Screening And Identification Of Molecular Markers Related To Reprouductive Traits Of Holstein Cows

The fertility of the dairy cows has gradually declined world widely. At the same time, our country is facing the biggest challenge to overcome this problem. Over the last 50 years, robust economic interests in high yielding dairy cows ignored other reproductive traits and thus, reproductive performance of dairy cows continued to decline in China.BovineSNP50 (54K) is a chip that had 54 001 SNPs spread across the entire bovine genome and had been used in conducting Genome-wide Association Studies. Depending on the results,2 SNPs (rs108938226 and rs41771122 belong to RAD54L and RRM1 gene respectively) were selected to conduct the second-stage experiments. At the mean time, PPARy2 and GPX4 gene were selected as candidate gene to analyze the correlation between the polymorphism and fertility traits in 925 Holstein cows from 2 different herds. The main results were shown as follows:1. Rs 108938226 was a C/T mutation located in intron 3 of RAD54L. By CRS-PCR, it caused variation of the restriction enzyme cutting site Sac I when C to be replaced by T. There were three genotypes (CC, CT and TT) in the population. The genotype frequency of rs 108938226 in total samples was CC>CT>TT, while the allele frequency was C>T. Association analysis showed that mutation at rs 108938226 affected on days to first service after calving (P＜0.05), but it had no effect on other reproductive traits. The previous results of GWAS showed that the SNP’s polymorphism can affect days to conception after 2nd calving (P＜0.05), but there was not significant difference in the study.2.There was a G/C mutation in the region of 20 bp uPstream of rs 108938226, and this mutation creates a new restriction enzyme site for Pst I. The SNP to be confirmed is rs211565358. There were three genotypes (GG, GC and CC) and the genotype frequency of the SNP in total samples was GG>GC>CC, and the allele frequency was G>C. Association analysis showed that mutation at rs211565358 affected on days to first service after 1st calving, first calving age, days to conception and calving interval (P<0.05) and had no significant effect on other reproductive traits.3. Rs41771122 was a C/T mutation located in intron 4 of RRM1, created a new restriction enzyme site for Dra I. There were three genotypes (CC,CT and TT) in the population. The genotype frequency of rs41771122 was CO＞CT>TT. The distribution of TT genotype was low in the two herds while the frequency of C allele was higher than T. Association analysis showed that rs41771122 had no significant effect on all reproductive traits in the study. Although rs41771122 had no significant effect on days to first service after 1st calving as the previous results of GWAS, the result that TT genotype was better than others was still consistent with the previous results of GWAS.4. There was a G/A mutation in the region of 137 bp uPstream of rs41771122, and this mutation created a new restriction enzyme site for HinfⅠ. To be confirmed, the SNP was rs211565358. there were three genotypes (GG, GA and AA) in the population. The genotype frequency of rs211565358 was GG>GA>AA, moreover, the number of AA genotypes was very low in the two herds while the allele frequency was G>A. Association analysis showed that rs211565358 had no significant effect on all reproductive traits in the study.5. A mutation located in intron 4 (T348G) of PPARy2 was found, and there were three genotypes (TT, TG and GG) in the population. The genotype frequency of the gene ranked as TT>TG>GG while the number of GG genotype was found lowest in the two herds and the allele frequency was T>G. Association analysis showed that this mutation had significant effect on services per conception for all parities, days to first service after 1st calving, days to conception after 1st calving and calving interval from 1st to 2nd calving (P<0.05). And in other traits, the mutation had no significant effect, but the TG genotype’s phenotypic value of the five reproductive traits all are smaller than other genotype. So the result indicated that the SNP may provide a slight effect with the reproductive traits of Holstein cows. The study detected all the other exons and partial intron region of PPARy2 gene, but no mutation was found.6. The study detected all exons and partial intron region of GPX4 gene and no mutation was found. Comparing with the sequences of other species, the results suggested that GPX4 gene was highly conserved in dairy cows.