Liver Transcriptome Analysis of the Large Yellow Croaker (Larimichthys crocea) during Fasting by Using RNA-Seq.
ABSTRACT: The large yellow croaker (Larimichthys crocea) is an economically important fish species in Chinese mariculture industry. To understand the molecular basis underlying the response to fasting, Illumina HiSeqTM 2000 was used to analyze the liver transcriptome of fasting large yellow croakers. A total of 54,933,550 clean reads were obtained and assembled into 110,364 contigs. Annotation to the NCBI database identified a total of 38,728 unigenes, of which 19,654 were classified into Gene Ontology and 22,683 were found in Kyoto Encyclopedia of Genes and Genomes (KEGG). Comparative analysis of the expression profiles between fasting fish and normal-feeding fish identified a total of 7,623 differentially expressed genes (P < 0.05), including 2,500 upregulated genes and 5,123 downregulated genes. Dramatic differences were observed in the genes involved in metabolic pathways such as fat digestion and absorption, citrate cycle, and glycolysis/gluconeogenesis, and the similar results were also found in the transcriptome of skeletal muscle. Further qPCR analysis confirmed that the genes encoding the factors involved in those pathways significantly changed in terms of expression levels. The results of the present study provide insights into the molecular mechanisms underlying the metabolic response of the large yellow croaker to fasting as well as identified areas that require further investigation.
Project description:The genetic map of a species is essential for its whole genome assembly and can be applied to the mapping of important traits. In this study, we performed RNA-seq for a family of large yellow croakers (Larimichthys crocea) and constructed a high-density genetic map. In this map, 24 linkage groups comprised 3,448 polymorphic SNP markers. Approximately 72.4% (2,495) of the markers were located in protein-coding regions. Comparison of the croaker genome with those of five model fish species revealed that the croaker genome structure was closer to that of the medaka than to the remaining four genomes. Because the medaka genome preserves the teleost ancestral karyotype, this result indicated that the croaker genome might also maintain the teleost ancestral genome structure. The analysis also revealed different genome rearrangements across teleosts. QTL mapping and association analysis consistently identified growth-related QTL regions and associated genes. Orthologs of the associated genes in other species were demonstrated to regulate development, indicating that these genes might regulate development and growth in croaker. This gene map will enable us to construct the croaker genome for comparative studies and to provide an important resource for selective breeding of croaker.
Project description:High-density single-nucleotide polymorphism (SNP) genotyping array is an essential tool for genetic analyses of animals and plants. Large yellow croaker (Larimichthys crocea) is one of the most commercially important marine fish species in China. Although plenty of SNPs have been identified in large yellow croaker, no high-throughput genotyping array is available. In this study, a high-throughput SNP array named NingXin-I with 600K SNPs was developed and evaluated. A set of 82 large yellow croakers were collected from different locations of China and re-sequenced. A total of 9.34M SNPs were identified by mapping sequence reads to the large yellow croaker reference genome. About 1.98M candidate SNPs were selected for further analyses by using criteria such as SNP quality score and conversion performance in the final array. Finally, 579.5K SNPs evenly distributed across the large yellow croaker genome with an average spacing of 1.19 kb were proceeded to array production. The performance of NingXin-I array was evaluated in 96 large yellow croaker individuals from five populations, and 83.38% SNPs on the array were polymorphic sites. A further test of the NingXin-I array in five closely related species in Sciaenidae identified 26.68-56.23% polymorphic SNP rate across species. A phylogenetic tree inferred by using the genotype data generated by NingXin-I confirmed the phylogenetic distance of the species in Sciaenidae. The performance of NingXin-I in large yellow croaker and the other species in Sciaenidae suggested high accuracy and broad application. The NingXin-I array should be valuable for quantitative genetic studies, such as genome-wide association studies (GWASs), high-density linkage map construction, haplotype analysis, and genome-based selection.
Project description:Pseudomonas plecoglossicida is a Gram-negative fish pathogen responsible for visceral granular disease in large yellow croaker, Larimichthys crocea. Here, we report the complete genome sequence of a verified virulent strain, XSDHY-P, that was isolated from spleen tissue of a diseased large yellow croaker.
