Hemorrhagic disease of the grass carp, Ctenopharyngodon idella, is a fatal disease in fingerlings and yearlings caused by a reovirus, GCRV. RNA-seq data from four diseased grass carp tissues (gill, intestine, liver and spleen) were obtained at 2h before and six times after (2h, 24h, 48h, 72h, 96h and 120h) GCRV challenge. A total of 7.25=C2=B10.18 million (M) clean reads and 3.53=C2=B10.37M unique reads were obtained per RNA-seq analysis. Compared with controls, there were 9060 unique differentially expressed genes (DEGs) in the four tissues at the six time points post-GCRV challenge. Hierarchical clustering analysis of the DEGs showed that the data from the six time points fell into three branches: 2h, 24h/48h, and 72h/96h/120h. Singular (SEA) and modular enrichment analyses of DEGs per RNA-seq dataset were performed based on gene ontology. The results showed that immune responses occurred in all four tissues, indicating that GCRV probably does not target any tissue specifically. Moreover, during the course of disease, disturbances were observed in lipid and carbohydrate metabolism in each of the organs. SEA of DEGs based on the Kyoto Encyclopedia of Genes and Genomes database was also performed, and this indicated that the complement system and cellular immunity played an important role during the course of hemorrhagic disease. The qPCR of pooled samples of duplicate challenge experiment were used to confirm our RNA-seq approach. Copyright =C2=A9 2014 Elsevier Ltd. All rights reserved.

Background: Grass carp is an important farmed fish in China that is affected by serious disease, especially hemorrhagic disease caused by grass carp reovirus (GCRV). The mechanism underlying the hemorrhagic symptoms in infected fish remains to be elucidated. Although GCRV can be divided into three distinct subtypes, differences in the pathogenesis and host immune responses to the different subtypes are still unclear. The aim of this study was to provide a comprehensive insight into the grass carp response to different GCRV subtypes and to elucidate the mechanism underlying the hemorrhagic symptoms. Results: Following infection of grass carp, GCRV-I was associated with a long latent period and low mortality (42.5%), while GCRV-II was associated with a short latent period and high mortality (81.4%). The relative copy number of GCRV-I remained consistent or decreased slightly throughout the first 7 days post-infection, whereas a marked increase in GCRV-II high copy number was detected at 5 days post-infection. Transcriptome sequencing revealed 211 differentially expressed genes (DEGs) in Group I (66 up-regulated, 145 down-regulated) and 670 (386 up-regulated, 284 down-regulated) in Group II. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed significant enrichment in the terms or pathways involved in immune responses and correlating with blood or platelets. Most of the DEGs in Group I were also present in Group II, although the expression profiles differed, with most DEGs showing mild changes in Group I, while marked changes were observed in Group II, especially the interferon-related genes. Many of the genes involved in the complement pathway and coagulation cascades were significantly up-regulated at 7 days post-infection in Group II, suggesting activation of these pathways. Conclusion: GCRV-I is associated with low virulence and a long latent period prior to the induction of a mild host immune response, whereas GCRV-II is associated with high virulence, a short latent period and stimulates a strong and extensive host immune response. The complement and coagulation pathways are significantly activated at 7 days post-infection, leading to the endothelial cell and blood cell damage that result in hemorrhagic symptoms.

The grass carp is an important farmed fish, accounting for 16% of global freshwater aquaculture, and has a vegetarian diet. Here we report a 0.9-Gb draft genome of a gynogenetic female adult and a 1.07-Gb genome of a wild male adult. Genome annotation identified 27,263 protein-coding gene models in the female genome. A total of 114 scaffolds consisting of 573 Mb are anchored on 24 linkage groups. Divergence between grass carp and zebrafish is estimated to have occurred 49-54 million years ago. We identify a chromosome fusion in grass carp relative to zebrafish and report frequent crossovers between the grass carp X and Y chromosomes. We find that transcriptional activation of the mevalonate pathway and steroid biosynthesis in liver is associated with the grass carp's adaptation from a carnivorous to an herbivorous diet. We believe that the grass carp genome could serve as an initial platform for breeding better-quality fish using a genomic approach. =20

Background: Grass carp (Ctenopharyngodon idella) is one of the most economically important freshwater fish, but its production is often affected by diseases that cause serious economic losses. To date, no good breeding varieties have been obtained using the oriented cultivation technique. The ability to identify disease resistance genes in grass carp is important to cultivate disease-resistant varieties of grass carp. Results: In this study, we constructed a non-normalized cDNA library of head kidney in grass carp, and, after clustering and assembly, we obtained 3,027 high-quality unigenes. Solexa sequencing was used to generate sequence tags from the transcriptomes of the head kidney in grass carp before and after grass carp reovirus (GCRV) infection. After processing, we obtained 22,144 tags that were differentially expressed by more than 2-fold between the uninfected and infected groups. 679 of the differentially expressed tags (3.1%) mapped to 483 of the unigenes (16.0%). The up-regulated and down-regulated unigenes were annotated using gene ontology terms; 16 were annotated as immune-related and 42 were of unknown function having no matches to any of the sequences in the databases that were used in the similarity searches. Semi-quantitative RT-PCR revealed four unknown unigenes that showed significant responses to the viral infection. Based on domain structure predictions, one of these sequences was found to encode a protein that contained two transmembrane domains and, therefore, may be a transmembrane protein. Here, we proposed that this novel unigene may encode a virus receptor or a protein that mediates the immune signalling pathway at the cell surface. Conclusion: This study enriches the molecular basis data of grass carp and further confirms that, based on fish tissue-specific EST databases, transcriptome analysis is an effective route to discover novel functional genes.

