Fontanesi et al. BMC Genomics 2010, 11:639 http://www.biomedcentral.com/1471-2164/11/639 To evaluate if CNVRs we identified in goats overlap with CNVRs reported in cattle, we compared our results with those obtained in four independent cattle experi- ments [49-52] carried out i) using aCGH including ~385,000 tiling oligonucleotides (177 CNVRs [51]) or ii) including 6.3 million of probes (304 CNVRs [52]) and iii) using the Illumina BovineSNP50 BeadChip contain- ing about 50K SNPs as reported by Matukumalli et al. ([49]; 79 CNVRs) and by Bae et al. ([50]; 368 CNVRs) (Additional file 4 and Additional file 5). Overlapping between aCGH results obtained in goat and cattle was highly significant (P < 0.0001) for both cattle experi- ments (17 and 11 goat CNVRs overlapped with cattle CNVRs identified by Liu et al. [51] and Fadista et al. [52], respectively). Only two goat CNVRs matched cattle CNVRs identified with the SNP panel [49,50], therefore overlapping was not significant. A similar bias on the common CNVRs between aCGH and SNP genotyping experiments was also evident comparing the cattle data obtained by the two methods (Additional file 5 and [51]). This could be due to resolution and genome cov- erage differences between the two platforms [51]. How- ever, merging all cattle CNVRs reported in the four different experiments (on the whole 764 unique cattle CNVRs were obtained [49-52], Additional file 4), overlap with goat CNVRs was highly significant (P < 0.0001), confirming the results obtained considering the different cattle datasets separately (Additional file 5). As the goat genome is not sequenced yet, we could not evaluate if the goat CNVRs have similar sequence characteristics in goat and cattle. Segmental duplications have been shown to significantly overlap with CNVRs in cattle [51,52] as well as in several other species [31,32, 34,35,37,39,46]. As segmental duplications might facili- tate non-allelic homologous recombination, it is likely that they are as well involved in the mechanisms that produce CNVs in goats. The overlapping CNVRs between goat and cattle might represent homoplastic recurrent interspecies CNVRs probably driven by geno- mic regions prone to instability present in the cattle- goat common ancestor that might have been retained in the genomes of both extant species. Indeed, cattle and goat share a common ancestor in the early Miocene about 17-23 Million years ago [4-6]. Similar reasoning could be considered for the CNVR that includes the ASIP gene [22] for which a recurrent CNVR has been reported in sheep [61], but not in cattle. Sheep and goat lineages separated about 6-14 millions of years before present [80]. Significant overlap of CNVRs among differ- ent species has been also observed comparing the human with both chimpanzee and rhesus macaque gen- omes [35-37]. These two non-human primate species diverged from the human lineage about 6 and 25 million years ago [81,82]. Together these results suggest that Page 6 of 15 certain genomic regions are prone to recurrent CNV formation and instability in both the primate and the Artiodactyla evolutionary lineages. However, a change in the formation process of CNVs and segmental duplica- tions in the human genome might be occurred in recent evolutionary history [83]. It will be interesting to evalu- ate if this has occurred in other lineages. A comparative analysis of CNVRs identified in cattle, goat, and sheep can open perspectives to evaluate the evolutionary mechanisms determining CNV formation during the mammalian evolution. Validation and gene content of CNVRs Several results suggest most of our CNVs are correctly identified. First of all, the number of CNVs was lower in the analysed Camosciata delle Alpi goat (no. = 8; Table 1). This was expected because the aCGH reference was a sample of genomic DNA of another goat of the same breed. Similar results were also reported in mouse and dog aCGH studies that used a reference genomic DNA of an animal of the same breed/line of others that have been analysed for CNV discovery [39,45]. In addition, an internal control included in the design of the tiling arrays was represented by a portion of BTA13 derived from the UMD 2.0 assembly that contains the ASIP and AHCY genes that we previously showed to be affected by CNV in goats [22]. All goats that were shown to have multiple copies of the ASIP and AHCY genes with different methods [22] reported evidence of gain in the aCGH experiment, compared to the reference Camos- ciata delle Alpi genomic DNA. The results for this region were also used to set an empirical threshold to call CNVs by using the CGHweb platform and multiple algorithms [77] (see Methods). Other three CNVRs identified on the goat chromo- somes corresponding to BTA4, BTA10, and BTA17 (Table 3 and Additional file 6) were directly validated with SQF-PCR as reported for the ASIP and AHCY genes [22]. The CNVR of BTA4 (CNVR no. 25) included a gene coding for the GTPase, IMAP family member 1 (GIMAP1; ENSBTAG00000001198) that was used to design the primers for PCR validation. Semiquantitative fluorescent multiplex PCR confirmed the gain of copies already reported in the goats used in the aCGH experi- ment and in additional goats of the Saanen, Girgentana and Murciano-Granadina breeds (Figure 3A) (Additional file 7). The evaluated CNVR of BTA10 (CNVR no. 61) was originally identified in a Murciano-Granadina goat by aCGH (Additional file 1 and Additional file 2). The bovine sequence coding for an unknown transcript (ENSBTAG00000027170) positioned in this region was used to design PCR primers for validation. However, analyzing the bovine genome by BLAT using the ENSBTAG00000027170 sequence, a large number of