Analysis of copy number variations in the sheep genome using 50K SNP BeadChip array

Jiasen Liu 0 2 3 Li Zhang 0 3 Lingyang Xu 0 3 Hangxing Ren 1 Jian Lu 0 3 Xiaoning Zhang 0 3 Shifang Zhang 0 3 Xinlei Zhou 0 3 Caihong Wei 0 3 Fuping Zhao 0 3 Lixin Du 0 3 0 National Center for Molecular Genetics and Breeding of Animal, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences , Beijing 100193 , People's Republic of China 1 Chongqing Academy of Animal Sciences , Chongqing 402460 , People's Republic of China 2 Institute of Animal Science, Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences , Hohhot, Inner Mongolia Autonomous Region 010031 , People's Republic of China 3 National Center for Molecular Genetics and Breeding of Animal, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences , Beijing 100193 , People's Republic of China Background: In recent years, genome-wide association studies have successfully uncovered single-nucleotide polymorphisms (SNPs) associated with complex traits such as diseases and quantitative phenotypes. These variations account for a small proportion of heritability. With the development of high throughput techniques, abundant submicroscopic structural variations have been found in organisms, of which the main variations are copy number variations (CNVs). Therefore, CNVs are increasingly recognized as an important and abundant source of genetic variation and phenotypic diversity. Results: Analyses of CNVs in the genomes of three sheep breeds were performed using the Ovine SNP50 BeadChip array. A total of 238 CNV regions (CNVRs) were identified, including 219 losses, 13 gains, and six with both events (losses and gains), which cover 60.35 Mb of the sheep genomic sequence and correspond to 2.27% of the autosomal genome sequence. The length of the CNVRs on autosomes range from 13.66 kb to 1.30 Mb with a mean size of 253.57 kb, and 75 CNVRs events had a frequency > 3%. Among these CNVRs, 47 CNVRs identified by the PennCNV overlapped with the CNVpartition. Functional analysis indicated that most genes in the CNVRs were significantly enriched for involvement in the environmental response. Furthermore, 10 CNVRs were selected for validation and 6 CNVRs were further experimentally confirmed by qPCR. In addition, there were 57 CNVRs overlapped in our new dataset and other published ruminant CNV studies. Conclusions: In this study, we firstly constructed a sheep CNV map based on the Ovine SNP50 array. Our results demonstrated the differences of two detection tools and integration of multiple algorithms can enhance the detection of sheep genomic structure variations. Furthermore, our findings would be of help for understanding the sheep genome and provide preliminary foundation for carrying out the CNVs association studies with economically important phenotypes of sheep in the future. - Background In recent years, genome-wide association studies (GWAS) have successfully uncovered single-nucleotide polymorphisms (SNPs) associated with complex diseases or traits [1]. With the rapid development of chip array-based genotyping techniques, thousands of genomic submicroscopic structural variations have been found in the human genome [2]. As a main genetic form of submicroscopic structural variation copy number variations (CNVs) are widely distributed in the human genome and influence gene expression, phenotypic variation and adaptation by disrupting genes and altering gene dosage [2-5]. Numerous studies showed that CNVs contributed to both disease susceptibility and phenotypic diversity [2,5]. Now, CNV is increasingly considered to be an important and abundant source of genetic variation and phenotypic diversity [5,6]. Investigations on CNVs have been successively carried out in human and other species [7-13]. In the domestic animals, there are involving in cattle [14-20], dog [21], chicken [22], pig [23,24], goat [25], sheep [26] and rabbit [27]. As for sheep, Fontanesi et al. [26] provided a first comparative map of CNVs of the sheep genome re ferred to the cattle genome using a cross-species array comparative genome hybridization(aCGH). However, the cross-species analysis based on heterologous hybridization couldnt identify all detectable CNVRs due to low homology between cattle probes and sheep DNA for some regions and doesnt show the CNVR distributions on the sheep genome. In addition to CGH, another major platform commonly used to identify CNVs is the SNP array. In SNP array, intensity values of SNPs derived from each sample are used to detect CNVs in each individual. Comparison with two panels, CGH array has excellent performance in signal-to noise ratios, while the SNP array based approach is more convenient for high-throughput analyses and follow-up association studies [28]. With the development of high density SNP arrays, higher resolution of genomic regions can be achieved [29]. Moreover, due to their low cost and high-density, commercial SNP arrays have been widely used for CNV detection in domestic animals, and CNV mapping and functional studies have made important progress. However, there are no reports on CNV detection of sheep based on SNP array data. In this study, we will investigate genome-wide CNV in three sheep populations. To pursue convincing results, we firstly employ the PennCNV program to analyze Ovine SNP50 genotyping data, and then use other algorithm program, cnvPartition, to validate CNVRs detected by PennCNV. To our knowledge, we will construct the first sheep CNV map based on SNP array data. This research will provide useful addition to the sheep CNV maps, and will provide potential genetic markers for further investigation on the roles of CNV in sheep productive traits and evolutionary adaptation. Results SNP genotyping The genomic DNA of 329 individual samples from three sheep breeds (German Mutton sheep, Dorper and Sunite sheep) were genotyped using Illumina OvineSNP50 Genotyping BeadChip according to the manufacturers protocol, and the PennCNV (http://www.openbioinformatics.org/ penncnv) software was used to identify the CNVs in the sheep genome (Table 1). According to the results of PennCNV, we defined the CNV call filtering criteria to exclude samples with low quality of signal intensity data. Table 1 Population size information in sheep copy number variation analysis No, of sheepa PennCNVb a: total samples before quality control by PennCNV and CNVpartition. b: The samples that passed the quality control of PennCNV in 329 individuals from three sheep breeds. c: The samples that passed the quality control of CNVpartition in 329 individuals from three sheep breeds. After applying the CNV quality control criteria detailed in the Methods section, 256 samples (157 German Mutton, 35 Dorper and 64 Sunite sheep) remained for further CNV analyses. Genome-wide surveys of sheep CNVs After filtering unreliable CNV calls, we discovered a total of 3624 CNV events (3416 losses and 208 gains), with an average number of 14.16 CNV events per individual. The average and m (...truncated)