| The growth traits of fish are closely related to the economic benefits of aquaculture,and they are paid more attention in aquaculture production and basic research.Like mammals,the growth of fish is regulated by the growth axis consisted of the hypothalamus-pituitary-liver.Meanwhile,the expression of growth axis related genes is regulated by many factors such as season,photoperiod,temperature,nutrients and salinity.Therefore,the research of these functional genes,genotypes and related regulatory mechanisms that significantly affecting fish growth has become a hot topic in genetics and breeding of aquatic animals.Large yellow croaker is a unique marine aquaculture fish in China.The cultivation and seedling production of which is the most in marine fish and occupies an important position in Chinese marine aquaculture industry.At present,the large yellow croaker industry has entered a stage of transformation and upgrading,and the industry is expecting more and more excellent large yellow croaker with fast growth,strong resistance and high quality.So far,to the best of our knowledge,the studies on molecular regulation mechanisms and genetic basis of growth traits of large yellow croaker are scare,and the transcriptome and epigenetic regulation information about the growth axis have not been reported.In this study,the growth regulation axis and epigenetic regulation of the large yellow croaker population with significant phenotypic growth difference were analyzed.In the experiment,the pituitary,hypothalamus and liver of " Fufa No.1 "new strains F4 generation of large yellow croaker from the maximal individuals(L group)and the minimal individuals(S group)of the same cultured population were sampled for RNA transcriptome sequencing to obtain differentially expressed genes,and the muscle samples were subjected to genome resequencing and DNA methylation sequencing to obtained differential SNP sites and methylation sites through bioinformatic method.Meanwhile,the SSR markers of differentially expressed genes related to growth were identified and validated.The detailed experimental results are summarized as follows:1.A total of 2,1737 differentially expressed genes were identified from transcriptome sequencing of hypothalamus,including 440 up-regulated genes and 1297 down-regulated genes(S vs L,the same below).The result of GO functional enrichment and KEGG pathway analysis revealed 24 significantly enriched pathways such as protein processing and TGF-β signaling pathway.A total of 22 differently expressed genes related to growth including SSR2,igf2,egf1,igfbp1,and cgrefl were screened for qRT-PCR,and their expression trends were basically consistent with high-throughput sequencing results.2.A total of 1,507 differentially expressed genes were identified from transcriptome sequencing data of pituitary gland,including 250 up-regulated genes and 257 down-regulated genes.The result of GO functional enrichment and KEGG pathway analysis revealed that neuroactive ligand-receptor interaction,cytokine-cytokine receptor interaction,cell cycle and other 7 significant enrichment metabolic pathways.Meanwhile,13 growth-related genes such as gh,ghr,ghrhr,rerg,igfbp2 were selected and validated through qRT-PCR experiment and the result revealed that the expression trend of these selected genes were basically consistent with high-throughput sequencing results.3.A total 3,167 differentially expressed genes were identified from transcriptome sequencing of the liver,including 59 up-regulated genes and 108 down-regulated genes.The result of GO functional enrichment and KEGG pathway analysis revealed 5 pathways including the purine metabolism,neural activity ligand-receptor interaction,and cell cycle pathways were significantly enriched.Three differently expressed growth-related genes(egfp6,irl and gas8)were selected for qRT-PCR.The expression trends were consistent with high-throughput sequencing results.4.From the above differentially expressed genes,five growth-related genes with SSR sequences were identified.Among them,a total of 37 SSR sequences were identified and ten SSRs had polymorphic loci.Furthermore,the igflr-11 locus was significantly correlated with the economic traits such as body height,body length and body weight of large yellow croaker(P<0.05)through analysis.At the same time,an SSR combination that significantly increased the breeding traits was identified through multi-SSR combination analysis method and applied in molecular marker-assisted breeding of large yellow croaker,which had a clear growth advantage in seed culture production than control.5.The muscle DNA of L and S groups were performed genome resequencing,and 118668000 and 177597126 high-quality clean reads were obtained,respectively.A total of 96.88%and 96.44%reads could match the reference genomes of large yellow croaker,respectively.Overall,6174502 genetic variation sites were identified,which included 5,232,484 SNPs and 942,018 indel loci.A total of 10 polymorphisms and SNPs linked to genes related to growth were screened for further growth trait correlation analysis.The results showed that two types(AA and AG)caused by the SNP locus mutation type A→G of egfr15 gene had significant difference in both full length and body height(P<0.05),and a extreamly significant difference in body weight traits(P<0.01).There were 3 types(GG,GT and TT)caused by the SNP locus mutation type G→T of the vegfb gene.Among them,GG and GT type had no significant difference in growth traits,however,the TT type showed significant differences in body length,overall length,body height and body weight(P<0.05).6.The DNA methylation genetic map of large yellow croaker was obtained through genomic bisulfite sequencing of L and S populations muscle samples,and 209509870 and 235955864 high quality clean reads were obtained,respectively.The analysis result revealed that more than 70%reads could match the reference genome and the whole genome methylation of large yellow croaker mainly occurred at the CG locus,which accounted for more than 90%of the methylation rate of all C loci.Further statistical analysis of differentially methylated regions(DMRs)revealed that there were 2204 differentially methylated genes of C loci,including 1506 up-regulated genes and 698 down-regulated genes;2171 differentially methylated genes of CG type,of which 1505 were up-regulated and 666 were down-regulated.The GO enrichment analysis and KEGG pathway analysis of CG-type differentially methylated genes showed that the DMRs-related genes in the upstream 2k region were mainly enriched in five significant enrichment pathways including endocytosis,cytokine-cytokine receptor interaction,and actin cytoskeleton regulation;gene-body DMRs-related genes are mainly enriched in 17 significant enrichment pathways such as Notch signaling pathway,MAPK,TGF-beta signaling pathway;DMRs-related genes in downstream 2k region genes were mainly enriched in the Notch signaling pathway,Jak-STAT signaling pathway,and protein processing in the endoplasmic reticulum.A total of the 13 DMRs-related genes were selected for qRT-PCR analysis.The result indicated that the differential expression genes in the upstream 2k region was negatively correlated with the level of methylation,while the differential expression genes in the genebody region was positively correlated body the level of methylation.In addition to,4 genes related to DMRs in the upstream 2k region were screened(fgf11,fgfrs2,egflp7 and pdgfra)to validate methylation levels and the results indicated that their methylation trends were consistent with high-throughput sequencing results.In this study,several functional genes,metabolic pathways,linkage molecular markers that related to growth traits were identified by transcriptome sequencing,genome resequencing and DNA methylation sequencing,which provided a basis for in-depth study of the molecular regulation mechanism of the growth traits of large yellow croaker. |
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