Sequence Analysis Of Partial Nramp1 Gene And The Association With Mastitis In Chinese Holstein Cattle

In this study,4 primer pairs were designed to amplify exon9,exon10 and exon11 of cattle natural resistance associated macrophage protein 1(Nramp1) gene. Polymorphismes of each fragment were detected by PCR-SSCP method for the first time.The correlation between the polymorphisms of cattle Nramp1 gene and mastitis in 344 Chinese Holstain cattle and the integrality of Nramp1 gene were analyzed.Results were as follows:Three SNPs,which located at SNP+7012,SNP+7401 and SNP+7455,were detected in exon9,exon10 and exon11 respectively.SNP+7012 locous resided in intron9,and the other SNPs(SNP+7401 and SNP+7455) loci in exon11.No SNPs loci were detected in amplified fragment for primer 1(exon9).Three genotypes named as MM,MN and NN were detected by PCR-SSCP method. Genotype frequencies were 0.756,0.198 and 0.046 respectively.The genotype distribution is in the station of Hardy-Weinberg equilibrium(P＞0.05).Two alleles were named as M and N whose frequencies were 0.854 and 0.146.The allele M was the dominant allele.The polymorphism information content(PIC) was 0.2183 which showed that the genetic diversity was with lower polymorphism(PIC＜0.25).One SNP site named 7012(C/T) was detected in intron9 by sequencing analysis(GenBank accession no:EU281620).Three genotypes,named as AA,AB and BB,were detected by PCR-SSCP method. Genotype frequencies were 0.610,0.314 and 0.076,respectively.The genotype distribution kept in Hardy-Weinberg equilibrium(P＞0.05).Two alleles were named as A and B whose frequency was 0.767 and 0.233.The allele A was the dominant allele. The polymorphism information content(PIC) was 0.2942 which showed that the genetic diversity was lower potymorphism(0.25＜PIC＜0.05).Two SNPs named 7401 (G/C) and 7455(A/C),which resulted in Pro356Ala and Leu374Met substitution, respectively,were detected in exon 10 by sequencing analysis.Partial sequence of intron12 was aquired by clone sequencing,and had been submitted to NCBI database(GenBank accession no:EF682067).The correlation between the polymorphisms of cattle Nramp1 gene and cattle mastitis was analyzed by GLM(General Linear Means,GLM) analysis.Our results showed that farms effect and season effect were significant prior to those of SCS (P＜0.01),Litter effect was prior ro those of SCS(P＜0.05),and genotype effect was not prior to SCS(P＞0.05).