| Mastitis is the most common and harmful disease in dairy cows.Except traditional treatment with antibiotic,using molecular biology method becomes a new way to improve the incidence of mastitis by cultivating high mastitis-resistant cows.Therefore,this study is going to select candidate genes for mastitis resistance,in order to establish a foundation for the cultivation of high mastitis-resistant cows.In this study,a total of 980 Holstein cows' blood samples were collected from 6 large and medium-sized dairy farms in Jiangsu province,and the DHI production performance measurement records of the sampled cows were collected.After extracting DNA from them,then selected 3 genes' promoter regions and part of coding regions' SNP mutations including interleukin enhancer binding factor 3(ILF3),interleukin 17A(IL17A)and cadherin 13(CDH13)in 20 cows with the method of direct sequencing,then detected the type of the SNP by time-of-flight mass spectrometry,The multivariate analysis of variance was used to analyze the relationship between these 3 genes' SNPs and 4 traits including somatic cell score,milk fat percentage,milk protein percentage and tested day milk yield.Expecting to provide a reference for the molecular marker selection of Chinese Holstein cow's mastitis resistance.The main results of the study are as follows:1.A total of 6 SNP loci were found in the promoter region of ILF3 gene,and 3 SNP loci were found in the coding region.5 linkage groups were selected by linkage disequilibrium analysis.1 SNP locus was selected from each of the 5 linkage groups for further study,they were promoter region-1487T>G,-774C>T and coding region 12647T>C,12746T>C,15214C>T.The dominant genotypes of these 5 SNP loci were TT,TT,CT,TT,CT,The dominant allelic genes were T,T,C,T,C respectively.A total of 9 haplotypes were generated at these 5 SNP loci,the highest frequency of haplotype was TTCCC.Analysis of variance showed that:the effect of-774C>T locus on milk protein percentage reached a significant level(P<0.05).The milk protein percentage of TT type cows were significantly higher than CC type and TC type.The effect of 12647T>C locus on tested day milk yield,milk fat percentage and SCS reached a significant level(P<0.05).The tested day milk yield of CC type individuals were significantly higher than CT type and TT type;CT type individuals had significantly higher milk fat percentage than CC type individuals;CC type individuals had extremely significant higher SCS than CT type,and CT type cows were extremely significant higher than TT type.The 15214C>T locus only had an extremely significant effect on SCS(P<0.01),and the SCS of CT and TT genotype individuals were extremely significant higher than CC type individuals.The effect of 12746T>C locus on tested day milk yield and milk protein percentage reached a significant level(P<0.05).The tested day milk yield of CC type cows were significantly higher than that of CT type,meanwhile CT type cows were significantly higher than TT type;the milk protein percentage of CC type cows were extremely significant higher than that of TT type.The-1487T>G locus did not have significant effect on the above 4 traits(P>0.05).There were 6 genotype combinations between 12647T>C locus and 15214C>T locus.The CCCC type cows had significantly higher tested day milk yield than the other 4 genotype cows except CCCT type.Meanwhile CCCT type cows were significantly higher than CTCT type and TTCC type cows(P<0.05);the milk protein percentage of CCCC type cows was extremely significant higher than that of other 4 genotype cows except CTCC(P<0.01);CCCT type individuals had extremely significant higher SCS than CCCC type individuals,CCCC type individuals were extremely significant higher than the CTCC and TTCC individuals in SCS.In addition,the SCS of CCTT and CTCT individuals were extremely significant higher than that of CTCC and TTCC individuals(P<0.01)2.There were 3 SNP loci in the promoter region of IL17A gene,which were-398T>A,-1578A>G and-1835G>A,and there were 2 SNP loci in the 3'UTR,which were 3164T>C and 3409G>C.The dominant genotypes of these 5 SNP loci were AT,AG,GG,CT,GG,the dominant allelic genes were A,G,G,C,G respectively.A total of 19 haplotypes were generated at these 5 SNP loci,the highest frequency of haplotype was GGACC.Analysis of variance showed that:the-1578A>G locus had a significant effect on the tested day milk yield and SCS of Holstein cattle(P<0.05).The tested day milk yield of GA type cows were extremely significant higher than that of AA type;the SCS of AA type cows were significantly higher than that of GA and GG type cows.All these 3 loci-398T>A?3164T>C and 3409G>C had an extremely significant effect on tested day milk yield(P<0.01).The tested day milk yield of AA cows were extremely significant higher than that of TT type;The tested day milk yield of CC and TC type cows were extremely significant higher than TT;CC type individuals had extremely significant higher tested day milk yield than GG type individuals.The-1835 G>A locus did not have significant effect on the above 4 traits(P>0.05).3.A total of 3 SNPs-2482G>C,-837G>A,-368C>G were found in the CDH13 gene,all of which were located in the promoter region.The dominant genotypes of these 3 SNP loci were GG,GG,CC,the dominant allelic genes were G,G,and C respectively.A total of 4 haplotypes were generated at these 3 SNP loci,the highest frequency of haplotype was GCG.Analysis of variance showed that:the-2482G>C locus had significant effects on all of these 4 traits(P<0.05).The tested day milk yield,milk protein percentage of CG and GG genotype cows were extremely significant higher than CC type.The milk fat percentage of CG type cows were extremely significant higher than that of GG type;the SCS of CC type cows were significantly higher than GG type.The-368C>G locus had a significant effect on tested day milk yield and milk protein percentage(P<0.05).The CC and GC type cows had significantly higher tested day milk yield than GG type;CC type cows had extremely significant higher milk protein percentage than GC type.The-837G>A locus had a significant effect on tested day milk yield,milk fat percentage and milk protein percentage(P<0.05),the tested day milk yield of GG type cows were significantly higher than GA type,while the milk fat percentage and milk protein percentage of GA type cows were extremely significant higher than GG type.4.The 12647T>C locus and 15214C>T locus of ILF3 gene had extremely significant effects on SCS(P<0.01).Their genotype combination of CTCC and TTCC individuals had extremely significant lower SCS than the other genotype individuals(P<0.01).The SCS of GA and GG type individuals from IL17A gene-1578A>G locus and GG type individuals from CDH13 gene-2482G>C locus were significantly lower than others(P<0.05),which could be used as a candidate gene of cow mastitis resistance for further study. |
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