Principles of genome evolution in the Drosophila melanogaster species group

Abstract

That closely related species often differ by chromosomal inversions was discovered by Sturtevant and Plunkett in 1926. Our knowledge of how these inversions originate is still very limited, although a prevailing view is that they are facilitated by ectopic recombination events between inverted repetitive sequences. The availability of genome sequences of related species now allows us to study in detail the mechanisms that generate interspecific inversions. We have analyzed the breakpoint regions of the 29 inversions that differentiate the chromosomes of Drosophila melanogaster and two closely related species, D. simulans and D. yakuba, and reconstructed the molecular events that underlie their origin. Experimental and computational analysis revealed that the breakpoint regions of 59% of the inversions (17/29) are associated with inverted duplications of genes or other nonrepetitive sequences. In only two cases do we find evidence for inverted repetitive sequences in inversion breakpoints. We propose that the presence of inverted duplications associated with inversion breakpoint regions is the result of staggered breaks, either isochromatid or chromatid, and that this, rather than ectopic exchange between inverted repetitive sequences, is the prevalent mechanism for the generation of inversions in the melanogaster species group. Outgroup analysis also revealed evidence for widespread breakpoint recycling. Lastly, we have found that expression domains in D. melanogaster may be disrupted in D. yakuba, bringing into question their potential adaptive significance.

Figure 1. Molecular Organization of Three Genomic Regions of the Right Arm of Chromosome 3 in D. melanogaster, D. yakuba, and in D. simulans

These three genomic regions harbor the breakpoints of the paracentric inversions 3R(7) and 3R(8), also known as In(3R)84F1;93F6–7, and have been reconstructed by BLAST analysis, in situ hybridization, resequencing, and whole-genome alignments at UCSC (http://genome.ucsc.edu/). According to the information in D. erecta and different outgroup species (Table 1), D. simulans (S) is the species that best represents the ancestral (A) configuration for all three regions. Reference genes at the different breakpoint regions have been colored red, blue, and orange. Between some of the reference genes, putatively expressed genes (green and yellow; [60]) and repetitive sequences (pink) are also present. Other surrounding genes are indicated in brown. Top, cytological coordinates of the regions in D. melanogaster (M). Long horizontal lines indicate chromosomes; solid pattern indicates key region; and dashed pattern indicates chromosomal stretch separating key regions. Cen, centromere; Tel, telomere. The head of each colored horizontal arrow represents the 3′ end of each gene or putative gene. Chromosomal segments included in the inversion 3R(7) and 3R(8) are indicated by dotted lines. For both inversions, the sequences between paired staggered breakpoints are indicated by short horizontal solid lines. Roman numerals indicate different chromosomal stretches spanning inversion breakpoints that were sequenced as a control. Vertical arrows indicate the localization in the ancestor (D. simulans) of the four breakpoints (a, b, c, and d) that are necessary to explain the inversion 3R(8) and the duplication of HDC14862, pfd800, and HDC12400 at 84E9 (3R:3862326–3867817; 3R:3874931–3876653) and 93F6–7 (3R:17554739–17562483) of D. melanogaster (see Figure 2). The gene configuration CG7918-CG34034-CG5849 has been disrupted independently in the lineages of D. melanogaster and D. yakuba (Y) by the inversions 3R(8) and 3R(7), respectively. In D. melanogaster, the gene pair CG2708 (Tom34)-CG31176 is also disrupted, whereas in D. yakuba, CG31286-CG1315 is disrupted. Inversion 3R(8) and its associated duplication event generate an apparently full copy of the putative expressed gene HDC14862 in 3R:93E10-F2 of D. melanogaster. This contains 56–59 bp from the 3′ UTR of the gene CG2708 (blue triangle) within one of its putative introns. HDC14862 is present as two different fragments both in D. simulans and D. yakuba (see main text for details). Further, the inversion 3R(7) has disrupted the antisense overlap of CG31286 and CG1315 in D. yakuba: the antisense configuration is conserved at 84A1 of D. melanogaster and D. simulans, as well as in other species (Table 1). Inversion 3R(7) was accompanied by a duplication of CG34034 and a complex pattern of rearrangement that also involved a fragment of the 5′ region of HDC14862. The two open reading frames (ORFs) of CG34034 are functional according to GENSCAN (http://genes.mit.edu/GENSCAN.html), although the putative protein sequences they encode differ substantially from that of their orthologs in D. melanogaster and D. erecta. Some stretches with significant homology with CG31286 are also detected adjacent to CG1315 in D. yakuba. The reference gene CG31286 is also tandemly duplicated and adjacent to CG34034. In D. yakuba, there are three copies of CG31286, two of them being pseudogenes (denoted as a red gradient). Only the copy immediately distal to HDC12143 is functional, although it apparently codes for only one of the two isoforms of its D. melanogaster ortholog. Genes and distances between them are not represented proportionally.

Figure 2. An Isochromatid Model with Staggered Single-Strand Breaks Can Give Rise to an Inversion Accompanied by Duplications at the Breakpoint Regions in Inverted Orientation

The mechanism is illustrated in relation to the inversion 3R(8), which is fixed in the lineage to D. melanogaster. (A) Ancestral state in D. simulans (Figure 1). (B) Two pairs of staggered single-strand breaks (a-b and c-d) result in long 5′-overhangs (C), which can then be filled in (grey dashed arrow); when followed by nonhomologous end joining, this may result in an inversion flanked by inverted duplications of the sequences between the paired single-strand breaks (D). Landmarks: A, CG2708; B, HDC14862 (3′); C, pfd800; D, HDC12400; E, HDC14861; F, HDC14861; G, CG31176; H, CG7918; I, HDC14862 (5′); J, CG34034; and K, CG5849. Color code as in Figure 1. Figure S2 illustrates the model for the formation of the inversion 3R(7).

Figure 3. Large-Scale Comparison of the Genomes of D. melanogaster and D. yakuba. Muller's Elements B and C (chromosome 2)

Similar plots are shown for Muller's element A (chromosome X) and Muller's elements D and E (chromosome 3) in Figure S4A and S4B, respectively. The outermost protein-coding genes of consecutive syntenic blocks are indicated. Following Bridges [116], syntenic blocks (defined as regions in which the relative gene order is globally conserved between D. melanogaster and D. yakuba) are numbered taking D. melanogaster as a reference and in an increasing order from the telomere of chromosome X (number 1) to the telomere of the right arm of chromosome 3 (number 58); an arrowhead indicates the orientation of the segments. Lines between chromosomes match homologous syntenic blocks between species. The pericentric inversion between Muller's elements B and C during the divergence of D. melanogaster and D. yakuba is shown by a color code, whereas syntenic blocks on the left and right arms of D. melanogaster appear in orange and green, respectively; the syntenic block that contains the centromere, number 26, is not colored. The solid triangles denote the gene CG6081, whose duplication accompanied the origin of the inversion 2L(2) (Figures 2, S2, and S3).