different processes such as cell transformation, apoptosis, DNA repair or embryogenesis, the molecular mechanistic basis for these and other effects is not completely understood and the number of controversial data on OA is increasing in the literature. RESULTS: In this study, we used suppression subtractive hybridization in SHSY5Y cells to identify genes that are differentially expressed after OA exposure for different times (3, 24 and 48 h). A total of 247 subtracted clones which shared high homology with known genes were isolated. Among these, 5 specific genes associated with cytoskeleton and neurotransmission processes (NEFM, TUBB, SEPT7, SYT4 and NPY) were selected to confirm their expression levels by real-time PCR. Significant down-regulation of these genes was obtained at the short term (3 and 24 h OA exposure), excepting for NEFM, but their expression was similar to the controls at 48 h. CONCLUSIONS: From all the obtained genes, 114 genes were up-regulated and 133 were down-regulated. Based on the NCBI GenBank and Gene Ontology databases, most of these genes are involved in relevant cell functions such as metabolism, transport, translation, signal transduction and cell cycle. After quantitative PCR analysis, the observed underexpression of the selected genes could underlie the previously reported OA-induced cytoskeleton disruption, neurotransmission alterations and in vivo neurotoxic effects. The basal expression levels obtained at 48 h suggested that surviving cells were able to recover from OA-caused gene expression alterations.

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Transcriptional profiling of bovine milk using RNA sequencing.

Publication Date: 2012 Jan 25 PMID: 22276848
Authors: Wickramasinghe, S. - Rincon, G. - Islas-Trejo, A. - Medrano, J. F.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Cow milk is a complex bioactive fluid consumed by humans beyond infancy. Even though the chemical and physical properties of cow milk are well characterized, very limited research has been done on characterizing the milk transcriptome. This study performs a comprehensive expression profiling of genes expressed in milk somatic cells of transition (day 15), peak (day 90) and late (day 250) lactation Holstein cows by RNA sequencing. Milk samples were collected from Holstein cows at 15, 90 and 250 days of lactation, and RNA was extracted from the pelleted milk cells. Gene expression analysis was conducted by Illumina RNA sequencing. Sequence reads were assembled and analyzed in CLC Genomics Workbench. Gene Ontology (GO) and pathway analysis were performed using the Blast2GO program and GeneGo application of MetaCore program. RESULTS: A total of 16,892 genes were expressed in transition lactation, 19,094 genes were expressed in peak lactation and 18,070 genes were expressed in late lactation. Regardless of the lactation stage approximately 9,000 genes showed ubiquitous expression. Genes encoding caseins, whey proteins and enzymes in lactose synthesis pathway showed higher expression in early lactation. The majority of genes in the fat metabolism pathway had high expression in transition and peak lactation milk. Most of the genes encoding for endogenous proteases and enzymes in ubiquitin-proteasome pathway showed higher expression along the course of lactation. CONCLUSIONS: This is the first study to describe the comprehensive bovine milk transcriptome in Holstein cows. The results revealed that 69% of NCBI Btau 4.0 annotated genes are expressed in bovine milk somatic cells. Most of the genes were ubiquitously expressed in all three stages of lactation. However, a fraction of the milk transcriptome has genes devoted to specific functions unique to the lactation stage. This indicates the ability of milk somatic cells to adapt to different molecular functions according to the biological need of the animal. This study provides a valuable insight into the biology of lactation in the cow, as well as many avenues for future research on the bovine lactome.

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miRdSNP: a database of disease-associated SNPs and microRNA target sites on 3'UTRs of human genes.

Publication Date: 2012 Jan 25 PMID: 22276777
Authors: Bruno, A. E. - Li, L. - Kalabus, J. L. - Pan, Y. - Yu, A. - Hu, Z.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Single nucleotide polymorphisms (SNPs) can lead to the susceptibility and onset of diseases through their effects on gene expression at the posttranscriptional level. Recent findings indicate that SNPs could create, destroy, or modify the efficiency of miRNA binding to the 3'UTR of a gene, resulting in gene dysregulation. With the rapidly growing number of published disease-associated SNPs (dSNPs), there is a strong need for resources specifically recording dSNPs on the 3'UTRs and their nucleotide distance from miRNA target sites. We present here miRdSNP, a database incorporating three important areas of dSNPs, miRNA target sites, and diseases. Description: miRdSNP provides a unique database of dSNPs on the 3'UTRs of human genes manually curated from PubMed. The current release includes 786 dSNP-disease associations for 630 unique dSNPs and 204 disease types. miRdSNP annotates genes with experimentally confirmed targeting by miRNAs and indexes miRNA target sites predicted by TargetScan and PicTar as well as potential miRNA target sites newly generated by dSNPs. A robust web interface and search tools are provided for studying the proximity of miRNA binding sites to dSNPs in relation to human diseases. Searches can be dynamically filtered by gene name, miRBase ID, target prediction algorithm, disease, and any nucleotide distance between dSNPs and miRNA target sites. Results can be viewed at the sequence level showing the annotated locations for miRNA target sites and dSNPs on the entire 3'UTR sequences. The integration of dSNPs with the UCSC Genome browser is also supported. CONCLUSION: miRdSNP provides a comprehensive data source of dSNPs and robust tools for exploring their distance from miRNA target sites on the 3'UTRs of human genes. miRdSNP enables researchers to further explore the molecular mechanism of gene dysregulation for dSNPs at posttranscriptional level. miRdSNP is freely available on the web at http://mirdsnp.ccr.buffalo.edu.

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Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis.

Publication Date: 2012 Jan 25 PMID: 22276739
Authors: Tu, J. - Ge, Q. - Wang, S. - Wang, L. - Sun, B. - Yang, Q. - Bai, Y. - Lu, Z.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. RESULTS: Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. CONCLUSIONS: By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.

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An evaluation of two-channel ChIP-on-chip and DNA methylation microarray normalization strategies.

Publication Date: 2012 Jan 25 PMID: 22276688
Authors: Adriaens, M. E. - Jaillard, M. - Eijssen, L. M. - Mayer, C. D. - Evelo, C. T.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The combination of chromatin immunoprecipitation with two-channel microarray technology enables genome-wide mapping of binding sites of DNA-interacting proteins (ChIP-on-chip) or sites with methylated CpG di-nucleotides (DNA methylation microarray). These powerful tools are the gateway to understanding gene transcription regulation. Since the goals of such studies, the sample preparation procedures, the microarray content and study design are all different from transcriptomics microarrays, the data pre-processing strategies traditionally applied to transcriptomics microarrays may not be appropriate. Particularly, the main challenge of the normalization of "regulation microarrays" is (i) to make the data of individual microarrays quantitatively comparable and (ii) to keep the signals of the enriched probes, representing DNA sequences from the precipitate, as distinguishable as possible from the signals of the un-enriched probes, representing DNA sequences largely absent from the precipitate. Results: We compare several widely used normalization approaches (VSN, LOWESS, quantile, T-quantile, Tukey's biweight scaling, Peng's method) applied to a selection of regulation microarray datasets, ranging from DNA methylation to transcription factor binding and histone modification studies. Through comparison of the data distributions of control probes and gene promoter probes before and after normalization, and assessment of the power to identify known enriched genomic regions after normalization, we demonstrate that there are clear differences in performance between normalization procedures. Conclusion: T-quantile normalization applied separately on the channels and Tukey's biweight scaling outperform other methods in terms of the conservation of enriched and un-enriched signal separation, as well as in identification of genomic regions known to be enriched. T-quantile normalization is preferable as it additionally improves comparability between microarrays. In contrast, popular normalization approaches like quantile, LOWESS, Peng's method and VSN normalization alter the data distributions of regulation microarrays to such an extent that using these approaches will impact the reliability of the downstream analysis substantially.

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The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis.

Publication Date: 2012 Jan 24 PMID: 22272770
Authors: De Paola, D. - Cattonaro, F. - Pignone, D. - Sonnante, G.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Plant microRNAs (miRNAs) are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA) libraries can detect even poorly expressed miRNAs. No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots. RESULTS: Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families) of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs) identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1. CONCLUSIONS: The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.

