The Single Nucleotide Polymorphisms Of Chicken 70-kilodalton Heat Shock Protein (HSP70) Gene

The heat shock protein 70, which coded by the 70-kilodalton heat shock protein (HSP70) gene, is very important for the genesis of the heat tolerance of chicken. In the research of resistance breeding, HSP70 gene can be considered as the candidate loci of improving the sensibility of heat shock. But in present, researches about single nucleotide polymorphisms (SNPs) exist in chicken HSP70 gene are very limited. In this research, 591 individuals from 20 different Chinese native chicken breeds were studied. Denaturing High Performance Liquid Chromatography (DHPLC) technology was used to screen unknown SNPs existed in chicken HSP70 gene sequence. From every typical DHPLC peak, 2 individuals were selected to sequence. Homologous analysis was carried out based on the sequence information of samples that amplified from same primer to identity new unknown SNPs. From the compare of the typical DHPLC peak and sequence information, the genotype of every individual in each SNPs site was deduced. The genotype and gene frequency of every SNPs site in different chicken breeds were calculated and the difference exist among different breeds were tested. Average heterozygosity of different breeds, genetic diversity and its associate parameters, standard genetic distance (Ds) and genetic identity parameters were calculated to measure the genetic variation exist among and between different chicken breeds in HSP70 gene. Based on the genotype of every individual in each SNPs sites, the PHASE program, which compiled by the theory of most likelihood, was used to affirm the haplotype and the distribution frequency of every haplotype in different chicken breed was calculated.In the PCR products amplified by primer A5, A6, A7 and A4, 10 SNPs sites (A258G, C276G, C507T, C1040A, G1044A, C1431A, T1476C, G1500A, A1529G and C1722T) were detected. All of these SNPs exist in the code region of HSP70 gene. Especially, the mutation of C-A happened in 1040 site lead to a substitution of the amino acid histidine for proline at residue 347 and the mutation of A-G happened in 1529 site lead to a substitution of the amino acid arginine for lysine at residue 510 in HSP70.From the compare of the typical DHPLC peak and sequence results, the genotype of every individual in each SNPs site were confirmed. To those not sequenced individuals, the genotypes in each SNPs site were confirmed according to their DHPLC peak, samepeak mean same genotype. Thus the relationship between genotypes and DHPLC peak was formed.When consider the distribution frequency of genotype and gene in each SNPs site, significant difference (P<0.05) or highly significant difference (PO.01) of mutation gene among different chicken breeds were found in eight sites (A258G C276G C507T G1044A T1476C G1500A A1529G C1722T). But these frequency differences were not accordant with the difference of each chicken breed's geographic latitude. Generally speaking, in 3 to 4 sites, high frequency of mutation gene were found and significant difference (P<0.05) exist among breeds in 10 chicken breeds, they were Xinghua, Silkie (Taihe) , Chongren Partridge, Yugan sooty, Qingyuan Partridge, Huiyang Bearded, Silkie (Guangdong) , Taoyuan, Taibai and Henan game. In A1529G and C1722T, the mutation allele frequency in chicken breeds distributed in two southern provinces, Guangdong and Jiangxi, were significantly higher (P<0.05) or even highly significantly higher (P<0.01) than chicken breeds distributed in northern provinces.The average heterozygosity of every chicken breed in each SNPs site of the HSP70 gene were not high, the highest one was 0.2885 which exist in Huiyang Bearded and the smallest one was 0.1813 which exist in Lueyang Black. The gene divergence (Hj) of each SNPs site were different greatly, the highest HT was 0.4971 that exist in C276G site. The HT values in G1500A and A258G sites were also high; they were 0.4900 and 0.4387 respectively. The Gst values of these sites were with the same trend. They indicated that high variation exist in these sites, not only within but also among bre...