Noxa, a pro-apoptotic protein, plays an important role in cell apoptosis. The researches about noxa gene were concentrated in mammalians, whereas the role and transcriptional regulatory mechanism of noxa in fish were still unclear. In this study, the expression pattern and transcriptional regulatory mechanism of noxa gene in grass carp were analyzed. Noxa was constitutively expressed in all the examined tissues but the relative expression level differed. After exposure to grass carp reovirus (GCRV), mRNA expression level of noxa was down-regulated at the early phase whereas up-regulated at the late phase of infection. Luciferase assays showed that the promoter region -867 +107 of noxa had high activity and the region -678 -603 was important in the response to GCRV infection. By deleting the predicted transcription factor binding sites, transcription factors FOXO1 and CEBPbeta were found important for noxa in response to GCRV infection. Moreover, the noxa promoter was biotin-labeled and incubated with nuclear extracts from GCRV infected cells. Mass spectrometry analysis showed that transcription factors FOXO1 and CEBPbeta were also enriched in the combined proteins. Therefore, the results suggested that transcription factors FOXO1 and CEBPbeta may play an important role in the regulation of noxa. Our study would provide new insight into the transcriptional regulatory mechanism of noxa in teleost fish. Copyright =C2=A9 2015 Elsevier Ltd. All rights reserved.

Transcription activator-like effector nucleases (TALENs) are used for gene knockout and genome-editing studies in zebrafish, and these techniques have the potential to be applied to other fish species. Here, we show that TALENs can directly knock out a green fluorescent protein (GFP) transgene in medaka by affecting translation and synthesis of the GFP. We constructed a transgenic plasmid (pGFP-RFP) carrying the GFP and red fluorescent protein (RFP) genes, and used a modified TALEN method to assemble a pair of TALENs for the core chromophore Y66 region of GFP. Embryo toxicity of TALEN messenger RNA (mRNA) was far lower than the linearized plasmid; meanwhile, 76.3% embryos, green fluorescence of embryos decreased significantly after co-injection of TALEN mRNA and the linearized plasmid, but red fluorescence showed no significant change. Real-time quantitative polymerase chain reaction and sequencing results showed that nearly 100% mutated GFP position was disrupted at the Y66 region of GFP in the co-injected medaka embryos, caused by TALENs. This led to random insertion-deletion of nucleotides, which affected the translation of GFP and disrupted GFP synthesis. This provides new experimental evidence for designing TALEN sites in genes for which only key functional domains are known. Our results show that a modified TALEN method can efficiently and specifically mediate a transgene knockout in medaka. This report may promote the application of TALENs in gene-editing studies of fish species other than zebrafish. =20

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4x haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3x haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.

BACKGROUND: Compensatory growth is accelerated compared with normal growth and occurs when growth-limiting conditions are overcome. Most animals, especially fish, are capable of compensatory growth, but the mechanisms remain unclear. Further investigation of the mechanism of compensatory growth in fish is needed to improve feeding efficiency, reduce cost, and explore growth-related genes.; RESULTS: In the study, grass carp, an important farmed fish in China, were subjected to a compensatory growth experiment followed by transcriptome analysis by RNA-sequencing. Samples of fish from starved and re-feeding conditions were compared with the control. Under starved conditions, 4061 and 1988 differentially expressed genes (DEGs) were detected in muscle and liver tissue when compared the experimental group with control group, respectively. After re-feeding, 349 and 247 DEGs were identified in muscle and liver when the two groups were compared. Moreover, when samples from experimental group in starved and re-feeding conditions were compared, 4903 and 2444 DEGs were found in muscle and liver. Most of these DEGs were involved in metabolic processes, or encoded enzymes or proteins with catalytic activity or binding functions, or involved in metabolic and biosynthetic pathways. A number of the more significant DEGs were subjected to further analysis. Under fasting conditions, many up-regulated genes were associated with protein ubiquitination or degradation, whereas many down-regulated genes were involved in the metabolism of glucose and fatty acids. Under re-feeding conditions, genes participating in muscle synthesis and fatty acid metabolism were up-regulated significantly, and genes related to protein ubiquitination or degradation were down-regulated. Moreover, Several DEGs were random selected for confirmation by real-time quantitative PCR.; CONCLUSIONS: Global gene expression patterns of grass carp during compensatory growth were determined. To our knowledge, this is a first reported for a teleost fish. The results will enhance our understanding of the mechanism of compensatory growth in teleost fish.=20