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Genetic diversity of human RNase 8.

Publication Date: 2012 Jan 24 PMID: 22272736
Authors: Chan, C. C. - Moser, J. M. - Dyer, K. D. - Percopo, C. M. - Rosenberg, H. F.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Ribonuclease 8 is a member of the RNase A family of secretory ribonucleases; orthologs of this gene have been found only in primate genomes. RNase 8 is a divergent paralog of RNase 7, which is lysine-enriched, highly conserved, has prominent antimicrobial activity, and is expressed in both normal and diseased skin; in contrast, the physiologic function of RNase 8 remains uncertain. Here, we examine the genetic diversity of human RNase 8, a subject of significant interest given the existence of functional pseudogenes (coding sequences that are otherwise intact but with mutations in elements crucial for ribonucleolytic activity) in non-human primate genomes. RESULTS: RNase 8 expression was detected in adult human lung, spleen and testis tissue by quantitative reverse-transcription PCR. Only two single-nucleotide polymorphisms and four unique alleles were identified within the RNase 8 coding sequence; nucleotide sequence diversity (pi = 0.00122 +/- 0.00009 per site) was unremarkable for a human nuclear gene. We isolated transcripts encoding RNase 8 via rapid amplification of cDNA ends (RACE) and RT-PCR which included a distal potential translational start site followed by sequence encoding an additional 30 amino acids that are conserved in the genomes of several higher primates. The distal translational start site is functional and promotes RNase 8 synthesis in transfected COS-7 cells. CONCLUSIONS: These results suggest that RNase 8 may diverge considerably from typical RNase A family ribonucleases and may likewise exhibit unique function. This finding prompts a reconsideration of what we have previously termed functional pseudogenes, as RNase 8 may be responding to constraints that promote significant functional divergence from the canonical structure and enzymatic activity characteristic of the RNase A family.

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The core and unique proteins of haloarchaea.

Publication Date: 2012 Jan 24 PMID: 22272718
Authors: Capes, M. D. - Dassarma, P. - Dassarma, S.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Since the first genome of a halophilic archaeon was sequenced in 2000, biologists have been advancing the understanding of genomic characteristics that allow for survival in the harsh natural environments of these organisms. An increase in protein acidity and GC-bias in the genome have been implicated as factors in tolerance to extreme salinity, desiccation, and high solar radiation. However, few previous attempts have been made to identify novel genes that would permit survival in such extreme conditions. RESULTS: With the recent release of several new complete haloarchaeal genome sequences, we have conducted a comprehensive comparative genomic analysis focusing on the identification of unique haloarchaeal conserved proteins that likely play key roles in environmental adaptation. Using bioinformatic methods, we have clustered 31,312 predicted proteins from nine haloarchaeal genomes into 4,455 haloarchaeal orthologous groups (HOGs). We assigned likely functions by association with established COG and KOG databases in NCBI. After identifying homologs in four additional haloarchaeal genomes, we determined that there were 784 core haloarchaeal protein clusters (cHOGs), of which 83 clusters were found primarily in haloarchaea. Further analysis found that 55 clusters were truly unique (tucHOGs) to haloarchaea and qualify as signature proteins while 28 were nearly unique (nucHOGs), the vast majority of which were coded for on the haloarchaeal chromosomes. Of the signature proteins, only one example with any predicted function, Ral, involved in desiccation/radiation tolerance in Halobacterium sp. NRC-1, was identified. Among the core clusters, 33 % was predicted to function in metabolism, 25 % in information transfer and storage, 10 % in cell processes and signaling, and 22 % belong to poorly characterized or general function groups. CONCLUSION: Our studies have established conserved groups of nearly 800 protein clusters present in all haloarchaea, with a subset of 55 which are predicted to be accessory proteins that may be critical or essential for success in an extreme environment. These studies support core and signature genes and proteins as valuable concepts for understanding phylogenetic and phenotypic characteristics of coherent groups of organisms.

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Comparative genomic analysis of the genus Staphylococcus including Staphylococcus aureus and its newly described sister species Staphylococcus simiae.

Publication Date: 2012 Jan 24 PMID: 22272658
Authors: Suzuki, H. - Lefebure, T. - Pavinski Bitar, P. - Stanhope, M. J.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Staphylococcus belongs to the Gram-positive low G+C content group of the Firmicutes division of bacteria. Staphylococcus aureus is an important human and veterinary pathogen that causes a broad spectrum of diseases, and has developed important multidrug resistant forms such as methicillin-resistant S. aureus (MRSA). Staphylococcus simiae was isolated from South American squirrel monkeys in 2000, and is a coagulase-negative bacterium, closely related, and possibly the sister group, to S. aureus. Comparative genomic analyses of closely related bacteria with different phenotypes can provide information relevant to understanding adaptation to host environment and mechanisms of pathogenicity. RESULTS: We determined a Roche/454 draft genome sequence for S. simiae and included it in comparative genomic analyses with 11 other Staphylococcus species including S. aureus. A genome based phylogeny of the genus confirms that S. simiae is the sister group to S. aureus and indicates that the most basal Staphylococcus lineage is Staphylococcus pseudintermedius, followed by Staphylococcus carnosus. Given the primary niche of these two latter taxa, compared to the other species in the genus, this phylogeny suggests that human adaptation evolved after the split of S. carnosus. The two coagulase-positive species (S. aureus and S. pseudintermedius) are not phylogenetically closest but share many virulence factors exclusively, suggesting that these genes were acquired by horizontal transfer. Enrichment in genes related to mobile elements such as prophage in S. aureus relative to S. simiae suggests that pathogenesis in the S. aureus group has developed by gene gain through horizontal transfer, after the split of S. aureus and S. simiae from their common ancestor. CONCLUSIONS: Comparative genomic analyses across 12 Staphylococcus species provide hypotheses about lineages in which human adaptation has taken place and contributions of horizontal transfer in pathogenesis.

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Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida.

Publication Date: 2012 Jan 24 PMID: 22272603
Authors: Hansen, G. A. - Ahmad, R. - Hjerde, E. - Fenton, C. G. - Willassen, N. P. - Haugen, P.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84 % identity to E. coli Spot 42. RESULTS: We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose we found a surprisingly large number of [greater than or equal to]2x differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered a sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose . CONCLUSIONS: We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin.

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A metabolomic strategy defines the regulation of lipid content and global metabolism by Delta-9 desaturases in Caenorhabditis elegans.

Publication Date: 2012 Jan 20 PMID: 22264337
Authors: Castro, C. - Sar, F. - Shaw, W. R. - Mishima, M. - Miska, E. A. - Griffin, J. L.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Caenorhabditis elegans provides a genetically tractable model organism to investigate the network of genes involved in fat metabolism and how regulation is perturbed to produce the complex phenotype of obesity. C. elegans possess the full range of desaturases, including the Delta9 desaturases expressed by fat-5, fat-6 and fat-7. They regulate the biosynthesis of monounsaturated fatty acids, used for the synthesis of lipids including phospholipids, triglycerides and cholesteryl esters. RESULTS: Liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to define the metabolome of all the possible knock-outs for the Delta9 desaturases, including for the first time intact lipids. Despite the genes having similar enzymatic roles, excellent discrimination was achievable for all single and viable double mutants highlighting the distinctive roles of fat-6 and fat-7, both expressing steroyl-CoA desaturases. The metabolomic changes extend to aqueous metabolites demonstrating the influence Delta9 desaturases have on regulating global metabolism and highlighting how comprehensive metabolomics is more discriminatory than classically used dyes for fat staining. CONCLUSIONS: The propagation of metabolic changes across the network of metabolism demonstrates that modification of the Delta9 desaturases places C.elegans into a catabolic state compared with wildtype controls.

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Discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias.

