| Sander lucioperca is a fierce carnivorous fish with delicious meat,high nutritional value,fast growth rate and strong disease resistance,its nutritional value and breeding benefits also far exceed those of the four major fish,and is deeply loved by farmers and consumers,and is a breeding species worth promoting.The Sander lucioperca has now been promoted on a large scale and is now farmed in northeast,north,northwest,south and east China.For the rapid development of Sander lucioperca farming,it is necessary to study the genetic analysis of growth traits of Sander lucioperca.In this study,we used RNA-Seq sequencing technology to analyze individual Sander lucioperca growth differences,to explore the relevant candidate genes and pathways that are beneficial to growth,and then cloned the excavated growth candidate genes GH and GHRH and analyzed their tissue expression characteristics to develop molecular markers for SNP related to growth traits,to improve the selection efficiency of superior varieties,and to lay a theoretical foundation for the selection and breeding of fast-growing varieties of Sander lucioperca.1.RNA-Seq sequencing was performed on 30 samples using the Illumina Hi Seq TM2500 platform and validation of the target candidate genes by RT-q PCR using large group and small group of Sander lucioperca brain,liver and muscle as material.A total of 192.43 Gb of Clean Data was obtained from transcriptome sequencing,with a Q30 of over 91.26% and an efficiency of over 92.78% for sequence alignment with the Sander lucioperca reference genome.The number of differentially expressed genes identified in brain,liver and muscle were 20,236 and 268 respectively,and the GO functional enrichment showed that the different expression gene set were mainly enriched in the processes of cell synthesis,growth,developmental metabolism and bioregulation.The KEGG pathways were very significant enriched in the Gn RH signalling pathway,neuroactive ligand-receptor interaction pathway,insulin,Apelin signalling pathway and PPAR signalling pathway,with down-regulated genes significantly enriched in fatty acid metabolism and fat deposition related pathways and up-regulated genes mainly enriched in metabolism related pathways.Among the differential genes,gh,ghrh,ghra,ghrhr,igf1,egr1,gadd45,igfbp1,rgs21,cpt1b,cebpb and other growth candidates were screened,and the results of RT-q PCR experiments and transcriptome sequencing were compared and analyzed,the results showed that the expression trends of eight selected up-regulated and eight down-regulated candidate genes were consistent with the transcriptomic results.The results provide a basis for the genetic analysis of growth traits in Sander lucioperca.2.GH and GHRH genes were cloned and their structural features and expression in various tissues were analysed.(1)The c DNA of GH gene is 926 bp in length,including 74 bp of 5′-UTR,237 bp of 3′-UTR and a coding region encoding 204 amino acids.The amino acid sequence similarity between GH and Perca flavescens and Perca fluviatilis was 97.04% and 94.05%,respectively.GH was expressed in all tissues of Perca flavescens,and the expression in the pituitary was extremely significantly higher than that in other tissues(P<0.01);GH gene expression was positively correlated with body mass of Perca flavescens,and the expression of GH gene in all four tissues was significantly higher than that in other tissues(P<0.01).The expression of GH gene was positively correlated with body mass in all four tissues,and the expression of GH gene was higher in the very large body mass group than in the very small group,and the difference was highly significant in pituitary and brain(P<0.01)and in muscle(P<0.05).(2)The c DNA of GHRH gene is 1100 bp,including 494 bp of 5′-UTR,180 bp of 3′-UTR and a coding region encoding 141 amino acids;the amino acid sequence similarity between GHRH of Sander lucioperca and Perca flavescens and Perca fluviatilis is 95.04% and 94.33%respectively.The expression of GHRH gene was positively correlated with body mass in Sander lucioperca,and the expression of GHRH gene in brain,liver and muscle was higher in the very large group than in the very small group,and there was a significant difference in brain(P<0.05).The results suggest that GH and GHRH genes can be used as candidate genes for Sander lucioperca growth selection,and also provide a reference for further analysis of the molecular mechanism of GH and GHRH genes in regulating Sander lucioperca growth and development.3.DNA direct sequencing was used to screen the SNP loci of GH and GHRH genes and identify SNP markers associated with growth traits.The results showed that three SNP loci(Ll-G44 A,L2-G78 A,L3-G116C)were found on the second exon of GH gene and one SNP loci(L4-T12A)was found on the second intron of Sander lucioperca.Comparative analysis showed that body mass,height,head length and muzzle length were significantly higher in the GG genotype at the L2-G78A locus than in the AA and AG genotypes(P<0.05),eye diameter was significantly higher in the AG genotype at the L1-G44A locus than in the AA genotype(P<0.05),and L3-G116C was significantly associated with tail shank length(P<0.05).The Sander lucioperca GHRH gene had one SNP loci(L5-T1993A)in the 5′-UTR region,and all growth traits were higher in the AT genotype than in the TT genotype at this locus,but there was no significant difference.The results showed that three SNP(Ll-G44A,L2-G78A and L3-G116C)in the GH gene contributed significantly to growth traits and could be used as candidate markers for molecular assisted breeding in Sander lucioperca. |
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