Pattern and frequency of CNVRs Following the methodological copy number pipeline, 37 CNVRs were identified by array CGH across all the four chromosomes queried (Figure 2) with the proportion of any given chromosome amenable to CNVR varying from 0.03% to 0.31%. In summary, 19 (51.4%) CNVRs were called in two animals and the remaining (48.6%) called in three or more animals. Concerning copy number status, 18 (48.6%) were called as gains and the remaining 19 (51.4%) called as losses (Table 1). Previously, it has been suggested that deletions are under stronger purifying selection than duplications [33]. If so, deletions Figure 2. Ideogram from the Sus scrofa (May 2008 assembly) representing the CNVRs detected in our study. Losses are depicted in red while gains are in green. Not all the 37 CNVRs are visible since some are very close to each other. doi:10.1371/journal.pone.0003916.g002 CNVs in the Pig Genome should be both less frequent and shorter than duplications. When comparing the length and number of gains versus losses in the CNVRs, practically the same number was detected (see above) while the total size of the gains was about 16 kb larger than the size of the losses (although according to the Wilcoxon rank sum test not statistically significant at p value #0.01). Copy number regions were discarded if (1) they overlapped more than one contig and (2) contained gaps due to the high error rate of this preliminary assembly. This was done because contigs can be misassigned and gaps may contain future contigs that can result in the disruption of the CNVRs called (see Figure 3 for an example of a CNVR called). The CNVRs ranged in size from 1.74 kb to 61.92 kb with a mean of 9.32 kb and a median of 6.89 kb, covering 429.269 kb (0.18%) of the 237.76 Mb of sequence addressed (Table 2). When querying the part of the genome covered by CNVRs for the greatest divergence in genome size between two animals among our set, it was found that animal B had the biggest net gain, spanning 57.56 kb over five CNVRs, while animal E had the biggest net loss with 2106.6 kb over eleven CNVRs. Comparison of these genomes disclosed a difference of 164.1 kb in size between these two animals. There are seven CNVRs that are apparently aberrant in the genomes of at least half of the test boars. However, these CNVRs are most likely to be aberrant regions in the reference boar since this is of a different breed (i.e. Hampshire), and probably more structural genomic variation is present between breeds than within the same breed, since all the test boars are of the Duroc breed. This hypothesis remains to be tested. Previous analyses have reported enrichment of CNVs near segmental duplications (sequences $1 kb in size with sequence identity $90% [34]) in humans [9–12,16–17], mice [20–23], and chimpanzees [19]. Segmental duplications have been mapped in the genomes of human [35], chimpanzee [36], and mouse [37], but the incomplete and highly error-prone preliminary assembly of the pig genome prevents us from drawing such a map. Consequently, only the available numbers can be focused on in this study. Among the regions found to contain CNVs, five (13.5%) overlapped segmental duplications (Table 1 and Methods). Despite the discovery of CNVRs overlapping segmental duplications, it is important to note that our array probe design is biased against the detection of CNVRs that coincide with sites of segmental duplication because it only allowed probes that had a unique match in the genome (Methods). Functional analysis In order to assess the gene content within the CNVRs reported, a sequence similarity $98% search between the pig Unigene database and the CNVRs was made and three pig Unigenes were retrieved (Table 1). Since a CNV can also affect gene expression at long distances [28] an additional search was performed for pig Unigenes that showed $98% sequence similarity with the contigs where the CNVRs are. Contigs with CNVRs range in length from 5.577 kb from 245.924 kb. Further there were nine new Unigenes in eight of the contigs (CNVR.1 - Ssc.28459; CNVR.11 - Ssc.25025; CNVR.12 - Ssc.26197; CNVR.13 - Ssc.26126; CNVR.14 - Ssc.42797, CNVR.25 - Ssc.8364; CNVR.31 - Ssc.38482, Ssc.14020; CNVR.33 - Ssc.63374) . Searching the Refseq mRNA database for vertebrate mammals, a gene was identified (95% sequence id) as part of the contig containing the CNVR 33. This Refseq mRNA corresponds to the ADRA2 gene, encoding the alpha2A-adrenergic receptor - a transmembrane receptor belonging to the rhodopsin family from which genes have been consistently reported to overlap CNV PLoS ONE | www.plosone.org 3 December 2008 | Volume 3 | Issue 12 | e3916