Publication Date: 2012 Jan 19 PMID: 22260749
Authors: Didion, J. P. - Yang, H. - Sheppard, K. - Fu, C. P. - McMillan, L. - Pardo-Manuel de Villena, F. - Churchill, G. A.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: High-density genotyping arrays that measure hybridization of genomic DNA fragments to allele-specific oligonucleotide probes are widely used to genotype single nucleotide polymorphisms (SNPs) in genetic studies, including human genome-wide association studies. Hybridization intensities are converted to genotype calls by clustering algorithms that assign each sample to a genotype class at each SNP. Data for SNP probes that do not conform to the expected pattern of clustering are often discarded, contributing to ascertainment bias and resulting in lost information - as much as 50% in a recent genome-wide association study in dogs. RESULTS: We identified atypical patterns of hybridization intensities that were highly reproducible and demonstrated that these patterns represent genetic variants that were not accounted for in the design of the array platform. We characterized variable intensity oligonucleotide (VINO) probes that display such patterns and are found in all hybridization-based genotyping platforms, including those developed for human, dog, cattle, and mouse. When recognized and properly interpreted, VINOs recovered a substantial fraction of discarded probes and counteracted SNP ascertainment bias. We developed software (MouseDivGeno) that identifies VINOs and improves the accuracy of genotype calling. MouseDivGeno produced highly concordant genotype calls when compared with other methods but it uniquely identified more than 786,000 VINOs in 351 mouse samples. We used whole-genome sequence from 14 mouse strains to confirm the presence of novel variants explaining 28,000 VINOs in those strains. We also identified VINOs in human HapMap 3 samples, many of which were specific to an African population. Incorporating VINOs in phylogenetic analyses substantially improved the accuracy of a Mus species tree and local haplotype assignment in laboratory mouse strains. CONCLUSION: The problems of ascertainment bias and missing information due to genotyping errors are widely recognized as limiting factors in genetic studies. We have conducted the first formal analysis of the effect of novel variants on genotyping arrays, and we have shown that these variants account for a large portion of miscalled and uncalled genotypes. Genetic studies will benefit from substantial improvements in the accuracy of their results by incorporating VINOs in their analyses.

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Transcriptional Ontogeny of the Developing Liver.

Publication Date: 2012 Jan 19 PMID: 22260730
Authors: Lee, J. S. - Ward, W. O. - Knapp, G. - Ren, H. - Vallanat, B. - Abbott, B. - Ho, K. - Karp, S. J. - Corton, J. C.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: During embryogenesis the liver is derived from endodermal cells lining the digestive tract. These endodermal progenitor cells contribute to forming the parenchyma of a number of organs including the liver and pancreas. Early in organogenesis the fetal liver is populated by hematopoietic stem cells, the source for a number of blood cells including nucleated erythrocytes. A comprehensive analysis of the transcriptional changes that occur during the early stages of development to adulthood in the liver was carried out. RESULTS: We characterized gene expression changes in the developing mouse liver at gestational days (GD) 11.5, 12.5, 13.5, 14.5, 16.5, and 19 and in the neonate (postnatal day (PND) 7 and 32) compared to that in the adult liver (PND67) using full-genome microarrays. The fetal liver, and to a lesser extent the neonatal liver, exhibited dramatic differences in gene expression compared to adults. Canonical pathway analysis of the fetal liver signature demonstrated increases in functions important in cell replication and DNA fidelity whereas most metabolic pathways of intermediary metabolism were under expressed. Comparison of the dataset to a number of previously published microarray datasets revealed 1) a striking similarity between the fetal liver and that of the pancreas in both mice and humans, 2) a nucleated erythrocyte signature in the fetus and 3) under expression of most xenobiotic metabolism genes throughout development, with the exception of a number of transporters associated with either hematopoietic cells or cell proliferation in hepatocytes. CONCLUSIONS: Overall, these findings reveal the complexity of gene expression changes during liver development and maturation, and provide a foundation to predict responses to chemical and drug exposure as a function of early life-stages.

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High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach.

Publication Date: 2012 Jan 19 PMID: 22260654
Authors: Allard, M. W. - Luo, Y. - Strain, E. - Li, C. - Keys, C. E. - Son, I. - Stones, R. - Musser, S. M. - Brown, E. W.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Next-Generation Sequencing (NGS) is increasingly being used as a molecular epidemiologic tool for discerning ancestry and traceback of the most complicated, difficult to resolve bacterial pathogens. Making a linkage between possible food sources and clinical isolates requires distinguishing the suspected pathogen from an environmental background and placing the variation observed into the wider context of variation occurring within a serovar and among other closely related foodborne pathogens. Equally important is the need to validate these high resolution molecular tools for use in molecular epidemiologic traceback. Such efforts include the examination of strain cluster stability as well as the cumulative genetic effects of sub-culturing on these clusters. Numerous isolates of S. Montevideo were shot-gun sequenced including diverse lineage representatives as well as numerous replicate clones to determine how much variability is due to bias, sequencing error, and or the culturing of isolates. All new draft genomes were compared to 34 S. Montevideo isolates previously published during an NGS-based molecular epidemiological case study. RESULTS: Intraserovar lineages of S. Montevideo differ by thousands of SNPs, that are only slightly less than the number of SNPs observed between S. Montevideo and other distinct serovars. Much less variability was discovered within an individual S. Montevideo clade implicated in a recent foodborne outbreak as well as among individual NGS replicates. These findings were similar to previous reports documenting homopolymeric and deletion error rates with the Roche 454 GS Titanium technology. In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data. CONCLUSIONS: Implementation of a validated pipeline for NGS data acquisition and analysis provides highly reproducible results that are stable and predictable for molecular epidemiological applications. When draft genomes are collected at 15X-20X coverage and passed through a quality filter as part of a data analysis pipeline, including sub-passaged replicates defined by a few SNPs, they can be accurately placed in a phylogenetic context. This reproducibility applies to all levels within and between serovars of Salmonella suggesting that investigators using these methods can have confidence in their conclusions.

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IntegromeDB: an integrated system and biological search engine.

Publication Date: 2012 Jan 19 PMID: 22260095
Authors: Baitaluk, M. - Kozhenkov, S. - Dubinina, Y. - Ponomarenko, J.
Journal: BMC Genomics

ABSTRACT: Background With the growth of biological data in volume and heterogeneity, web search engines become key tools for researchers. However, general-purpose search engines are not specialized for the search of biological data. Description Here, we present an approach at developing a biological web search engine based on the Semantic Web technologies and demonstrate its implementation for retrieving gene- and protein-centered knowledge. The engine is available at www.integromedb.org. Conclusions The IntegromeDB search engine allows scanning data on gene regulation, gene expression, protein-protein interactions, pathways, metagenomics, mutations, diseases, and other gene- and protein-related data that are automatically retrieved from publicly available databases and web pages using biological ontologies. To perfect the resource design and usability, we welcome and encourage the community feedback.

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Sequencing and comparative genomic analysis of 1227 Felis catus cDNA sequences enriched for developmental, clinical and nutritional phenotypes.

Publication Date: 2012 Jan 18 PMID: 22257742
Authors: Irizarry, K. J. - Malladi, S. B. - Gao, X. - Mitsouras, K. - Melendez, L. - Burris, P. A. - Brockman, J. A. - Al-Murrani, S. W.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The feline genome is valuable to the veterinary and model organism genomics communities because the cat is an obligate carnivore and a model for endangered felids. The initial public release of the Felis catus genome assembly provided a framework for investigating the genomic basis of feline biology. However, the entire set of protein coding genes has not been elucidated. RESULTS: We identified and characterized 1227 protein coding feline sequences, of which 913 map to public sequences and 314 are novel. These sequences have been deposited into NCBI's genbank database and complement public genomic resources by providing additional protein coding sequences that fill in some of the gaps in the feline genome assembly. Through functional and comparative genomic analyses, we gained an understanding of the role of these sequences in feline development, nutrition and health. Specifically, we identified 104 orthologs of human genes associated with Mendelian disorders. We detected negative selection within sequences with gene ontology annotations associated with intracellular trafficking, cytoskeleton and muscle functions. We detected relatively less negative selection on protein sequences encoding extracellular networks, apoptotic pathways and mitochondrial gene ontology annotations. Additionally, we characterized feline cDNA sequences that have mouse orthologs associated with clinical, nutritional and developmental phenotypes. Together, this analysis provides an overview of the value of our cDNA sequences and enhances our understanding of how the feline genome is similar to, and different from other mammalian genomes. CONCLUSIONS: The cDNA sequences reported here expand existing feline genomic resources by providing high-quality sequences annotated with comparative genomic information providing functional, clinical, nutritional and orthologous gene information.

