Genetic Polymorphism Of TLR2 Gene And Correlation With Mastitis In Dairy Cattle

Mastitis is one of the most prevalent and complicated disease which causes huge economic losses to the dairy industry worldwide.Its pathogenesis is the invasion of the host by microorganisms,which causes inflammatory response of the body. The genetic correlations ranging from 0.60 to 0.80 between Somatic cell score(SCS) and mastitis. The incidence of mastitis can be significantly reduced by reducing somatic cells in milk,which enables indirect selection for resistance to mastitis. TLR2( Toll-like receptors 2) gene a member of Toll receptor-like protein gene family,which plays an important role in pathogen defence.In this study, we investigated the genetic polymorphism in exon 2 of TLR2 gene among Chinese Holstein(331), Luxi cattle(66)and Bohai Black cattle(30)using PCR-RFLP, CRS-RFLP methods and gene sequencing. The association were observed between genotypes or haplotypes and SCS in there restriction enzymes polymorphic sites in Chinese Holstein by SAS for GLM model.The results were as follows:Parital sequence of encode region and the 3′-untranslating region of TLR2 gene was amplified,sequencing result indicated,there are a mutation of G→C in position 2191 and a mutation of T→C in position 3008 of mRNA. Four SNPs (c.398G>A,c.651G>A, c.827A>G,c.2191C>G) in encode region and a SNP (c.3008T>C) in the 3′-untranslating region of TLR2 gene, corresponding to restriction endonuclease HaeⅢ,PstI,Hin1Ⅱ,Rsal和HhaⅠby PCR-RFLP and CRS-RFLP analysis.Three genotypes AA (90bp/ 21bp/19bp),AB(109bp/90bp/21bp/19bp) and BB(109bp/21bp) were detected in HaeⅢpolymorphism locus among the three bovine breeds,allele A was predominant. Luxi cattle with the highest the polymorphism information content (PIC) value.which was moderate polymorphism.Genotypic frequencies and allelic frequencies for the various polymorphisms at HaeⅢlocus were found to be significantly different among the three analyzed populations based on (χ2 = 58.90,df = 2,P<0.01) and (χ2 = 58.04,df = 4, P<0.01). Only two genotypes AA(221bp) and AB(221bp/138bp/93bp) were detected in PstI polymorphism locus among the three bovine breeds,allele A is predominant. There were no significant differences for genotypic and allelic frequencies at PstI locus among the three bovine breeds. Three genotypes AA(194bp),AB(194bp/172bp/ 22bp) and BB(172bp/22bp) were detected in Hin1Ⅱp olymorphism locus among the three bovine breeds, allele A in Chinese Holstein and Luxi cattle was predominant, while allele B in Bohai Black cattle is predominant.The PIC value ranging from 0.25 to 0.5 was moderate polymorphism among the three bovine breeds. Bohai Black cattle with the largest variation at Hin1Ⅱpolymorphism locus. Genotypic frequencies and allelic frequencies for the various polymorphisms at Hin1Ⅱlocus were found to be significantly different among the three analyzed populations based on (χ2= 71.56,df=2,P<0.01)and (χ2=73.94,df=4,P<0.01).Three genotypes AA (165 bp),AB(165bp/146bp/19bp) and BB(146bp/19bp) were detected in Rsal polymerphis- -m locus among the three bovine breeds, allele A in Chinese Holstein and Bohai Black cattle was predominant, while allele B in Luxi cattle is predominant.The PIC value ranging from 0 to 0.25 was low polymorphism in Chinese Holstein, while moderate polymorphism in Luxi cattle and Bohai Black cattle. Luxi cattle with the largest variation at Rsal polymorphism locus. Genotypic frequencies and allelic frequencies for the various polymorphisms at Rsal locus were found to be significantly different among the three analyzed populations based on (χ2= 316.404 ,df=2,P<0.01) and (χ2= 258.413,df=4,P<0.01).Three genotypes AA (444bp), AB (444 bp /426bp/18bp) and BB(426bp/18bp) were detected in HhaI polymorphis- -m locus in Luxi cattle and Chinese Holstein.but the genotype BB were not detected in Bohai Black cattle. allele A was predominant among the three bovine breeds. The PIC value ranging from 0.25 to 0.5 was moderate polymorphism in Luxi cattle, while low polymorphism in other two bovine breeds.Moreover, there were significant differencesfor genotypic frequencies allelic frequencies between Luxi cattle and in Chinese Holstein and Bohai Black cattle (P<0.01).Statistical result ofχ2 test indicated that five polymorphic sites reached Hardy-Weinberg equilibrium (P>0.05) except for HhaⅠpolymorphism in Luxi cattle.The association between the five polymorphism sites residing in second extron of TLR2 gene and SCS were analyzed by GLM model using SAS.The results showed only the genotypes of Hin1Ⅱhad significant effect on SCS trait among 331 Chinese Holstein. The SCS values for genotype BB were significantly lower than genotype AA and AB (P<0.01), The SCS values for genotype BB were lower than genotype AB (P<0.05). The average allele substitution effect of Hin1Ⅱfor SCS was 0.741, with allele A increasing SCS over allele B.Five TLR2 SNPs were used for haplotype reconstruction, which revealed seven possible haplotypes and nine possible haplotype combinations, The frequency of H1H1 is the highest(45.62%)and the SCS of H1H4 is the lowest(2.82).The correlation was detected between different haplotype combina- -tions and SCS.