Jianjun Fu

A consensus linkage map of the grass carp (Ctenopharyngodon idella) based on microsatellites and SNPs

BackgroundGrass carp (Ctenopharyngodon idella) belongs to the family Cyprinidae which includes more than 2000 fish species. It is one of the most important freshwater food fish species in world aquaculture. A linkage map is an essential framework for mapping traits of interest and is often the first step towards understanding genome evolution. The aim of this study is to construct a first generation genetic map of grass carp using microsatellites and SNPs to generate a new resource for mapping QTL for economically important traits and to conduct a comparative mapping analysis to shed new insights into the evolution of fish genomes.ResultsWe constructed a first generation linkage map of grass carp with a mapping panel containing two F1 families including 192 progenies. Sixteen SNPs in genes and 263 microsatellite markers were mapped to twenty-four linkage groups (LGs). The number of LGs was corresponding to the haploid chromosome number of grass carp. The sex-specific map was 1149.4 and 888.8 cM long in females and males respectively whereas the sex-averaged map spanned 1176.1 cM. The average resolution of the map was 4.2 cM/locus. BLAST searches of sequences of mapped markers of grass carp against the whole genome sequence of zebrafish revealed substantial macrosynteny relationship and extensive colinearity of markers between grass carp and zebrafish.ConclusionsThe linkage map of grass carp presented here is the first linkage map of a food fish species based on co-dominant markers in the family Cyprinidae. This map provides a valuable resource for mapping phenotypic variations and serves as a reference to approach comparative genomics and understand the evolution of fish genomes and could be complementary to grass carp genome sequencing project.

Molecular cloning and expression analysis of the liver-expressed antimicrobial peptide 2 (LEAP-2) gene in grass carp.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a novel antimicrobial peptide (AMP) recently found in vertebrates, and exhibits distinct amino acid sequence, secondary structure and expression pattern from other peptides. In this study, the LEAP-2 gene and its full-length cDNA were cloned from grass carp. Grass carp LEAP-2 gene consists of two introns and three exons. The translated product contains 92 amino acids, including a 26 amino acids signal peptide and a mature peptide of 41 amino acids. Grass carp LEAP-2 gene was expressed in a wide range of tissues except blood, with the highest level of transcripts found in liver. Upon induction by Aeromonas hydrophila, its expression was significantly up-regulated in liver, gill, skin, muscle, spleen, blood, head kidney, heart and intestine, but down-regulated in trunk kidney and brain. The transcript level was high in embryos at the 16-cell stage but declined gradually afterwards, suggesting that LEAP-2 transcripts in early embryos might be maternal. Mature peptides obtained by in vitro expression displayed selective antimicrobial activities. These results together further our understanding of the physiological function of LEAP-2 in vertebrates.

Next-generation sequencing identified microRNAs that associate with motile aeromonad septicemia in grass carp.

MicroRNAs (miRNAs) modulate many biological processes and can be up-or down-regulated in a variety of diseases states, including in bacterial infection. In this study, we characterized miRNAs associated with susceptibility or resistance to motile Aeromonas hydrophila in grass carp (Ctenopharyngodon idella), a commercially important farmed fish species in China. Using Illumina next-generation sequencing, we detected 185 miRNAs in A. hydrophila-susceptible (SGC) and -resistant grass carp (RGC) during immune activation. Twenty-one miRNAs were found to be differentially expressed between SGC and RGC, with 23.8% (5 out 21) exhibiting elevated expression in SGC. Further investigation of a selected five miRNAs indicated differences in the timing and tissue sources of expression. Target genes likely regulated by these miRNAs were identified using computational prediction combined with transcriptome expression data. MiRNA targets of differentially expressed mRNAs included those with functions important for bacterial resistance, such as tlr4 and nfil3-6. In addition, we demonstrated that nfil3-6 and tlr4 genes are direct targets of let-7i and cid-miRn-118. The present study suggested that the multiple miRNAs, displaying diverse kinetics and tissue sources on antibacterial immune processes.

