| Heat stress in dairy cows refers to the sum of non-specific responses of dairy cows to high temperature environments that are not conducive to physiological activities.High temperature acts on the hypothalamicpituitary-adrenal axis,the hypothalamic-pituitary-thyroid axis,and the hypothalamic-pituitary-gonadal axis,resulting in changes in the body’s homeostasis,which in turn leads to a series of physiological changes.Studies in recent years have shown that heat stress has a particularly serious impact on reproduction.It is reported that under the condition of consistent breeds,breeding techniques and feeding conditions,the conception rate of dairy cows in estrus in spring and autumn is about 60%,while heat stress in summer is about 60%.The impact of the stimulus dropped to 44%.Therefore,a comprehensive and in-depth study of the relationship between heat stress response and reproductive traits of dairy cows,and screening of stressresistant genetic markers for reproductive traits of dairy cows under heat stress conditions is of great significance for reducing economic losses caused by heat stress and cultivating heat-resistant breeds.In this experiment,the research group used the results of genome-wide association analysis(GWAS)on the stress resistance of Chinese Holstein reproductive traits at the individual level in the early stage,and obtained candidate genes related to heat stress response for large group verification and screening for molecular markers.Candidate genes are RAD51,PCNA,PLCB1,FOXL2,PLCD4,CLVS1,LOC112449637 and PRND,respectively.In this study,three DNA pools were constructed using the semen DNA of 60 unrelated Chinese Holstein bulls,and the 2000 bp upstream regulatory regions of eight genes including RAD51,PCNA,PLCB1,FOXL2,PLCD4,CLVS1,LOC112449637 and PRND were analyzed by sanger sequencing.,downstream 1000 bp and all coding regions were detected by SNP,and 1060 Chinese Holstein cows were individually genotyped by KASP technology.Finally,28 SNPs of 8 genes were grouped for genotyping.The results of single-marker association analysis showed that 8 loci located in FOXL2,PLCB1,PLCD4,RAD51,LOC112449637 genes,including SNP4,SNP5,SNP6,SNP7,SNP10,SNP13,SNP27,SNP28,were significantly correlated with reproductive traits stress resistance(P< 0.05).Based on pig ovary transcriptome data,we found that FOXL2,PLCB1,RAD51,PCNA and other four genes were significantly down-regulated under heat stress(P<0.05).Therefore,we further explored the possible regulatory methods of SNP4 and SNP5 located upstream of FOXL2 gene,SNP7 located in the 3’UTR region of RAD51 transcript,and SNP10 located in exon 10 of PLCB1 gene.Affect the upstream binding of transcription factors MNT,NFIX and FOXL2 gene,which leads to changes in gene expression.Correlation analysis found that FOXL2 gene was significantly correlated with MNT and NFIX gene expression;SNP7 was located in the RAD51 gene transcript and bta-mi R-132 seed In the sequence binding region,different genotypes have different binding abilities between m RNA and mi RNA;using the previous transcriptome data of the research group,SNP calling of 234 cattle found that SNP10 was significantly correlated with PLCB1 gene expression(P<0.05),which may be used as a quantitative Trait loci come into play.In conclusion,this study revealed that there are abundant genetic variations in stress resistance-related genes of reproductive traits in Chinese Holstein cattle under heat stress conditions.The stress resistancerelated molecular markers of reproductive traits in Holstein cattle lay a foundation for revealing the genetic mechanism of heat stress in dairy cattle,and provide a theoretical basis for the molecular breeding of heat stress resistance in Chinese Holstein cattle. |
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