Targeted Resequencing Of GWAS Loci Reveals Novel Functional Genes For Milk Production Traits

Dairy industry is one of the most important parts of animal husbandry which represents the advancement of agriculture. As the most important economic trait of cattle, milk production trait, including milk yield, milk fat yield, milk protein yield, milk fat percentage and milk protein percentage is a research hotspot of molecular breeding. Seeking and verifying the functional genes of milk production traits is very important. We have conducted Genome-wide association study (GWAS) of milk production traits in Chinese Holstein cattle population, and indentified105genome-wide significant loci which affected milk production traits in dairy cattle.In the study, we obtained127218SNPs in the significant regions of GWAS by high-throughput pooling sequencing,735of which are located in the coding region and418are located in the UTR region. Through bioinformatics analysis and screening, we selected200SNPs for further research. The experimental population contained734cattle from17bull families in Beijing and15bull families in Shanghai. The SNPs were genotyped by Sequenom MassARRAY iPLEX genotyping technology. Then we analyzed the association between the5milk production traits and the SNPs using mixed model based single locus regression analyses. A total of40significant SNPs located in33genes were identified,17of which are consistent with the results of our previous GWAS.7genes which affected all the five milk production traits are DGAT1, HEATR7A, VPS28, CPSF1, PPP1R16A, GPT and LOC509113.Of the40significant SNPs,35are located on BTA14. In order to increase the statistical power, we use the BEAGLE software to infer the genotypes of another1917China Holstein cattle which are half-sib sisters of the genotyped734cows. After analyzing the combined population, we found66significant SNPs distributed in53genes. There are38SNPs overlaped with our first association study, and31genes are including in the first association study results. Of the66significant SNPs,49were on BTA14,2were on BTA1,2were on BTA3,2were on BTA11and11were on BTA20.We conducted real-time quantitative PCR of20genes randomly chosen from the31genes which are significantly effected milk production traits in both results of the association study. The samples are8tissue (heart, liver, lung, kidney, mammary gland, ovarian, uterine and muscle) from4Chinese Holstein cattle during lactating. The mRNA expression level of mammary gland is much higher than the other seven tissues in15genes. The most two highly expressed gene in mammary gland are ribosomal protein L8(RPL8) and eukaryotic elongation factor gene1D (EEF1D). Western blot was used to verify the function of these two genes. The results are similar to the RT-PCR. By detecting the methylation level of CpG island in the UTR region of EEF1D, we found that the methylation level is the lowest in mammary gland. This suggested that the expression of EEF1D is regulated by DNA methylation. The results of the promoter activity analysis experiment of the significant SNPs in EEF1D showed that the promoter activity of wild type is much higher than mutate type. EMSA experiment was employed to analyze the significant SNPs of RPL8and showed that the mutant probe can combine with more transcription factors compared with the wild probe. The results showed that EEFID and RPL8gene may be potential candidate genes which affect milk production traits in Chinese Holstein cattle. The expression level of EEFID might be regulated by DNA methylation and SNP located in the UTR region, the SNP located in the promoter region of RPL8may be a functional variant which affected milk production traits in dairy cattle.