| The PCR-SSCP method was used to analyse the Nramp1 gene(exon 2 and intron 6) andγ-IFN (all exons)gene polymorphisms in Hezuo pig.It aims to get molecular genetics information and provide the scientific basis for molecular marker of disease resistance of Hezuo pig.The results as following:Nramp1 gene exon 2,intron 6 andγ-IFN gene exon 4 appeared polymorphic.The result of PCR-SSCP polymorphism examination of the Nramp1 exon 2:Cloning and sequencing indicated that there were two polymorphic loci 62 T→C and 92 A→G and this mutation did not induce the changes of encoding amino acid.It was controlled by alleles gene of A and B,There were three genotypes,namely AA,BB and AB in Nramp1 exon 2.Frequency of A allele was 0.6519 and it was predominant.The result of PCR-SSCP polymorphism examination of the Nramp1 intron 6:Sequencing analysis showed that ten new nucleotide polymorphisms in intron 6,which were the single nucleotide substitution of C→T at the position of 99,225,304,307,313, A→G at the position of 105,C→A at the position of 190,T→G at the position of 298,T→C at the position of 306,and 295-296bp was detected one insertion:AA and CC genotype -→T,BB genotype -→G,There were five genotypes,namely AA,AB,BB,AC and CC,Allele B was predominant.Sequencing analysis showed that five nucleotide polymorphisms inγ-IFN exon 4,In which:5284bp A→G,5341bp G→A,5371bp A→G are new nucleotide polymorphisms but this mutation did not induce the changes of encoding amino acid.One single nucleotide mutation G→A at 5301bp and T→C at 5330bp resulted in two amino acid change Ser/Asn and Tyr/His,There were four genotypes AA,AB,AC and AD were detected,Allele A was predominant.Statistical results showed that the Nramp1 exon 2 SNPs sites in Hezuo pig was at Hardy-Weinberg disequilibrium(P<0.05),PIC was intermediate polymorphism;Nramp1 intron 6 SNPs sites in Hezuo pig was at Hardy-Weinberg equilibrium(P>0.05),PIC was high polymorphism;γ-IFN exon 4 SNPs sites in Hezuo pig was at Hardy-Weinberg equilibrium(P>0.05),and PIC was low polymorphism. |
