Screening And Validation Of Candidate Genes For Litter Size In East Friesian Sheep And Hu Sheep Hybrid Population Based On GWAS

The dairy sheep industry has shown a trend of rapid development in various countries in the world.The dairy group formed by crossing East Friesian sheep and local breed Hu sheep(East Friesian and Hu crossbred sheep),which has broadened our country’s sheep industry to use both milk and meat and adapted to the local ecological environment.However,the breeding of dairy sheep breeds for reproductive performance faces some challenges.In fact,the economic benefits obtained by the double-lamb population of sheep are far more than that of the single-lamb population.Therefore,in order to improve the reproductive traits of hybrid dairy sheep,improving the lambing rate of sheep has become one of the main goals of breeding.In this study,blood samples were collected from Hu sheep,East Friesian sheep,and hybrid dairy sheep(f1,f2).DNA was extracted and analyzed for quality testing,while collecting lambing data and pedigree information for the corresponding samples.Secondly,the hybrid second-generation sheep were selected for genome-wide association studies(GWAS)to find candidate regions and important mutation loci that affect the litter size.Finally,the mutation distribution frequency and polymorphism parameters of the candidate loci were calculated in the populations,and then the correlation analysis was performed with the litter size to verify the results of the GWAS.It will provide a reliable basis for breeding of the hybrid dairy sheep with multiple-lamb trait,and promote the healthy and efficient development of dairy sheep industry.The results obtained in this study are as follows:1.Statistical analysis and GWAS of F2 populationThe second generation of the hybrid sheep was genotyped using Mol Breeding Geno Baits(?)sheep 40K chip.There were 245 individuals in the F2 population who recorded litter size in the first parity,the average of litter size was 1.97,and the standard deviation was0.872.After genotype imputation based on the genome resequencing data of Hu sheep and East Friesian as a reference,661,109 loci were finally obtained for GWAS.All of loci are distributed in sheep chromosome 6,and their candidate regions are located at 24845169-45005517(NC＿040257.1).In the results of GWAS,it was found that the key genes significantly related to the litter size of East Friesian and Hu crossbred sheep were BMPR1B,UNC5C,BANK1,CCSER1,PDLIM5,RAP1GDS1,TSPAN5,GRID2,KCNIP4,etc.c.746A>G of BMPR1B gene may also be a major locus affecting lambing in East Friesian and Hu crossbred sheep.2.Polymorphism detection of c.746A>G locus of BMPR1B gene in Hu sheep,East Friesian and the crossed sheepBy detecting distribution and calculating the genetic diversity parameters,it can be concluded that Hu sheep only contains BB genotype(mutant homozygote)and B+type(mutant heterozygous),the frequency of allele B is 0.970,and the frequency of+allele is0.030;East fresian sheep and second-generation sheep only contain++genotype(wild homozygote)and B+genotype,and the allele frequency of+was higher than that of B.The F1 population contained++,BB and B+genotype,while the frequency of B+genotype was0.939,showing a high degree of heterozygosity.Compared with B+genotype of Hu sheep,the second parity,third parity and average of BB genotype were significantly increased by 0.56,0.39,and 0.25 in the results of the association analysis,respectively.In addition,the difference in litter size of first parity was not significant.In the first-generation population,c.746A>G significantly increased the litter size of second parity,third parity and average.Besides,the litter size of second and average parity of B+genotype of the first parity in F2 group were significantly increased by 0.75,0.44 and 0.67 compared with++genotype.3.Polymorphism identification of 90bp deletion mutation(Del-90bp)in BMPR1BAccording to the genotype distribution of Del-90bp,Hu sheep contained only ID(deletion heterozygous)and DD(deletion homozygous).Allele D has a higher gene frequency than I.East fresian sheep,F1 and F2 populations all contained three genotypes of II,ID and DD.The four populations at this locus were all in line with Hardy-Weinberg equilibrium(p>0.05),the Hu sheep had low genetic polymorphism(PIC<0.25),and the other three had moderate polymorphism(0.25<PIC<0.5).4.Linkage disequilibrium analysis and association analysis between c.746A>G and Del-90bp of BMPR1B geneThe results of linkage disequilibrium analysis showed that c.746A>G and Del-90bp had strong linkage relationship(r~2>0.33)in Hu sheep and F1.According to the strong linkage relationship,the 90bp deletion in sheep was used as the standard to detect Fec B mutation.It was showed that the detection accuracy in Hu sheep and F1 population reached 100%and95.58%,respectively.The results of association analysis showed that in the Hu sheep population,individuals of B+/DD and BB/ID genotype had higher litter size than other combinations,while B+/DD had fewer samples,and the effect on lambing needed to be verified by larger samples.In the F1 population,litter size of BB/DD genotype in all of parities were higher than other genotype combinations,which can be used as the most dominant genotype for multi-lamb breeding.In addition,the selection of ID genotype can significantly increase the litter size of F1 without Fec B mutation.In conclusion,the candidate regions that significantly affect the litter size of East Friesian and Hu crossbred sheep were located through GWAS,and it was proved that c.746A>G locus was one of the main loci affecting hybrid sheep.The Del-90bp locus of BMPR1B gene in the candidate region is linked with Fec B mutation and has an interactive effect on the litter size.This study provides a theoretical basis for improving the lambing rate of East Friesian and Hu crossbred sheep and promoting the development of precision breeding of sheep reproduction.