Genome-Wide Association Study Of Body Weights In Hu Sheep And Related SNPs Validation And Rapid Detection Methods

In sheep,body weight（BW）is a critical economic trait for meat production.Currently,genome-wide association studies（GWAS）have been applied to identify candidate genes for many quantitative traits.This progress provided an opportunity to increase the selection efficacy,especially for the traits that cannot be easily improved by conventional selection methods.Currently,there is no GWAS report in Hu sheep.Therefore,GWAS is needed to screen functional SNPs and candidate genes related to body weight traits to improve the production performance of Hu sheep.1.Whole-genome association analysis of body weight traits in Hu sheepThe 240 Hu sheep selected in the experiment were from the parent（G1）and progeny（G2）of Hu sheep nucleus herd Culture Department in HuzhouTaihu Lake Culture Cooperative.Genotyping of individual SNPs was performed using the Ovine SNP50BeadChip.Subsequently,the data are input into Bead Studio software for related analysis.After the genotypes of the software output are sorted and proofread,statistical analysis is performed,and the inaccurate SNP loci are eliminated or corrected by the software to obtain the characteristics of all the genome-wide data.After genotypic data were processed for quality control.The general linear model（GLM）and mixed linear model（MLM）of TASSEL software（http://www.maizegenetics.net）were used for GWAS analysis of SNPs（11,12）,and the SNPs related to the phenotype of the core traits of Hu sheep were mined.The GLM model corrected two confounding factors of gender and group structure.The aims of this study are locating candidate genes for weight traits in Hu sheep,and also providing important theoretical basis and reference for exploring functional genes for weight traits in sheep.The genomic DNA was detected by the whole genome-based detection platform,and the following quality control management was performed on 240 individual samples and 54241 SNP sites using Plink1.09 software.After quality control,228 sheep and 46209 SNPs were used for the final analysis.To reduce the false-positive rate caused by multiple tests,P-values were adjusted for multiple-testing to a 0.05 Bonferroni-corrected significance threshold（P<1.422×10-6）.GWAS identified eleven SNPs significantly affecting the weight of Hu sheep.The results of GLM analysis showed that one SNP（P<1.422×10-6）was significantly correlated with the birth weight.The most significant SNP was OAR6₆1075514.1（P=1.34×10-6）,located at chromosomes 6.In addition,the result showed seven SNPs（P<1.422×10-6）that were significantly correlated with the yearling weight.OAR2₁54444578.1（P=9.07×10-7）andOAR2₄9338577.1（P=1.07×10-6）were located on chromosome 2,whiles00122.1（P=1.14×10-7）,s37471.1（P=1.23×10-7）,and OAR3₁67468293.1（P=3.75 × 10-7）on chromosomes 3,OAR6₁10120561.1（P=1.19×10-6）on chromosomes 6;DU223894₅56.1（P=6.60×10-7）on chromosome 14.The results of the MLM analysis showed that three SNPs（P<1.422×10-6）were significantly correlated with the yearling weight.OAR18₁7516069.1（P=2.13×10-70）was identified on chromosome 18 and OARX₁03999250.1（P=1.86×10-57）and OARX₁03934729.1（P=1.32×10-15）on chromosomes 27.Four SNPs obtained from GLM analysis occurred within a gene or in a regulatory region near a gene,such as OAR6₆1075514.1 in the 5’ untranslated region of PCDH7;s00122.1 in exon 10 of CNGA3:OAR6₁10120561.1 in ARHGAP24;DU223894₅56.1 in LOC105613615.In addition,four SNP loci were significantly associated with genome-wide levels in the intergenic region:OAR2₁54444578.1 located 500 kbp upstream of FIGN,OAR2₄9338577.1 located 36 kbp upstream of LOC101123612;s37471.1 located 80kbp upstream of CCND2,OAR3₁67468293.1 located 14.5 kbp upstream of PPM1H.Three SNPs obtained from MLM analysis were located in the intergenic region,OARX₁03934729.1 located 742kbp upstream of FMR1.2.Population verification of candidate functional SNPs for body weight traits in Hu sheepA total of 11 SNPs were associated with the weight trait of G1 and G2 generation of the nucleus herd of Hu sheep at a Bonferroni-corrected genome-wide significance threshold of 5%.The sequences around 11 SNPs were amplified to detect additional SNPs for the further population genetics analysis and association analysis between the SNPs and the BW traits of 202 female individuals of Hu sheep G3 generation nucleus herd.Analysis of genetic parameters and population genetics including analysis of gene frequency,polymorphic information content,and locus heterozygosity.The GLM procedure in SPSS 19.0 software（SPSS,Inc.）was performed to evaluate the SNP-phenotype association of G3 generation nucleus herd.A total of 51 SNPs were detected,except that only the predicted SNPs were detected at DU223894₅56.1 and OARX₁03934729.1,while some other mutations were identified near the target SNP loci in the amplification products.Five SNPs significantly associated