De novo Assembly of the Grass Carp Ctenopharyngodon idella Transcriptome to Identify miRNA Targets Associated with Motile Aeromonad Septicemia

Background De novo transcriptome sequencing is a robust method of predicting miRNA target genes, especially for organisms without reference genomes. Differentially expressed miRNAs had been identified previously in kidney samples collected from susceptible and resistant grass carp (Ctenopharyngodon idella) affected by Aeromonas hydrophila. Target identification for these differentially expressed miRNAs poses a major challenge in this non-model organism. Results Two cDNA libraries constructed from mRNAs of susceptible and resistant C. idella were sequenced by Illumina Hiseq 2000 technology. A total of more than 100 million reads were generated and de novo assembled into 199,593 transcripts which were further extensively annotated by comparing their sequences to different protein databases. Biochemical pathways were predicted from these transcript sequences. A BLASTx analysis against a non-redundant protein database revealed that 61,373 unigenes coded for 28,311 annotated proteins. Two cDNA libraries from susceptible and resistant samples showed that 721 unigenes were expressed at significantly different levels; 475 were significantly up-regulated and 246 were significantly down-regulated in the SG samples compared to the RG samples. The computational prediction of miRNA targets from these differentially expressed genes identified 188 unigenes as the targets of 5 conserved and 4 putative novel miRNA families. Conclusion This study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The transcriptome assembly data represent a substantial increase in the genomic resources available for C. idella and will provide insights into the gene expression profile analysis and the miRNA function annotations in further studies.

Expression of complement component C7 and involvement in innate immune responses to bacteria in grass carp

Activation of the complement system, which forms a major part of the innate immune system, results in the formation of the terminal complement complex. The complement component, C7, plays an integral role in the assembly of this complex within target cell membranes. In this study, C7 was isolated and characterized from grass carp, an important cultured fish in China. The predicted amino acid sequence of C7 cDNA (2644 bp) exhibited 55.4 and 48.3% homology with trout C7-1 and zebrafish C7, respectively. The grass carp C7 gene was consisted of 18 exons and 17 introns. C7 gene expression was detected in the trunk kidney, liver, head kidney, skin, spleen, heart and intestine. Significant changes in C7 transcript expression (>20-fold) were detected following Aeromonas hydrophila infection, indicating C7 involvement in innate immune responses to bacteria in teleost fish.

Molecular cloning, characterization and expression patterns of HSP60 in the grass carp (Ctenopharyngodon idella)

HSP60 is a highly immunogenic molecule, which is able to activate a large number of T cell types and is implicated in a variety of autoimmune diseases. The grass carp (Ctenopharyngodon idella), a freshwater fish species of the family Cyprinidae, accounts for the third biggest value (USD 4.8 billion) at single species level of major cultured fish species in the world. Here, we isolated and characterized the HSP60 cDNA from grass carp (designated as CiHSP60). Its cDNA was 2434 bp in length and encoded a putative protein of 575 amino acids. BLAST analysis revealed that the CiHSP60 gene shared a high similarity with other known HSP60 sequences. CiHSP60 contained all three classical HSP60 family signatures. The mRNA of CiHSP60 was constitutively expressed in all tested tissues of untreated grass carp, including brain, muscle, trunk kidney, liver, head kidney, skin, spleen, heart, gill, intestine, and fin, with the highest expression level in the blood. CiHSP60 transcript was present in unfertilized eggs, which suggests that CiHSP60 transcription is maternally inherited. Fluorescent real-time quantitative RT-PCR was used to examine the expression of the CiHSP60 gene in grass carp after the challenge with the bacterium Aeromonas hydrophila. A clear time-dependent expression pattern of CiHSP60 was found after the bacterial challenge, and the mRNA expression reached a maximum level at three days post challenge, and returned to control levels after seven days. The upregulated mRNA expression of CiHSP60 in grass carp after bacterial challenge indicates that the HSP60 gene is inducible and involved in immune responses. These results suggest that CiHSP60 plays an important role in A. hydrophila-related diseases and in early embryonic development stages in grass carp.

