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Large genome structural variations can impact genome regulation and integrity. Repeat-rich regions like pericentric heterochromatin are vulnerable to structural rearrangements although we know little about how often these rearrangements occur over evolutionary time. Repetitive genome regions are particularly difficult to study with genomic approaches, as they are missing from most genome assemblies. However, cytogenetic approaches offer a direct way to detect large rearrangements involving pericentric heterochromatin. Here, we use a cytogenetic approach to reveal large structural rearrangements associated with the X pericentromeric region of Drosophila simulans. These rearrangements involve large blocks of satellite DNA—the 500-bp and Rsp-like satellites—which colocalize in the X pericentromeric heterochromatin. We find that this region is polymorphic not only among different strains, but between isolates of the same strain from different labs, and even within individual isolates. On the one hand, our observations raise questions regarding the potential impact of such variation at the phenotypic level and our ability to control for such genetic variability. On the other hand, this highlights the very rapid turnover of the pericentric heterochromatin most likely associated with genomic instability of the X pericentromere. It represents a unique opportunity to study the dynamics of pericentric heterochromatin, the evolution of associated satellites on a very short time scale, and to better understand how structural variation arises.

 

Repetitive elements (REs) are integral to the composition, structure, and function of eukaryotic genomes, yet remain understudied in most taxonomic groups. We investigated REs across 601 insect species and report wide variation in RE dynamics across groups. Analysis of associations between REs and protein-coding genes revealed dynamic evolution at the interface between REs and coding regions across insects, including notably elevated RE–gene associations in lineages with abundant long interspersed nuclear elements (LINEs). We leveraged this large, empirical data set to quantify impacts of long-read technology on RE detection and investigate fundamental challenges to RE annotation in diverse groups. In long-read assemblies, we detected ∼36% more REs than short-read assemblies, with long terminal repeats (LTRs) showing 162% increased detection, whereas DNA transposons and LINEs showed less respective technology-related bias. In most insect lineages, 25%–85% of repetitive sequences were “unclassified” following automated annotation, compared with only ∼13% inDrosophilaspecies. Although the diversity of available insect genomes has rapidly expanded, we show the rate of community contributions to RE databases has not kept pace, preventing efficient annotation and high-resolution study of REs in most groups. We highlight the tremendous opportunity and need for the biodiversity genomics field to embrace REs and suggest collective steps for making progress toward this goal.

 

Advances in genomic technology led to a more focused pattern for the distribution of chromosomal proteins and a better understanding of their functions. The recent development of the CUT&RUN technique marks one of the important such advances. Here we develop a modified CUT&RUN technique that we termed nanoCUT&RUN, in which a high affinity nanobody to GFP is used to bring micrococcal nuclease to the binding sites of GFP-tagged chromatin proteins. Subsequent activation of the nuclease cleaves the chromatin, and sequencing of released DNA identifies binding sites. We show that nanoCUT&RUN efficiently produces high quality data for the TRL transcription factor in Drosophila embryos, and distinguishes binding sites specific between two TRL isoforms. We further show that nanoCUT&RUN dissects the distributions of the HipHop and HOAP telomere capping proteins, and uncovers unexpected binding of telomeric proteins at centromeres. nanoCUT&RUN can be readily applied to any system in which a chromatin protein of interest, or its isoforms, carries the GFP tag. 

Y chromosomes across diverse species convergently evolve a gene-poor, heterochromatic organization enriched for duplicated genes, LTR retrotransposons, and satellite DNA. Sexual antagonism and a loss of recombination play major roles in the degeneration of young Y chromosomes. However, the processes shaping the evolution of mature, already degenerated Y chromosomes are less well-understood. Because Y chromosomes evolve rapidly, comparisons between closely related species are particularly useful. We generated de novo long-read assemblies complemented with cytological validation to reveal Y chromosome organization in three closely related species of the Drosophila simulans complex, which diverged only 250,000 years ago and share >98% sequence identity. We find these Y chromosomes are divergent in their organization and repetitive DNA composition and discover new Y-linked gene families whose evolution is driven by both positive selection and gene conversion. These Y chromosomes are also enriched for large deletions, suggesting that the repair of double-strand breaks on Y chromosomes may be biased toward microhomology-mediated end joining over canonical non-homologous end-joining. We propose that this repair mechanism contributes to the convergent evolution of Y chromosome organization across organisms. 

Abstract The first insect genome assembly (Drosophila melanogaster) was published two decades ago. Today, nuclear genome assemblies are available for a staggering 601 insect species representing 20 orders. In this study, we analyzed the most-contiguous assembly for each species and provide a “state-of-the-field” perspective, emphasizing taxonomic representation, assembly quality, gene completeness, and sequencing technologies. Relative to species richness, genomic efforts have been biased toward four orders (Diptera, Hymenoptera, Collembola, and Phasmatodea), Coleoptera are underrepresented, and 11 orders still lack a publicly available genome assembly. The average insect genome assembly is 439.2 Mb in length with 87.5% of single-copy benchmarking genes intact. Most notable has been the impact of long-read sequencing; assemblies that incorporate long reads are ∼48× more contiguous than those that do not. We offer four recommendations as we collectively continue building insect genome resources: 1) seek better integration between independent research groups and consortia, 2) balance future sampling between filling taxonomic gaps and generating data for targeted questions, 3) take advantage of long-read sequencing technologies, and 4) expand and improve gene annotations. 

