Long-read based assembly and synteny analysis of a reference Drosophila subobscura genome reveals signatures of structural evolution driven by inversions recombination-suppression effects

Abstract

Background: Drosophila subobscura has long been a central model in evolutionary genetics. Presently, its use is hindered by the lack of a reference genome. To bridge this gap, here we used PacBio long-read technology, together with the available wealth of genetic marker information, to assemble and annotate a high-quality nuclear and complete mitochondrial genome for the species. With the obtained assembly, we performed the first synteny analysis of genome structure evolution in the subobscura subgroup.

Results: We generated a highly-contiguous ~ 129 Mb-long nuclear genome, consisting of six pseudochromosomes corresponding to the six chromosomes of a female haploid set, and a complete 15,764 bp-long mitogenome, and provide an account of their numbers and distributions of codifying and repetitive content. All 12 identified paracentric inversion differences in the subobscura subgroup would have originated by chromosomal breakage and repair, with some associated duplications, but no evidence of direct gene disruptions by the breakpoints. Between lineages, inversion fixation rates were 10 times higher in continental D. subobscura than in the two small oceanic-island endemics D. guanche and D. madeirensis. Within D. subobscura, we found contrasting ratios of chromosomal divergence to polymorphism between the A sex chromosome and the autosomes.

Conclusions: We present the first high-quality, long-read sequencing of a D. subobscura genome. Our findings generally support genome structure evolution in this species being driven indirectly, through the inversions' recombination-suppression effects in maintaining sets of adaptive alleles together in the face of gene flow. The resources developed will serve to further establish the subobscura subgroup as model for comparative genomics and evolutionary indicator of global change.

Keywords: Adaptation; Genome structure evolution; Global change; Inversion fixation and polymorphism; Inversion originating mechanisms; Spatiotemporally fluctuating selection.

Fig. 1

De novo assembly of a D. subobscura genome from long-read PacBio sequencing data. The six chromosomes are referred to by their corresponding letter (i.e., A, J, U, E, O and dot) and Muller element (i.e., A, D, B, C, E and F, respectively; in parentheses) designations. Chromosomes are shown oriented from centromere (C) to telomere (T). Each chromosome panel includes (top) a scheme of the reconstructed pseudochromosome and their component forward (sepia) and reverse (black) scaffolds with labels (e.g., s062) on them; (center) a drawing of the Kunze-Mühl and Müller [12] reference standard karyotype, modified to take into account that the ch-cu strain used for genome sequencing is structurally O_3 + 4 (or O_ms + 4; see the results and discussion section) and (bottom) a ruler indicating the sections (from 1 to 100) and subsections (each from A to E) of the Kunze-Mühl and Müller [12] map. A 1 Mb-scale bar is shown below the dot

Fig. 2

D. subobscura mitogenome content and organization. Shown are protein coding genes (black), rRNA genes (red), tRNA genes (white), and the AT-rich (control) region (crosshatched). Arrowheads indicate gene direction

Fig. 3

Spatial distribution of two major categories of repetitive DNA along the five large acrocentric pseudochromosomes of the D. subobscura assembly. Dotted lines represent 95% confidence intervals around the expected average if repeats were distributed at random

Fig. 4

SyMAP comparative chromosome synteny analysis between D. subobscura (Ds; central gold horizontal bars) and each of D. melanogaster (Dm; upper grey) and D. guanche (Dg; bottom purple). Bands connecting homologous chromosomes denote noninverted (pink) and inverted (green) synteny blocks. Labeled ticks on chromosomes indicate proximal (p) and distal (d) inversion breakpoints. Labels for breakpoints in the proximal region of the A chromosome are provided in the upper right panel of the figure (h1_p to h4_d), along with the optimal reversal scenario for the transition between the standard sequence of D. subobscura and the arrangement of D. guanche in this region inferred using the GRIMM algorithm. The eight synteny blocks of that transition are designated by positive (noninverted) and negative (inverted) numbers, and the corresponding four intermediate hypothetical inversions (yellow) by letter “h” subscripted 1–4. Cytological map positions and pseudochromosome coordinates of inversions breakpoints are given in Additional file 21: Table S10

Fig. 5

Reconstructed most parsimonious chromosomal rearrangement history of the D. subobscura species subgroup. Shown are the continental lineage (brown) leading to D. subobscura, and the two derived island lineages (blue) D. guanche and D. madeirensis. The inferred ancestral chromosomal arrangement configuration of the subgroup is shown at the root. Arrangements at the terminal nodes can be fixed (black), polymorphic (green) or extinct (red). Worth to mention is the case of Oms. This arrangement was previously known as Og, because, based on cytogenetic evidence, it was thought to have originated in the lineage of D. guanche. The breakpoint sequence synteny analysis conducted herein, however, indicates that the arrangement originated in the mainland before the split of D. madeirensis, where it became fixed, and D. subobscura, where superimposed on it originated separately inversions 4 and ST, and then became extinct. Accordingly, D. subobscura presently polymorphic O inversions 4 and ST may be more appropriately referred to O_ms + 4 and O_ms + ST, rather than O_3 + 4 and O_ST, because arrangement 3 is ancestral to ms