Molecular cloning, expression analysis, and potential food intake attenuation effect of peptide YY in grass carp (Ctenopharyngodon idellus)

The peptide YY (PYY) is a 36 amino acid peptide involved in the food intake control in vertebrates. We have cloned and characterized a PYY gene from grass carp Ctenopharyngodon idellus. The full-length cDNA encodes a precursor protein of grass carp PYY (gcPYY) that consists of a putative 28-amino acid signal peptide, a 36-amino acid mature peptide, an amidation-proteolytic site, and a 30-amino acid carboxy-terminal extension. The gcPYY gene is comprised of 4 exons interspaced by 3 introns as seen in PYYs from other species. Amino acid alignment and gene structure comparison indicate that the structure of PYY is well preserved throughout vertebrate phylogeny. The tissue distribution and postprandial changes in gcPYY mRNA expression were evaluated by real-time PCR, which showed that the gcPYY is expressed abundantly in the central nervous system, with significantly increased expression following a single meal. During embryogenesis, the presence of gcPYY mRNA was detected in early developing embryos, and high expression levels were observed when most larvae completed their switch from endogenous nourishment to exogenous feeding. Reduced food intake by juveniles during a single meal after giving perpheral injection of gcPYY1-36 suggests a potentially important role of PYY in the food intake attenuation in grass carp.

Two novel homologs of simple C-type lectin in grass carp (Ctenopharyngodon idellus): Potential role in immune response to bacteria

C-type lectins play important roles in glycoprotein metabolism, multicellular integration and immunity. Based on their overall domain structure, they can be classified as different groups which possess different physiological functions. In this study, two novel simple C-type lectins were identified from grass carp (Ctenopharyngodon idellus), an important cultured fish in China. GcCL1 and gcCL2 share an essentially identical gene structure, a conserved promoter region shorter than 300 bp and an amino acid identity of 81.2%. Phylogenetic analysis indicated they may be products of gene duplication and could be classified as a new clade of group VII C-type lectins. Both of them were expressed in the eleven tissues examined, with the spleen having the highest abundance of transcript. The gcCL1 transcript was more abundant than gcCL2 in the majority of tissue samples from 2-yr-old grass carps, and was lower than those of gcCL2 before 15 days post-hatching. The expression of both genes was significantly up-regulated in spleen, muscle, skin, gills and hepatopancreas after induction by Aeromonas hydrophila. This is the first report that the expression of group VII C-type lectins could be induced by a pathogen, and indicates these lectins may be involved in the immune response to bacteria in fish.

Identification of immune-relevant genes by expressed sequence tag analysis of head kidney from grass carp (Ctenopharyngodon idella)

Grass carp is the third largest aquaculture species in global production. However, genomic research of this species has been limited. To identify immune-related genes in grass carp, a normalized full-length cDNA library was constructed from head kidney tissues, and 6432 randomly selected clones were sequenced. 5289 high quality expressed sequence tags (EST) were generated and assembled into 2687 unigenes. Among them, 1585 unigenes showed significant similarity with known sequences in public databases, whereas the remaining 1102 unigenes appeared to be novel sequences with unknown functions. In particular, 136 immune-related genes were identified to encode immunoglobulins, FcRgamma, IFN-related proteins, various CD markers, MHCs, complements and other important immune-related factors; a majority of these genes are reported in grass carp for the first time. Sequence analysis indicated that grass carp has at least three subtypes of immunoglobulin light chains, namely L1, L2 and L3. Furthermore, FCRgamma was found to broadly express in different tissues. Our study constitutes the first EST analysis of lymphatic tissue in grass carp, and could pave the way for further research of immune-related genes and functional genomics in grass carp.

MicroRNA-induced negative regulation of TLR-5 in grass carp, Ctenopharyngodon idella

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in antibacterial defence in fish has not been fully determined. Here, we identified that nine miRNAs are differentially expressed in kidney between susceptible and resistant grass carp strains. Analysis of spatial and temporal miRNA expression patterns suggests that cid-miRn-115 and miR-142a-3p are potential regulators of anti-bacterial activity. Overexpressing of cid-miRn-115 and miR-142a-3p results in a visible change in Ctenopharyngodon idella kidney (CIK) cells immune effector activity. Bioinformatics analysis and overexpressing assay shows that cid-miRn-115 and miR-142a-3p directly regulate tlr5 expression. cid-miRn-115 and miR-142a-3p overexpressing leads to a significant decrease in tlr5 expression in CIK, thereby repressing its downstream genes, such as il-1β, il-8 and tnf-α. These findings provide a novel insight into the determination of anti-bacterial compounds in grass carp.