| Large yellow croaker(Larimichthys crocea)is an economically important marine fish species in China.The aquaculture production of large yellow croaker reached approximately 198 thousand tons in 2018,which was the highest annual production among maricultured fish in China,and its industrial output value exceeded 10 billion yuan.However,problems arose with the expansion of the culture industry.Lower growth rate,small size,poor flesh quality,and loss of environmental adaptability were prevalent in the cultured population of large yellow croaker.It is especially sensitive to hypoxia stress,and the oxygen concentration must be above 5 mg/L for the aquaculture of large yellow croaker.Therefore,genetic improvement for important economic traits has been considered a potentially effective means to overcome these problems.High-quality genomic and transcriptomic are useful for fine mapping and positional cloning of quantitative trait loci associated with economically important traits,such as growth and hypoxia-resistance.To obtain a higher-quality genome assembly of large yellow croaker,we applied a hybrid assembly method by using long molecule sequences from Single Molecule Real-Time(SMRT)sequencing and short read sequences from next-generation sequencing.The 25-fold coverage of the long molecule sequences and a 563-fold coverage of short read sequences were used for genome assembly.The new version of genome assembly was 672 Mb,with a contig N50 of 282.69 kb and a scaffold N50 of 6.55 Mb,containing 26,100 protein-coding genes.The overall assembly statistics of our new genome version were dramatically better than the original version,with a 4.5-fold increase in contig N50,a 6.4-fold increase in scaffold N50.Importantly,more than 99.88% of the high-quality reads could be mapped to the improved assembly.Moreover,all 248 highly conserved genes tested and 99.6% of orthologous genes of Actinopterygii also could be mapped to the improved assembly.This improved assembly revealed 142 novel gene families that were not found in the original genome assembly,and 68 novel expanded gene families(P < 0.05),some of which are associated with olfactory detection(olfactory receptor genes: OR2B11 and OR4C5)and detoxification(cytochrome P450 genes: CYP2J2,CYP2J6,and CYP2K1).We also identified expansions in several immune-relevant genes,including macrophage mannose receptors(MRC1and MRC2),chemokines(CCL2,CCL3,and CCL5)and antimicrobial peptide(Hamp).Finally,24 pseudochromosomes of large yellow croaker were reassembled using 4,778 high-quality singlenucleotide polymorphisms(SNPs),representing 90% of the genome assembly(604.4 Mb).These data indicated that the new versions of the genome assembly and chromosome map were substantially better than the original versions.The location information of 20 hypoxia-related genes were identified,and HIF-1α and VEGF were cloned in large yellow croaker.These results reveal the molecular and genetic basis of fish adaptation to environmental stress and provide valuable resources for the genetic improvement of large yellow croaker.To understand the molecular responses of large yellow croaker to hypoxia stress,RNA-Seq technology was used to study the gene expression profiles in brain,gills,heart,spleen and kidney of large yellow croaker at different time points after hypoxia stress.A total of 8,402,1,546,2,746,2,499 and 3,685 differentially expressed genes(DEGs)were identified in brain,gills,heart,spleen and kidney(Fold change >2 and false discovery rate < 0.001),respectively.Expression changes of several selected genes in each tissue were further validated by the quantitative real-time PCR.KEGG enrichment showed that signaling pathways with the highest number of DEGs were“Neuroactive ligand-receptor interaction”,“Metabolic pathway”(P=0.0098),“Focal adhesion”(P=0.000008),“Axon guidance”(P=0.0120)and “Metabolic pathway”(P=0.0014)in brain,gills,heart,spleen and kidney,respectively.Based on GO and KEGG enrichments,the candidate DEGs related to hypoxia stress were collected in brain,gills,heart,spleen and kidney of hypoxic large yellow croakers.The master regulator of hypoxic response,HIF-1α,was up-regulated in gills,heart,spleen and kidney at 6h after hypoxia,with 2.46-,2.49-,4.51-and 2.66-fold increases,respectively.Moreover,HIF-1signaling pathway related genes,such as LDHA,VEGFA and ABCG2,were also up-regulated in these tissues.These results suggest that HIF-1 signaling pathway may play important roles in regulating gene expression in gills,heart,spleen and kidney responses to hypoxia stress.The expression level of HIF-1α in the brain of hypoxic large yellow croakers was not significantly changed after hypoxia,while genes involved in hypothalamic-pituitary-adrenal(HPA)axis and hypothalamic-pituitary-thyroid(HPT)axis were significantly changed,indicating that the neuroendocrine-immune/metabolism regulatory networks may help the fish brain to avoid cerebral inflammatory injury and maintain energy balance under hypoxia stress.Fish often shift their metabolisms in response to hypoxia stress.In this study,the expression of LDHA rapidly increased in gills,heart,spleen and kidney after hypoxia,together with the upregulation of other glycolysis-related genes,FDA and GAPDH in brain,heart,spleen and kidney,HK1 in heart and spleen,PKM in brain and kidney,indicating a fast adaptive activation of anaerobic ATP-generating pathway.The expression pattern of genes in tricarboxylic acid(TCA)cycle was not consistent in different tissues of hypoxic large yellow croakers.In brain,genes involved in the TCA cycle(PDC-E1,SCS,and FH)were down-regulated under hypoxia,indicating that the TCA cycle was inhibited by hypoxia in brain.In contrast,two key enzymes in TCA cycle,DH2 and OGDH,showed significant increased expression in gills,heart and kidney.The upregulation of these two key enzymes in the TCA cycle suggests that the aerobic oxidation would still be maintained in gills,heart and kidney under hypoxia,possibly because these tissues need more energy to accelerate gas exchange and blood circulation.Finally,we found that hypoxia also inhibited several innate immune pathways in brain,gills,heart and spleen,protein synthesis in brain and kidney,ion transport in gills,and programmed cell death in spleen and kidney,suggesting an energy-saving strategy for reducing oxygen consumption.These data afford new information to understand the tissue-specific molecular responses of bony fish to hypoxia stress.The hypoxia-related candidate genes identified in this study,provide valuable information for breeding hypoxia-tolerant large yellow croakers. |
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