Detecting Copy Number Variation Loci Related To Egg Performance And Their Mechanism

Chicken,as an important economic animal,provides high-quality animal protein such as eggs and meat for human beings.Egg performance that include laying traits and egg quality traits,belong to quantitative traits controlled by multiple genetic loci.Through the method of the genome-wide association study,it displayed a large number of SNPs associated with egg performance traits;however,some SNPs could not maintain good repeatability in application,they could not repeat the desired effect in other populations or generations.Recent studies have found that CNV and SNP are complementary in explaining genetic variation in the genome.CNV belong to DNA sequence structural variation.Compared with the genetic effect of base mutation,the genetic effect of the structural variation is greater.Therefore,CNV as a new type of molecular marker is of great importance for laying hen breeding.The aim of this study was to detect CNVs related to egg performance traits in chicken genome and explore the mechanism of their effects on egg performance.The candidate loci were selected from the published CNV maps as well as the chicken quantitative trait loci（QTLs）related with egg performace traits.The main results were as follows:（1）Two probes were designed for each locus.The measurment results showed that only 3 of the 17 loci had dispserse copy number distribution,and the corresponding probes were Locus02-A,Locus05-A,Locus14-A/B.The Locus05-A corresponds to QTL of egg quality.According to the Galgal5 version of the reference genome in NCBI,this site is located in the first exon of the TMEM86 A gene transcript（XM₄26403.6）.Locus14-B is nearby the QTL of the age at first egg and the site is located in the intron of the PCDHA gene cluster.（2）The Locus14-B copy types were significantly（P<0.05）associated with the number of eggs at 250 days and age at first egg in the Xinhua E-strain.Among the upper 1/4 quantile of age at first egg distribution,the number of individuals with acquired Locus14-B copies is larger;in the lower 1/4 quantile of age at first egg distribution,the number of individuals with loss copies of Locus14-B is larger;the individuals with normal copies of Locus14-B are mainly concentrated in the range from the upper 1/4 quantile to the lower 1/4 quantile.Reverse PCR was used to demonstrate the existence of tandem repeats between different copies of Locus14-B,and during that formation procress,a 260 bp fragment deletion occurred in one copy.Through PCR amplification with a large number of primers,we identified six new variable exons in PCDHA gene cluster.The distribution of the variable exons in Xinhua E-strain,Chahua chicken,Tulufan chicken,and Tibetan chicken were divergent,and the phenomenon may be resulted from the evolutionary selection.In addition,the individuals with gained copies reflected higher expression levels of PCDHA gene cluster in the pituitary gland.Since the PCDHA gene cluster is involved in neural development,we speculated that individuals with more Locus14-B copies have more variable exons,it participates in the neuron development via dose effects to affect the the hen laying ability.（3）We found the copy types of Locus05-A were significantly associated with egg yolk weight and eggshell strength（p<0.05）.The RT-qPCR results showed that the expression of TMEM86 A gene was extremely high in the liver,and the copies were negatively correlated with the expression of TMEM86 A gene.After the treatment of the limited feeding on the laying hens,the levels of triglyceride and total cholesterol in the plasma were decreased,as well was the egg production performance,but the limited feeding treatment promoted the expression of the TMEM86 A gene in the liver.Besides,the TMEM86 A gene exhibited different expression patterns in granulosa cells and theca cells of follicles.In granulosa cells,the treatment of FSH,EGF and VEGF could regulate the expression of TMEM86 A gene.Among them,EGF up-regulated the expression of TMEM86 A gene in granulosa cells of small yellow follicle,and inhibited its expression in granulosa cells before ovulation.The expression pattern is similar to that in the granulosa layer of the sized follicles.（4）The promoter of TMEM86 A gene was used for prediction of the methylation status,which showed three CpG islands distributing in this region.Through bisulfite sequencing of chicken liver,granulosa layer and theca layer,28 CpG sites were identified in the promoter of TMEM86 A.Those CpG sites were distributed distally away from the transcription start site（TSS）.Furthermore,7 transcription factors targeting the TMEM86 A promoter were selected by prediction for RT-qPCR analysis.The RT-qPCR result showed that the limited feeding treatment also changed the expression of SP1,CREB1 and ARNT genes.Finally,2 transcription factors,including SP1 and CREB1,were confirmed to change the promoter activity of the TMEM86 A promoter and affect TMEM86 A expression.In addition,the JASPAR software predicted that there are more than one hundred SP1 binding sites distributing in the promoter region of TMEM86 A gene,and two of them were overlaped with the 28 CpG sites.The site-mutant vectors were constructed and were transfected into DF1 cells,which showed obviously（p<0.01）decreased fluorescence activity,indicating the regulation of the two CpG sites on the transcription of the TMEM86 A gene.In conclusion,we speculated that the promoter methylation mediates the regulation of SP1 on TMEM86 A gene expression.