Genome-wide Association Study For Numbers Of Thoracic And Lumbar Vertebrae In Dezhou Donkey And Functional Study Of Candidate Genes

The variation in numbers of vertebrae,especially the numbers of thoracolumbar vertebrae,greatly affects the carcass length and meat production.Dezhou donkey,originated in Shandong province,is one of the most famous donkey breeds in China.It is characterized by its large body size,good adaptability,and resistance of roughage.It has been introduced into other places of China as superior breeding stocks for genetic improvement of other local donkey breeds.There was also variation of thoracolumbar numbers in Dezhou donkey population,the common number of thoracic vertebrae(TVn)is 18,while 17 and 19 were also reported;there are two types for the numbers of lumbar vertebrae(LVn):5 and 6.The aim of this study was to identify candidate genes affecting TVn and LVn in Dezhou donkey and investigate the function of candidate genes.A genome-wide association study(GWAS)was conducted using whole genome sequence data imputed from low-coverage genome sequencing,and the validation of associated genetic variation was performed in another donkey populations.For the candidate genes,detecting the expression profile during mouse embryonic somite development and the role on the MC3T3-E1 osteoblasts differentiation.These candidate genes and molecular markers will lay a theoretical foundation for cultivating new breeding of multiple thoracolumbar donkeys in the future.The main results are as follows:(1)GWAS of the TVn and LVn in Dezhou donkey.In order to explore the candidate genes affecting TVn and LVn of Dezhou donkey,a GWAS was conducted based on low coverage sequencing and genotype imputation.The average sequencing depth of 347 samples was 4×.The STITCH and Beagle software were used to impute genotype,and the accuracy of imputing was evaluated using high-depth sequencing data of 18 samples.The results showed that the average genotype correlation coefficient was 0.98,and the average genotype concordance was 98.44%.Finally,the linear mixed model of GEMMA software and logistic mixed model of GMMAT software were used for association analysis.For TVn,we identified 38 genome-wide significant and 64 suggestive SNPs,which relate to 7 genes(NLGN1 DCC,SLC26A7,TOX,WNT7A,LOC123286078,and LOC123280142).For LVn,we identified 9 genome-wide significant and 38 suggestive SNPs,which relate to 8 genes(GABBR2,FBXO4,LOC123277146,LOC123277359,BMP7,B3GAT1,EML2,and LRP5).For the TVn+LVn,we identified 24 suggestive SNPs,which relate to 7 genes(NLGN4,LOC123286078,SLC35F6,ENPP5,PDZRN3,MLLT3,ZNF536).Among them WNT7A,BMP7 and LRP5 involved in the Wnt and TGF-β signaling pathways and play important role in embryonic development and bone formation and could be good candidate genes for TVn and LVn.(2)Association analysis of significantly associated SNPs in new Dezhou donkey populations.According to the SNPs and haplotype information obtained by GWAS of thoracolumbar numbers,Four SNPs associated with TVn and five SNPs associated with LVn were selected as research objects.The genotypes of 159 Dezhou donkey populations were statistically analyzed by MALDI-TOF-MS,and their association with thoracolumbar numbers was analyzed.The results showed that 4 SNPs were still significantly associated with TVn and another 4 SNPs were significantly associated with LVn in this new population using general linear model and logistic regression model,which further confirmed the association between these SNPs and thoracolumbar numbers.The reliability of GWAS analysis is verified.(3)The expression profile of candidate genes in different developmental stages of mouse embryo.Among the candidate genes identified by GWAS,it has been reported that WNT7A,LRP5 and BMP7 play a role in bone and somite development.The vertebrae are derived from somite,which is produced from the presomitic mesoderm(PSM).We detected the expression profile of WNT7A,LRP5 and BMP7 in different developmental stages of mouse embryo.The expression of WNT7A continued to upregulated from 7.5dpc to 12.5dpc during embryonic development;LRP5 mainly expressed in the later stage of somite development;The expression pattern of BMP7 was firstly increased and then decreased,and it is highly expressed in the formation stage of thoracolumbar vertebrae.So it can be inferred that the candidate genes screened by GWAS may play a role in the development of mouse somite based on the expression profiles.(4)The role of candidate genes on the osteogenic differentiation of MC3T3-E1 cells.The spine development of vertebrate is an endochondral osteogenesis process,including mesenchymal stem cells gradually form bone after undergoing a series of differentiation,maturation and apoptosis.Osteoblast differentiation is an important process for the bone formation.Therefore,WNT7A,BMP7 and LRP5 were selected to investigate the role on the osteogenic differentiation of MC3T3-E1 cells.In vitro MC3T3-E1 cells were induced differentiation,at the same time were infected with overexpressed plasmid of WNT7A gene.By detecting the activity of alkaline phosphatase(ALP)and the expressions of major osteoblast differentiation marker genes SPP1 and RUNX2,it was found that compared to the wild cells,the activity of ALP was enhanced and the expression of marker genes was up-regulated.The results of overexpressed LRP5 gene was consistent with WNT7A,promoting osteoblast differentiation.When WNT7A and LRP5 knock-down in MC3T3-E1 cells,ALP activity were reduced and the expression of marker genes were down-regulated.BMP7 gene was not detected in this cell,so there was no more exposition about this gene.These results indicate that candidate genes WNT7A and LRP5 can significantly promote the osteoblast differentiation.The functional study of these candidate genes has potential significance to reveal the major regulatory genes for the numbers of thoracolumbar vertebrae in Dezhou donkey.