suppression of endogenous retroelements in mammals. If this mechanism is attenuated, the repeated DNA sequences may threaten genome integrity: demethylation leading to an open chromatin structure at repeated sequences may cause structural and numerical variations [10]. Multiple examples of correlations between methylation state and genome structural variation have recently come to light in cancer cells, where disrupted methylation patterns are common [10,27]. Furthermore, it was recently observed that hypomethylated blocks in tumor cell lines correspond to fragile regions of the genome and synteny break- points in the mouse [28]. This correlation suggests a common source of instability independent from genomic sequence and related to the epigenetic state of the DNA. O’Neill and colleagues showed that the genome of a hybrid between two species of Australian wallaby (marsupials) was hypomethylated when com- pared to the parental species [29]. In these hybrids a hypomethy- lated retroviral element was abnormally replicated causing an evident centromeric expansion. The same group also reported double-minute chromosome formation in mouse interspecific hybrids (M. musculus6M. caroli) [30]. Together with our findings this observation indicates that changes in methylation levels may explain perturbations of the uniform rate of genome evolution. Other mammal species (dog, mouse and rat), display very rearranged karyotypes and it will be important to investigate if the scenario we described in the gibbon is common to these species as well. Nevertheless, at the moment, the resolution of the synteny breakpoints for these species is still very far from the one needed to carry out an analysis comparable to the one we performed on the gibbon genome. We have presented here a scenario that may explain the genome reshuffling observed in gibbon species: hypomethylation of certain Alu elements may predispose them to recombination. We are currently investigating the magnitude of the genome hypomethylation in gibbon repeat elements, and whether repeats other than Alu are involved. At the moment we can only speculate about the possible causes of the difference in levels of methylation of Alus that we observed in the gibbon. One hypothesis is linked to the observation that CpG methylation is disrupted in hybrids [29,30]. Population genetics theories propose that speciation may occur after hybrid recombination, followed by inbreeding and reproductive isolation due to the new genetic make-up. This idea is well accepted for plants, and it has recently been proposed for gibbon species [31]. Hybridization may have gradually disturbed the apparatus responsible for the methylation of repeats in the hybrids, leading to higher numbers of chromosomal rearrangements [30]. Very recently the implications of a specific class of small RNAs (piRNAs) in methylation of repeats have been discovered. A rapid divergence of these sequences during speciation could therefore explain the reduction in the cytosine methylation efficiency in cross-species hybrids [32]. Materials and Methods Random sampling simulation The statistical analysis of the breakpoints repeat and duplication content was performed with the help of a C# application written in-house [11]. Tracks of genome-wide repeat content for different subcategories of repeats and for segmental duplications content were prepared for input to the simulation software using data from http://genome.ucsc.edu human genome (hg18 release). The measure we used counted up the existence of at least one element of the corresponding track in each region of the set and returned a detailed report for the set. To attain the simulation, the program Evolutionary Breakpoints in Gibbon reallocates randomly al the regions maintaining the chromosome of origin and size as the initial counterpart. The same measurements were taken for each random set after a reiteration of 5,000 times. The resulting sampling distribution was then plotted to compare the original set of regions with the global genomic landscape. The track relative to gibbon specific segmental duplication was built as result of our in silico analysis of the trace archives. Subsequently the latter tracks were used in order to perform different overlap measurements with the set of 57 breakpoints. When mapped on the human genome and the regions with ambiguous mapping are removed, the dataset corresponds to 120 regions of about 500 bp size (on average). Another set which we called ‘‘stringent set’’ was also used to determine the overlap with hSD. In this set all the breakpoints form two BACs (CH271-298N13 and CH271-372B11) known to be centromeric in the gibbon and containing multiple breakpoints, were excluded. In silico segmental duplication detection Gibbon reads were downloaded from the NCBI Trace Archives and screened for quality. A total of 24,350,447 reads that passed quality screening were mapped to the human genome (build NCBI 36.1, UCSC hg18) using Pash [33,34]. In order to remove highly ambiguous mappings, reads mapping to .500 locations with a score within 6% of its top mapping score were removed from consideration. Furthermore, reads that overlapped by .75% with repeats, as identified by RepeatMasker [35], were removed from consideration. A total of 15,518,707 mapped reads remained after filtering. Putative gibbon segmental duplications were identified follow- ing the method outlined in Bailey, et al., 2002 [16]. The number of gibbon mapped reads was determined in 5000 bp windows across the human genome. The mean (31.11) and standard deviation (18.75) of mapped read counts was calculated across windows not overlapping with human segmental duplications. A read count cutoff of 3 standard deviations from the mean was applied meaning any 5000 bp region with .87 mapped reads was identified as a putative gibbon segmental duplication. This resulted in 1630 identified gibbon segmental duplications. Array CGH 32,855 BACs, spanning 95% of the human euchromatic genome, have been assembled and re-arrayed into 384-well microtiter dishes [36,37]. DNA was purified, amplified using the DOP-PCR method, and spotted on CMT-GAPS coated glass slides (Corning, UltraGaps). Genomic DNA from NLE was obtained from blood and anonymous human reference DNA was obtained from Children’s Hospital Oakland Research Institute. Labeling and hybridization were performed essentially as described by [38]. Hybridization images were generated by scanning the slides on a 4000B scanner (Axon). The images were first processed using GenePix Pro 5.1 (Axon Instruments). The primary experimental data (GenePix Results files) were subjected to fully standardized data-analysis (flagged spots removal, background subtraction and loess normalization) by uploading them to the BASE micro-array analysis software installation [39] which performs standard normalization. The final output was human chromosome specific plots of Log2ratio values vs chromosome location as well as a whole genome view. Fluorescence in situ hybridization (FISH) Chromosome preparations were obtained from peripheral blood following standard procedures. Briefly, blood was incubated with cell culture media and phytohemagglutinin (GIBCO) for PLoS Genetics | www.plosgenetics.org 8 June 2009 | Volume 5 | Issue 6 | e1000538