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Microarray analysis on germfree mice elucidates the primary target of a traditional Japanese medicine juzentaihoto: Acceleration of IFN-alpha response via affecting the ISGF3-IRF7 signaling cascade.

Publication Date: 2012 Jan 18 PMID: 22257721
Authors: Munakata, K. - Takashima, K. - Nishiyama, M. - Asano, N. - Mase, A. - Hioki, K. - Ohnishi, Y. - Yamamoto, M. - Watanabe, K.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The traditional Japanese medicine juzentaihoto (JTX) is a pharmaceutical grade multi-herbal medicine widely used for the prevention of cancer metastasis and infection in immuno-compromized patients in Japan. The effect of JTX has been supposed to be intimately affected by the immunological properties of host and enteric microflora. The influence of JTX on the gene expression profile in the large and intestine was investigated by microarray analyses using mice of different strains with or without enteric microflora. RESULTS: In all types of mice, including germfree (GF) animals, the genes most affected by two-week oral JTX treatment were the type 1 interferon (IFN)-related genes including Stat1, Isgf3g and Irf7, which play a critical role in the feedback loop of IFN-alpha production cascade. In IQI specific pathogen free (SPF) mice JTX increased the steady state level of the expression of IFN-related genes, but had the opposite affect in IQI GF and BALB/c SPF mice. Promoter analysis suggests that tandem repeated $IRFF (the promoter sequences for interferon regulatory factors) may be a primary target for JTX action. Pre-treatment of JTX accelerated the effects of an oral IFN "inducer" 2-amino-5-bromo-6-methyl-4-pyrimidinol (ABMP) (up-regulation of IFN-alpha production in IQI strain and down-regulation in BALB/c mice), which is in good accordance with the effect of JTX on gene expression of type 1 IFN-related genes. CONCLUSIONS: Microarray analysis revealed that the target of JTX might be the transcription machinery regulating the steady-state level of genes involved in the ISGF3-IRF7 cascade, whose effect is bi-directional in a strain- and microbiota-dependent manner.

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Centromere-associated repeat arrays on Trypanosoma brucei chromosomes are much more extensive than predicted.

Publication Date: 2012 Jan 18 PMID: 22257693
Authors: Echeverry, M. C. - Bot, C. - Obado, S. O. - Taylor, M. C. - Kelly, J. M.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: African trypanosomes belong to a eukaryotic lineage which displays many unusual genetic features. The mechanisms of chromosome segregation in these diploid protozoan parasites are poorly understood. Centromeres in Trypanosoma brucei have been localised to chromosomal regions that contain an array of ~147 bp AT-rich tandem repeats. Initial estimates from the genome sequencing project suggested that these arrays ranged from 2 - 8 kb. In this paper, we show that the centromeric repeat regions are much more extensive. RESULTS: We used a long-range restriction endonuclease mapping approach to more accurately define the sizes of the centromeric repeat arrays on the 8 T. brucei chromosomes where unambiguous assembly data were available. The results indicate that the sizes of the arrays on different chromosomes vary from 20 to 120 kb. In addition, we found instances of length heterogeneity between chromosome homologues. For example, values of 20 and 65 kb were obtained for the arrays on chromosome 1, and 50 and 75 kb for chromosome 5. CONCLUSIONS: Our results show that centromeric repeat arrays on T. brucei chromosomes are more similar in size to those of higher eukaryotes than previously suspected. This information provides a firmer framework for investigating aspects of chromosome segregation and will allow epigenetic features associated with the process to be more accurately mapped.

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Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA.

Publication Date: 2012 Jan 18 PMID: 22257641
Authors: Mastrokolias, A. - den Dunnen, J. T. - van Ommen, G. B. - 't Hoen, P. A. - van Roon-Mom, W. M.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Transcriptome analysis is of great interest in clinical research, where significant differences between individuals can be translated into biomarkers of disease. Although next generation sequencing provides robust, comparable and highly informative expression profiling data, with several million of tags per blood sample, reticulocyte globin transcripts can constitute up to 76% of total mRNA compromising the detection of low abundant transcripts. We have removed globin transcripts from 6 human whole blood RNA samples with a human globin reduction kit and compared them with the same non-reduced samples using deep Serial Analysis of Gene Expression. RESULTS: Globin tags comprised 52-76% of total tags in our samples. Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples. In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples. Removing globin transcripts allowed us to also identify 2112 genes that could not be detected in the non-globin reduced samples, with roles in cell surface receptor signal transduction, G-protein coupled receptor protein signalling pathways and neurological processes. CONCLUSIONS: The reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

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Estimation of linkage disequilibrium in four US pig breeds.

Publication Date: 2012 PMID: 22252454
Authors: Badke, Y. M. - Bates, R. O. - Ernst, C. W. - Schwab, C. - Steibel, J. P.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The success of marker assisted selection depends on the amount of linkage disequilibrium (LD) across the genome. To implement marker assisted selection in the swine breeding industry, information about extent and degree of LD is essential. The objective of this study is to estimate LD in four US breeds of pigs (Duroc, Hampshire, Landrace, and Yorkshire) and subsequently calculate persistence of phase among them using a 60 k SNP panel. In addition, we report LD when using only a fraction of the available markers, to estimate persistence of LD over distance. RESULTS: Average r2 between adjacent SNP across all chromosomes was 0.36 for Landrace, 0.39 for Yorkshire, 0.44 for Hampshire and 0.46 for Duroc. For markers 1 Mb apart, r2 ranged from 0.15 for Landrace to 0.20 for Hampshire. Reducing the marker panel to 10% of its original density, average r2 ranged between 0.20 for Landrace to 0.25 for Duroc. We also estimated persistence of phase as a measure of prediction reliability of markers in one breed by those in another and found that markers less than 10 kb apart could be predicted with a maximal accuracy of 0.92 for Landrace with Yorkshire. CONCLUSIONS: Our estimates of LD, although in good agreement with previous reports, are more comprehensive and based on a larger panel of markers. Our estimates also confirmed earlier findings reporting higher LD in pigs than in American Holstein cattle, especially at increasing marker distances (> 1 Mb). High average LD (r2 > 0.4) between adjacent SNP found in this study is an important precursor for the implementation of marker assisted selection within a livestock species.Results of this study are relevant to the US purebred pig industry and critical for the design of programs of whole genome marker assisted evaluation and selection. In addition, results indicate that a more cost efficient implementation of marker assisted selection using low density panels with genotype imputation, would be feasible for these breeds.

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Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression.

Publication Date: 2012 Jan 17 PMID: 22251412
Authors: Vining, K. J. - Pomraning, K. R. - Wilhelm, L. J. - Priest, H. D. - Pellegrini, M. - Mockler, T. C. - Freitag, M. - Strauss, S.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. RESULTS: We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. CONCLUSIONS: We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

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Postmortem cardiac tissue maintains gene expression profile even after late harvesting.

