Gibbon Chromosomal Inversion Table 2. cont. Species N. gabriellae Cross-genus PCR Species S. syndactylus H. leuconedys H. moloch H. agilis H. lar H. muelleri H. albibarbis H. pileatus ID 94241 ID SS901 HL307 HMO894 15353 9088 8136 212067 HP120 material DNA Material blood blood blood blood blood blood blood blood origin/collector/storage Los Angeles Zoo origin/collector/storage Gibbon Conservation Center Gibbon Conservation Center Gibbon Conservation Center Henry Doorly Zoo Gladys Porter Zoo Gladys Porter Zoo Louisiana Purchase Gardens Zoo Gibbon Conservation Center captive/wild born captive born sibling to 95141 captive/wild born captive born wild born captive born captive born captive born captive born wild born captive born Abbreviation: EPRC; Endangered Primate Rescue Center; GBP; Gene Bank of Primates doi:10.1371/journal.pone.0004999.t002 Lernould [17] reported in their study that northern and southern white-cheeked gibbons share the same form of chromosome 7. The PCR test with the breakpoint primers did not generate an amplification product from the NLS samples, while amplification with the confirmation primers was obtained. This result indicates that the five NLS do not carry the inversion found in NLE. Multi-species sequence analysis of the HSA 4-22 breakpoint site DNA sequences of the amplification product of the undisrupted region in different gibbon species (H. agilis (agile gibbon), H. moloch, S. syndactylus and H. leuconedys) gave us the opportunity to reconstruct and analyze the inferred ancestral chromosomal site, which in NLE was then disrupted by the inversion. This analysis demonstrated that the ancestral locus contains two Alu sequences, which are also present in human. The inversion breakpoint disrupted one of the Alu elements that recombined with a LTR (Long Terminal Repeat) element located on the segment homologous to human chromosome 4 (Figure S1). From the study of the other synteny breakpoints of the northern white- cheeked genome, we know that Alu elements are the most represented repeats at the breakage sites (Carbone unpublished data). This is not surprising, as it is well known that Alu-Alu recombination events are often responsible for chromosomal rearrangements in primate evolution and human genomic disorders [21,22]. Interestingly, one example of a gene deletion due to an Alu-Alu recombination event has been recently character- ized in detail in gibbons [23]. Following inspection of the human reference assembly revealed that this breakpoint most likely did not result in a gene disruption on chromosome 22 or chromosome 4 in the northern white- cheeked gibbon. Discussion In this study we present the analysis of a chromosomal inversion that differentiates the northern white-cheeked gibbon (NLE) from the other gibbon taxa. This chromosomal rearrangement has Figure 2. FISH on different gibbon species. A) FISH experiments on different gibbon species using as probe CH271-263C9 (red) and CH271- 457L13 (green) which span the reciprocal breakpoints of the inversion on NLE7. B) BAC clone CH271-457L13 shows a peculiar pattern in hoolock with cross-hybridization on almost all the centromeres. Abbreviation used in the figure: NGAB is N. gabriellae, HLA is H. lar, HMO is H. moloch, HMU is H. muelleri, HPL is H. pileatus, HLE is H. leuconedys, SSY is S. syndactylus. doi:10.1371/journal.pone.0004999.g002 PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4999