Detecting Genetic Variations In Five Important Genes And Their Effect On Litter Size In Sheep

With China’s economic development and the implementation of the "Belt and Road" strategy,the rapid and effective improvement of economic traits is the main driving force for the development of the sheep industry,which is severely constrained by low sheep fertility.Candidate genes and molecular markers affecting sheep reproductive traits are important for the improvement of sheep lambing numbers.Molecular genetic markers(e.g.InDel,SNP,etc.)play an important role in modern molecular breeding of livestock and poultry.Therefore,the aim of this study was to investigate known lambing-related genes(BMPRIB,GHR,PRL,POU1F1,PROP1)and genetic variants of the screened DLG gene family for genotyping and association analysis with lambing number in sheep,and the following results were obtained:1.Ovine BMPRIB gene polymorphisms(InDel,SNP)and their effect on sheep litter size.The ovine BMPRIB gene is first major gene that was found in Booroola Merino sheep and regulates granulosa cell proliferation and plays a crucial role in early follicle growth.In this study,the distribution of the 90-bp deletion within the first intron of BMPRIB was analyzed in total of 3648 samples of 54 various sheep individuals.The seven breeds(Australian White(AUW),Small Tail-Han(STH),Guiqian semi-fine wool(GQSFW),Luxi Blackhead(LXBH),Yuan sheng Dairy(DHS),Lanzhou fat-tailed(LZFT),Weining(WN))were genotyped using PCR-based genotyping,and 47 breeds(n=677)worldwide(including STH sheep)were genotyped via whole genome sequencing(WGS)method.Genotyping outcomes revealed that the 90-bp polymorphism was polymorphic in 45 of 54 breeds.Meanwhile,the results of PCR-RFLP method exhibited that p.Q249 R SNP was polymorphic in LXBH,STH and LZFT breeds.Moreover,Del-90-bp significantly affected litter size in GQSFW sheep(P < 0.05),and ewes with the ID genotype had greater litter sizes.Linkage disequilibrium(LD)results displayed that these two polymorphisms are not linked in in all breeds.2.GHR and PRL gene InDel polymorphisms and their effect on litter size.The GHR and PRL genes regulate the most important processes related to reproduction.Meanwhile,these genes lack studies of InDel polymorphisms and their association with litter size.A total of 3220 sheep samples of 41 breeds enrolled in this study.A total of 16 pairs of primers were designed for the above four genes to detect novel InDel loci.After the DNA genotyping,among the GHR,PRL,PROP1 and POU1F1 genes,two InDels,including P2-del-23 bp site and P8-del-23 bp site in introns 3 and 4,within the GHR gene and one P1-ins-23 bp InDel in intron 2 of the PRL gene were polymorphic.However,polymorphisms in POU1F1 and PROP1 genes were monomorphic in four specialized breeds.According to results of the association analysis,the P1-ins-23 bp locus in the PRL gene had higher significant correlation with litter size in AUW and DHS sheep(P < 0.05),and ewes with the DD and II genotypes had greater litter size,respectively.Furthermore,P2-del-23 bp locus was associated with first parity litter size in AUW sheep,and ewes with II genotype exhibited higher litter size compared to ID and DD genotype.The P8-del-23 bp locus in the GHR gene was significantly related to litter size in AUW and DHS sheep(P <0.05),and individuals with DD genotype showed lower litter size compared to others.3.Ovine DLG gene family: InDel polymorphisms of the DLG1 and DLG5 genes and their effect on sheep litter size.The GWAS study revealed that the DLG1 gene can be candidate gene that might associated with litter size in sheep.In this work,total of 2793 samples of 41 different sheep breeds have been used.A total of 32 pairs of primers were designed based on the complete sequencing of DLG1,DLG3,DLG4,DLG5 genes.DNA pool results showed that four insertion/deletions in the DLG1 gene,including DLG-P2-del-40bp(rs1085899974;intron 5),DLG1-P3-del-24bp(rs605180748;intron 10),DLG1-del-P7-8bp(rs599156010;intron 5)and DLG1-del-P16-8bp(rs589774584;downstream),while one polymorphism in the DLG5-P3-ins-13 bp within the DLG5 gene were detected in sheep population.However,polymorphisms within the DLG3 and DLG4 genes were not polymorphic in the tested population.In addition,there were not InDels for DLG2 gene.The DLG-P2-del-40 bp InDel has been associated(P< 0.05)with fourth parity litter size in AUW sheep.Furthermore,the significant association of the DLG1-P7-del-8bp and DLG1-P16-del-8bp InDels with the second and fourth parity litter size in DHS ewes,individuals with ID genotype showed greater litter size(P < 0.05).The DLG5-P3-ins-13 bp locus had a significant association with the second parity litter size(P = 0.010)and average litter size(P =0.024)in AUW ewes,and ewes ID genotype had higher litter size in AUW sheep.In addition,the results of chi-square test displayed that the current InDel had a different distribution between the mothers of single-lamb and multi-lamb at second parity litter sizes(P = 0.020).4.Ovine DLG gene family: DLG1 gene promoter activity and effect of functional SNPs on sheep litter size.Considering the effect of DLG1 polymorphisms on litter size,it was postulated that there will be other regulatory polymorphisms in promoter region of this new gene.Firstly,m RNA expression of DLG1 gene exhibited expression in several tissues,including ovary.Secondly,in this experiment,promoter vectors of different lengths were successfully constructed,and the DLG1 promoter vectors in two HEK293 T and CHO-K1 cell lines were examined.The fluorescence activity from p GL3-pro2 to p GL3-pro3 showed a highly significant(P < 0.0001)activity,suggesting that a core promoter region located between-746 and-354(-392 nt)before the transcription start site.Transcription factor binding site analyses also showed that the-392 nt core promoter position resulted in binding affinities for several transcriptional factors.Further,the study revealed that the three SNPs(rs408285706(SNP1),rs401998328(SNP2),rs401085093(SNP3))in core promoter region using WGS data of 123 AUW sheep.Furthermore,the association analysis revealed that SNP1(rs408285706)and SNP3(rs401085093)were associated with litter size in AUW sheep.For SNP3,the individuals with the TT genotype had greater litter size compared to other one.The mutant G allele can cause the REV-Erbalpha transcription factor not to bind to the promoter region and the results of luciferase reporter gene experiments show that the G allele of SNP1 leads to increased DLG1 promoter activity and higher individual lambing in AUW sheep with the reference A allele.In summary,this study used whole-genome resequencing and PCR genotyping to screen InDels and SNPs in known reproductive-related genes(e.g.,BMPRIB,GHR,PRL)and the DLG gene family(DLG1,DLG5)in 3648 individuals from 54 different sheep breeds,and analyzed multiple genetic loci that significantly associated with sheep litter size.These findings could contribute to marker-assisted selection in different sheep population,indicating this approach could be useful for further research in sheep breeding.