Author Summary Mammalian genomes are remarkably stable (with few exceptions). In humans, wrong recombination events occur quite rarely, manifesting themselves in genomic disorders or cancer. On exceptional occasions, the rate of genome evolution has been accelerated by genome-wide reshuffling events giving rise to some highly derivative karyotypes. The genomes of gibbon species (Hylobatidae) are an example of accelerated genome structural evolu- tion; gibbons display a rate of chromosome evolution 10– 20 fold higher than the default rate found in mammals (one chromosome change every 4 million years). As we are interested in investigating the possible genetic causes of this phenomenon, we sequenced a considerable number of chromosomal breakpoints in the northern white- cheeked gibbon genome and analyzed the genomic features of these sites. We observe that the gibbon breakpoints are mostly associated with endogenous retrotransposons called Alus, which are normally abundant in the genomes of primates. Furthermore, our analysis revealed that gibbon Alus have a lower content of methylated CpG when compared to the orthologous human Alus. In mammals, CpG methylation is known to be responsible for keeping retrotransposons in a repressed state and protect genome integrity. We therefore suggest that a glitch in the methylation apparatus might have driven the higher genome recombination in gibbons. In search of an explanation for the abundance of evolutionary chromosomal changes in gibbons, we have now characterized the sequence and molecular structure of 57 breakpoint sites in the northern white-cheeked gibbon (Nomascus leucogenys leucogenys, NLE). We had previously created a high-resolution physical map of the break of synteny regions for this species [11], using the human genome as a reference. This map allowed us to localize the breakpoints within an 80 Kbp range. We have identified an association between the breakpoints and Alu retroelements, and we find that Alu elements in the gibbon are undermethylated in comparison to their human orthologues. Our findings suggest that epigenetic activity of Alu sequences may have facilitated karyotypic evolution and disruption of the uniform rate of chromosomal changes in gibbon species. Results Identification and sequencing of 57 gibbon breakpoints To identify the breakpoints at the sequence level we selected 80 Bacterial Artificial Chromosome (BAC) from the Nomascus leucogenys leucogenys (NLE) genomic BAC library (CHORI-271) spanning the breakpoints of translocations and inversions. These BACs were selected from a high-resolution map that we constructed [11] and from a complementary list of gibbon BACs identified as spanning breakpoints by BAC End Sequencing (BES). Out of these 80 BACs, 23 were sequenced using a shotgun approach and assembled to high quality sequence (Table S1). The final assembled sequences were individually aligned by BLAT [12] to the most recent human genome assembly (hg18), and we identified the breakpoints between human and gibbon at the base pair level (Table S2). As we sequenced the BACs, we discovered multiple breakpoints inside the same clone in 8 of the 23 BACs including two cases previously reported by us. The complex structure of three of these BACs may be explained by their centromeric location in the gibbon (Table S2). In a few instances the presence of human segmental duplications did not allow for Evolutionary Breakpoints in Gibbon unambiguous mapping. To enrich our breakpoint dataset in a cost effective way, we pooled and shotgun sequenced at lower coverage the remaining 57 gibbon BACs (Protocol S1). This approach added 33 breakpoints to our dataset: 25 at the base pair level and 7 at the resolution of a small insert clone (about 6 Kbp) (Table S2). The remaining breakpoints could not be identified, due either to densely repeated regions or to lack of coverage. This brought the final number of breakpoints to 68 (57 at the base pair level). These results indicate that the frequency of breakpoints is higher than BES mapping alone can estimate. Hence assembly of the gibbon genome will be necessary to pinpoint all the breakpoints. Gibbon breakpoints show overlap with human- and gibbon-specific segmental duplications In a previous study we uncovered a significant association between gibbon break of synteny regions, identified at a resolution of 80 Kbp, and human segmental duplications (hSD) [11]: 42% of the breakpoints were found to overlap with at least one hSD. In the current study we were able to identify the breakpoints at a higher resolution. This allowed us to further examine their relationship with SDs by measuring the correlation between a 1 Kbp window (2/+500 bp) including the breakpoint mapped on human and hSDs. We found that 15% of the breakpoints overlap with at least one hSD, which is significant (p = 0.0002) based on a random sampling simulation (performed as described in Materials and Methods) (Figure S1A). Recent studies have shown that a burst in duplication occurred in humans and chimpanzees after their divergence from other hominoids [13–15]. Thus we assume that the hSDs do not always correspond to gibbon SDs (gSD). As an assembled gibbon genome is not yet available, we used two methods to identify gSDs. First, we performed array-comparative genomic hybridization (array-CGH) of gibbon genomic DNA against human genomic DNA. This experiment allowed identifi- cation of large (.300 Kb) duplications/deletions that distinguish the two species. Second, following the method described by Bailey et al. [16], we mapped gibbon reads from the Trace Archives onto the human genome and identified putative gSD regions by detecting a higher depth of coverage by the reads (supporting online material). Of the gSDs identified by array-CGH, 37% were also identified as putative gSDs based on read coverage. Using the random sampling simulation approach mentioned above, we noticed that the overlap between the gibbon breakpoints and the gSDs is extremely large and statistically significant (Figure S1B), more than the overlap observed for the hSD. Examples of gibbon segmental duplications in breakpoints that could be detected by FISH are shown in Figure 1A. Even though the array-CGH and read-coverage-based gSD datasets do not show exact correspon- dence, we observed a significant correlation (p = 7.63e-9 by Fisher’s Exact Test). We also validated, by Fluorescence in situ Hybridization (FISH), 11 duplications and 11 deletions identified by both methods (Figure 1B). Of note, the array-CGH results showed an excess of deletions in the gibbon relative to human (data not shown). We verified that 30% of the deletions are regions that are present in human at a higher copy number than in gibbon, confirming the occurrence of abundant human-specific duplication events. Gibbon breakpoints disrupt genes We looked at the relationship between breakpoints and genes. When mapped onto the human genome, 53% (36 out of 68) of the breakpoints occur within a gene and 19% occur within non-coding transcripts (Table S2). We hypothesize that when a breakpoint disrupts a gene, the selective pressure on the sequence should be reduced as a consequence of loss of function, unless the truncated PLoS Genetics | www.plosgenetics.org 2 June 2009 | Volume 5 | Issue 6 | e1000538