Evolutionary Breakpoints in Gibbon Figure 1. Analysis of gibbon specific segmental duplications. (A) Fluorescence in situ hybridization (FISH) experiments on NLE nuclei and metaphases using gibbon BACs spanning breakpoints which overlap with gSD. The fluorescent signals show a pattern typical of repeated sequences; (B) Images from the Array-CGH experiment using gibbon (test) versus human (reference) genomic DNA. Human chromosomes 2 and 3 are shown; duplications are represented in green and deletions in red. The duplications were validated by FISH on metaphases and nuclei of both human and gibbon using as probes the human BACs from the 32Kset. Duplicated regions present a higher depth of coverage of Trace Archives reads on the human genome as illustrated in the lateral panels. doi:10.1371/journal.pgen.1000538.g001 protein is rescued and still functional. As a measure of relaxed selective constraint on these disrupted genes, we calculated the dN/dS ratio between non-synonymous (dN) and synonymous (dS) substitutions between human and gibbon (using macaque as the outgroup). This analysis was carried out only on the 23 fully sequenced BACs (Protocol S2). The same method was applied to an equal number of randomly selected gibbon BACs sequenced by the NIH intramural sequencing center (NISC) comparative vertebrate sequencing project [9] (Table S3). This analysis showed a significant increase (p = 0.01, Mann-Whitney’s U test) in the dN/ dS ratio of gibbon genes when the breakpoint BACs are compared to the NISC BACs (Figure S2). It is worth noting that the p value becomes even smaller when the genes at ,50 Kbp distance are considered, indicating a possible position effect. To confirm this trend, we sampled additional gibbon genes located at 500 Kbp and 1 M bp from the breakpoints, and found no differences when gibbon was compared to macaque (Table S4). Frequently, genes affected by the breakpoints are part of clusters: the ABCC family on HSA 16, the ABCA family on HSA 17, the growth hormone cluster on HSA 17, RFPL on HSA 22, MUC4 and MUC20 on HSA3, PLSCR (phospholipid scramblase) on HSA 3. The association between breakpoints and gene-clusters has at least two biological implications. First, gene clusters result from duplication events that may cause genome instability through NAHR. Second, the presence of other genes with redundant functions could mitigate natural selection against chromosomal rearrangements that disrupt genes. Gibbon breakpoints are enriched in interspersed and simple repeats The role of repeats in evolutionary or disease-causing chromosomal rearrangements is well documented [17–20]. We identified repeats within 150 bp of the 57 sequenced breakpoints with Repeat Masker. 81% of the breakpoints co-localized with at PLoS Genetics | www.plosgenetics.org 3 June 2009 | Volume 5 | Issue 6 | e1000538