Whole Genome Analysis Of Chinese Cattle And The Dosage Effects Of The Skeletal Muscle Involving Genes

Genomic variation is the important basis of phenotypic differences in animals. In cattle,the database of genomic variation in several breeds have been published, and manyquantitative trait loci （QTL）, such as muscle growth, fat metabolism, fecundity and diseaseresistance, have been identified by bioinformatics analysis. However, up to date, the studieson genome-wide sequencing of Chinese native cattle breeds have not yet been reported.Currently, three platforms, SNP （Single nucleotide polymorphism） array, CGH （Comparativegenomic hybridization） array and whole-genome sequencing, are routinely used for variabilityscreens in many species. Of these, whole-genome sequencing is standing out due to itscomprehensiveness and accuracy. Thus, in the present study, we detected the whole genomevariations of two phenotypically distinct Chinese cattle breeds, Nanyang and Qinchuan, andcompared the sequence differences between the two breeds. In addition, we also identified thedosage effects of CNVs （Copy number variations） on gene expressions and phenotypic traits.Moreover, we firstly reported a novel10-bp indel polymorphism of three continuous-repeatregion within bovine Pax7（Paired box7） gene promotor, and analyzed its dosage effects onpromotor activity, mRNA level and growth traits. The main results of the present study areincluding:1. Detection and analysis of whole genome variations in Chinese cattleIn this study, we used Solexa technology to sequence genomic DNA from Nanyang andQinchuan bulls, which were covered with an average mapping depth of9.37-fold and11.76-fold, respectively. In total,67Gbp sequence data was generated from the two genomes.Putative SNPs were identified while mapping the aligned reads to the reference assembly（Bos_taurus_UMD₃.1）, a total sets of9,010,096and6,965,062SNPs were detected in theNanyang and Qinchuan genomes, of which,50.83%and29.02%were novel SNPs,respectively. Gene annotation results revealed that Nanyang cattle possessed more geneticvariation than Qinchuan cattle, including SNPs and small indels, and the differences were alsofound in exon, intron and intergenic region. Phylogenetic analysis found that the Qinchuancattle was nearly related with the Bos taurus, while the Nangyang cattle formed amonophyletic group with the European Bos indicus. In addition, aligning the Nanyang againstthe Qinchuan genome, we detected2,907cases of CNV, corresponding to0.37%of the cattle genome. Of these CNVs, the number of loss was higher than gain cases; the greatest andlowest number of CNVs was discovered on chromosome X and25, respectively. Nearly27%（783） CNVs were encompassed495Ensembl genes. The GO analysis demonstrated that many CNVgenes were enriched in environment adaptability and immune system.2. CNV validation and muscle development involving genes screeningTo verify the accuracy of our assessment of CNVs from genome sequencing, quantitativePCR （qPCR） was conducted for a randomly selected subset of20CNVs, including fourgroups （Gain-GENIC, Gain-NON-GENIC, Loss-GENIC and Loss-NON-GENIC）.90%ofour qPCR results （i.e.,18of20validated） agreed with the CNV predictions in these regions,and only Chr7_CNVR₁61and Chr13_CNVR₁410, harboring the SSBP2and SNTA1genes,respectively, were not congruent with the predictions from our genome sequencing results. Tofurther evaluate CNV between breeds, the same20CNV regions were quantified in10Nanyang and10Qinchuan individuals. The confirmation results in the group of copy numbergains showed that average copy numbers of Nanyang were larger than those of Qinchuanbreed. In contrast, mean copy numbers of Nanyang were always lower than those of Qinchuanin the case of CNV losses. By GO and pathway analysis, six genes involved in muscledevelopment were prelimiarily determined, of which, FHL1、MICAL-L2and MYH3geneswere highly expressed in muscle, and they could be considered as candidate copy numbervariation genes involving in muscle development.3. Dosage effect analysis of candidate CNV genes involving muscle developmentComparing the CNV genes with CattleQTL database, we found that the candidate genes（FHL1、MICAL-L2and MYH3） were associated with several important quantitative trait loci,such as intramscular fat content, rump angle, body height and fat thickness at the12th rib.This indicated that the genes may play critical roles in shaping phenotypes. In addition, CNVdistributions of FHL1、MICAL-L2and MYH3genes showed remarkable differences amongfour cattle breeds, especially in Nanyang cattle, the relative copy numbers were found todiffer more extent than the other tested populations. These observations implicated that theCNV loci may expose potential effects on phenotype in different breeds, underscoring itsdiversity in genetic copy numbers. Dosage effect analysis revealed that the copy number gainsof FHL1and MYH3gene could promote the transcript level, and significantly influenced thegrowth traits of Nanyang cattle. However, MICAL-L2gene copy number gains could inhibitthe mRNA level, and was significantly associated with Nanyang growth traits. 4. Identification of the promoter variation in bovine Pax7gene and population geneticanalysisIn the present study, we firstly discovered a novel10-bp （TCGTCTCCCC） short copynumber variation locus in promoter region of bovine Pax7gene. By polyacrylamide gelelectrophoresis, two copy number types,3copies and2copies, were identified in fiveChinese cattle breeds. Statistical results showed that distributions of10-bp genotypes weredifferent among five cattle breeds, and the10-bp locus showed significant associations withgrowth traits of Nanyang cattle. Therefore, the10-bp indel polymorphism of bovine Pax7gene could be considered as promising marker for cattle breeding programs.5. Mechanism and dosage effect analysis of the Pax7gene10-bp indel variationIn order to study the effect of Pax7gene10-bp indel variation on promoter activity, weconstructed the plasmids with different10-bp copies, including pGL3-pro2-3copies,pGL3-pro2-2copies, pGL3-pro2-1copy and pGL3-pro2-Δ10-bp. Dual-luciferase reporterresults showed that the Pax7promoter activity is lowest in pGL3-pro2-3copies vector,compared with the pGL3-pro2-2copies or pGL3-pro2-1copy. In addition, overexpression ofZNF219gene demonstrated that the copy number of10-bp sequence could alter the bindingstrength of the transcription factor ZNF219, and finally influence promoter activity of Pax7gene. Moreover, the10-bp locus can change the mRNA expression level of bovine Pax7geneand its down-stream genes （Id2、Id3and CXCR4）. All these results suggested that the smallcopy number variations in promoter region of functional gene could influence the promoteracitivity by dosage effects, and then alter gene expression level and phenotypic traits.