Publication Date: 2012 Jan 17 PMID: 22251372
Authors: Gupta, S. - Halushka, M. K. - Hilton, G. M. - Arking, D. E.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Gene expression studies can be used to help identify disease-associated genes by comparing the levels of expressed transcripts between cases and controls, and to identify functional genetic variants (expression quantitative loci or eQTLs) by comparing expression levels between individuals with different genotypes. While many of these studies are performed in blood or lymphoblastoid cell lines due to tissue accessibility, the relevance of expression differences in tissues that are not the primary site of disease is unclear. Further, many eQTLs are tissue specific. Thus, there is a clear and compelling need to conduct gene expression studies in tissues that are specifically relevant to the disease of interest. One major technical concern about using autopsy-derived tissue is how representative it is of physiologic conditions, given the effect of postmortem interval on tissue degradation. RESULTS: In this study, we monitored the gene expression of 13 tissue samples harvested from a rapid autopsy heart (non-failed heart) and 7 from a cardiac explant (failed heart) through 24 hours of autolysis. The 24 hour autopsy simulation was designed to reflect a typical autopsy scenario where a body may begin cooling to ambient temperature for ~12 hours, before transportation and storage in a refrigerated room in a morgue. In addition, we also simulated a scenario wherein the body was left at room temperature for up to 24 hours before being found. A small fraction (<2.5%) of genes showed fluctuations in expression over the 24 hr period and largely belong to immune and signal response and energy metabolism-related processes. Global expression analysis suggests that RNA expression is reproducible over 24 hours of autolysis with 95% genes showing <1.2 fold change. Comparing the rapid autopsy to the failed heart identified 480 differentially expressed genes, including several types of collagens, lumican (LUM), natriuretic peptide A (NPPA) and connective tissue growth factor (CTGF), which allows for the clear separation between failing and non-failing heart based on gene expression profiles. CONCLUSIONS: Our results demonstrate that RNA from autopsy-derived tissue, even up to 24 hours of autolysis, can be used to identify biologically relevant expression pattern differences, thus serving as a practical source for gene expression experiments.

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The architecture and ppGpp-dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression.

Publication Date: 2012 Jan 17 PMID: 22251276
Authors: Ramachandran, V. K. - Shearer, N. - Jacob, J. J. - Sharma, C. M. - Thompson, A.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (STEX), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wild-type S. Typhimurium and a ppGpp null strain under growth conditions which model STEX. In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium STEX primary transcriptome than previously recognised. RESULTS: Here we report the precise mapping of transcriptional start sites (TSSs) for 78% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34% and 20% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment. CONCLUSIONS: The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research.

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Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes.

Publication Date: 2012 PMID: 22248082
Authors: Sharbati, S. - Sharbati, J. - Hoeke, L. - Bohmer, M. - Einspanier, R.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Small interfering and non-coding RNAs regulate gene expression across all kingdoms of life. MicroRNAs constitute an important group of metazoan small RNAs regulating development but also disease. Accordingly, in functional genomics microRNA expression analysis sheds more and more light on the dynamic regulation of gene expression in various cellular processes. RESULTS: We have developed custom RT-qPCR arrays allowing for accurate quantification of 31 small RNAs in triplicate using a 96 well format. In parallel, we provide accurate normalisation of microRNA expression data based on the quantification of 5 reference snRNAs. We have successfully employed such arrays to study microRNA regulation during human monocyte differentiation as well as Salmonella infection. Besides well-known protagonists such as miR-146 or miR-155, we identified the up-regulation of miR-21, miR-222, miR-23b, miR-24, miR-27a as well as miR-29 upon monocyte differentiation or infection, respectively. CONCLUSIONS: The provided protocol for RT-qPCR arrays enables straight-forward microRNA expression analysis. It is fully automatable, compliant with the MIQE guidelines and can be completed in only 1 day. The application of these arrays revealed microRNAs that may mediate monocyte host defence mechanisms by regulating the TGF-beta signalling upon Salmonella infection. The introduced arrays are furthermore suited for customised quantification of any class of small non-coding RNAs as exemplified by snRNAs and thus provide a versatile tool for ubiquitous applications.

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Genome-wide association scan and phased haplotype construction for quantitative trait loci affecting boar taint in three pig breeds.

Publication Date: 2012 Jan 13 PMID: 22244367
Authors: Gregersen, V. R. - Conley, L. N. - Sorensen, K. K. - Guldbrandtsen, B. - Velander, I. H. - Bendixen, C.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Boar taint is the undesirable smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone and skatole (3-methyl-indole). The steroid androstenone is a sex pheromone produced in the testis of the boars. Skatole is produced from tryptophan by bacteria in the intestine of the pigs. In many countries pigs are castrated as piglets to avoid boar taint, however, this is undesirable for animal welfare reasons. Genetic variations affecting the level of boar taint have previously been demonstrated in many breeds. In the study presented in this paper, markers and haplotypes, which can be applied to DNA-based selection schemes in order to reduce or eliminate the boar taint problem, are identified. RESULTS: Approximately 30,000 SNPs segregating in 923 boars from three Danish breeds; Duroc, Landrace, and Yorkshire, were used to conduct genome wide association studies of boar taint compounds. At 46 suggestive quantitative trait loci (QTL), 25 haplotypes and three single markers with effects were identified. Furthermore, 40% of the haplotypes mapped to previously identified regions. Haplotypes were also analysed for effects of slaughter weight and meat content. The most promising haplotype was identified on Sus scrofa chromosome 1. The gain in fixed effect of having this haplotype on level of androstenone in Landrace was identified to be high (1.279 micro gram/g). In addition, this haplotype explained 16.8% of the phenotypic variation within the trait. The haplotype was identified around the gene CYB5A which is known to have an indirect impact on the amount of androstenone. In addition to CYB5A, the genes SRD5A2, LOC100518755, and CYP21A2 are candidate genes for other haplotypes affecting androstenone, whereas, candidate genes for the indolic compounds were identified to be SULT1A1 and CYP2E1. CONCLUSIONS: Despite the small sample size, a total of 25 haplotypes and three single markers were identified including genomic regions not previously reported. The haplotypes that were analysed showed large effects on trait level. However, little overlap of QTL between breeds was observed.

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Characterisation of full-length cDNA sequences provides insights into the Eimeria tenella transcriptome.

Publication Date: 2012 Jan 13 PMID: 22244352
Authors: Amiruddin, N. - Lee, X. W. - Blake, D. P. - Suzuki, Y. - Tay, Y. L. - Lim, L. S. - Tomley, F. M. - Watanabe, J. - Sugimoto, C. - Wan, K. L.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Eimeria tenella is an apicomplexan parasite that causes coccidiosis in the domestic fowl. Infection with this parasite is diagnosed frequently in intensively reared poultry and its control is usually accorded a high priority, especially in chickens raised for meat. Prophylactic chemotherapy has been the primary method used for the control of coccidiosis. However, drug efficacy can be compromised by drug-resistant parasites and the lack of new drugs highlights demands for alternative control strategies including vaccination. In the long term, sustainable control of coccidiosis will most likely be achieved through integrated drug and vaccination programmes. Characterisation of the E. tenella transcriptome may provide a better understanding of the biology of the parasite and aid in the development of a more effective control for coccidiosis. RESULTS: More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs). Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR) analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis. CONCLUSIONS: This paper describes the generation and characterisation of full-length cDNA sequences from E. tenella second generation merozoites and provides new insights into the E. tenella transcriptome. The data generated will be useful for the development and validation of diagnostic and control strategies for coccidiosis and will be of value in annotation of the E. tenella genome sequence.

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Sequence based polymorphic (SBP) marker technology for targeted genomic regions: its application in generating a molecular map of the Arabidopsis thaliana genome.

