| As seasonal estrous specie,the estrous activity of Sheep(Ovis aries)starts in the autumn with gradually shortened light,and ends at the turn of winter and spring,which is considered as a typical rhythmic biological activity.The circadian rhythm(Clock)have been proved important in most of the rhythmic biological activities and regulated by a variety of genes(Clock genes)with rhythmic expression characteristics.To date,several canonical circadian Clock genes have been discovered including Clock,Bmall,Cry1/2,Per1/2/3,etc.Previous researches reveal that these genes are crucial to the regulation of the seasonal estrous in mammals,but the detailed mechanism of Clock in most of the mammals especially sheep is still largely unknown.To reveal the relationship between circadian Clock genes and the seasonal estrous of sheep,firstly,Sequenom MassARRAY(?)SNP was applied to genotype 6 single nucleotide polymorphism sites(SNPs)of Clock,Cry1 and Cry2 in year-round estrous rams(Small Tail Han sheep,Hu sheep,and Cele Black sheep)and seasonal estrous rams(Tan sheep,Sunite sheep,and Tibetan sheep),then the correction was calculated between the SNPs and the litter size of Small Tail Han sheep.Secondly,based on the resequencing data of previous study,2 different decision trees machine learning methods(Random Forest and XGBoost)were Performed to analysis the relationship between 21 SNPs of Clock、Cry1/2、Per 1/2/3 and the litter size of Small Tail Han sheep.At last,qPCR was applied to investigate the expression profile of Clock、Bmal1、Cry 1/2 and Per 1/2 in different tissues of HPG axis(brain,cerebellum,hypothalamus,pituitary,testis,epididymis,vas deferens and adrenal gland)in Small Tail Han Ram and Sunite Ram,the main results were as follows:1.Population genetic analysis showed that both genotype frequency and allele frequency were extremely significant different between year-round estrous and seasonal estrous sheep breeds(P<0.01)in all SNPs except for the g.175357583C>T locus of Cry1,all 6 SNPs were at moderate polymorphism(0.25<PIC<0.5)and were under Hardy-Weinberg equilibrium(P>0.05)in most of the sheep populations.Association analysis indicated that there was no significant correlation between the 6 SNPs and the litter size of Small Tail Han sheep(P>0.05).2.The analysis of the decision tree machine learning methods showed that,the minimum prediction error of Random Forest was obtained when Ntree is 200,000 and mtry is 0.1,the minimum prediction error of XGBoost was obtained when Ntree is when nround is 2,000 and colsample is 0.8.The results of Random Forest showed that:the top 5 ranked loci were g.175355119T>C locus of Cry1,g.2852655 T>C of Per2,g.43677274 T>C of Per3,g.27346502 A>G locus of Per1 and g.175357583C>T locus of Cry1.The results of XGBoost showed that:the top 5 ranked loci wereg.175355119T>C locus of Cry1,g.2852655 T>C locus of Per2,g.70875941C>T locus of Clock,g.27346502 A>G locus of Per1 and g.70873128G>A locus of Clock g.175355119T>C locus of Cry1 ranked first in the analysis of both two methods.3.Clock,Bmal1,Cry1/2 and Per 1/2 were detected in all 8 tissues of Small Tail Han Ram and Sunite Ram.The expression of Clock in hypothalamus,pituitary and epididymis in Small Tail Han Ram were significantly higher than that in Sunite Ram(0.01<P<0.05);the expression of Bmall in the brain,cerebellum,hypothalamus,pituitary,testis and epididymis in Small Tail Han Ram were extremely significant higher than that in Sunite Ram(P<0.01);the expression of Cry1 in the pituitary,testis,and adrenal gland in Small Tail Han Ram were significant lower than that in Sunite Ram(0.01<P<0.05);the expression of Cry2 in the brain,cerebellum,hypothalamus,pituitary and adrenal gland in Small Tail Han Ram were significant higher than that in Sunite Ram(0.01<P<0.05);the expression of Per1 in pituitary and epididymis in Small Tail Han Ram were extremely significant lower than that in Sunite Ram(P<0.01);the expression of Per2 in brain,hypothalamus,pituitary,testis and epididymis in Small Tail Han Ram were significant lower than that in Sunite Ram(0.01<P<0.05);the expression of Per2 in the vas deferens and adrenal gland were significant higher than that in Sunite Ram(0.01<P<0.05).In general,the SNPs of Clock,Cry1/2 genes were genotyped in year-round and seasonal estrous sheep breeds and population genetic analysis were Performed.Based on this,2 decision tree machine learning methods was applied to investigated the relationship between 21 SNPs of Clock,Cry1/2,Per 1/2/3 and the litter size of Small Tail Han sheep in which found that there may be a certain effect between g.175355119T>C locus of Cry1 and the litter size of Small Tail Han sheep.Furthermore,tissue expression pattern of Clock,Bmall,Cry 1/2,Per 1/2 in Small Tail Han Ram and Sunite Ram was investigated,which can path the way for the in-depth research on seasonal estrus of sheep. |
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