Molecular characterization of two natural hotspots in the Drosophila buzzatii genome induced by transposon insertions

Abstract

Transposable elements (TEs) have been implicated in the generation of genetic rearrangements, but their potential to mediate changes in the organization and architecture of host genomes could be even greater than previously thought. Here, we describe the naturally occurring structural and nucleotide variation around two TE insertions in the genome of Drosophila buzzatii. The studied regions correspond to the breakpoints of a widespread chromosomal inversion generated by ectopic recombination between oppositely oriented copies of a TE named Galileo. A detailed molecular analysis by Southern hybridization, PCR amplification, and DNA sequencing of 7.1 kb surrounding the inversion breakpoints in 39 D. buzzatii lines revealed an unprecedented degree of restructuring, consisting of 22 insertions of ten previously undescribed TEs, 13 deletions, 1 duplication, and 1 small inversion. All of these alterations occurred exclusively in inverted chromosomes and appear to have accumulated after the insertion of the Galileo elements, within or close to them. The nucleotide variation at the studied regions is six times lower in inverted than in noninverted chromosomes, suggesting that most of the observed changes originated in only 84,000 years. Galileo elements thus seemed to promote the transformation of these, otherwise normal, chromosomal regions in genetically unstable hotspots and highly efficient traps for transposon insertions. The particular features of two new Galileo copies found indicate that this TE belongs to the Foldback family. Together, our results strengthen the importance of TEs, and especially DNA transposons, as inducers of genome plasticity in evolution.

Figure 1

Physical map of the distal and proximal 2j breakpoint regions in the st-1 and j-1 lines. Thick lines represent the single-copy A, B, C, and D sequences. TE insertions are represented as empty boxes. Hatched and black rectangles correspond, respectively, to the AB and CD probes used for the Southern hybridization analysis. Small arrows represent primers used in the PCR amplification. Some of the restriction sites found in this region are shown: C, ClaI; D, DraI; H, HindIII; P, PstI; S, SalI.

Figure 2

Schematic representation of the structures found at the proximal (A) and distal (B) breakpoints of inversion 2j in the 30 2j lines studied. All different structures are shown, except for that of j-16 in the proximal breakpoint, which differs from jz³–4 by the absence of d6 deletion. Thick lines represent the single-copy A, B, C, and D sequences. TEs are represented as colored boxes and sharp ends correspond to the ITRs. Insertions and deletions are delimited by green and red lines, respectively, and are named with an i or a d followed by a number. Target site duplications flanking the insertions are shown above them. Blue lines indicate the inversion of an internal segment. Arrows below the diagrams inform on the orientation of some homologous segments. Segments sequenced in each structure are enclosed within clear rectangles. Only the D. buzzatii lines representative of each structural variant are shown. Lines sharing the same structure in the proximal breakpoint are jq⁷–1, jq⁷–2, and jq⁷–3; j-1, j-2, j-3, j-4, j-5, j-6, j-7, j-14, j-15, j-20, j-21, and jq⁷–4; j-9, j-11, j-12, j-13, j-18, and j-22 (deletion d2 was detected during j-12 sequencing and we do not know whether it is present in other lines or not); jz³–1, jz³–2, and jz³–3. Lines sharing the same structure in the distal breakpoint are j-1, j-2, j-3, j-4, j-5, j-6, j-7, j-13, j-15, j-20, j-21, jz³–2, jq⁷–1, and jq⁷–3; j-8, j-11, j-12, j-14, j-16, j-17, j-18, j-22, jz³–1, jz³–3, and jq⁷–4. Hyp are hypothetical structures not found in our sample of 2j lines. Small black arrows are PCR primers used in the study.

Figure 3

Nucleotide polymorphism at the breakpoint regions of inversion 2j. Nucleotide position is represented above the sequences. The breakpoints are taken as start point of A, B, C, D, distal breakpoint insertion, and proximal breakpoint insertion sequences. Nucleotides identical to the first sequence are indicated by a dot and missing data by a question mark. Deletions and insertions are indicated by minus and plus signs, respectively, and their size in base pairs is shown below. Gross deletions affecting the sequenced regions are named as in Fig. 2 and are included in rectangles. TE insertions and target site duplications are not shown. In 2st lines there is a 18-bp stretch between A and B sequences resembling Galileo footprints (Cáceres et al. 1999) that is not represented here either. Positions A65 to A101 in st-3 and st-8 accumulate multiple nucleotide changes with regard to the other lines and are shown in italics.

Figure 4

Neighbor-joining phylogenetic tree of the breakpoint sequences of inversion 2j based on the A, B, C, and D sequence data for the nine 2st and 12 2j Drosophila buzzatii lines. The Ma-4 Drosophila martensis line was used as outgroup. Bootstrap values in percentage out of 500 replicates are indicated for the main nodes.