Publication Date: 2012 Jan 13 PMID: 22244314
Authors: Sahu, B. B. - Sumit, R. - Srivastava, S. K. - Bhattacharyya, M. K.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Molecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions. Advancements in sequencing technology have made possible the identification of single nucleotide polymorphisms (SNPs) for any genomic regions. Here a sequence based polymorphic (SBP) marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described. RESULTS: A ~3X genome coverage sequence of the Arabidopsis thaliana ecotype, Niederzenz (Nd-0) was obtained by applying Illumina's sequencing by synthesis (Solexa) technology. Comparison of the Nd-0 genome sequence with the assembled Columbia-0 (Col-0) genome sequence identified putative single nucleotide polymorphisms (SNPs) throughout the entire genome. Multiple 75 base pair Nd-0 sequence reads containing SNPs and originating from individual genomic DNA molecules were the basis for developing co-dominant SBP markers. SNPs containing Col-0 sequences, supported by transcript sequences or sequences from multiple BAC clones, were compared to the respective Nd-0 sequences to identify possible restriction endonuclease enzyme site variations. Small amplicons, PCR amplified from both ecotypes, were digested with suitable restriction enzymes and resolved on a gel to reveal the sequence based polymorphisms. By applying this technology, 21 SBP markers for the marker poor regions of the Arabidopsis map representing polymorphisms between Col-0 and Nd-0 ecotypes were generated. CONCLUSIONS: The SBP marker technology described here allowed the development of molecular markers for targeted genomic regions of Arabidopsis. It should facilitate isolation of co-dominant molecular markers for targeted genomic regions of any animal or plant species, whose genomic sequences have been assembled. This technology will particularly facilitate the development of high density molecular marker maps, essential for cloning genes based on their genetic map positions and identifying tightly linked molecular markers for selecting desirable genotypes in animal and plant breeding experiments.

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Transcriptomic analysis of Chinese bayberry (Myrica rubra) fruit development and ripening using RNA-Seq.

Publication Date: 2012 Jan 13 PMID: 22244270
Authors: Feng, C. - Chen, M. - Xu, C. J. - Bai, L. - Yin, X. R. - Li, X. - Allan, A. C. - Ferguson, I. B. - Chen, K. S.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Chinese bayberry (Myrica rubra Sieb. and Zucc.) is an important subtropical fruit crop and an ideal species for fruit quality research due to the rapid and substantial changes that occur during development and ripening, including changes in fruit color and taste. However, research at the molecular level is limited by a lack of sequence data. The present study was designed to obtain transcript sequence data and examine gene expression in bayberry developing fruit based on RNA-Seq and bioinformatic analysis, to provide a foundation for understanding the molecular mechanisms controlling fruit quality changes during ripening. RESULTS: RNA-Seq generated 1.92 G raw data, which was then de novo assembled into 41,239 UniGenes with a mean length of 531 bp. Approximately 80% of the UniGenes (32,805) were annotated against public protein databases, and coding sequences (CDS) of 31,665 UniGenes were determined. Over 3,600 UniGenes were differentially expressed during fruit ripening, with 826 up-regulated and 1,407 down-regulated. GO comparisons between the UniGenes of these two types and interactive pathways (Ipath) analysis found that energy-related metabolism was enhanced, and catalytic activity was increased. All genes involved in anthocyanin biosynthesis were up-regulated during the fruit ripening processes, concurrent with color change. Important changes in carbohydrate and acid metabolism in the ripening fruit are likely associated with expression of sucrose phosphate synthase (SPS) and glutamate decarboxylase (GAD). CONCLUSIONS: Mass sequence data of Chinese bayberry was obtained and the expression profiles were examined during fruit ripening. The UniGenes were annotated, providing a platform for functional genomic research with this species. Using pathway mapping and expression profiles, the molecular mechanisms for changes in fruit color and taste during ripening were examined. This provides a reference for the study of complicated metabolism in non-model perennial species.

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Rapid gene-based SNP and haplotype marker development in non-model eukaryotes using 3'UTR sequencing.

Publication Date: 2012 Jan 12 PMID: 22239826
Authors: Koepke, T. - Schaeffer, S. - Krishnan, V. - Jiwan, D. - Harper, A. - Whiting, M. - Oraguzie, N. - Dhingra, A.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Sweet cherry (Prunus avium L.), a non-model crop with narrow genetic diversity, is an important member of sub-family Amygdoloideae within Rosaceae. Compared to other important members like peach and apple, sweet cherry lacks in genetic and genomic information, impeding understanding of important biological processes and development of efficient breeding approaches. Availability of single nucleotide polymorphism (SNP)-based molecular markers can greatly benefit breeding efforts in such non-model species. RNA-seq approaches employing second generation sequencing platforms offer a unique avenue to rapidly identify gene-based SNPs. Additionally, haplotype markers can be rapidly generated from transcript-based SNPs since they have been found to be extremely utile in identification of genetic variants related to health, disease and response to environment as highlighted by the human HapMap project. RESULTS: RNA-seq was performed on two sweet cherry cultivars, Bing and Rainier using a 3' untranslated region (UTR) sequencing method yielding 43,396 assembled contigs. In order to test our approach of rapid identification of SNPs without any reference genome information, over 25% (10,100) of the contigs were screened for the SNPs. A total of 207 contigs from this set were identified to contain high quality SNPs. A set of 223 primer pairs were designed to amplify SNP containing regions from these contigs and high resolution melting (HRM) analysis was performed with eight important parental sweet cherry cultivars. Six of the parent cultivars were distantly related to Bing and Rainier, the cultivars used for initial SNP discovery. Further, HRM analysis was also performed on 13 seedlings derived from a cross between two of the parents. Our analysis resulted in the identification of 84 (38.7%) primer sets that demonstrated variation among the tested germplasm. Reassembly of the raw 3'UTR sequences using upgraded transcriptome assembly software yielded 34,620 contigs containing 2243 putative SNPs in 887 contigs after stringent filtering. Contigs with multiple SNPs were visually parsed to identify 685 putative haplotypes at 335 loci in 301 contigs. CONCLUSIONS: This approach, which leverages the advantages of RNA-seq approaches, enabled rapid generation of gene-linked SNP and haplotype markers. The general approach presented in this study can be easily applied to other non-model eukaryotes irrespective of the ploidy level to identify gene-linked polymorphisms that are expected to facilitate efficient Gene Assisted Breeding (GAB), genotyping and population genetics studies. The identified SNP haplotypes reveal some of the allelic differences in the two sweet cherry cultivars analyzed. The identification of these SNP and haplotype markers is expected to significantly improve the genomic resources for sweet cherry and facilitate efficient GAB in this non-model crop.

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Lipopolysaccharide priming enhances expression of effectors of immune defence while decreasing expression of pro-inflammatory cytokines in mammary epithelia cells from cows.

Publication Date: 2012 Jan 12 PMID: 22235868
Authors: Gunther, J. - Petzl, W. - Zerbe, H. - Schuberth, H. J. - Koczan, D. - Goetze, L. - Seyfert, H. M.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Udder infections with environmental pathogens like Escherichia coli are a serious problem for the dairy industry. Reduction of incidence and severity of mastitis is desirable and mild priming of the immune system either through vaccination or with low doses of immune stimulants such as lipopolysaccharide LPS was previously found to dampen detrimental effects of a subsequent infection. Monocytes / macrophages are known to develop tolerance towards the endotoxin LPS (endotoxin tolerance, ET) as adaptation strategy to prevent exuberant inflammation. We have recently observed that infusion of 1 ug of LPS into the quarter of an udder effectively protected for several days against an experimentally elicited mastitis. We have modelled this process in primary cultures of mammary epithelial cells (MEC) from the cow. MEC are by far the most abundant cells in the healthy udder coming into contact with invading pathogens and little is known about their role in establishing ET. RESULTS: We primed primary MEC cultures for 12 h with LPS (100 ng/ml) and stimulated three cultures either 12 h or 42 h later with 10E7/ml particles of heat inactivated E. coli bacteria for six hours. Priming-related alterations in the global transcriptome of those cells were quantified with Affymetrix microarrays. LPS priming alone caused differential expression of 40 genes and mediated significantly different response to a subsequent E. coli challenge of 226 genes. Expression of 38 genes was enhanced while that of 188 was decreased. Higher expressed were anti-microbial factors (beta-defensin LAP, SLPI), cell and tissue protecting factors (DAF, MUC1, TGM1, TGM3) as well as mediators of the sentinel function of MEC (CCL5, CXCL8). Dampened was the expression of potentially harmful pro-inflammatory master cytokines (IL1B, IL6, TNF-alpha and immune effectors (NOS2, matrix metalloproteases). Functional network analysis highlighted the reduced expression of IL1B and of IRF7 as key to this modulation. CONCLUSION: LPS-primed MEC are fitter to repel pathogens and better protected against misguided attacks of the immune response. Attenuated is the exuberant expression of factors potentially promoting immunopathological processes. MEC therefore recapitulate many aspects of ET known so far from professional immune cells.

