Sequence Analysis of the Segmental Duplication Responsible for Paris Sex-Ratio Drive in Drosophila simulans

Abstract

Sex-ratio distorters are X-linked selfish genetic elements that facilitate their own transmission by subverting Mendelian segregation at the expense of the Y chromosome. Naturally occurring cases of sex-linked distorters have been reported in a variety of organisms, including several species of Drosophila; they trigger genetic conflict over the sex ratio, which is an important evolutionary force. However, with a few exceptions, the causal loci are unknown. Here, we molecularly characterize the segmental duplication involved in the Paris sex-ratio system that is still evolving in natural populations of Drosophila simulans. This 37.5 kb tandem duplication spans six genes, from the second intron of the Trf2 gene (TATA box binding protein-related factor 2) to the first intron of the org-1 gene (optomotor-blind-related-gene-1). Sequence analysis showed that the duplication arose through the production of an exact copy on the template chromosome itself. We estimated this event to be less than 500 years old. We also detected specific signatures of the duplication mechanism; these support the Duplication-Dependent Strand Annealing model. The region at the junction between the two duplicated segments contains several copies of an active transposable element, Hosim1, alternating with 687 bp repeats that are noncoding but transcribed. The almost-complete sequence identity between copies made it impossible to complete the sequencing and assembly of this region. These results form the basis for the functional dissection of Paris sex-ratio drive and will be valuable for future studies designed to better understand the dynamics and the evolutionary significance of sex chromosome drive.

Keywords: D. simulans; meiotic drive; segmental duplication; sex-ratio.

Figure 1

General organization of the sex-ratio duplication dot plot comparison of the duplication on the X^SR6 chromosome of D. simulans (abscissa) with the homologous region in D. melanogaster (ordinate). The D. simulans sequence was obtained from BACs 58j14 and 46o6, which do not overlap (limits showed by the vertical dotted line). The black arrows show Hosim1-SR sequences (no homolog in D. melanogaster genome), separated by fragments with homologs in the second intron of Trf2 (IST). Two horizontal dotted arrows show the limits of the duplicated fragment.

Figure 2

Sequence identity between the two copies of the sex-ratio duplication on the X^SR6 chromosome. The analysis was performed using a 50 bp sliding window with a step size of 10 bp. The gray box represents the fragment with 100% identity; the striped box represents the region containing the DDSA traces described in Figure S3. The stars show the position of markers sequenced in the population study of Derome et al. (2008), and the triangle shows the position of the small deletion in the distal copy of CG32712.

Figure 3

(A) Parsimonious scenario explaining the pattern of sequence variation observed between the two copies of the sex-ratio duplication carried by the chromosome X^SR6. The vertical dotted lines show the limits of the 10 kb fragment with 100% sequence identity. The stars show the position of markers sequenced in Derome et al. (2008). The triangles show the position of CG32712 (the white triangle stands for the deleted allele brought by recombination). The vertical gray/white strips represent the repeated motifs of the junction region. (B) Interpretation of Figure S1 in Derome et al. (2008). X^M01 is a sex-ratio chromosome from Madagascar, carrying a combination of haplotypes commonly found there. For each marker (stars in Figure 3A), the ancestral sequence is symbolized in light gray. Alleles supposed to have been brought by recombination are in medium and dark gray (proximal recombination) and in black (distal recombination).

Figure 4

(A) Schematic representation of the canonical IST (Intronic Sequence of Trf2), found in the published D. simulans genome and in the distal copy of Trf2 on chromosome X^SR6. (B) Organization of the junction region on chromosome X^SR6, observed in BAC 58j14 (top) and BACs 67l12 and 46o6 (bottom). It consists of alternating Hosim1-SR elements and direct tandems of rIST. Fragments (a–c) amplified by PCR to control the organization on DNA from (X^SR6)_ST8 males (sequence of primers in Table S1). NNN: gap in sequence assembly

Figure 5

Comparison of Hosim1-SR with the canonical Hosim1: Schematic representation of the nucleotide alignment. Terminal inverted repeats (TIR): TAGTGTTGGGT. The white boxes show the position of the main deletions in Hosim1-SR, with their size below (in bp). The star shows the localization of the intron presented in Figure S7. (a) Position of primers used to amplify both Hosim1 and Hosim1-SR transcripts (Figure 7), (b) position of primers used to estimate the number of canonical Hosim1 (Figure 6A), (c) position of primers used to estimate the total number of elements (Hosim1 + Hosim1-SR, Figure 6A), and the total amount of transcripts (Figure 6B).

Figure 6

Quantification of Hosim1 copy number (A) and Hosim1 transcripts (B) by real-time PCR. The values in (X^SR6)_ST8 males were estimated relative to that in (X^ST8)_ST8 males. Vertical bars: confidence interval (95%). (A, left) “Canonical” elements (see text). (A, right) Canonical elements plus Hosim1-SR. The reference genes were RPL17 and GAPDH. (B) Total amount of testicular transcripts (= Hosim1 + Hosim1-SR). Reference genes were light and RPII140.

Figure 7

Detection of Hosim1 transcripts by RT-PCR. The primers used straddle the deletion of 79 bp specific of Hosim1-SR [Figure 5,(a)], so this element must produce a shorter band (699 bp) than the canonical Hosim1 (784 bp). Even shorter fragments were obtained from cDNAs revealing an intron of 67nt (see text). Amplification of Trf2 with primers straddling an intron was used to control the lack of DNA contamination in the cDNA samples. NC, negative control (no cDNA nor DNA); SL, SmartLadder DNA ladder (Eurogentec); SR6, (X^SR6)_ST8 males; ST, (X^ST8)_ST8 males.

Figure 8

Detection of IST transcripts by RT-PCR. The IST and rIST probes were designed within the region shown in light gray in Figure 4. Amplification of Trf2 gene marker was used to control the lack of DNA in the cDNA samples (see Figure 7). SL, SmartLadder DNA ladder (Eurogentec); XSR6, (X^SR6)_ST8 males; XST8, (X^ST8)_ST8 males.