with birth weight of Hu sheep female individuals were identified:G→T mutation at 30bpupstream of s00122.1,T→C mutation at 173bp upstream of OAR6₁10120561.1,G→A mutation at 134bp upstream of OAR6₁10120561.1,C→G mutation at 116bpupstream of OAR6₁10120561.1,and G>A mutation at 165bp downstream of OAR6₁0120561.1.Three SNPs significantly associated with the yearling weight of Hu sheep female individuals were identified,including G→T mutation at 24bp downstream of s00122.1,A→G mutation at 166bpupstream OAR6₁10120561.1,and TT insertion at 62bpupstream of OAR2₄9338577.1We further located functional SNPs near s00122.1 and OAR6₁10120561.1,and listed their CNGA3 and ARHGAP24 as candidate genes associated with body weight traits in growth and development of Hu sheep.3.Effects of five functional weight candidate SNPs on gene transcription activityWe selected 5 SNP loci associated with weight traits in 3 amplified detection regions,including T→C at173bpupstream of OAR6₁10120561.1 and G→A at 134bpupstream of OAR6₁10120561.1,were significantly associated with the birth weight of Hu sheep female individuals（P<0.05）.G→A mutation at 166-bp upstream of OAR6₁10120561.1 was significantly associated with the yearling weight of Hu sheep female（P<0.05）,TT insertion at 62bpupstream of OAR2₄9338577.1 and s00122.1 upstream 99bp T→C.Candidate functional SNP loci significantly associated with birth weight or yearling weight in the wild-type and mutant homozygous sheep were selected.The different haplotypes amplification product was cloned into the pGL4.10 dual luciferase expression vector.After 24 h,the luciferase activity was measured on a microplate reader using the Dual-Luciferase（?） reporter assay system.Quantitative PCR was carried out for the detection and quantification of the expression of different haplotype luciferase hRluc gene.Eight haplotypes（173bp,166bp,and 134bpupstream of OAR6₁10120561.1）,CAA,CAG,CGA,CGG,TAA,TGG,TGA,and TGQ were constructed recombinant plasmids.24 h after cell transfection,dual-fluorescence activity was detected.The results showed that the relative fluorescence activity of haplotype TGA was significantly lower than that of haplotypes CAG and CGA（P<0.05）.Moreover,the expression ofhRluc gene of different haplotypes in porcine kidney cellswas detected by qPCR.The expression of luc2 mRNA in haplotype TGA was significantly lower than that of haplotypes CAG and CGA（P<0.05）.T→C mutation at 173 bp upstream locus could be identified as functional SNP for the growth traits of Hu sheep as a molecular marker for later breeding.4.Strip rapid detection system for functional SNP genotyping of body weight traits in Hu sheepWe use the 173bpT→C upstream of OAR6₁10120561.1 as a functional SNP for the growth triat of Hu sheep as a molecular marker for future breeding screening.By designing modified primers and combining the quick and easy characteristics of the result detection of nucleic acid test strips,a nucleic acid test strip reaction system capable of screening the aforementioned mutation sites was established,and a rapid detection method for different genotypes was established.The results show that the detection system can effectively distinguish wild homozygous TT individuals,mutant homozygous CC individuals,and mutant heterozygous TC individuals.At the same time,the use of nucleic acid test strips can quickly judge the test results,reduce the threshold of genotype detection test technology,get rid of the constraints of professional instruments and operations,improve detection efficiency,expand the application group of detection technology.Based on the quick and easy display of nucleic acid test strips for nucleic acid amplification results,we combine nucleic acid test strips with the thermostatic amplification RAA amplification system.this study laid the experimental foundation for the rapid development and effective identification of different meat visualization test kits.In a conclusion,GWAS identified eleven SNPs significantly affecting the bobdy weight of Hu sheep.A total of 51 SNPs were detected,except that only the predicted SNPs were detected at DU223894₅56.1 and OARX₁03934729.1,while some other mutations were identified near the target SNP loci in the amplification products.We further located functional SNPs near s00122.1 and OAR6₁10120561.1,and listed their CNGA3 and ARHGAP24 as candidate genes associated with body weight traits in growth and development of Hu sheep.The effects of eight homozygous haplotypes on transcriptional activity and luciferase hRluc gene expression were explored.We use the 173bpT→C upstream of OAR6₁10120561.1 as a functional SNP for the growth shape of Hu sheep as a molecular marker for future breeding screening.At the same time,a nucleic acid test strip detection system that can effectively distinguish wild homozygous TT individuals,mutant homozygous CC individuals,and mutant heterozygous TC individuals at this site was established.