| To explore the regulatory mechanism of tilapia's cold tolerance traits and select the molecular markers related to tilapia's cold tolerance traits,GIFT tilapia fry from a national-level tilapia breeding farm of Nanning,Guangxi is studied by selection in low temperature stress,PCR-SSCP testing,DNA sequencing and correlation analysis;the genotype frequencies,number of alleles,allele frequencies and Hardy-Weinberg balance of polymorphism samples are recorded;the correlation between the genotype and cold tolerance traits of polymorphism samples is analyzed with chi-square test;the correlation between single nucleotide polymorphism(SNP)and cold tolerance traits is preliminarily discussed and the potential SNP loci relating to tilapia's cold tolerance are selected.The results are as follows:1.2000 GIFT tilapia fry with strong build,5.5g±0.5g in weight and 6.5cm±0.5cm in length are selected and divided into four experime±ntal groups(500 each)A,B,C and D for low temperature stress experiment.The stress temperatures for the four experimental groups fall respecti,vely to 8?,10?,12? and 14? and then remain stable.Select 16 samples of low temperature intolerance and 16 of low temperature tolerance from each group to study the correlation between the genotype and cold tolerance traits of tilapia polymorphism samples in different low temperature stresses.Then 21 pairs of specific primers designed with the sequence of the five cold resistance related candidate genes of tilapia preliminarily selected are used to carry out PCR-SSCP testing of the DNA of the samples from each experimental group.The result shows that,in gene HSP70,the first pair of primers HP70-1 show belt-type polymorphism in the electrophoretogram of samples at the final constant temperature of 8?,10? and 12?;the fourth pair of primers HP70-4 do in the electrophoretogram of samples at the final constant temperature of 8?,the sixth pair of primers HP70-6 in the electrophoretogram of samples at the final constant temperature of 8?,10?,12? and 14? and the thirteenth pair of primers TCP1-4 of gene TCP-1-eta in the electrophoretogram of samples at the final constant temperature of 8?,12? and 14?.2.The analysis of correlation between mutational locus gene polymorphism and cold tolerance traits show that the locus of the first pair of primers HP70-1 at the final constant temperature of 10? and the loci of the sixth pair of primers HP70-6 at the final constant temperature of 8? and 14? in gene HSP70 as well as the loci of the thirteenth pair of primers TCP 1-4 at the final constant temperature of 8?,12? and 14? in gene TCP-1-eta are all at Hardy-Weinberg balance in both the low temperature intolerance group and the low temperature tolerance group.Except the distribution of genotypes AA and AC at the loci of the first pair of primers HP70-1 at the final constant temperature of 10? in gene HSP70 and genotypes AA and AB at the loci of the thirteenth pair of primers TCP 1-4 at the final constant temperature of 8?,12? and 14? in gene TCP-1-eta,the distribution of genotypes of all mutational loci at Hardy-Weinberg balance has significant correlation with cold tolerance traits.It is preliminarily speculated that genotype AA at the mutational loci is the dominant genotype in the low temperature intolerance group and genotypes AB and AC are dominant genotype in the low temperature tolerance group,which have some influence on the cold tolerance traits of tilapia.Genes HSP70 and TCP-1-eta can be inferred to be the potential cold tolerance-related genes of tilapia.3.Comparative analysis of the sequencing results shows that,among the cold tolerance-related candidate genes,35 SPN loci are found in gene HSP70 and 8 SNP loci are found in gene TCP-l-eta.A total of 43 SNP loci can be initially considered as the cold tolerance-related candidate SNP loci of tilapia.Among them,11 SNP loci are in the 8?experimental group,4 SNP loci in the 10 ? experimental group,18 SNP loci in the 12 ?experimental group and 10 SNP loci in the 14? experimental group.4.The analysis of correlation between SNP and cold tolerance traits show that haplotype GC and CA in the domain of one linkage disequilibrium monomer formed by 11 SNP loci of the 8? experimental group have no significant correlation with tilapia's cold tolerance traits and the polymorphism of six SNP loci has significant correlation with tilapia's cold tolerance traits;the haplotype CCG and TGA in the domain of one linkage disequilibrium monomer formed by 4 SNP loci of the 10? experimental group have significant correlation with tilapia's cold tolerance traits and the polymorphism of three SNP loci has significant correlation with tilapia's cold tolerance traits;the haplotype TT and CC,AT and GC in the domain of two linkage disequilibrium monomers formed by 18 SNP loci of the 12? experimental group have no significant correlation with tilapia's cold tolerance traits and the polymorphism of one SNP locus has significant correlation with tilapia's cold tolerance traits;the haplotype TT and CC in the domain of one linkage disequilibrium monomer formed by 10 SNP loci of the 14? experimental group have significant correlation with tilapia's cold tolerance traits and the polymorphism of six SNP loci has significant correlation with tilapia's cold tolerance.In this study,two potential cold tolerance-related genes of tilapia are inferred and 16 SNP loci significantly correlated with tilapia's cold tolerance traits are selected.It has provided molecular genetics reference for solving the problem of tilapia's safe overwintering,selective breeding of cold-tolerant tilapia varieties,achieving the optimal combination of tilapia's important economic characters and good genes as well as enhancing China's tilapia breeding technique system. |
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