Hoolock Hylobates Hylobates agilis agilis Hylobates agilis unko Hylobates albibarbis Hylobates lar lar Hylobates lar carpenteri Hylobates lar entelloides Hylobates lar vestitus Symphalangus syndactylus continentis Symphalangus Symphalangus syndactylus syndactylus doi:10.1371/journal.pone.0004999.t001 Table 1. classification of gibbons as described in [3,9,29] Genus Nomascus Species Common name Nomascus concolor concolor Western black gibbon Nomascus leucogenys siki Southern white-cheeked gibbon Nomascus concolor lu Nomascus nasutus Nomascus hainanus Nomascus leucogenys leucogenys Nomascus gabriellae Hoolock hoolock Hoolock leuconedys Hylobates klossii Hylobates pileatus Hylobates moloch Hylobates muelleri muelleri Eastern Mu¨ ller’s gibbon Hylobates muelleri funereus Northern M u¨ller’s gibbon Hylobates muelleri abbotti Abbott’s gray gibbon Laotian black gibbon Eastern black gibbon Hainan gibbon Northern white-cheeked gibbon Buff-cheeked gibbon Western hoolock gibbon Eastern hoolock gibbon Kloss’ gibbon Pileated or capped gibbon Javan, silvery, or moloch gibbon Mountain agile gibbon Lowland agile gibbon Bornean white-bearded gibbon Malayan lar gibbon Carpenter’s lar gibbon Mainland lar gibbon Sumatran lar gibbon Sumatran siamang Malayan siamang detection of most syntenic homologies with human and the great apes [8,11–16]. This observation has drawn the interest of many scientists in the field of genome evolution. Nearly, all modes of chromosomal rearrangements observed in the karyotypic diver- gence of mammals have been recognized in gibbons (pericentric and paracentric inversions, chromosomal fission, Robertsonian and tandem fusion, reciprocal translocations). This feature is even more striking if one considers that the chromosomes of more distantly related primates, like Old World monkeys, share more similarities with humans than gibbons. With the aim of identifying genetic bases of such instability we recently created a high-resolution map of synteny disruptions between the northern white-cheeked gibbon (Nomascus leucogenys leucogenys, NLE) and the human [14]. The northern white-cheeked gibbon is a critically endangered species (IUCN 2008, www. iucnredlist.org) with possibly less than 500 individuals left in the wild [T Nadler unpublished data], with successful captive breeding programs taking place. We report here the analysis of one particular inversion on NLE chromosome 7 described in the literature as ‘‘polymorphic’’ for individuals from the genus Nomascus. This assumption is based on scarce observations [17,18] mostly because of difficulties in performing large scale cytogenetic studies on species from the genus since there are very low numbers of unrelated captive individuals [19,20]. By taking advantage of the breakpoint sequence availability, we designed a test to discern presence or absence of this inversion simply by Gibbon Chromosomal Inversion PCR. This approach does not require high quality DNA or chromosome preparations, and therefore additional individuals can be targeted, including those of which only low quality DNA is available. From our study samples we were able to show that only northern white-cheeked gibbons carry this inversion, with no evidence of it being polymorphic in this subspecies. As a result, our PCR test can become one of the tools for guiding some of the housing strategies in zoos and conservation centers with the advantage of not requiring cytogenetic experts. Results Cross-species analysis of breakpoint spanned by BAC CH271-263C9 With the aim of looking at the mechanisms underlining the abundance of chromosomal rearrangements in gibbons, we recently established a physical map of synteny breakpoints between the northern white-cheeked gibbon and human. We identified 67 gibbon BAC (Bacterial Artificial Chromosome) clones spanning gibbon-human synteny breakpoints. We addi- tionally fully sequenced a sample of these BACs to identify the position of the breakpoints at the base pair level. This sample included clone ‘‘CH271-263C9’’ (sequence accession CT954303) whose BES (BAC End Sequences) map onto human chromosomes 22 (HSA 22) and 4 (HSA 4), respectively. This BAC was identified by screening high density filters containing the NLE genomic BAC library (CHORI-271) using the mapping information obtained by array-painting [14]. Using FISH (fluorescence in situ hybridiza- tion), we verified that clone CH271-263C9 localizes on NLE chromosome 7b (NLE7b; Figure 1A) in correspondence of sequences homologous to human chromosome 22. We identified this junction to result from the inversion on NLE7 described by Couturier and Lernould [17]. In their study, chromosome banding was used to compare four crested gibbon taxa (N. gabriellae [buff- cheeked gibbon], N. l. leucogenys, N. l. siki [southern white-cheeked gibbon], N. hainanus [Hainan gibbon]) and to identify taxon- specific karyotypic traits. We decided to investigate the origin of this inversion in the gibbon lineage using a cross-species approach which takes advantage of the availability of the breakpoint site sequence. Our strategy is quite straightforward: two ‘‘breakpoint primers’’ (BP_primers) are designed on both sides of the break- point originally identified in NLE. When these primers are tested on the genomic DNA, they will generate an amplification product only if the breakpoint is present. An additional PCR experiment is required to rule out that the absence of an amplification product is due to a technical artifact: the breakpoint forward primer is combined with a ‘‘human specific’’ reverse primer (confirma- tion_primers), designed in the corresponding non-disrupted region of the human chromosome (Figure 1B). In the scenario where a breakpoint identified in NLE is not shared by another gibbon taxon, the PCR with the breakpoint primers should be negative as opposed to a positive result with the confirmation primers. Using this method, many samples can easily be tested per experiment, contingent on the availability of genomic DNA samples of minimal quality and quantity. In our first experiment, we used genomic DNA from nine individuals representing nine gibbon species in all four genera (Table 2, Figure 1C): N. leucogenys, N. gabriellae, Symphalangus syndactylus (siamang), Hoolock leuconedys (eastern hoo- lock gibbon), Hylobates moloch (Javan gibbon), H. lar (lar gibbon), H. muelleri (Mueller’s gibbon), H. pileatus (pileated gibbon) and H. albibarbis (white-bearded gibbon). The PCR results on this first set of samples revealed that the junction HSA 4-22 was present exclusively in NLE (Figure 1C). All the other species lacked breakpoint amplification whereas the PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4999