Gibbon Chromosomal Inversion Figure 1. FISH and cross-species PCR with BAC clone CH271-263C9. A) Cohybridization of the whole chromosome paint of human chromosome 22 (red) and BAC CH271-263C9 (green) on NLE and NGAB (N. gabriellae) metaphases. These two species carry a different form of chromosome 7 (7b and 7a) due to an inversion which occurred in NLE; B) the image shows the cross-species PCR strategy; C) gel image summarizing the result of the cross-species PCR on different gibbon taxa using the primers illustrated in B. The weak band for H. pileatus is the result of a PCR artifact. doi:10.1371/journal.pone.0004999.g001 region syntenic to human HSA 22 was amplified. We were not successful in amplifying the region homologous to HSA 4. Most significantly, the HSA 22-4 breakpoint was also absent in N. gabriellae indicating that this breakpoint may correspond with the cytogenetic inversion described by Couturier and Lernould [17]. Our findings were further confirmed by FISH using two differentially-labeled BACs (CH271-263C9 & CH271-457L13) as probes. These gibbon clones map in silico to the same combination of syntenic regions on human chromosomes 22 and 4, as determined from the gibbon BES. The FISH results revealed that the two BACs map on different locations on the same chromosome for NLE, but co-localize for six other species representatives of three genera (Figure 2A). This is consistent with the two probes spanning the reciprocal breakpoints resulting from the NLE-specific inversion. Surprisingly clone CH271-457L13 generates an unusual pattern on the hoolock gibbon chromo- somes, hybridizing to the centromeric regions of most chromo- somes (Figure 2B). In humans and in the other gibbons this clone does not map on centromeric regions. As we were intrigued by this pattern we sequenced clone CH271-457L13 (at low coverage) using a shotgun approach and we looked for possible traces of satellites or repeats. The only satellite sequence we were able to identify was HSATI in the portion of the clone mapping on HSA 22. To understand if this satellite was responsible for the FISH pattern in Hoolock, we chose human BAC clones overlapping the same genomic region and performed further FISH analyses on hoolock gibbon chromosomes. None of the BACs produced the same pattern observed for clone CH271-457L13, instead a single spot was detected (data not shown). We therefore assumed that the gibbon clone contains some additional gibbon specific sequences (most likely repeats) that are present at high concentration at the centromere in the hoolock gibbon. This is additional evidence that regions overlapping with synteny breakpoints are often rich in repeats and present some kind of plasticity whose outcome can vary in different species [14]. Junction HSA 22-4 is specific for NLE Our preliminary investigation showed that this inversion is specific for NLE in our small set of samples. We then needed to exclude the possibility that this inversion is polymorphic in NLE and/or closely related taxa. We did not find any study including an adequate number of individuals to reach a conclusion. Nevertheless this rearrangement has been defined polymorphic [18]. We, therefore, tested the ‘‘BP_primers’’ and the ‘‘confirma- tion_primers’’ on 51 crested gibbon DNA samples obtained from blood, tissue or hair (Table 2, materials and methods). The results of this extensive investigation confirmed that the breakpoint HSA22-4 is exclusive for the northern white-cheeked gibbon. Our sample also includes five southern white-cheeked gibbons (N. l. siki, NLS), three of which were ‘‘wild-born’’ (Table 2). Couturier and PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4999