IGF-1/IGF-2 Gene Cloning,Expression And Development Of Their SNPs Markers Of Sander Lucioperca

Pikeperch(Sander lucioperca)has become an important aquaculture object and also an important fishing sport fish species in the world.Nowdays,the lack of good breeding has seriously hindered the development of pikeperch culture industry.With the development and application of DNA molecular marker technology,molecular marker assisted breeding(MAS)has become effective approach of cultivating new varieties.So,this study mainly cloned growth related genes(IGF-1,IGF-2)of pikeperch by RACE,and developed their SNPs associated with growth,and aimed to to lay the foundation for MAS of Pikeperch.1 Molecular cloning and gene expression analysis of IGF-1 gene in pikeperch(Sander lucioperca)The full-length cDNA sequence of insulin-like growth factors-1(IGF-1)was isolated from the liver of pikeperch(Sander lucioperca L.)by 5 ’ and 3 ’rapid-amplification of cDNA ends(RACE).The cDNA is 883 bp,which contains an229 bp 5’-UTR,a 93 bp 3’-UTR including a polyA tail,and a 561 bp open reading frame(ORF)that encodes 183 amino acids.The IGF-1 protein of pikeperch possesses the same characteristic motifs and architectural design as IGF-1s of other fish species.Comparing with other fish IGF-1s,it is most homologous to the Larimichthys crocea and Siniperca chuatsi,where the cDNAs identity is 99%.Real-time polymerase chain reaction(RT-PCR)by using a pair of the IGF-1 gene-specific primers revealed the expression of IGF-1 in seven kind tissues(heart,liver,spleen,brain,gill,intestines,muscle)of pikeperch.IGF-1 was expressed in all tissues tested,of which its expression was the highest in liver,and the lowest in muscle.2 Molecular cloning and gene expression analysis of IGF-2 gene in pikeperch The full-length cDNA sequence of insulin-like growth factors-2(IGF-2)was isolated from the liver of pikeperch(S.lucioperca)by 5’and 3’rapid-amplification of cDNA ends(RACE).The cDNA is 1210 bp,which contains an 127 bp 5’-UTR,a 435 bp 3’-UTR including a polyA tail,and a 648 bp open reading frame(ORF)that encodes215 amino acids.The IGF-2 protein of pikeperch possesses the same characteristic motifs and architectural design as IGF-1s of other fish species.Comparing with other fish IGF-2s,it is most homologous to the Siniperca chuatsi,where the cDNAs identity is 95%.Real-time polymerase chain reaction(RT-PCR)by using a pair of the IGF-2gene-specific primers revealed the expression of IGF-2 in seven kind tissues(heart,liver,spleen,brain,gill,intestines,muscle)of pikeperch.IGF-1 was expressed in tissues tested,of which its expression was highest in the liver,lowest in the muscle and the intestines.3 Development of SNPs in GH-IGFs axis of pikeperch and their correlation analyses with growth trait The growth of higher organism is regulated by GH-IGFs axis,which mainly includes GH,IGF,IGFR,IGFBP and GHR genes.In this study,after IGF-1,IGF-2,GHR and IGF-1R DNA sequences were amplified,these sequences were compared with that of other perciformes fish to predict introns.The results showed that there were 5exons and 4 introns in IGF-1,and the exons were 277 bp,159bp,255 bp,39bp,and147bp;the introns were 1412 bp,8000kb,1282 bp and 709 bp.IGF-2 contained four exons and three introns,the exons were 204 bp,149bp,191 bp and 666 bp,and the introns were 862 bp,397bp and 1435 bp.The sequences of above genes in 16 individuals were compared.We found there were 2 SNP in intron 1 of IGF-1,and 1 SNP in intron 3of IGF-2,1 SNP in mRNA of GHR,and 1 SNP in intron of IGF-1R.Four SNPs(SL1-A612 C,SL2-A2914 AA,SL3-C1967 C,SL4-A122C)were genotyped by KASP(Kompetitive Allele Specific PCR)in randomly mixed group(298 individuals).The SPASS 17.0 was used to analyze the correlation between SNPs and growth traits of pikeperch.Results uncovered 3 SNPs had significant relation with no less one growth trait,in which 1 SNPs(IGF-2-SL2-A2914AA)had significant relation with total length of pikeperch.4 Correlation analyses of SNPs developed from the transcriptome and the growth trait of pikeperch In this study,8 SNPs were successfully verified in 17 SNPs potential for growth trait(SL5-C1425 T,SL6-C670 G,SL7-C370 C,SL8-C643 G,SL9-C710 G,SL10-C1652 T,SL11-C1870 C and SL12-C2397C).6 SNPs(SL6-C670 G and SL8-C643 G except)weregenotyped by KASP in randomly mixed group(298 individuals).The results showed that the advantage of genotypes were AA of SL7-C370 C,AA of SL9-C710 G,CT and TT of SL11-C1870,CT and TT of SL12-C2397 C and CC of SL10-C1652 T.The Results uncovered 5 SNPs had significant relation with no less one growth trait,in which 1SNP(SL9-C710G)had significant relation with total lengh of pikeperch.