| In recent years,tilapia has gradually become one of the largest freshwater fish farmed in China,but the growth rate of tilapia in different strains or different families of the same strain is greatly different,which affects the cultivation time,yield and production of tilapia Specifications increase the cost of farming and reduce the economic benefits of the tilapia industry.Therefore,it is of great significance for the development of tilapia industry to strengthen the selection of traits of tilapia by molecular assisted breeding technology.In this study,two strains of tilapia were used as the research object,and the relationship between the polymorphisms of 5’ flanking regions and coding regions of major appetite-related genes ghrelin,NPY,prepro-orexin and major growth-related genes GHR1,GHR2,and IGF-I with tilapia growth was explored,SNPs loci related to growth traits were screened,and a preliminary analysis of SNPs loci regulating fish growth was performed.This study includes the following three aspects:1.Screening of SNPs for tilapia appetite-related regulatory genes and analysis of their association with growth.In the experiment,preliminary screening of SNPs was performed on the parent individuals of Jifu tilapia(JF population)and Egyptian Nile tilapia(AJ population),and the secondary screening of SNPs was performed on the condition that SNPs caused changes in sequence function.Correlation analysis was performed between the screened SNPs and the growth characteristics of the offspring.The results show:(1)After preliminary screening,a total of 12 SNPs loci were found in 3 appetite-related genes in the JF population,and a total of 8 SNPs loci were found in the AJ population.(2)After secondary screening,7 loci were retained in the 3 appetite-related genes in the JF population.Among them,only the S1 locus was retained in the ghrelin gene,only the S4 locus was retained in the NPY gene,and a total of 5 were retained in the prepro-orexin gene.In the AJ population,a total of 5 loci were retained,of which only the S’1 locus was retained in the ghrelin gene,only the S’2 locus was retained in the NPY gene,and a total of 3 SNPs loci were retained in the prepro-orexin gene.(3)In the JF population,the genotype CC with pure S7,S8,and S9 loci was significantly greater than the heterozygous genotype CG in all traits(P<0.05);The heterozygous genotype CT was significantly larger in all traits than the pure sum genotype CC(P<0.05);only the S1 locus produced significant differences in each trait in three different genotypes,showing TT>CT>CC(P<0.05).No SNPs loci associated with growth traits were found in the AJ population.(4)After analyzing the linkage diplotype of the SNPs loci in the prepro-orexin gene,5 SNPs loci in the JF population constituted 9 diplotypes,of which the D7 diplotype was significant in various growth indicators Higher than D2,D4,D5,D9(P<0.05).3 SNPs loci in the AJ population constituted 6 types of diplotypes,but no diplotypes associated with growth traits were found.2.Screening of growth-related regulator genes SNPs in tilapia and analysis of their association with growth.In the experiment,preliminary screening of SNPs was performed on the parent individuals of JF tilapia and AJ tilapia,and the secondary screening of SNPs was performed on the condition that SNPs caused changes in sequence function.Correlation analysis was performed between the screened SNPs and the growth characteristics of the offspring.The results show:(1)After preliminary screening,a total of 19 SNPs loci were found in 3 growth-related genes in the JF population,and a total of 13 SNPs loci were found in the AJ population.2)After secondary screening,9 loci were retained in the 3 growthrelated genes in the JF population,among which 7 loci were retained in the GHR1 gene;2 loci were retained in the GHR2 gene;no SNPs were retained in the IGF-I gene.A total of 5 loci were retained in the AJ population,of which 4 loci were retained in the GHR1 gene;only the N’10 locus was retained in the GHR2 gene;IGF No SNPs loci were retained in the-I gene.(3)In the JF population,the N3,N4,N7,N8,and N9 loci in all traits except body length showed that the genotype individuals after mutation were significantly greater than those before the mutation.(P<0.05);The N17 locus showed that the genotype individuals who were homogeneous before mutation were significantly larger than those who were heterozygous(P<0.05).In the AJ population,only genotype individuals with heterozygous body length and head length at the N’10 locus were significantly larger than those before the mutation and the pure and genotype individuals(P<0.05).(4)After analyzing the linkage diplotype of the SNPs loci in the GHR1 and GHR2 genes,the 7 SNPs loci in the GHR1 gene of the JF population constitute 7 types of diplotypes,of which E1 and E2 are in the height of the body and tail tail The length and body traits were significantly larger than E7(P<0.05);the two SNPs loci in the GHR2 gene constituted 6 types of diplotypes.of which E12 and E13 were significantly larger than E9 in all traits(except body weight,P>0.05),E10 and E11(P<0.05).4 SNPs loci in the GHR1 gene of the AJ population constituted 7 diplotypes in total,but no diplotypes were found to be associated with growth traits.3.Study on the regulation of fish growth based on SNPs of S1(C-226T)locus of ghrelin gene.In the experiment,specific functional prediction was made on the 5 ’flanking region of the ghrelin gene,and real-time quantitative analysis of ghrelin genes of different genotype individuals was performed for the three genotypes CC,CT,and TT at the C-226T locus.Based on the effect of mutation on sequence function,the C-226T site was analyzed from the perspective of actual function.The results show:(1)The 5’ flanking region of the ghrelin gene contains a variety of important promoter elements,including core initiator elements(Inr),TATA box,CAAT box,GAGA sequence,CAC sequence and specificity Specificity protein 1(Sp1);5’ flanking region of ghrelin gene may contain Activator protein-2(AP2),Myogenic differentiation(MyoD),Octamer transcription factor 1(Oct-1),Activating transcription factor 1(ATF1),Zinc finger protein 217(ZNF 217)and serum response factor(SRF)binding site.In addition,a mutation in the TT genotype of C-226T will result in an additional Oct-1 transcription factor binding site in the 5’ flanking region of the ghrelin gene.(2)After 12 hours of fasting,the relative expression of ghrelin gene in individuals with TT genotype at the C-226T locus was 1.65 times that of CC genotype,and there was a significant difference between the two groups(P<0.05).The relative expression level of TT genotype individuals was 1.22 times that of CT genotype individuals(P>0.05),and the relative expression level of CT genotype individuals was 1.35 times that of CC genotype individuals(P>0.05).(3)Based on the above results,make the following inference:When the ghrelin gene C-226T site is expressed as the TT genotype,the promoter region increases the Oct-1 transcription factor binding site,which improves the binding efficiency of Oct-1,Which greatly enhances the activity of the ghrelin promoter and increases the expression of ghrelin,thereby stimulating the frequency of fish eating and causing significant growth traits such as weight;on the contrary,if the C-226T locus is CC genotype,Promoter activity,ghrelin expression,frequency of fish feeding,and growth traits were not improved.Therefore,the TT genotype of C-226T in the ghrelin gene has great potential for molecular marker development and can be used for molecular assisted breeding of tilapia individuals with excellent growth traits. |
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