Project description:Elongation of very long chain fatty acids protein 6 (Elovl6) is a key enzyme in fatty acid synthesis, which participates in converting palmitate (C16:0) to stearate (C18:0). Although studies of Elovl6 have been carried out in mammals, the nutritional regulation of elovl6 in fish remains poorly understood. In the present study, the cloning and nutritional regulation of elovl6 were determined in large yellow croaker. Sequence and phylogenetic analysis revealed that the full-length cDNA of elovl6 was 1360 bp, including an open reading frame of 810 bp encoding a putative protein of 269 amino acid that possesses the characteristic features of Elovl proteins. The transcript level of elovl6 was significantly increased in the liver of croaker fed the diets with soybean oil (enriched with 18: 2n-6, LA) or linseed oil (enriched with 18: 3n-3, ALA) than that in croaker fed the diet with fish oil (enriched with 20: 5n-3 and 22: 6n-3). Correspondingly, the elovl6 expression in croaker's hepatocytes treated with ALA or LA was remarkably increased compared to the controls. Furthermore, the transcription factors including hepatocyte nuclear factor 1α (HNF1α), CCAAT-enhancer-binding protein β (CEBPβ), retinoid X receptor α (RXRα), and cAMP response element-binding protein 1 (CREB1) greatly enhanced promoter activity of elovl6 in large yellow croaker, and the expression of transcription factors is consistent with the changes of elovl6 expression in response to fatty acids in vivo and in vitro. In conclusion, this study revealed that elovl6 expression in large yellow croaker could be upregulated by dietary ALA or LA via the increased transcriptional expression of transcription factors including hnf1α, cebpβ, rxrα, and creb1.
Project description:High-density genetic maps are essential for genome assembly, comparative genomic analysis and fine mapping of complex traits. In this study, 31,191 single nucleotide polymorphisms (SNPs) evenly distributed across the large yellow croaker (Larimichthys crocea) genome were identified using restriction-site associated DNA sequencing (RAD-seq). Among them, 10,150 high-confidence SNPs were assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 5451.3 cM with an average distance of 0.54 cM between loci. This represents the densest genetic map currently reported for large yellow croaker. Using 2889 SNPs to target specific scaffolds, we assigned 533 scaffolds, comprising 421.44 Mb (62.04%) of the large yellow croaker assembled sequence, to the 24 linkage groups. The mapped assembly scaffolds in large yellow croaker were used for genome synteny analyses against the stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes). Greater synteny was observed between large yellow croaker and stickleback. This supports the hypothesis that large yellow croaker is more closely related to stickleback than to medaka. Moreover, 1274 immunity-related genes and 195 hypoxia-related genes were mapped to the 24 chromosomes of large yellow croaker. The integration of the high-resolution genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits in large yellow croaker.
Project description:The piscidin family, which includes potent antimicrobial peptides with broad-spectrum activity, plays an important role in the innate immune system of fish. In this study, we cloned piscidin-5-like type 3 (<i>Lcpis5lt3</i>) in large yellow croaker (<i>Larimichthys crocea</i>). Multiple alignments with other known piscidins revealed amino acid conservation throughout the fish, especially at the signal peptide (22 amino acids). The phylogenetic tree confirmed that <i>Lcpis5lt3</i> and large yellow croaker piscidin-5-like proteins were grouped together to form a branch. Quantitative real-time PCR revealed that <i>Lcpis5lt3</i> was expressed in a wide range of tissues, including the brain, muscle, gill, head kidney, intestine, kidney, liver, and spleen. The highest mRNA expression level of <i>Lcpis5lt3</i> was found in the spleen. After <i>Vibrio alginolyticus</i> infection, mRNA expression was rapidly upregulated in the liver, head kidney, gill, kidney, and intestine at 4, 8, 12, and 24 h post infection (hpi), whereas there were no significant changes in the spleen. The antimicrobial spectrum showed that the synthetic mature peptide of <i>Lcpis5lt3</i> exhibited different activity <i>in vitro</i> against various bacteria, such as <i>Aeromonas hydrophila</i>, <i>V. anguillarum</i>, <i>V. alginolyticus</i>, <i>V. parahaemolyticus</i>, <i>Staphylococcus aureus</i>, and <i>Listeria monocytogenes</i>. In addition, survival rates from the <i>in vivo</i> assay indicated that the synthetic peptide of <i>Lcpis5lt3</i> increased the survival rate of large yellow croaker after <i>V. alginolyticus</i> challenge, resulting in a decline in bacterial burden and mRNA expression levels of interleukin-1?, interleukin-10, and tumor necrosis factor-?. These data suggest that <i>Lcpis5lt3</i> plays an important role in innate immunity in large yellow croaker and might represent a potential therapeutic agent against pathogen invasion.
