SNPs Detection Of Genes In Control Of Food Intake And The Association With Growth Traits In Largemouth Bass (Micropterus Salmoides)

The largemouth bass(Micropterus salmoides), common named Californian bass, was subordinate to Micropterus, which has been one of important cultivated fish in China. Largrmouth bass is a typical carnivorous fish, and its traditional breeding way depends on ice fresh fish, for which was not only restricted by the insufficient marine fishery resources, also the risk of introduction of pathogens. And make it difficult to carry out factory farming and production of non pollution of aquatic products. In recent years, the use of artificial diets in largemouth bass feeding has developed a lot along with the development of fish feed industry and breeding industry. However, the further development of largemouth bass aquaculture in our country is seriously restricted with the phenomenon of slow growth of artificial feed in the middle and later breeding of largemouth bass generally occurred. So the research emphasis of the researchers is to foster a new strain of largemouth bass with better using of artificial feed and growth tarits. Marker assistant selection(MAS) is a pretty important way in genetic improvement which could provide an effective way to selective breeding by analysis the association with the candidate gene and the growth traits. According to the research reports, fish feeding is influenced by many internal and external factors, of which appetite condition is the main internal factor, when the farmed fish is in a state of excitement of appetite, their feed intake and efficiency will be improved accordingly. Fish feeding is mainly regulated through the interaction of the brain and peripheral signals, and appetite related genes are the main gene involved in fish feeding, genetic mutation of those genes can affect the growth traits. In this study, we choose ghrelin,pacap and npy as the candidate genes to learn the growth traits of largemouth bass, this research would offer some effective markers to the molecular marker assisted breeding, and bring helps to the selection of new strain of largemouth bass.The main research content as follows:1. SNPs Detection of Ghrelin Gene and Its Association with Growth Traits in Largemouth Bass(Micropterus salmoides)Ghrelin is one of the vital brain- gut peptides in vertebrates. In 1999, a noval 28-amino acid gut-brain peptide named ghrelin was first isolated from the mammalian stomach. Similar to the synthetic molecules of growth hormone secretagogues, ghrelin was proved to be an endogenous ligand for growth hormone secretagogue receptor and involved in growth hormone secretion, food intake, carbohydrate metabolism, and energy homeostasis. So, ghrelin gene is a potential candidate gene for fish growth traits. In this study, we detected the polymorphism of the promoter of largemouth bass ghrelin gene by DNA sequencing in 327 individuals, for exploring the association between the polymorphism of ghrelin and growth traits of largemouth bass. Two single nucleotide polymorphism(SNP) mutations were identified(S1:A-642 C,S2:A-639C) of the gene. The genotype and gene frequency of 327 largemouth bass were further assayed with the method of Sna Pshot, at site S1, there three kinds of genotypes named AA, AC and CC, the gene frequency were 0.067, 0.685 and 0.248 respectively. There were only two genotypes AA and AC on the locus of S2, of which gene frequency were 0.850 and 0.150 respectively. Genetic structure was analyzed by POPGEN E32 software, the results showed that the two locus were in Hardy-Weinberg equilibrium, the effective number, expected heterozygosity and observed heterozygosity of S1 were 1.9362, 0.4843 and 0.6831 respectively, which of S2 were 1.1620, 0.1396 and 0.1508. The sites of S1 and S2 formed five diplotypes(D1, D2, D3, D4 and D5).Association analysis showed that individuals with genotype AC were significantly higher than those of individuals with genotype CC in body depth and total length at site S1(P<0.05). At site S2, the individuals of genotype with AA significantly showed a higher value on head length compared to the genotype of AC(P<0.05). The five diplotypes consisted of the two SNPs were different on some growth traits, the body weight, total length and body depth of diplotypes D1 showed significantly higher than those of D3(P<0.05). And diplotype D1 also showed a significantly higher value of body weight, body width and body depth compared with genotype D4(P<0.05). The SNP markers founded in ghrelingene promoter would be candidate genetic markers, and it suggested that S1:A-642 C and S2:A-639 C could be two molecular markers for providing helps to the molecular marker assisted breeding of largemouth bass.2. SNPs Detection of PACAP Gene and Its Association with Growth Traits in Largemouth Bass(Micropterus salmoides)Pituitary adenylate cyclase-activating polypeptide(PAC AP), a member of glucagon/secretes peptide family, is a pleiotropic hormone that enhances and stimulates the secretion of growth hormone, gonadotropin, prolactin and somatolactin. The single nucleotide polymorphism(SNP) in PACAP gene could affect its function or translation and transcription. Studying the association between the SNP and growth traits can provide candidate markers for maker-assisted selection. We detected the polymorp hism of PACAP gene by DNA sequencing to explore the association between the polymorphism of PACAP and growth traits of largemouth bass(Micropterus salmoides). Five pairs of primers were designed to amplify the PACAP gene sequence, and found a SNP A-2282 C site in the forth intron, and has 3 genotypes named as AA, AC and CC, respectively. The genotype and gene frequency of this SNP in 327 largemouth bass were further assayed with the method of Sna Pshot, the result showed that gene frequency of genotypes AA, AC and CC were 0.034, 0.645 and 0.321, respectively, and the frequency of A allele and C allele was 0.3558 and 0.6442. C hi-square test