A molecular perspective on a complex polymorphic inversion system with cytological evidence of multiply reused breakpoints.

Genome sequence comparison across the Drosophila genus revealed that some fixed inversion breakpoints had been multiply reused at this long timescale. Cytological studies of Drosophila inversion polymorphism had previously shown that, also at this shorter timescale, some breakpoints had been multiply reused. The paucity of molecularly characterized polymorphic inversion breakpoints has so far precluded contrasting whether cytologically shared breakpoints of these relatively young inversions are actually reused at the molecular level. The E chromosome of Drosophila subobscura stands out because it presents several inversion complexes. This is the case of the E1+2+9+3 arrangement that originated from the ancestral Est arrangement through the sequential accumulation of four inversions (E1, E2, E9 and E3) sharing some breakpoints. We recently identified the breakpoints of inversions E1 and E2, which allowed establishing reuse at the molecular level of the cytologically shared breakpoint of these inversions. Here, we identified and sequenced the breakpoints of inversions E9 and E3, because they share breakpoints at sections 58D and 64C with those of inversions E1 and E2. This has allowed establishing that E9 and E3 originated through the staggered-break mechanism. Most importantly, sequence comparison has revealed the multiple reuse at the molecular level of the proximal breakpoint (section 58D), which would have been used at least by inversions E2, E9 and E3. In contrast, the distal breakpoint (section 64C) might have been only reused once by inversions E1 and E2, because the distal E3 breakpoint is displaced >70 kb from the other breakpoint limits.