Polymorphism Analysis Of GHSR And Ghrelin Gene In Oreochromis Niloticus And Screening Of Their Growth Related SNPs

Nile tilapia（Oreochromis niloticus, GIFT strain）, whose growth rate is remarkable higher than other breeds of tilapia, is the most widely cultured and the highest-yielding tilapia species in China at present. Recent researches showed that O. niloticus GIFT strain has great different growth rate among individuals of the same family or among different families. This indicated that O. niloticus GIFT strain has an abundant genetic diversity and enormous breeding potential. In this research, two growth regulation related genes（Growth Hormone Secretagogue Receptor（GHSR） gene and ghrelin gene）of O. niloticus GIFT strain were studied for screening of the growth-related SNPs.The results will help with maintaining of the stability of the growth trait of O. niloticus GIFT strain, preventing germplasm recession from inbreeding, and breeding of new strain of fast-growing O. niloticus. The SNPs were obtained through gene cloning and polymorphism analysis. Afterwards, the association between the SNPs and growth traits of O. niloticus GIFT strain was studied. We aimed to find the growth-related SNPs in the two genes of O. niloticus GIFT strain, which could be applied to molecular assisted selection（MAS）of Nile tilapia. The results were as follows:1. Cloning and polymorphic analysis of GHSR gene and ghrelin gene in Nile tilapia.The cDNA sequences of GHSR â… a and GHSR â… b were obtained. The complete c DNA sequence of GHSR-Ia was 4060 bp, which consisted of a 5′-noncoding region（1003bp）, a 3′-noncoding region（1905bp） and an open reading frame（ORF, 1152bp）coding 384 amino acids. The complete c DNA sequence of GHSR-â… b was 4268 bp,which consisted of a 5′-noncoding region（1003bp）, a 3′-noncoding region（2371bp） and an open reading frame（ORF, 894bp） coding 298 amino acids. The phylogenetic tree indicated that the O. niloticus had the nearest evolutionary relationship with the O.hornorum and O. mossambicus. They clustered with Sparus macrocephalus（Cichlaidae）in the phylogenetic tree, and displayed a far evolutionary relationship with Oncorhynchus mykiss, Cyprinus carpio var. Jian and Homo sapiens. The genomic sequence（3206bp） of GHSR gene of Nile tilapia was obtained, which contained a5′-noncoding region（1589bp）, a 3′-noncoding region（231bp） and a coding region（1386bp）. The coding region of GHSR gene contained 2 exons and 1 intron. The m RNA of GHSR gene encoded GHSR â… a（384 amino acids） and GHSRâ… b（298 amino acids）according to the two different ways of shear, respectively. The similarity of GHSR genomic sequence between Nile tilapia and O. mossambicus（Gen Bank No:EU910220.1） was as high as 98.54%, while it was 88.70% between Nile tilapia and Haplochromis burtoni（Gen Bank No: XM₀05926046.1）.78 sequences of GHSR gene（2857bp in length） were obtained from two populations of Nile tilapia（fast-growing population and base population）. 340 polymorphic sites which contained 63 single nucleotide polymorphism loci and 277 parsimony informative sites and 39 kinds of haplotypes were found in GHSR gene. The nucleotide diversity（pi）, the average number of nucleotide differences（K） and the haplotype diversity（Hd） of GHSR gene were 0.0217, 57.923 and 0.00133, respectively. The genetic parameters(single nucleotide polymorphism（SNP）, haplotype（H）, nucleotide diversity（pi）, haplotype diversity（HD） and average number of nucleotide differences（K）,of base population were higher than those of fast-growing population in Nile tilapia.It indicated that the genetic diversity of the fast-growing population of Nile tilapia had declined in the breeding process.77 sequences of ghrelin gene fragment（841bp） were obtained from two populations of Nile tilapia（fast-growing population and base population）. 38 polymorphic sites which contained 31 single nucleotide polymorphism loci and 7 parsimony informative sites and 13 kinds of haplotypes were found. The nucleotide diversity（pi）, the average number of nucleotide differences（K） and the haplotype diversity（Hd） were 0.00402,2.7717 and 0.722, respectively. The number of single nucleotide polymorphism loci（SNP） of the fast-growing population is less than that of the base population in ghrelingene. The nucleotide diversity（pi） and average number of nucleotide differences（K） of the fast-growing population is higher than those of the base population, which might be attributed to the high levels of inbreeding in the breeding process of fast-growing population.2. Screening of SNPs associated with growth trait in GHSR gene of Oreochromis niloticus.Five SNPs locus（S1（A-409G）, S2（G-424T）, S3（T-553A）, S4（T-1114A） and S5（A-1168C）） were found in GHSR gene of two populations of Nile tilapia（fast-growing population and base population）, which were located in the 5′ flanking region of the gene. The results of association analysis between the genotypes of SNPs and the growth traits of the offspring of Nile tilapia showed that S4 site had significantly correlation with growth trait. The individuals belong to genotype AA or AT in S4 site had significantly higher growth traits（body length, body height, head length, caudal peduncle length, caudal peduncle depth, and larger body weight） than the individuals belong to genotype TT（P<0.05）. The 5 SNPs sites composed 7 diplotypes（D1-D7）. The body weight, body length, body height, head length and caudal peduncle depth of individuals belong to diplotypes D3 and D5 were significantly larger or higher than that of the individuals belong to diplotypes D4 and D7（P<0.05）. The growth traits of individuals belong to diplotypes D1（except for body width）, D2（except for body width and the shank length） and D6（except for body height） had no significantly difference compared with individuals belong to the other diplotypes. The dominant diplotypes in the generation of fast-growth population and base population were diplotypes D3（46.25%） and D4（35.44%）, respectively. In conclusion, the genotypes AA and AT in S4 site, diplotypes D3 and D5 of GHSR gene were closely related to high growth rate in Nile tilapia, which can be used as candidate molecular markers for tilapia breeding.3. Screening of SNPs related with growth in ghrelin gene of Oreochromis niloticusThree SNPs locus(S1（A+134G）, S2（A+145G）, S3（A+155G） were found in the ghrelin gene of two populations of Nile tilapia（fast-growing population and base population）, which were located in the first intron of ghrelin gene. The three SNPs were complete genetic linkage, which have the same genetic diversity parameters,frequencies of alleles and genotypes. The dominant genotypes of the three SNP loci were the same in the offspring of fast-growing population and base population. The frequencies of dominant genotypes in the offspring of fast-growing population were significantly greater than those in the offspring of base population. The result of correlation analysis between genotypes of SNPs and growth traits of the offspring in two populations showed that growth traits（body weight, body length, body height, head length and caudal peduncle depth） were significantly different in different genotypes（S1:GG > AG, S2:TT > AT, S3:AA > AT; P<0.05）. The growth traits（body weight, body length, body height, head and tail handle height） of D1 diplotype（S1: GG, S2: TT,S3:AA） were significantly higher than those of D2 diplotype（S1:AG, S2:AT, S3:AT）.The above results showed that D1 diplotype of ghrelin gene was closely related to the growth performance of Nile tilapia, which can be used as candidate molecular markers for breeding for improved growth in tilapia.