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Journal: Functional & integrative genomics

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Since the first two complete bacterial genome sequences were published in 1995, the science of bacteria has dramatically changed. Using third-generation DNA sequencing, it is possible to completely sequence a bacterial genome in a few hours and identify some types of methylation sites along the genome as well. Sequencing of bacterial genome sequences is now a standard procedure, and the information from tens of thousands of bacterial genomes has had a major impact on our views of the bacterial world. In this review, we explore a series of questions to highlight some insights that comparative genomics has produced. To date, there are genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. However, the distribution is quite skewed towards a few phyla that contain model organisms. But the breadth is continuing to improve, with projects dedicated to filling in less characterized taxonomic groups. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides bacteria with immunity against viruses, which outnumber bacteria by tenfold. How fast can we go? Second-generation sequencing has produced a large number of draft genomes (close to 90 % of bacterial genomes in GenBank are currently not complete); third-generation sequencing can potentially produce a finished genome in a few hours, and at the same time provide methlylation sites along the entire chromosome. The diversity of bacterial communities is extensive as is evident from the genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. Genome sequencing can help in classifying an organism, and in the case where multiple genomes of the same species are available, it is possible to calculate the pan- and core genomes; comparison of more than 2000 Escherichia coli genomes finds an E. coli core genome of about 3100 gene families and a total of about 89,000 different gene families. Why do we care about bacterial genome sequencing? There are many practical applications, such as genome-scale metabolic modeling, biosurveillance, bioforensics, and infectious disease epidemiology. In the near future, high-throughput sequencing of patient metagenomic samples could revolutionize medicine in terms of speed and accuracy of finding pathogens and knowing how to treat them.

Concepts: DNA, Gene, Genetics, Bacteria, Organism, Virus, Genome, Escherichia coli

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Next-generation DNA sequencing technologies, such as RNA-Seq, currently dominate genome-wide gene expression studies. A standard approach to analyse this data requires mapping sequence reads to a reference and counting the number of reads which map to each gene. However, for many transcriptome studies, a suitable reference genome is unavailable, especially for meta-transcriptome studies which assay gene expression from mixed populations of organisms. Where a reference is unavailable, it is possible to generate a reference by the de novo assembly of the sequence reads. However, the high cost of generating high-coverage data for de novo assembly hinders this approach and more importantly the accurate assembly of such data is challenging, especially for meta-transcriptome data, and resulting assemblies frequently suffer from collapsed regions or chimeric sequences. As an alternative to the standard reference mapping approach, we have developed a k-mer-based analysis pipeline (DiffKAP) to identify differentially expressed reads between RNA-Seq datasets without the requirement for a reference. We compared the DiffKAP approach with the traditional Tophat/Cuffdiff method using RNA-Seq data from soybean, which has a suitable reference genome. We subsequently examined differential gene expression for a coral meta-transcriptome where no reference is available, and validated the results using qRT-PCR. We conclude that DiffKAP is an accurate method to study differential gene expression in complex meta-transcriptomes without the requirement of a reference genome.

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Although water buffaloes are the main milk-producing animals in Indian subcontinent, only limited attempts have been made to identify canonical pathways and gene regulatory networks operating within the mammary glands of these animals. Such information is important for identifying unique transcriptome signatures in the mammary glands of diseased animals. In this report, we analyzed the transcription profile of 3 prepubertal buffalo mammary glands and identified common genes (mean FPKM > 0.2 in all samples) operating in the glands. Among 19,994 protein coding genes, 14,678 genes expressed and 5316 unique genes did not express in prepubertal buffalo mammary glands. Of these 14,678 expressed genes, 79% comprised a ubiquitous transcriptome that was dominated by very lowly expressed genes (51%). The percentage of rarely, moderately, and abundantly expressed genes was 25%, 2%, and 1%, respectively. Gene Ontology (GO) terms reflected in the expression of common genes (mean FPKM > 5.0) for molecular function were related to binding and catalytic activity. Products of these genes were involved in metabolic and cellular processes and belong to nucleic acid binding proteins. The canonical pathways for growth of mammary glands included integrin signaling, inflammation, GnRH and Wnt pathways. KEGG enriched pathways revealed many pathways of cancer including ribosome, splisosome, endocytosis, and ubiquitin-mediated proteolysis, pathways for viral infection, and bacterial invasion of epithelial. Highly expressed genes (mean FPKM > 500 included beta-actin (ACTB), beta-2 microglobulin (B2M), caseins (CSN2, CNS3), collagens (COL1A1, COL3A1), translation elongation factors (EEF1A1, EEF1G, EEF2), keratins (KRT15, KRT19), major histocompatibility complex genes (CD74, JSP.1), vimentin (VIM), and osteopontin (SPP1). Interestingly, expression of milk protein genes in prepubertal glands opens possible roles of these genes in development of mammary glands. We report the whole transcriptomic signature of prepubertal buffalo mammary gland and indicated its molecular signature is similar to cancer type.

