| Mastitis is known as the most common inflammatory disease of economic and animal welfare concern in dairy animals. Selection of dairy cattle with genetic resistence to mastitis has been the prime concern of animal breeders in order to lower the risk of this economically important disease. The candidate gene approach is considered to be a powerful method and is predicted to discover a single nucleotide polymorphism (SNP) even with a minor effect provided that the putative gene is a true causative gene. The present study recruited5genes elated to the JAK-STAT signaling pathway (JAK2, STAT5A, STAT5B, CD4and LAGS3). The present research used an integrated approach with the following objectives i.e.1) to analyze the association of SNPs in these genes on serum cytokines, mastitis and production traits in Chinese Holstein cattle,2) to analyze gene expression in different genotypes and in different groups of cattle i.e. healthy control and mastitis in the study genes, and3) to evaluate the DNA methylation level in mastitis and healthy cows in the genes under study, in order to develop effective genetic markers for selection of cows with genetic resistance to mastitis development. Initially pool DNA sequencing revealed totally60SNPs, of which27SNPs were selected for association study in a population of468Holstein dairy cattle, followed by mRNA expression evaluation of the significant SNPs and the DNA methylation evaluation in the CpG island in promoter region of the study genes. Brief achievements of the study are mentioned below.1) JAK2:All of the identified5SNPs of JAK2gene were novel. Amongst these5SNPs, the first two SNPs (SNP1and SNP2) were located in the exon16and20. These two SNPs were missense that was predicted to change the amino acid from isoleucine to valine and from lysine to asparagine, respectively. The other3SNPs (SNP3, SNP4and SNP5) were located in3’flanking region. Association study results showed that all of these five SNPs were significantly associated with serum cytokines and mastitis traits. The results of mRNA expression were consistent with association study results, the genotype of SNPs associated with higher SCC/SCS showed higher mRNA expression level. As for haplotype analysis,4haplotypes were revealed amongst the5SNPs in JAK2gene. The DNA methylation evaluation of JAK2promoter revealed higher methylation level of CpG sites and lower gene expression in healthy control compared with the mastitis cows. DNA methylation was negatively correlated with gene expression in JAK2gene. Six active transcription factors were identified on the CpG sites in the promoter region of JAK2.2) STAT5A:Two SNPs (SNP6A43046497C and SNP7G43047829A) were revealed in the intron9of STAT5A gene. Of these2SNPs, SNP6was found to be associated with IL-6. The wild type AA genotypes showed higher level of IL-6compared to the other genotypes. The mRNA expression of the AA genotypes was significantly higher than the other genotypes. The2SNPs showed two haplotypes and were in LD. The DNA methylation results showed that the promoter of STAT5A CPG sites were hyper methylated and showed significantly higher gene expression in healthy control compared with mastitis cows. Two active transcription factors were identified on the CpG sites in the promoter region of STAT5A.3) STAT5B:Total three SNPs (SNP8G43047829A, SNP9A43673888G, and SNP10T43660093C) were identified in STAT5B gene. Among these3SNPs, SNP8was already known SNP and was located in intron4, whereas, the other two SNPS (SNP9and SNP10) were novel and located on exon16and exon19, respectively. The first2SNPs (SNP8and SNP9) were significantly associated with serum cytokines and mastitis traits. The wild type AA genotype of SNP8was associated with lower level of SCC, SCS and IL-4than the AG and GG genotypes. The results of mRNA expression level of the SNP8were in agreement with the association study results.4) CD4:CD4gene revealed a number of SNPs. Totally,14SNPs in CD4gene were studies in the present study. Of these14SNPs,11SNPs (from SNP14to SNP24), were located in intron and exon of the CD4and the other3SNPs (SNP11-SNP14) were in2kb promoter region. The11SNPs (SNP14to SNP24) showed2haplotypes and were in strong LD. The results of association study showed that the11SNPs in LD (SNP14-SNP24) were significantly associated with fat%, whereas, SNP12was associated with SCC. The DNA methylation evaluation showed that the CpG sites of CD4promoter region had higher methylation and lower mRNA expression in mastitis cows compared to the healthy controls. These results suggest that DNA methylation was negatively correlated with gene expression in CD4gene. Four active transcription factors were identified on the CpG sites in the promoter region of JAK2. 5) LAG3:Three SNPs were identified in LAG3gene, including SNP25(A104032765C) located in exon4, and SNP26(C104028410T) and SNP27(C104028580A) located in3’flanking region. All of these three SNPs were novel. SNP25was missense that was predicted to cause amino acid substitution from threonine to proline. The results of association study showed that SNP26and SNP27were significantly associated with serum cytokines and production traits. The CpG island in promoter region of LAG3was unmethylated therefore, no further analysis were performed for LAG3promoter methylation.6) Integrated analysis of association, mRNA expression and DNA methylation study:We integrated the three projects and did grand analysis in order to get a broader picture of the effect of study genes on mastitis resistance traits. SNP1and SNP2in JAK2gene, SNP6in STAT5A and the11SNPs (SNP14-SNP24) in LD in CD4gene showed significant effect on mRNA expression and DNA methylation. Moreover, the effects of SNP and DNA methylation integration of the above SNPs were also significant on mastitis related traits. As for correlation analysis, the DNA methylation levels of CD4showed significantly higher positive correlation with mastitis traits.In conclusion, the results of association study, gene expression, methylation analysis and the integration of these three projects data analysis strongly qualifies JAK2, STAT5A/B, CD4and LAG3to be used as functional candidate genes in mastitis resistance studies. SNP1and SNP2in JAK2, SNP6in STAT5A and11SNPs (SNP14-24) in LD in CD4gene were significant on all criteria i.e. association study, mRNA expression and the DNA methylation analysis, which suggest that all these SNPs could be powefull functional genetic markers against mastitis development. The Aberrant DNA methylation level between healthy control and mastitis in CD4, JAK2and STAT5A could be used as epigenetic markers of mastitis resistance. |
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