Expression Of Seasonal Estrus-related Gene And Its Association Analysis Between Their Polymorphism And Reproductive Traits In Sheep

Most of sheep are seasonal estrus breeds,but only a few are year-round estrus breeds in China.Excavating key genes related to seasonal estrus in sheep has become a prerequisite for regulating seasonal breeding and breeding of year-round estrus breeds.This study aimed to analysis of expression patterns of seasonal estrus candidate genes KISS1,RFRP3,DIO2 and DIO3 in different breeds(year-round estrus of Small Tail Han sheep and seasonal estrus of Prairie Tibetan sheep)and different reproductive periods(luteal and follicular periods at Small Tail Han sheep)at reproduction-related tissues.At the same time,selected the KISS1 and DIO2 genes polymorphism in year-round estrous(Small Tail Han sheep,Cele black sheep and Hu sheep)and seasonal estrous(Tan sheep,Sunite sheep and Prairie Tibetan sheep)sheep breeds and correlated with sheep reproductive traits.In order to find key genes and loci associated with seasonal estrus in sheep,providing a new loci marker-assisted selection of high fecundity traits in sheep.The main results were as follows:1.Seasonal estrus-related gene expression characteristics in sheepThe KISS1,RFRP3,DIO2,and DIO3 genes are primarily expressed in reproductive-related tissues.The KISS1 and RFRP3 genes mainly regulate the synthesis and secretion of GnRH in the hypothalamus,while the DIO2 and DIO3 genes mainly regulate the TH levels in the hypothalamus to affect the seasonal estrus of animals.Compared between the Small Tailed Han sheep and Prairie Tibetan sheep two breeds in different tissues,the expression of KISS1 gene was higher in hypothalamus,brain,pituitary and thyroid of Small Tailed Han sheep than that in Prairie Tibetan sheep(P<0.05);The expression of DIO3 gene was higher in hypothalamus,pituitary,ovary,uterus and thyroid of Small Tailed Han sheep than that in Prairie Tibetan sheep(P<0.05);But the expression of RFRP3 gene was higher in in hypothalamus,ovary and thyroid of Prairie Tibetan sheep than that in Small Tailed Han sheep(P<0.05);The expression of DIO2 gene was higher in hypothalamus,pituitary,ovary and uterus of Prairie Tibetan sheep than that in Small Tailed Han sheep(P<0.05).Compared between different reproductive periods,the expression of KISS1 in hypothalamus and pineal gland was significantly higher(P<0.05)in follicular period than that in luteal period;The expression of DIO3 in follicular phase of pineal gland,hypothalamus,uterus,pituitary was significantly higher(P<0.05)in follicular period than that in luteal period;But the expression of RFRP3 in ovary was significantly higher(P<0.05)in luteal period than that in follicular period;The expression of DIO2 in pituitary,uterus,hypothalamus,pineal gland,fallopian tube and ovary was significantly higher(P<0.05)in luteal period than that in follicular period;Therefore,the regulation of KISS1 and RFRP3 genes in sheep may be achieved by regulating the secretion of GnRH in the hypothalamus.The DIO2 and DIO3 genes may affect the seasonal estrus of sheep by regulating the concentration of T3 and T4 in the hypothalamus.2.Analysis of polymorphism and estrus characteristic of KISS1 and DIO2 genes in sheepBoth genotype frequency and allele frequency at g.1317523C>T and g.88484603G>C loci of KISS1 and DIO2 genes were extremely different between year-round estrous and seasonal estrous sheep(P<0.01);Those two loci had moderate polymorphism(0.25<PIC<0.5)in six populations of sheep;Bioinformatics analysis of the g.1317523C>T site revealed that the amino acids changed from alanine to glycine after mutation.This amino acid change causes a change in the tertiary structure of the protein.which may change the protein function.It is speculated that g.1317523C>T and g.88484603G>C loci might have certain regulation effects on seasonal reproductive traits of sheep.3.Association between KISS1 and DIO2 gene polymorphism and litter size in Small Tail Han sheepThe polymorphism of four SNPs of KISS1 and DIO2 had no correlation with the litter size in Small Tail Han sheep(P>0.05),while the litter size of TT was higher than GT and GG in the first three births at g.1317523C>T locus of KISS1.It is speculated that this site mutation increases the litter size ability of Small Tail Han sheep to a certain extent.Therefore,the KISS1 and DIO3 genes play a driving role,and the RFRP3 and DIO2 genes play an inhibitory role in the seasonal estrus of sheep.The g.1317523C>T and g.88484603G>C loci of the KISS1 and DIO2 genes can be used as an auxiliary marker for perennial estrus sheep.The g.1311578G>T locus of KISS1 gene has a potential regulatory effect on litter size traits in sheep.The results of this study laid the foundation for an intensive study of the role of KISS1,RFRP3,DIO2 and DIO3 genes in seasonal estrus in sheep.