showed that the locus was in Hardy-Weinberg equilibrium in the largemouth bass population,the effective number of the locus was 1.8465, expected heterozygosity and observed heterozygosity was 0.5409 and 0.6442. The least squire method(LSM) was used to analysis the association of genotype with growth traits, and variance analysis indicated the mean values of AC genotype group was higher than that of AA and CC groups in body weight, body length, head length, head length, body height and caudal peduncle length. AC genotype were significantly higher than AA in caudal peduncle length and body width(P<0.05). The depth with AC genotype was extremely significantly higher than group with CC(P<0.01), and those with AC genotype were significantly higher than those with AA genotype(P<0.05). Th locus could be a candidate molecular marker for largemouth bass breeding.3. Cloning and Analysis of NPY Gene DNA and cDNA Sequence of Northern and Florida Subs pecies of Largemouth BassNeuropeptide Y plays an important role in the body of the feeding activity. In mammals, NPY is one of the most potent endogenic orexigenic factor. In order to learnthe structure and the function of NPY gene in the largemouth bass, the NPY c DNA of northern and Florida subspecies of largemouth bass were cloned by RT-PCR and RACE techniques, the result reveals that the NPY c DNA of the two subspecies all include an ORF encoding 99 amino acids and a same 5′-flanking sequence. Using genomewalker technology, a 3561 bp and a 3565 bp genomic sequence of NPY of northern subspecies of largemouth bass were isolated. DN A Exon- intron structure analysis suggested that both sequence contained three introns, four exons. By bioinformatics analysis with MATINSPECTO R, the basic transcriptional regulatory elements such as TATA Box、CAAT Box、CCAAT Box、GATA Box were found. This study found six single site differences between the two genomic DNA sequence, a 4 bp insert segments was identified in promote sequence of Florida subspecies of largemouth bass compared with the northern′s. The homology with NPY genes from other species was also compared, the largemouth bass has the highest nucleotide homology with mandarin fish and grouper, reach to 90% and 88% respectively, and the amino acid homology were 93% and 95%. This research provides help to the further study of neuropeptide Y in largemouth bass.4. A preliminary study on the association between SNPs and growth traits of largemouth bass NPY geneNeuropeptide Y is the most powerful feeding factor in the regulation network of fish feeding. The single nucleotide polymorphism(NPY) may affect the hormone secretion and fish feeding, which could be correlated to growth traits. Study the association between the SNP and growth trait can provide candidate markers for marker-assisted selection. In this study, we detected a SNP in the 5 ’non encoding region of NPY by direct sequencing method. Randomly selected 327 tail Largemouth bass breeded by the same batch and farmed in the same ponds, Using snapshot method to analysis the SNP type. Popgene32 software was used to analyze the genetic structure, using SPSS to obtain the association between different genotypes and growth traits. The result showed that gene frequency of genotypes AA, AC and CC were 0.471, 0.376 and 0.153, respectively, and the frequency of A allele and C allele was 0.7676 and 0.2324. Chi-square test showed that the locus was in Hardy-Weinberg equilibrium in the largemouth bass population,the effective number of the locus was 1.9282, expected heterozygosity and observed heterozygosity was 0.4491 and 0.4498. The average of body mass, body width, length and body height of AC genotype increased by 6.51%,2.56%, 2.18% and 3.34%, respectively, compared to the AA genotype group. increased by 15.37%, 7.97%, 4.09% and 5.12%, respectively, compared to the group with AA genotype. However, there was no significant correlation(P>0.05) between differe nt genotypes in growth traits. The SNP founded in this study could provides a reference for further study of the sites in largemouth bass.5. Effect of the Polymorphis m of Three Genes and Its Com Bined Genotypes on Growths Traits in Largemouth BassThis study aimed to further study the relationship among Ghrelin, PACAP and NPY with the growth traits of largemouth bass. DN A sequencing was applied in this study to detect the polymorphisms of these genes, and analyze the association of polymorphisms and combined genotypes with the growth traits. The result showed that nine combined genotypes were found of Ghrelin-PACAP and Ghrelin-NPY genes respectively, there were four genotypes combined by PACAP and NPY, combined genotypes of these three genes had nine and six kinds of different genotypes. The further study showed that genotypes of ACAC and AAAC combined by Ghrelin-PACAP genes took advantage of the growth traits, analyzing the combined genotypes of PACAP and site S1 of Ghrelin, advantageous genotype ACAC was 14.1%, 4.3% and 3.6% higher than disadcantageous genotype CCCC in body weight, body width and body length, when analyzing the combined genotypes of PACAP and site S2 of Ghrelin, advantageous genotype AAAC was 16.2%, 4.5% and 3.3% higher than disadcantagous genotype ACAC in body weight, body width and total length respectively. The growth traits of advantageous combined genotype of PACAP and NPY were better than others, while there were no statistically significant correlations. During the combined genotypes of PACAP and NPY, the body depth and total length of ACAC were significantly higher than those of CCAA(P<0.05), ACAC was 12.4%, 4.3% and 3.3% higher than CCAA in body weight, body width and body length respectively. And the genotype AAACAC was 26.8%, 8.5% and 4.2% higher than CCCCAA, of which three genes. So we make a judgment that the more single advantageous genotypes pyramiding, the larger effect on growths traits of largemouth bass. The study provides basic data of gene polymerization technology in aquatic animal genetic improvement.