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Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples.

Publication Date: 2012 Jan 11 PMID: 22235840
Authors: Mullen, M. P. - Creevey, C. J. - Berry, D. P. - McCabe, M. S. - Magee, D. A. - Howard, D. J. - Killeen, A. P. - Park, S. D. - McGettigan, P. A. - Lucy, M. C. - Machugh, D. E. - Waters, S. M.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. RESULTS: In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9 % (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom(R) MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). CONCLUSIONS: The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.

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A searchable cross-platform gene expression database reveals connections between drug treatments and disease.

Publication Date: 2012 Jan 10 PMID: 22233519
Authors: Williams, G.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Transcriptional data covering multiple platforms and species is collected and processed into a searchable platform independent expression database (SPIED). SPIED consists of over 100,000 expression fold profiles defined independently of control/treatment assignment and mapped to non-redundant gene lists. The database is thus searchable with query profiles defined over genes alone. The motivation behind SPIED is that transcriptional profiles can be quantitatively compared and ranked and thus serve as effective surrogates for comparing the underlying biological states across multiple experiments. RESULTS: Drug perturbation, cancer and neurodegenerative disease derived transcriptional profiles are shown to be effective descriptors of the underlying biology as they return related drugs and pathologies from SPIED. In the case of Alzheimer's disease there is high transcriptional overlap with other neurodegenerative conditions and rodent models of neurodegeneration and nerve injury. Combining the query signature with correlating profiles allows for the definition of a tight neurodegeneration signature that successfully highlights many neuroprotective drugs in the Broad connectivity map. CONCLUSIONS: Quantitative querying of expression data from across the totality of deposited experiments is an effective way of discovering connections between different biological systems and in particular that between drug action and biological disease state. Examples in cancer and neurodegenerative conditions validate the utility of SPIED.

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Differential expression patterns of conserved miRNAs and isomiRs during Atlantic halibut development.

Publication Date: 2012 Jan 10 PMID: 22233483
Authors: Bizuayehu, T. T. - Lanes, C. F. - Furmanek, T. - Karlsen, B. O. - Fernandes, J. M. - Johansen, S. D. - Babiak, I.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) play a major role in animal ontogenesis. Size variants of miRNAs, isomiRs, are observed along with the main miRNA types, but their origin and possible biological role are uncovered yet. Developmental profiles of miRNAs have been reported in few fish species only and, to our knowledge, differential expressions of isomiRs have not yet been shown during fish development. Atlantic halibut, Hippoglossus hippoglossus L., undergoes dramatic metamorphosis during early development from symmetrical pelagic larval stage to unsymmetrical flatfish. No data exist on role of miRNAs in halibut metamorphosis. RESULTS: miRNA profiling using SOLiD deep sequencing technology revealed a total of 199 conserved, one novel antisense, and one miRNA* mature form. Digital expression profiles of selected miRNAs were validated using reverse transcription quantitative PCR. We found developmental transition-specific miRNA expression. Expression of some miRNA* exceeded the guide strand miRNA. We revealed that nucleotide truncations and/or additions at the 3' end of mature miRNAs resulted in size variants showing differential expression patterns during the development in a number of miRNA families. We confirmed the presence of isomiRs by cloning and Sanger sequencing. Also, we found inverse relationship between expression levels of sense/antisense miRNAs during halibut development. CONCLUSION: Developmental transitions during early development of Atlantic halibut are associated with expression of certain miRNA types. IsomiRs are abundant and often show differential expression during the development.

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Comprehensive transcriptome analysis reveals novel genes involved in cardiac glycoside biosynthesis and mlncRNAs associated with secondary metabolism and stress response in Digitalis purpurea.

Publication Date: 2012 PMID: 22233149
Authors: Wu, B. - Li, Y. - Yan, H. - Ma, Y. - Luo, H. - Yuan, L. - Chen, S. - Lu, S.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Digitalis purpurea is an important ornamental and medicinal plant. There is considerable interest in exploring its transcriptome. RESULTS: Through high-throughput 454 sequencing and subsequent assembly, we obtained 23532 genes, of which 15626 encode conserved proteins. We determined 140 unigenes to be candidates involved in cardiac glycoside biosynthesis. It could be grouped into 30 families, of which 29 were identified for the first time in D. purpurea. We identified 2660 mRNA-like npcRNA (mlncRNA) candidates, an emerging class of regulators, using a computational mlncRNA identification pipeline and 13 microRNA-producing unigenes based on sequence conservation and hairpin structure-forming capability. Twenty five protein-coding unigenes were predicted to be targets of these microRNAs. Among the mlncRNA candidates, only 320 could be grouped into 140 families with at least two members in a family. The majority of D. purpurea mlncRNAs were species-specific and many of them showed tissue-specific expression and responded to cold and dehydration stresses. We identified 417 protein-coding genes with regions significantly homologous or complementary to 375 mlncRNAs. It includes five genes involved in secondary metabolism. A positive correlation was found in gene expression between protein-coding genes and the homologous mlncRNAs in response to cold and dehydration stresses, while the correlation was negative when protein-coding genes and mlncRNAs were complementary to each other. CONCLUSIONS: Through comprehensive transcriptome analysis, we not only identified 29 novel gene families potentially involved in the biosynthesis of cardiac glycosides but also characterized a large number of mlncRNAs. Our results suggest the importance of mlncRNAs in secondary metabolism and stress response in D. purpurea.

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Gene fragmentation in bacterial draft genomes: extent, consequences and mitigation.

Publication Date: 2012 Jan 10 PMID: 22233127
Authors: Klassen, J. L. - Currie, C. R.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Ongoing technological advances in genome sequencing are allowing bacterial genomes to be sequenced at ever-lower cost. However, nearly all of these new techniques concomitantly decrease genome quality, primarily due to the inability of their relatively short read lengths to bridge certain genomic regions, e.g., those containing repeats. Fragmentation of predicted open reading frame (ORFs) is one possible consequence of this decreased quality. In this study we quantify ORF fragmentation in draft microbial genomes and its effect on annotation efficacy, and we propose a solution to ameliorate this problem. RESULTS: A survey of draft-quality genomes in GenBank revealed that fragmented ORFs comprised > 80% of the predicted ORFs in some genomes, and that increased fragmentation correlated with decreased genome assembly quality. In a more thorough analysis of 25 Streptomyces genomes, fragmentation was especially enriched in some protein classes with repeating, multi-modular structures such as polyketide synthases, non-ribosomal peptide synthetases and serine/threonine kinases. Overall, increased genome fragmentation correlated with increased false-negative Pfam and COG annotation rates and increased false-positive KEGG annotation rates. The false-positive KEGG annotation rate could be ameliorated by linking fragmented ORFs using their orthologs in related genomes. Whereas this strategy successfully linked up to 46% of the total ORF fragments in some genomes, its sensitivity appeared to depend heavily on the depth of sampling of a particular taxon's variable genome. CONCLUSIONS: Draft microbial genomes contain many ORF fragments. Where these correspond to the same gene they have particular potential to confound comparative gene content analyses. Given our findings, and the rapid increase in the number of microbial draft quality genomes, we suggest that accounting for gene fragmentation and its associated biases is important when designing comparative genomic projects.

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SNP mining in C. clementina BAC end sequences; transferability in the Citrus genus (Rutaceae), phylogenetic inferences and perspectives for genetic mapping.