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Terminal heat stress has detrimental effect on the growth and yield of wheat. Very limited information is available on heat stress-associated active proteins (SAAPs) in wheat. Here, we have identified 159 protein groups with 4271 SAAPs in control (22 ± 3 °C) and HS-treated (38 °C, 2 h) wheat cvs. HD2985 and HD2329 using iTRAQ. We identified 3600 proteins to be upregulated and 5825 proteins to be downregulated in both the wheat cvs. under HS. We observed 60.3% of the common SAAPs showing upregulation in HD2985 (thermotolerant) and downregulation in HD2329 (thermosusceptible) under HS. GO analysis showed proton transport (molecular), photosynthesis (biological), and ATP binding (cellular) to be most altered under HS. Most of the SAAPs identified were observed to be chloroplast localized and involved in photosynthesis. Carboxylase enzyme was observed most abundant active enzymes in wheat under HS. An increase in the degradative isoenzymes (α/β-amylases) was observed, as compared to biosynthesis enzymes (ADP-glucophosphorylase, soluble starch synthase, etc.) under HS. Transcript profiling showed very high relative fold expression of HSP17, CDPK, Cu/Zn SOD, whereas downregulation of AGPase, SSS under HS. The identified SAAPs can be used for targeted protein-based precision wheat-breeding program for the development of ‘climate-smart’ wheat.

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Water temperature affects the survival, growth, immunity, reproduction, and productivity of farmed fish. The temperature beyond suitable range will disrupt the normal physiological activity. Common carp (Cyprinus carpio L.) is a representative eurythermic fish; they are able to sense and respond to changes in water temperature by adjusting their physiology. To investigate the miRNAs in common carp at different temperatures, nine liver small-RNA libraries (5 °C, 17 °C, and 30 °C, each group have three biological repetitions) were constructed and sequenced using high-throughput sequencing. A total of 110 known miRNAs were identified. Twenty-nine known miRNAs were differentially expressed compared with in control group. GO and KEGG analysis indicated that the miRNAs may play important roles in metabolism and environment information processing. Specifically, we considered the insulin-signaling and glycerophospholipid metabolism pathway, and the results show that in 30 °C, miR-301a, miR-203b-5p, and miR-210-3p were upregulated; their target genes which are the mechanistic targets of the rapamycin kinase (mtor) gene and the protein kinase AMP-activated catalytic subunit alpha 1 (prkaa1) gene in the insulin-signaling pathway were downregulated. And miR-9-5p, miR-27d, miR-92b-3p, and miR-155 were upregulated; their target genes, 1-acylglycerol-3-phosphate O-acyltransferase 3 (agpat3), CDP-diacylglycerol-inositol 3-phosphatidyltransferase (cdipt), glycerol-3-phosphate acyltransferase mitochondrial (gpam), and phosphatidylglycerophosphate synthase 1 (pgs1), in glycerophospholipid metabolism pathway were downregulated. But in 5 °C, the situation was opposite. These findings suggest that significant changes occur in energy metabolism and metabolic processes with components of the cell membrane in different temperatures, which significantly advance our understanding of the regulatory mechanisms underlying the physiological change of temperature stress-induced in liver, specifically with regard to miRNAs. These data provide a foundation for further studies of the role of miRNAs in environmental adaptation in fish.

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Wheat, one of the most broadly cultivated and consumed food crops worldwide, can accumulate high Cd contents in their edible parts, which poses a major hazard to human health. Cd accumulation ability differs among varieties in wheat, but the underlying molecular mechanism is largely unknown. Here, key genes responsible for Cd accumulation between two contrasting wheat genotypes (low-Cd accumulation one L17, high-Cd accumulation one H17) were investigated. Total 1269 were differentially expressed genes (DEGs) in L17 after Cd treatment, whereas, 399 Cd-induced DEGs were found in H17. GO-GO network analysis showed that heme binding was the most active GO, and metal binding was the second one that associated with other GOs in response to Cd stress in both genotypes. Pathway-pathway network analysis showed that phenylpronanoid biosynthesis and glutathione metabolism were the top pathways in response to Cd stress in both genotypes. Furthermore, we found that DEGs related to ion binding, antioxidant defense mechanisms, sulfotransferase activity, and cysteine biosynthetic process were more enriched in L17. In conclusion, our results not only provide the foundation for further exploring the molecular mechanism of Cd accumulation in wheat but also supply new strategies for improving phytoremediation ability of wheat by genetic engineering.