Project description:BACKGROUND: Chemosensory receptors, which are all G-protein-coupled receptors (GPCRs), come in four types: odorant receptors (ORs), vomeronasal receptors, trace-amine associated receptors and formyl peptide receptor-like proteins. The ORs are the most important receptors for detecting a wide range of environmental chemicals in daily life. Most fish OR genes have been identified from genome databases following the completion of the genome sequencing projects of many fishes. However, it remains unclear whether these OR genes from the genome databases are actually expressed in the fish olfactory epithelium. Thus, it is necessary to clone the OR mRNAs directly from the olfactory epithelium and to examine their expression status. RESULTS: Eighty-nine full-length and 22 partial OR cDNA sequences were isolated from the olfactory epithelium of the large yellow croaker, Larimichthys crocea. Bayesian phylogenetic analysis classified the vertebrate OR genes into two types, with several clades within each type, and showed that the L. crocea OR genes of each type are more closely related to those of fugu, pufferfish and stickleback than they are to those of medaka, zebrafish and frog. The reconciled tree showed 178 duplications and 129 losses. The evolutionary relationships among OR genes in these fishes accords with their evolutionary history. The fish OR genes have experienced functional divergence, and the different clades of OR genes have evolved different functions. The result of real-time PCR shows that different clades of ORs have distinct expression levels. CONCLUSION: We have shown about 100 OR genes to be expressed in the olfactory epithelial tissues of L. crocea. The OR genes of modern fishes duplicated from their common ancestor, and were expanded over evolutionary time. The OR genes of L. crocea are closely related to those of fugu, pufferfish and stickleback, which is consistent with its evolutionary position. The different expression levels of OR genes of large yellow croaker may suggest varying roles of ORs in olfactory function.
Project description:Vaccination is an effective and safe strategy for combating bacterial diseases in fish, but the mechanisms underlying the early immune response after vaccination remain to be elucidated. In the present study, we used RNA-seq technology to perform transcriptome analysis of spleens from large yellow croaker (Larimichthys crocea) induced by inactivated trivalent bacterial vaccine (Vibrio parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila). A total of 2,789 or 1,511 differentially expressed genes (DEGs) were obtained at 24 or 72 h after vaccination, including 1,132 or 842 remarkably up-regulated genes and 1,657 or 669 remarkably down-regulated genes, respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichments revealed that numerous DEGs belong to immune-relevant genes, involved in many immune-relevant pathways. Most of the strongly up-regulated DEGs are innate defense molecules, such as antimicrobial peptides, complement components, lectins, and transferrins. Trivalent bacterial vaccine affected the expressions of many components associated with bacterial ligand-depending Toll-like receptor signaling pathways and inflammasome formation, indicating that multiple innate immune processes were activated at the early period of vaccination in large yellow croaker. Moreover, the expression levels of genes involved in antigen processing were also up-regulated by bacterial vaccine. However, the expression levels of several T cell receptors and related CD molecules and signal transducers were down-regulated, suggesting that the T cell receptor signaling pathway was rapidly suppressed after vaccination. These results provide the comprehensive insights into the early immune response of large yellow croaker to vaccination and valuable information for developing a highly immunogenic vaccine against bacterial infection in teleosts.
Project description:Whole-genome single-nucleotide polymorphism (SNP) markers are valuable genetic resources for the association and conservation studies. Genome-wide SNP development in many teleost species are still challenging because of the genome complexity and the cost of re-sequencing. Genotyping-By-Sequencing (GBS) provided an efficient reduced representative method to squeeze cost for SNP detection; however, most of recent GBS applications were reported on plant organisms. In this work, we used an EcoRI-NlaIII based GBS protocol to teleost large yellow croaker, an important commercial fish in China and East-Asia, and reported the first whole-genome SNP development for the species. 69,845 high quality SNP markers that evenly distributed along genome were detected in at least 80% of 500 individuals. Nearly 95% randomly selected genotypes were successfully validated by Sequenom MassARRAY assay. The association studies with the muscle eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content discovered 39 significant SNP markers, contributing as high up to ?63% genetic variance that explained by all markers. Functional genes that involved in fat digestion and absorption pathway were identified, such as APOB, CRAT and OSBPL10. Notably, PPT2 Gene, previously identified in the association study of the plasma n-3 and n-6 polyunsaturated fatty acid level in human, was re-discovered in large yellow croaker. Our study verified that EcoRI-NlaIII based GBS could produce quality SNP markers in a cost-efficient manner in teleost genome. The developed SNP markers and the EPA and DHA associated SNP loci provided invaluable resources for the population structure, conservation genetics and genomic selection of large yellow croaker and other fish organisms.
Project description:Brain transcriptome at 0h, 1h, 3h, 6h, 12h, 24h, 48h after hypoxia stress Large yellow croakers (body weight at 90-100 g) were purchased from the mariculture farm in Ningde, Fuzhou, China. The fish were maintained at 25 °C in aerated water tanks (dissolved oxygen concentration: 7.8±0.5 mg per liter) with a flow through seawater supply. After 7 days of acclimation, these fish were used for the following experiments. Hypoxic time-course experiments were conducted at 25 °C using published method 30, by bubbling nitrogen gas into an aquarium. The desired pO2 was controlled by using dissolved oxygen meter (, Canada). At the onset of the time course, the oxygen content of the tank was lowered from an aerated pO2 of 100% (7.8 mg per liter) down to 20% (1.6±0.2 mg per liter) over a 30-min period. At the 1-, 3-, 6-, 12-, 24-, and 48-h time points, fish were sampled and sequenced.