Selection Signature Analysis And Gene Function Verification For Ketosis Resistance In Chinese Holstein Cattle

Ketosis is a polygenic controlled metabolic disease with a high incidence in Chinese Holstein cattle,which not only causes damage to the physical condition of cows,but also causes certain economic losses to the livestock breeding enterprise.Ketosis is a low heritability trait.Therefore,genotype imputation technology was used through the two-step genotype imputation method of Fimpute and Beagle software.Treating the 150 K gene chip of 190 Chinese Holstein cattle as a reference panel,the 50 K gene chip of 2488 Chinese Holstein cattle was imputed in the NAGRP database.1.Taking cows suffering from ketosis or healthy binary traits as phenotypes,a genome-wide association analysis was performed on 115787 markers of 2678 individuals after genotype imputation by the universal linear model of GEMMA software.A total of 25 ketosis-related sites were scanned.And candidate genes were sifted around these sites,including genes such as ALDH1L2,ACAD10,and AK3.At the same time,using the ?-hydroxybutyric acid content in blood of cow as a phenotype,a genome-wide association analysis was performed on the 140668 markers of 190 cows with the multiple regression model of Plink software.A total of 191 significant sites were screened,and there were 13 candidate genes related to ketosis,including ACADM,PECR,FN1 and other genes.2.In order to screen out the positive selection signature of high ketosis resistance during the artificial selection process,ketosis-infected and healthy cows groups were regarded as reference and experimental population,respectively.Three methods suitable for case-control positive selection signature analysis were used including Fst,XPEHH and XPCLR method with the significant threshold of the selection signature top 1%.A total of 122 positive selection regions related to ketosis were screened by Fst method.A total of 870 positive selection significant sites were detected by XPEHH method.There were 1253 positive selection signature regions related to ketosis screened by XPCLR method.And 81 candidate genes related to ketosis were co-localized by three methods.GO and KEGG functional enrichment analysis showed that these genes were mainly concentrated in metabolic pathways such as glucose metabolism,fatty acid metabolism and tricarboxylic acid cycle.Candidate genes scanned at least by two methods were closely related to ketone metabolism,including ACAA2,ACADS,ACSL3,ACSL6,APOA1,HMGCL,IDH3 A,PC,PCK1.3.Functional verification was performed on the APOA1 gene scanned by this study and CPT1 A gene reported in the literature.SNP(g.-572 A>G)in the promoter region of APOA1 gene and SNP(g.-108 G>T)in the promoter region of CPT1 A gene were significantly correlated with ketosis by sequencing and correlation analysis(P <0.05).The qRT-PCR showed that the expression level of APOA1 and CPT1 A genes in ketosis cattle was higher than that in healthy cattle(P <0.05).The double luciferase report experiment showed that the promoter activity of mutant(GG)individuals located in the core promoter region of APOA1 gene was significantly higher than that of wild type(AA)individuals(P <0.05).The promoter activity of mutant(TT)individuals located in the core promoter region of CPT1 A gene was significantly higher than that of wild type(GG)individuals(P <0.05).Differences in the activity of the APOA1 gene and CPT1 A gene promoters of individuals with different genotypes lead to different gene expression levels,which in turn affects the occurrence of ketosis.In summary,the SNP on the APOA1 gene(g.-572 A>G)and the SNP on the CPT1 A gene(g.-108 G>T)can be used as genetic markers for cow ketosis resistance.