Estimation Of Genetic Parameters And Identification Of Candidate Genes For Growth Traits In Dezhou Donkey

Dezhou donkey is one of the high-quality large donkey breeds in China,with strong disease resistance,fast growth rate,coarse feeding tolerance and strong environmental adaptability.Due to the improvement of mechanization level and the lack of relevant research on economic performance,the number of donkeys in China has declined sharply,some of the good traits have also been degraded,and the industrial development was seriously lagging behind other animal breeds.In this study,genetic parameter estimation,genome-wide association analysis based on low depth sequencing data,transcriptome analysis and functional study of candidate genes were carried out to provide scientific basis for the utilization and development of genetic resources in Dezhou donkey.The main results of this paper are as follows:1.In this study,ASReml-R software was used to test the significance of different effects on the growth traits of Dezhou donkeys,and a single-trait animal model was used to estimate the genetic parameters of body weight and body size at different growth stages(birth,6months and 12 months of age).The results showed that the year-season effect of birth and the measuring age effect had significant effects on the growth traits of foals(P < 0.01),but gender effect only had significant effect on a few early growth traits.The heritability of growth traits at 6 months of age ranged from 0.09 to 0.41,higher than that of the corresponding traits at birth(0.02-0.19)and 12 months of age(0.13-0.18).The two-trait animal model was used to analyze the correlation of body weight at different stages,and it was found that the genetic and phenotypic correlations of body weight between 6 months and12 months were the highest,reaching 0.95 and 0.70,respectively.The estimation results of genetic parameters between different traits at the same stage showed that the correlation between body weight and body size was high,among which the genetic correlation between body weight and body size at 6 months was 0.77 to 0.96,and the phenotypic correlation was0.57 to 0.79.2.In order to explore the candidate genes affecting the early growth traits of Dezhou donkey,we conducted genome-wide association analysis of growth traits at different stages based on low depth sequencing and genotype imputation.The average sequencing depth of613 samples with growth traits was 3.22×.Firstly,the Basevar software was used to obtain the positional information of SNPs within the Dezhou donkey genome.Secondly,STITCH and Beagle software were used to impute genotype,and the accuracy of imputing was evaluated using high-depth sequencing data of 30 samples.The results showed that both Cor and IQS were higher than 0.90.Finally,the mixed linear model of GEMMA software was used for genome-wide association analysis and FDR method was used for multiple correction of original P values.A total of 34 SNPs significantly correlated with growth traits of Dezhou donkeys were screened.Twenty-six candidate genes including MYF5,LCORL and ZFAT were identified by annotation.3.In order to identify the differentially expressed genes affecting the growth rate of Dezhou donkeys,three foals with fast growth(group H)and three foals with slow growth(group L)were selected and sequenced by RNA-seq technique.A total of 269 differentially expressed genes were found,among which 241 up-regulated genes and 28 down-regulated genes were found in group H.In order to verify the accuracy of quantitative analysis of genes in the transcriptome,20 genes were selected for fluorescence quantitative PCR.The results showed that the correlation coefficient of the results obtained by the two methods was greater than 0.8,and the transcriptome sequencing data was accurate.Through the analysis of KEGG pathway enrichment and PPI,we found that the differentially expressed genes mainly comments to calcium signaling pathways,secretion of digestive glands,digestion and absorption of fat,minerals and vitamins.Therefore,we believe that the difference in the early growth rate of Dezhou donkey is partly caused by the differentially expressed genes involved in the above pathways.4.LCORL is a key candidate gene affecting the growth traits of Dezhou donkey and other species.In order to verify whether LCORL gene is involved in the regulation of growth and development of Dezhou donkey as a transcription factor,DAP-seq technology was used to capture and analyze its downstream regulation genes.In addition,Blast was used to search the binding sites of LCORL in promoter regions of all genes in donkey genome,and then potential target genes were screened.In this study,most of the annotation results of DAP method were distal intergene regions and no downstream target genes were screened,while30 target genes were found by Blast method.According to the function description of target genes,we selected three genes,CHD8,TLK1 and GAS1,and verified them by yeast single hybridization test.The results showed that LCORL may be involved in the regulation of cell cycle and mitosis by combining GAS1 and TLK1,thus affecting the growth and development of animals.This study was the first to estimate the genetic parameters of the growth traits of Dezhou donkey and carry out GWAS based on low depth sequencing,providing reference data and molecular markers for the development of a new breed breeding program of Dezhou donkey and the establishment of genomic selection methods.The KEGG pathway related to the growth rate of Dezhou donkey through transcriptome analysis and the downstream target genes of LCORL provided new ideas for the precise study of the genetic mechanism of the growth and development of the Dezhou donkey.