Publication Date: 2012 Jan 10 PMID: 22233093
Authors: Ollitrault, P. - Terol, J. - Garcia-Lor, A. - Berard, A. - Chauveau, A. - Froelicher, Y. - Belzile, C. - Morillon, R. - Navarro, L. - Brunel, D. - Talon, M.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: With the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers. However, SNP chip data generally suffers from ascertainment biases caused by the SNP discovery and selection process in which a small number of individuals are used as discovery panels. The ongoing International Citrus Genome Consortium sequencing project of the highly heterozygous Clementine and sweet orange genomes will soon result in the release of several hundred thousand SNPs. The primary goals of this study were: (i) to estimate the transferability within the genus Citrus of SNPs discovered from Clementine BACend sequencing (BES), (ii) to estimate bias associated with the very narrow discovery panel, and (iii) to evaluate the usefulness of the Clementine-derived SNP markers for diversity analysis and comparative mapping studies between the different cultivated Citrus species. RESULTS: Fifty-four accessions covering the main Citrus species and 52 interspecific hybrids between pummelo and Clementine were genotyped on a GoldenGate array platform using 1,457 SNPs mined from Clementine BES and 37 SNPs identified between and within C. maxima, C. medica, C. reticulata and C. micrantha. Consistent results were obtained from 622 SNP loci. Of these markers, 116 displayed incomplete transferability primarily in C. medica, C. maxima and wild Citrus species. The two primary biases associated with the SNP mining in Clementine were an overestimation of the C. reticulata diversity and an underestimation of the interspecific differentiation. However, the genetic stratification of the gene pool was high, with very frequent significant linkage disequilibrium. Furthermore, the shared intraspecific polymorphism and accession heterozygosity were generally enough to perform interspecific comparative genetic mapping. CONCLUSIONS: A set of 622 SNP markers providing consistent results was selected. Of the markers mined from Clementine, 80.5% were successfully transferred to the whole Citrus gene pool. Despite the ascertainment biases in relation to the Clementine origin, the SNP data confirm the important stratification of the gene pools around C. maxima, C. medica and C. reticulata as well as previous hypothesis on the origin of secondary species. The implemented SNP marker set will be very useful for comparative genetic mapping in Citrus and genetic association in C. reticulata.

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Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III effector HopZ2.

Publication Date: 2012 Jan 9 PMID: 22230763
Authors: Lewis, J. D. - Wan, J. - Ford, R. - Gong, Y. - Fung, P. - Nahal, H. - Wang, P. W. - Desveaux, D. - Guttman, D. S.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Identification of protein-protein interactions is a fundamental aspect of understanding protein function. A commonly used method for identifying protein interactions is the yeast two-hybrid system. RESULTS: Here we describe the application of next-generation sequencing to yeast two-hybrid interaction screens and develop Quantitative Interactor Screen Sequencing (QIS-Seq). QIS-Seq provides a quantitative measurement of enrichment for each interactor relative to its frequency in the library as well as its general stickiness (non-specific binding). The QIS-Seq approach is scalable and can be used with any yeast two-hybrid screen and with any next-generation sequencing platform. The quantitative nature of QIS-Seq data make it amenable to statistical evaluation, and importantly, facilitates the standardization of experimental design, data collection, and data analysis. We applied QIS-Seq to identify the Arabidopsis thaliana MLO2 protein as a target of the Pseudomonas syringae type III secreted effector protein HopZ2. We validate the interaction between HopZ2 and MLO2 in planta and show that the interaction is required for HopZ2-associated virulence. CONCLUSIONS: We demonstrate that QIS-Seq is a high-throughput quantitative interactor screen and validate MLO2 as an interactor and novel virulence target of the P. syringae type III secreted effector HopZ2.

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Sculpting the maturation, softening and ethylene pathway: The influences of microRNAs on tomato fruits.

Publication Date: 2012 PMID: 22230737
Authors: Zuo, J. - Zhu, B. - Fu, D. - Zhu, Y. - Ma, Y. - Chi, L. - Ju, Z. - Wang, Y. - Zhai, B. - Luo, Y.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs), a ubiquitous class of short RNAs, play vital roles in physiological and biochemical processes in plants by mediating gene silencing at post-transcriptional (PTGS) level. Tomato is a model system to study molecular basis of fleshy fruit ripening and senescence, ethylene biosynthesis and signal transduction owing to its genetic and molecular tractability. To study the functions of miRNAs in tomato fruit ripening and senescence, and their possible roles in ethylene response, the next generation sequencing method was employed to identify miRNAs in tomato fruit. Bioinformatics and molecular biology approaches were combined to profile the miRNAs expression patterns at three different fruit ripening stages and by exogenous ethylene treatment. RESULTS: In addition to 7 novel miRNA families, 103 conserved miRNAs belonging to 24 families and 10 non-conserved miRNAs matching 9 families were identified in our libraries. The targets of many these miRNAs were predicted to be transcriptional factors. Other targets are known to play roles in the regulation of metabolic processes. Interestingly, some targets were predicted to be involved in fruit ripening and softening, such as Pectate Lyase, beta-galactosidase, while a few others were predicted to be involved in ethylene biosynthesis and signaling pathway, such as ACS, EIN2 and CTR1. The expression patterns of a number of such miRNAs at three ripening stages were confirmed by stem-loop RT-PCR, which showed a strong negative correlation with that of their targets. The regulation of exogenous ethylene on miRNAs expression profiles were analyzed simultaneously, and 3 down-regulated, 5 up-regulated miRNAs were found in this study. CONCLUSIONS: A combination of high throughput sequencing and molecular biology approaches was used to explore the involvement of miRNAs during fruit ripening. Several miRNAs showed differential expression profiles during fruit ripening, and a number of miRNAs were influenced by ethylene treatment. The results suggest the importance of miRNAs in fruit ripening and ethylene response.

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Transcriptome changes during fruit development and ripening of sweet orange (Citrus sinensis).

Publication Date: 2012 PMID: 22230690
Authors: Yu, K. - Xu, Q. - Da, X. - Guo, F. - Ding, Y. - Deng, X.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: The transcriptome of the fruit pulp of the sweet orange variety Anliu (WT) and that of its red fleshed mutant Hong Anliu (MT) were compared to understand the dynamics and differential expression of genes expressed during fruit development and ripening. RESULTS: The transcriptomes of WT and MT were sampled at four developmental stages using an Illumina sequencing platform. A total of 19,440 and 18,829 genes were detected in MT and WT, respectively. Hierarchical clustering analysis revealed 24 expression patterns for the set of all genes detected, of which 20 were in common between MT and WT. Over 89% of the genes showed differential expression during fruit development and ripening in the WT. Functional categorization of the differentially expressed genes revealed that cell wall biosynthesis, carbohydrate and citric acid metabolism, carotenoid metabolism, and the response to stress were the most differentially regulated processes occurring during fruit development and ripening. CONCLUSION: A description of the transcriptomic changes occurring during fruit development and ripening was obtained in sweet orange, along with a dynamic view of the gene expression differences between the wild type and a red fleshed mutant.

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Rootstock-regulated gene expression patterns associated with fire blight resistance in apple.

Publication Date: 2012 Jan 9 PMID: 22229964
Authors: Jensen, P. J. - Halbrendt, N. - Fazio, G. - Makalowska, I. - Altman, N. - Praul, C. - Maximova, S. N. - Ngugi, H. K. - Crassweller, R. M. - Travis, J. W. - McNellis, T. W.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Desirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response. RESULTS: Rootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchard-grown apple trees, consisting of 'Gala' scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. 'Gala' scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while 'Gala' on M.27 and B.9 showed the highest rates of necrosis. 'Gala' scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown 'Gala' scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population. CONCLUSIONS: Rootstocks had significant effects on the fire blight susceptibility of 'Gala' scions, and rootstock-regulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.

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RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug.

Publication Date: 2012 Jan 6 PMID: 22226239
Authors: Mamidala, P. - Wijeratne, A. J. - Wijeratne, S. - Kornacker, K. - Sudhamalla, B. - Rivera-Vega, L. J. - Hoelmer, A. - Meulia, T. - Jones, S. C. - Mittapalli, O.
Journal: BMC Genomics

ABSTRACT: BACKGROUND: Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance. RESULTS: We performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid. CONCLUSIONS: We developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in C. lectularius.

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