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Wheat can adapt to most agricultural conditions across temperate regions. This success is the result of phenotypic plasticity conferred by a large and complex genome composed of three homoeologous genomes (A, B, and D). Although drought is a major cause of yield and quality loss in wheat, the adaptive mechanisms and gene networks underlying drought responses in the field remain largely unknown. Here, we addressed this by utilizing an interdisciplinary approach involving field water status phenotyping, sampling, and gene expression analyses. Overall, changes at the transcriptional level were reflected in plant spectral traits amenable to field-level physiological measurements, although changes in photosynthesis-related pathways were found likely to be under more complex post-transcriptional control. Examining homoeologous genes with a 1:1:1 relationship across the A, B, and D genomes (triads), we revealed a complex genomic architecture for drought responses under field conditions, involving gene homoeolog specialization, multiple gene clusters, gene families, miRNAs, and transcription factors coordinating these responses. Our results provide a new focus for genomics-assisted breeding of drought-tolerant wheat cultivars.

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Increasing data demonstrate that circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) play important roles in tumorigenesis. However, the mechanisms in colorectal cancer (CRC) remain unclear. Here, hundreds of significantly expressed circRNAs, and thousands of lncRNAs as well as mRNAs were identified. By qRT-PCR, one abnormal circRNA, lncRNA, and three mRNAs were verified in 24 pairs of tissues and blood samples, respectively. Then, by GO analysis, we found that the gene expression profile of linear counterparts of upregulated circRNAs in human CRC tissues preferred positive regulation of GTPase activity, cellular protein metabolic process, and protein binding, while that of downregulated circRNAs of CRC preferred positive regulation of cellular metabolic process, acetyl-CoA metabolic process, and protein kinase C activity. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that p53 signaling pathway was an important pathway in upregulated protein-coding genes, whereas cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathway was the top enriched KEGG pathway for downregulated transcripts. Furthermore, lncRNA-mRNA co-expression analysis demonstrated that downregulated lncRNA uc001tma.3 was negatively with CDC45 and positively with ELOVL4, BVES, FLNA, and HSPB8, while upregulated lncRNA NR_110882 was positively with FZD2. In addition, lncRNA-transcription factor (TF) co-expression analysis showed that the most relevant TFs were forkhead box protein A1 (FOXA1), transcription initiation factor TFIID submint 7 (TAF7), and adenovirus early region 1A(E1A)-associated protein p300 (EP300). Our findings offer a fresh view on circRNAs and lncRNAs and provide the foundation for further study on the potential roles of circRNAs and lncRNAs in colorectal cancer.

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Transcription factors (TFs) fine-tune the host defense transcriptome in response to pathogen invasions. No information is available on Zingiber zerumbet (Zz) TFs involved in defense response against Pythium myriotylum. Here, we provide a global identification, characterization, and temporal expression profiling of Zz TFs following an incompatible interaction with P. myriotylum using a transcriptome sequencing approach. We identified a total of 903 TFs belonging to 96 families based on their conserved domains. Evolutionary analysis clustered the Zz TFs according to their phylogenetic affinity, providing glimpses of their functional diversities. High throughput expression array analysis highlighted a complex interplay between activating and repressing transcription factors in fine-tuning Zz defense response against P. myriotylum. The high differential modulation of TFs involved in cell wall fortification, lignin biosynthesis, and SA/JA hormone crosstalk allows us to envisage that this mechanism plays a central role in restricting P. myriotylum proliferation in Zz. This study lays a solid foundation and provides valuable resources for the investigation of the evolutionary history and biological functions of Zz TF genes involved in defense response.

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Nitrogen is an essential macronutrient for plant growth and reproduction. In durum wheat, an appropriate nitrogen soil availability is essential for an optimal seed development. miRNAs contribute to the environmental change adaptation of plants through the regulation of important genes involved in stress processes. In this work, nitrogen stress response was evaluated in durum wheat seedlings of Ciccio and Svevo cultivars. Eight small RNA libraries from leaves and roots of chronically stressed plants were sequenced to detect conserved and novel miRNAs. A total of 294 miRNAs were identified, 7 of which were described here for the first time. The expression level of selected miRNAs and target genes was analyzed by qPCR in seedlings subjected to chronic (Ciccio and Svevo, leaves and roots) or short-term (Svevo roots) stress conditions. Some miRNAs showed an immediate stress response, and their level of expression was either maintained or returned to a basal level during a long-term stress. Other miRNAs showed a gradual up- or downregulation during the short-term stress. The newly identified miRNA ttu-novel-106 showed an immediate strongly downregulation after nitrogen stress, which was negatively correlated with the expression of MYB-A, its putative target gene. PHO2 gene was significantly upregulated after 24-48-h stress, corresponding to a downregulation of miR399b. Ttu-miR399b putative binding sites in the 5' UTR region of the Svevo PHO2 gene were identified in the A and B genomes. Both MYB-A and PHO2 genes were validated for their cleavage site using 5' RACE assay.