Detection And Validation Of Functional SNP In Coding Regions Of The Whole Genome Of Domesticated Sheep

Sheep(Oris aries)is one of the earliest domesticated animals.After long-term natural and artificial selection,about 1,400 landraces and developed breeds with rich germplasm characteristics have been formed worldwide.They not only provide human beings with important living materials such as meat,fur,skin and milk,but also play an important part in human social development and cultural transmission.According to the economic type,sheep breeds can be divided into specialized breeds such as meat,wool and milk,as well as dual-purpose breeds such as meat-wool and wool-meat types.There are obvious differences among all breeds in body appearance,environmental adaptation and production performance.In recent years,with the development of molecular genetics,molecular biology,especially genomics,great progress has been made in the genetic basis of sheep breed characteristics by means of candidate gene method,QTL mapping,genome-wide association analysis(GWAS)and selection signals,and a large number of major genes and molecular markers for economic traits have been obtained,which can be applied to marker-assisted selection.However,due to the large number and wide distribution of sheep breeds and the difficulty in obtaining production performance data,only a few functional cause mutation sites related to production performance or adaptability have been identified so far.Resequencing technology can effectively detect single nucleotide polymorphism(SNP),insertion and deletion(InDel),copy number variation(CNV)and structural variation(SV)in the whole genome.Among them,SNP is the most abundant type of genetic variation in the biological genome,which is usually two allelic polymorphisms and easy to detect.In this study,677 individuals from 86 domestic sheep breeds worldwide were selected as research objects.By using genome resequencing,bioinformatics analysis and gene editing techniques,SNPs in the whole genome of domestic sheep were detected and annotated,and the number and distribution of functional SNPs in the coding region were counted and analyzed.Functional SNP in FGF5 gene was selected for knockout and verification,and the following results were obtained:(1)A total of 348.25 Gb of high-quality Clean Data was obtained by resequencing the whole genome of 10 Small-tailed Han sheep.The average depth of individual sequencing was 10.89×,and the average comparison rate was 99.77%.(2)Using Oar＿rambouillet＿v1.0 version of sheep genome as reference genome,a database of SNP and InDel genetic variation was constructed,including 677 individuals of 86 domestic sheep breeds.The geographical locations of breed origin included China,Asia,Africa,Europe,America and Oceania.(3)A total of 41,466,519 biallelic SNP were obtained from 26 sheep autochromosomes by SNP extraction and filtering,and the number of SNP on chromosome 1 was the largest(4,703,059).Chromosome 26 had the fewest SNP,with 790,471.(4)According to the intersection of the two database annotations in NCBI and Ensembl,a total of 877 nonsense mutations and 88,513 missense mutation sites were obtained in the coding region of 677 individuals;Ensembl database(release 109)VEP online SIFT predicted that there were 25,781 deleterious mutations in the missense mutations;and 54.48% of the deleterious mutation sites had a MAF(Minor Allele Frequency)less than 0.01.(5)25,781 deleterious missense mutation sites were annotated to 9,436 genes,and 877 nonsense mutations were annotated to 724 genes.By intersecting all the annotated candidate genes with known genes in the OMIA(ONLINE MENDELIAN INHERITANCE IN ANIMALS),311 genes related to animal performance or diseases were obtained.They include genes related to wool production traits(FGF5,DSG4,MLPH and MC1 R,etc.),growth and development(MC4R,LMBR1,GRM1 and PTEN,etc.),meat quality(COL9A2,BMPR1 B,GYS1 and GHR,etc.)and reproductive traits(APAF1,PTEN and TSHR,etc.).(6)Allele frequency and genotype frequency analysis were performed for the three deleterious missense mutation loci(6:105914565,6:105922731 and 6:105935295)annotated to FGF5 gene in 39 breeds.The results showed that,at 6:105922731 locus,the frequency of the T allele was 1 in the Valais Blacknose sheep,that is,all individuals were TT homozygous in the Valais Blacknose sheep;and the frequency of T was 0.9167 in the Valais red sheep breed,that is,6individuals contained 1 heterozygote and 5 homozygotes in the Valais red sheep breed.(7)Mice modified with 6:105922731(FGF5,c.434C>T)locus were obtained by CBE gene editing system;The results of phenotypic observation and hair length measurement showed that there was no difference in hair length between wild type(CC)and mutant heterozygote(CT),but there was significant difference between them compared with mutant homozygote(TT),indicating that the T base of c.434C>T locus in FGF5 gene was related to the hair length of mice and showed a recessive inheritance pattern.This study provided candidate genes and functional SNP loci for revealing the genetic basis of environmental adaptability and phenotypic differences of important economic traits in different domestic sheep breeds.Meanwhile,it also provided molecular targets for the establishment of detection methods to quickly eliminate low-frequency deleterious alleles.Therefore,it has important significance in scientific research and production practice.