A Snapshot of CNVs in the Pig Genome Joa˜ o Fadista, Marianne Nygaard, Lars-Erik Holm, Bo Thomsen, Christian Bendixen* Group of Molecular Genetics and Systems Biology, Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, Tjele, Denmark Abstract Recent studies of mammalian genomes have uncovered the extent of copy number variation (CNV) that contributes to phenotypic diversity, including health and disease status. Here we report a first account of CNVs in the pig genome covering part of the chromosomes 4, 7, 14, and 17 already sequenced and assembled. A custom tiling oligonucleotide array was used with a median probe spacing of 409 bp for screening 12 unrelated Duroc boars that are founders of a large family material. After a strict CNV calling pipeline, 37 copy number variable regions (CNVRs) across all four chromosomes were identified, with five CNVRs overlapping segmental duplications, three overlapping pig unigenes and one overlapping a RefSeq pig mRNA. This CNV snapshot analysis is the first of its kind in the porcine genome and constitutes the basis for a better understanding of porcine phenotypes and genotypes with the prospect of identifying important economic traits. Citation: Fadista J, Nygaard M, Holm L-E, Thomsen B, Bendixen C (2008) A Snapshot of CNVs in the Pig Genome. PLoS ONE 3(12): e3916. doi:10.1371/ journal.pone.0003916 Editor: Juergen Kroymann, Max Planck Institute for Chemical Ecology, Germany Received July 16, 2008; Accepted November 14, 2008; Published December 16, 2008 Copyright: 2008 Fadista et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors of this paper did not receive any commercial funding for this project. This work was conducted as part of the SABRETRAIN Project, funded by the Marie Curie Host Fellowships for Early Stage Research Training, as part of the 6th Framework Programme of the European Commission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Christian.Bendixen@agrsci.dk Introduction The pig (Sus scrofa) is a cetartiodactyl mammal from a different clade than rodents and primates and last shared a common ancestor with humans approx. 83 million years ago [1]. The porcine genome has an estimated size of 2.7 Gb, consisting of 18 autosomes and the X and Y sex chromosomes [2]. Genomic comparisons between the pig and human have unravelled more structural resemblance than, for example, mouse and human [2– 4]. Pig is also a more trustworthy animal model for human disease since its physiological and anatomical resemblance is far greater than any other laboratory species. Consequently, the pig has been used progressively as a model within the human health research in e.g. obesity, cardiovascular disease, arthritis, diabetes, hyperten- sion, cancer, organ transplantation, and Alzheimer’s disease [5–8]. Further to the biomedical relevance, the pig is of great agricultural importance as the main source of animal protein world-wide (Porcine sequencing white paper). Recently it has been reported that structural variation, like copy number variants (CNVs), is genome-wide present not only in humans [9–17] but also in chimpanzees [18–19], mice [20–23], nematodes [24], fruit fly [25], and cow [26]. CNVs represent segments of DNA larger than 1 kb present at a variable copy number in comparison with a reference genome [27] and they can be responsible for altered gene expression [28] leading to striking phenotypic variance including disease associated traits [29–30]. Despite numerous studies, no assessment of the extent and impact of CNVs in the pig genome has been made until now. Based on a pig family material comprising 14 boars, 700 sows and about 12,000 offsprings this paper presents a preliminary analysis of CNVs detected in the genomes of twelve of the boar founders compared to one unrelated Hampshire boar, using high density tiling-path oligonucleotide array comparative genomic hybridization technology (array CGH) [31]. The designed arrays encompass part of the chromosomes 4, 7, 14, and 17 from the August 2007 preliminary assembly release with a median probe spacing of 409 bp. After a stringent pipeline, the analysis led to the identification of 37 copy number variable regions. Chosen CNVs were further confirmed by RT-PCR [32]. As the first of its kind in pig, this study examines the extent and pattern of CNVs in the pig genome, important for future studies associating phenotype to genome architecture. Results Study design Array CGH was carried out using an array comprising 384,979 oligonucleotide probes covering the preliminary pig genome assembly for part of the chromosomes 4, 7, 14, and 17 with a median probe spacing of 409 bp. Copy number variation was assessed by equating the log2ratio of signal intensity between the reference and test samples. Given the relative type of these comparative data, it was not possible to unequivocally ascertain the real status of the CNVs not RT-PCR validated, and hence whether they were deletions or duplications in the reference or in the test samples. Therefore, the status of the copy number variations reported here is in relation to the reference sample. Since our criteria of CNV detection (Methods and Figure 1) only permit to call a CNV if it is detected in at least two animals, we will be referring to copy number variable regions or CNVRs (merging of overlapping CNVs in two or more animals) instead of CNVs. The possibility that true CNVs exist within the loci discovered only in a single animal is acknowledged, since they may comprise sporadic cases, but in order to minimize the false positive rate, we focused only on CNVs found in two or more animals. Another strong reason to discard CNVs found in only one animal relates to the fact that some CNVs may be somatic and not germline [10,60–61]. Therefore, in this study, as previously [10], a CNV was considered to be ‘‘germline’’ if it was detected in at least two animals. PLoS ONE | www.plosone.org 1 December 2008 | Volume 3 | Issue 12 | e3916