Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution

Abstract

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Keywords: codon bias; evolution of heterochromatin; gene size; melting characteristics; transposons.

Figure 1

The Drosophila F element has maintained its heterochromatic properties in four different Drosophila species. (Left) Immunofluorescent staining of polytene chromosomes using H3K9me2-specific antibodies shows that the D. melanogaster, D. erecta, D. mojavensis, and D. grimshawi F elements (colored arrows) are enriched in H3K9me2 (a mark of heterochromatin). (Right) Phylogenetic tree of the Drosophila genomes sequenced by the Drosophila 12 Genomes Consortium (Powell 1997). The colored stars next to the species names in the phylogenetic tree denote the species analyzed in this study; the same color scheme is used in this and subsequent figures.

Figure 2

Sequence improvement of the D. mojavensis F element scaffold. One of the gaps in the D. mojavensis CAF1 assembly is located within the initial coding exon of the B and E isoforms of the putative ortholog of unc-13 in D. mojavensis (red arrow). The improved assembly added 434 bases to resolve the 25-bp gap in this region (bottom) and allows us to produce annotation for the entire coding exon. Another gap was resolved by incorporating a 1.2-kb scaffold (scaffold_6641, chartreuse yellow rectangle) from the CAF1 assembly into the improved F element assembly (black arrow). This scaffold contains an internal coding exon for the A and D isoforms of unc-13. The remaining gaps and low quality regions were resolved by additional sequencing. Changes between the CAF1 and the improved assemblies are summarized in the “Difference with D. mojavensis CAF1 Assembly” track (red rectangles). The “GEP Gene Annotations” track (green) shows the manual gene annotations for all the isoforms of unc-13 in D. mojavensis based on the improved sequence. The “FlyBase Gene Annotations” evidence track (blue) shows the GLEAN-R gene predictions currently maintained by FlyBase.

Figure 3

The repetitive element landscapes of the F and the base of the D elements in D. melanogaster (red), D. erecta (orange), D. mojavensis (blue), and D. grimshawi (purple). (A) WindowMasker analysis (low complexity repeats and transposons); (B) tantan analysis (simple and low complexity repeats); (C) Tandem Repeats Finder; (D) RepeatMasker analysis (transposon density). Within each species, the F element generally shows a higher repeat density (particularly transposable elements) than the euchromatic reference regions from the D elements. Except for tandem repeats, the base (light orange), extended (olive), and telomeric (green) regions from the D. erecta D element generally show similar repeat density.

Figure 4

Distributions of 13-mers and dinucleotide repeats in the regions analyzed. (A) Consistent with the WindowMasker results, more 13-mers are found to be repeated (present at a higher frequency) on the D. mojavensis F element (dark blue line) than the other analysis regions. The genomic sequence in each analysis region is partitioned into overlapping 13-mers and the frequency of each 13-mer is tabulated using Tallymer. The values on the x-axis correspond to the number of times that a particular 13-mer is found in the analysis region whereas the y-axis correspond to the total number of 13-mers (of all sequences) that appear at each frequency. For example, approximately 10⁶ 13-mers appear only once in each analysis region. (B) Cumulative dinucleotide repeats analysis shows a higher frequency of dinucleotide repeats on the D. mojavensis and D. grimshawi F elements (dark blue and purple lines, respectively) than on the D. melanogaster and D. erecta F elements (dark red and orange lines, respectively). A pseudocount of one has been added to the cumulative distribution plots in order to show a continuous distribution in the semi-log plot.

Figure 5

Violin plots of gene characteristics for each analysis region. A violin plot is composed of a boxplot and a kernel density plot: the black dot denotes the median; the darker regions and the thin white box denote the range between the first (Q1) and third (Q3) quartiles [i.e., the interquartile range (IQR)]. Whiskers extending from the white box span from Q1-1.5☓IQR to Q3+1.5☓IQR; the data points beyond the whiskers are outliers. For violin plots using a log scale, a pseudocount of one was added to all data points. The larger coding spans of F element genes can be attributed not only to larger introns (often containing repeats), but also to larger coding regions. The larger coding regions reflect the higher number of coding exons.

Figure 6

F element genes exhibit different patterns of codon bias in D. mojavensis and D. grimshawi compared to D. melanogaster and D. erecta. (A) Distributions of Effective Number of Codons (Nc). (B) Distributions of Codon Adaptation Index (CAI). (C) Scatterplots of Nc vs. CAI show that, similar to the base of the D elements, codon bias in the D. grimshawi F element genes can be attributed primarily to selection rather than mutational biases, as indicated by a LOESS regression line (red line) with negative slope (see main text). The dotted line in each Nc vs. CAI scatterplot demarcates the CAI value for a gene with no codon bias relative to the species-specific reference gene sets constructed by the program scnRCA (see File S1).

Figure 7

Distance–Distance Plots of robust distance (RD) vs. Mahalanobis distance (MD) show both common and species-specific outliers. The horizontal and vertical lines correspond to the cutoff values for outliers (97.5% quantile of the χ² distribution, see File S1). Values greater than the cutoff values identify outliers. Triangles in the upper right quadrant are outliers based on both RD and MD. Triangles in the upper left quadrant are outliers only based on RD. The dashed line corresponds to points with equal RD and MD values. F element genes that reside in a Polycomb domain in D. melanogaster are highlighted in gray.

Figure 8

Metagene analyses show that F element genes have a lower median 9-bp melting temperature (T_m) than genes at the base of the D element. The 9-bp T_m was calculated using a sliding-window of 9 bp and a step size of 1 bp. The T_m for each coding span was subsequently normalized to 3 kb to create the metagene profile (see File S1).

Figure 9

F element gene movements in the four Drosophila species analyzed in this study. (A) Placement of the 12 F element wanderer genes [five on the F element in D. melanogaster and D. erecta (top left), and seven on the F element in D. mojavensis (top right)]. (B) Schematic representations of the wanderer gene hotspots on the D. mojavensis and D. grimshawi F elements where most of the wanderer genes are found. The genes PRY and Or13a (blue boxes) have moved from other Muller elements to the D. mojavensis F element. The gene yellow-h (purple box) has moved from the F element to the A element in D. mojavensis. Assignment of the D. virilis ortholog of Or13a to the A element (denoted by an asterisk) is based on the placement of the other seven genes found in that scaffold (13050) (see File S1). Placement of PRY on the Y chromosome is based on Koerich et al. (2008).