Fig 1.
Drosophila miranda is a model species to study sex chromosome evolution.
A. Male (left) and female (right) D. miranda. B. Mitotic chromosome squashes of male D. miranda. Both the ancestral X (XL/XR) and the Y chromosome (YD/neo-Y) show large blocks of dark staining (Giemsa), indicative of heterochromatin. The acrocentric rods are the neo-X, and chromosomes 2 and 4. C. Polytene chromosomes of a female D. miranda stained for HP1 (heterochromatin protein 1). Note the large blocks of heterochromatin (arrows) on chromosomes 2 and 4. D. Karyotype evolution in D. miranda. Chromosomal fusions between the sex chromosomes and autosomes have resulted in both the reversal of Yanc to an autosome as well as the independent de novo formation of new sex chromosomes from autosomes at two distinct evolutionary time points (XR and YD were formed about 15 MY ago, and the neo-X and neo-Y originated about 1.5 MY ago). Genome analysis allows us to reconstruct the temporal dynamics and molecular processes involved in sex chromosome evolution in this species. chr, chromosome; dot, dot chromosome; XL and XR, left and right arm of the X chromosome; YD, Y chromosome resulting from the unfused D element; HP1, heterochromatin protein 1; MY, million years; MYA, million years ago; Yanc, ancestral Y chromosome.
Fig 2.
Assembly and validation of Drosophila miranda genome.
A. Overview of assembly pipeline. The steps include assembly of male PacBio reads followed by scaffolding using Hi-C, and extensive QC using BioNano reads and BAC clone sequencing followed by gene and repeat annotation. B. Hi-C linkage density map. Chromatin interaction maps allow recovery of entire chromosome arms. Note that the Y-linked contigs were scaffolded separately from X-linked and autosomal contigs. Unlinked regions with many contacts indicate repetitive regions. C. Comparison of current (Dmir2.0) versus old (Dmir1.0) D. miranda assembly. Note that the Y/neo-Y was not assembled in Dmir1.0, and the dot plot indicates homology between our neo-Y assembly and the neo-X. Other repeat-rich regions, such as the large pericentromeric block on AD, are also missing from D.mir1.0. D. BAC clone mapping for assembly verification. BAC clones are color coded according to how many genomic regions they map to in our assembly; green lines indicate stitch points of scaffolds based on Hi-C contacts, and the black line gives the local repeat content along the genome. Three hundred sixty-one sequenced BAC clones (97%) map contiguously and uniquely to our genome assembly. BAC, bacterial artificial chromosome; F, female; M, male; QC, quality control; Repeat %, local repeat content.
Table 1.
Assembly statistics.
Fig 3.
Gene and repeat content of Drosophila miranda genome assembly.
Shown is the gene content, repeat content, H3K9me3 enrichment and density of the most abundant satellites (21-bp repeat and 99-bp repeat), the telomeric transposable elements, and the most abundant transposons (BEL, CR1, Gypsy, Helitron, R1) across the D. miranda genome assembly. A cartoon of the chromosomes is drawn, with color indicating the Muller element (see Fig 1D), and the shaded regions are heterochromatic. Gene and repeat content are shown in 40-kb sliding windows, and H3K9me3 enrichment and satellite and TE abundance are shown in 10-kb sliding windows. ch, chromosome; H3K9me3, trimethylation of histone 3 lysine 9.
Fig 4.
Recovery of telomeres and identification of putative centromere repeats for each chromosome.
A. Presence of telomere repeats at or near the ends of most chromosome arms. Shown is enrichment of telomere repeats and H3K9me3 marks in 10-kb nonoverlapping sliding windows. B. Alignment of chromosome ends and telomere repeats. Colors indicate the percent similarity between the alignments and the direction of the lines indicates the direction of the match. C. Histogram of most abundant satellites in Drosophila miranda genome. Repeat categories refer to the size of the repeat unit. Note that the 84-bp repeat is a higher-order variant of four units of the 21-bp repeat. D. Consensus sequence of 21-bp and 99-bp repeats. Gray shading indicates AA/TT/AT repeats that occur at a 10-bp periodicity. E. Comparison of the centromeric repeat from different chromosomes. Shown are alignments of regions from Muller B and Muller C, with high density of the 99-bp and 21-bp tandem centromeric repeats, respectively. F. Location of putative centromere repeats in pericentromeric regions, and H3K9me3 enrichment. H3K9me3 enrichment is reduced at the putative centromeric repeats (S13 Fig). Note that, for the acrocentric chromosome 2, we recover the entire centromere, including the telomere. ch, chromosome; H3K9me3, trimethylation of histone 3 lysine 9; TE, transposable element.
Fig 5.
A. Global neo-sex chromosome alignments show large homologous blocks between the neo-sex chromosomes along the long arm of the Y/neo-Y. B and C. Zoom-in of selected homologous regions along the neo-sex chromosomes. Neo-sex-linked regions often contain blocks of homologous genes, reflecting their recent evolutionary origin, but note the dramatic repeat accumulation (shown in gray) at both intergenic and gene regions on the neo-Y, greatly increasing its size.
Fig 6.
Karyotype evolution in Drosophila miranda.
A. Chromosome arm homology in D. miranda. Genes in D. miranda are color coded according to their location in D. melanogaster (see Fig 1). B. Sequence composition of the D. miranda dot chromosome. Shown is the origin of dot genes (color coded as in Fig 1), the repeat and H3K9me3 content, as well as the location of sequenced BAC clones. Ppr-Y and kl-3 are genes located on the ancestral Y of Drosophila. C. Origin of the D. miranda Y/neo-Y. Shown are the location of centromeric and telomeric repeats, H3K9me3 enrichments, the color coded location of single-copy neo-Y genes (with black corresponding to unknown ancestral location in D. pseudoobscura), the location of homologous Y-linked genes identified in D. pseudoobscura, mapping of Y-derived sequencing reads from D. pseudoobscura, and the location of the rDNA genes. The inferred ancestry of the Y/neo-Y chromosome is shown as a cartoon, with the short arm presumably corresponding to the Y chromosome shared with D. pseudoobscura and the long arm representing the neo-Y. D. Our genomic analysis allows us to reconstruct sex chromosome evolution in D. miranda (see text). BAC, bacterial artificial chromosome; chr, chromosome; H3K9me3, trimethylation of histone 3 lysine 9; rDNA, ribosomal DNA; Yanc, ancestral Y chromosome.