Detection Of Copy Number Variations And Genetic Effects In Chinese Cattle

CNVs (Copy Number Variations) seem to have stronger impact on phenotype andspecies evolution. Given the importance of CNVs and their high rates of mutation, interest inCNV detection has extended to domesticated animals. In cattle, several CNV datasets hadbeen published. And the results show that CNV were associated with, or affect, cattle’s healthand production traits under recent selection. However, up to date, few studies have confirmedthe genome-wide presence of CNVs in Chinese native cattle breeds. Here we selected15breeds in three main bovine groups in China (twelve B. taurus, one B. grunniens, and twoBubalus bubalis ones) to conduct a genome-wide CNV analysis based on comparativegenomic hybridization (CGH) arrays and further examined their effects on gene expressionand growth traits of cattle. Furthermore, based on maternal and paternal lineages in Chinesedomesticated bulls, we had a close look at CNVR distributions. Finally, we validated theinfluence of CNV to genes, such as position effects and gene interruption. The main results ofthis study included:1. We identified470copy number variable regions (CNVRs), covering2.13%of thebovine genome, in taurine, together with127ones in yak and148ones in buffalo. Totally,605integrated CNVRs were identified, with more “loss” events than both “gain” and “both”ones, and clearly clustered them into three cattle groups. Interestingly, we confirmed theiruneven distributions across chromosomes, and the divided differences of mitochondrion DNAcopy number (gain: taurine/indicine, loss: yak&buffalo). Furthermore, we confirmed that253CNVRs spaned716cattle genes and427CNVRs overlapped with cattle quantitative traitloci (QTLs), respectively.2. To evaluate accuracy of the copy number assignments by CGH, quantitative realtime-PCR was used to validate the CGH results with nine selected CNVRs (using14primerpairs). The results showed that out of14qPCR assays,10ones (71%) confirm the predictionsby array CGH. Positive rate was66.5%. On the other hand, we chose confirmed CNVRs fordetailed functional analysis. We demonstrated that CNVR14had significantly negative effectson expression of PLA2G2D gene, and both CNVR14and CNVR237were associated withbody measurements in Chinese cattle, suggesting their key effects on gene expression andcattle traits.3. We describe CNVs by CGH in domesticated Chinese bulls, combining subgroups of paternal Y chromosome haplotypes (Y1, Y2and Y3) and maternal mitochondrial DNAgroups of taurine and zebu. Among470CNVRs, there were72CNVRs shared by three Ysubgroups, and more than200ones by taurine-zebu groups. These cluster analysis revealedthat CNVR can reflected the background of sampls. Domestication history had left imprint ofCNVRs in the cattle genome, because we found apparent lineage-specific distributions ofCNVRs which characterized Y-chromosome subgroups and taurine-zebu groups, andconfirmed CNV117in a large sample size to distinguish haplotype Y1. Finally, we found20positive and6negative co-occurrences of CNVRs, which can be explained by similarselection direction and isolated domestication, respectively.4. In cattle, ADIPOQ gene is located in the vicinity of the quantitative trait locus (QTL)affecting marbling, the ribeye muscle area and fat thickness on BTA1. In this study, a novelvariable duplication in the bovine ADIPOQ promoter region was identified and genotyped inChinese cattle breeds. Using a reporter assay, we identified that the variation was exactly inthe core region of ADIPOQ promoter. Furthermore, we also demonstrated that variableduplication decreased basal transcriptional activity of ADIPOQ gene in3T3-L1and C2C12cells. Finally, we also demonstrated that the increased dup suppressed mRNA expression ofthe gene in adipose and muscle tissues. An association analysis indicated that the incrementalvariable duplication was associated with body measurements.5. Nucleophosmin (NMP) was involved in the biological processes of cell growth,differentiation and programmed cell death. In cattle, a novel variation in NPM1gene codingregion had been identified and genotyped previously. In this study, we analyzed the12bp-delsequence and confirmed that it was a triplet base repeat element (GAT/A). We constructedtwo vectors: pEGFP-C1-NPM10R/14R (contain different GAT repeats) and transected intocells. The results showed that expression level of pEGFP-C1-NPM1-14R was much higherthan pEGFP-C1-NPM1-10R in cells. Furthermore, expression of Ki67, MyoG and PPAR-γ,which was all positively correlated with cell proliferation and differentiation, was higher inthe cell with pEGFP-C1-NPM1-14R than pEGFP-C1-NPM1-10R. So we confirmed that therepeat sequence decreased expression level of NPM1gene and impaired its function.Our results provide a valuable resource of genomic structural variations, and are usefulto understand paternal and maternal contributions to population structure as a result ofdomestication and migration in Chinese cattle.