Chromosome-Level Assembly of Drosophila bifasciata Reveals Important Karyotypic Transition of the X Chromosome

Abstract

The Drosophila obscura species group is one of the most studied clades of Drosophila and harbors multiple distinct karyotypes. Here we present a de novo genome assembly and annotation of D. bifasciata, a species which represents an important subgroup for which no high-quality chromosome-level genome assembly currently exists. We combined long-read sequencing (Nanopore) and Hi-C scaffolding to achieve a highly contiguous genome assembly approximately 193 Mb in size, with repetitive elements constituting 30.1% of the total length. Drosophila bifasciata harbors four large metacentric chromosomes and the small dot, and our assembly contains each chromosome in a single scaffold, including the highly repetitive pericentromeres, which were largely composed of Jockey and Gypsy transposable elements. We annotated a total of 12,821 protein-coding genes and comparisons of synteny with D. athabasca orthologs show that the large metacentric pericentromeric regions of multiple chromosomes are conserved between these species. Importantly, Muller A (X chromosome) was found to be metacentric in D. bifasciata and the pericentromeric region appears homologous to the pericentromeric region of the fused Muller A-AD (XL and XR) of pseudoobscura/affinis subgroup species. Our finding suggests a metacentric ancestral X fused to a telocentric Muller D and created the large neo-X (Muller A-AD) chromosome ∼15 MYA. We also confirm the fusion of Muller C and D in D. bifasciata and show that it likely involved a centromere-centromere fusion.

Keywords: Muller element; Nanopore; centromere; chromosome.

Figure 1

Evolutionary relationships and karyotype transitions of obscura group flies. The ancestral karyotype of the obscura group (shown here as Drosophila subobscura) consists of five large and one small pair of telocentric chromosomes, referred to as Muller elements A-F (reviewed in Schaeffer 2018), and shown color coded. Significant karyotypic changes have occurred across the obscura group (highlighted with gray boxes) with chromosomal fusions and centromere movement altering chromosome structure (Bracewell et al. 2019). Drosophila bifasciata represents an important karyotype to understand evolutionary transitions since Muller A (the X chromosome), B and E are thought to be metacentric and Muller A is unfused (Moriwaki and Kitagawa 1955). In D. bifasciata, it is thought that Muller C and D fused, although C-D fusions are only present in some obscura subgroup species (Buzzati-Traverso and Scossiroli 1955). Shown phylogenetic relationships adapted from (Gao et al. 2007) with subgroup designations shown along the branches.

Figure 2

Chromosome-level genome assembly of Drosophila bifasciata using Hi-C. A) Hi-C heatmap showing long-range contacts and scaffolding of the genome assembly. Green and blue squares denote contigs and chromosomes, respectively. Euchromatic chromosome arms and heterochromatic pericentromeres for each chromosome show distinct and primarily isolated associations that resemble a ‘checkerboard’ pattern. Note that chromosome arms on opposite sides of a pericentromere often show associations on the diagonal confirming their placement (yellow arrow) while pericentromeres show finer-scale associations with their chromosome arms (blue arrow). B) Shown is the D. bifasciata genome assembled into Muller elements (color coded as in Figure 1), scaffolding stitch points, gene density (genes per 100 kb) and repeat content (proportion of bases repeat-masked in 100 kb non-overlapping windows). Boxes around highly repetitive regions indicate putative pericentromere boundaries (defined as ≥40% repeat-masked sequence in sliding windows away from the center).

Figure 3

Transposable elements enriched in pericentromeres. Genomic distribution of common transposable element (TE) families in the D. bifasciata genome assembly. For each Muller element, TEs are arranged in horizontal tracks of decreasing abundance from top to bottom with the total TE abundance (black line) plotted on top. Shown is the proportion of bases repeat-masked per TE family in 100 kb non-overlapping windows.

Figure 4

Muller element evolution and synteny. Comparisons of synteny between D. bifasciata and D. athabasca Muller elements A (X) (red), B (green), and E (purple) with each line representing a protein-coding gene. Genes previously identified as pericentromeric in D. athabasca (Bracewell et al. 2019) are shown in black. Only Muller A (X) genes shown for D. athabasca.