The rapid evolution of repetitive DNA sequences, including satellite DNA, tandem duplications, and transposable elements, underlies phenotypic evolution and contributes to hybrid incompatibilities between species. However, repetitive genomic regions are fragmented and misassembled in most contemporary genome assemblies. We generated highly contiguous de novo reference genomes for the Drosophila simulans species complex ( D. simulans , D. mauritiana , and D. sechellia ), which speciated ∼250,000 yr ago. Our assemblies are comparable in contiguity and accuracy to the current D. melanogaster genome, allowing us to directly compare repetitive sequences between these four species. We find that at least 15% of the D. simulans complex species genomes fail to align uniquely to D. melanogaster owing to structural divergence—twice the number of single-nucleotide substitutions. We also find rapid turnover of satellite DNA and extensive structural divergence in heterochromatic regions, whereas the euchromatic gene content is mostly conserved. Despite the overall preservation of gene synteny, euchromatin in each species has been shaped by clade- and species-specific inversions, transposable elements, expansions and contractions of satellite and tRNA tandem arrays, and gene duplications. We also find rapid divergence among Y-linked genes, including copy number variation and recent gene duplications from autosomes. Our assemblies provide a valuable resource for studying genome evolution and its consequences for phenotypic evolution in these genetic model species. 

Study of repetitive DNA elements in model organisms highlights the role of repetitive elements (REs) in many processes that drive genome evolution and phenotypic change. Because REs are much more dynamic than single‐copy DNA, repetitive sequences can reveal signals of evolutionary history over short time scales that may not be evident in sequences from slower‐evolving genomic regions. Many tools for studying REs are directed toward organisms with existing genomic resources, including genome assemblies and repeat libraries. However, signals in repeat variation may prove especially valuable in disentangling evolutionary histories in diverse non‐model groups, for which genomic resources are limited. Here, we introduce RepeatProfiler, a tool for generating, visualizing, and comparing repetitive element DNA profiles from low‐coverage, short‐read sequence data. RepeatProfiler automates the generation and visualization of RE coverage depth profiles (RE profiles) and allows for statistical comparison of profile shape across samples. In addition, RepeatProfiler facilitates comparison of profiles by extracting signal from sequence variants across profiles which can then be analysed as molecular morphological characters using phylogenetic analysis. We validate RepeatProfiler with data sets from ground beetles (Bembidion), flies (Drosophila), and tomatoes (Solanum). We highlight the potential of RE profiles as a high‐resolution data source for studies in species delimitation, comparative genomics, and repeat biology.

 

Glowing fireflies dancing in the dark are one of the most enchanting sights of a warm summer night. Their light signals are ‘love messages’ that help the insects find a mate – yet, they also warn a potential predator that these beetles have powerful chemical defenses. The light comes from a specialized organ of the firefly where a small molecule, luciferin, is broken down by the enzyme luciferase. Fireflies are an ancient group, with the common ancestor of the two main lineages originating over 100 million years ago. But fireflies are not the only insects that produce light: certain click beetles are also bioluminescent. Fireflies and click beetles are closely related, and they both use identical luciferin and similar luciferases to create light. This would suggest that bioluminescence was already present in the common ancestor of the two families. However, the specialized organs in which the chemical reactions take place are entirely different, which would indicate that the ability to produce light arose independently in each group. Here, Fallon, Lower et al. try to resolve this discrepancy and to find out how many times bioluminescence evolved in beetles. This required using cutting-edge DNA sequencing to carefully piece together the genomes of two species of fireflies (Photinus pyralis and Aquatica lateralis) and one species of click beetle (Ignelater luminosus). The genetic analysis revealed that, in all species, the genes for luciferases were very similar to the genetic sequences around them, which code for proteins that break down fat. This indicates that the ancestral luciferase arose from one of these metabolic genes getting duplicated, and then one of the copies evolving a new role. However, the genes for luciferase were very different between the fireflies and the click beetles. Further analyses suggested that bioluminescence evolved at least twice: once in an ancestor of fireflies, and once in the ancestor of the bioluminescent click beetles. More results came from the reconstituted genomes. For example, Fallon, Lower et al. identified the genes ‘turned on’ in the bioluminescent organ of the fireflies. This made it possible to list genes that may be involved in creating luciferin, and enable flies to grow brightly for long periods. In addition, the genetic information yielded sequences from bacteria that likely live inside firefly cells, and which may participate in the light-making process or the production of potent chemical defenses. Better genetic knowledge of beetle bioluminescence could bring new advances for both insects and humans. It may help researchers find and design better light-emitting molecules useful to track and quantify proteins of interest in a cell. Ultimately, it would allow a detailed understanding of firefly populations around the world, which could contribute to firefly ecotourism and help to protect these glowing insects from increasing environmental threats. 

Genome size is implicated in the form, function, and ecological success of a species. Two principally different mechanisms are proposed as major drivers of eukaryotic genome evolution and diversity: polyploidy (i.e., whole-genome duplication) or smaller duplication events and bursts in the activity of repetitive elements. Here, we generated de novo genome assemblies of 17 caddisflies covering all major lineages of Trichoptera. Using these and previously sequenced genomes, we use caddisflies as a model for understanding genome size evolution in diverse insect lineages.

We detect a ∼14-fold variation in genome size across the order Trichoptera. We find strong evidence that repetitive element expansions, particularly those of transposable elements (TEs), are important drivers of large caddisfly genome sizes. Using an innovative method to examine TEs associated with universal single-copy orthologs (i.e., BUSCO genes), we find that TE expansions have a major impact on protein-coding gene regions, with TE-gene associations showing a linear relationship with increasing genome size. Intriguingly, we find that expanded genomes preferentially evolved in caddisfly clades with a higher ecological diversity (i.e., various feeding modes, diversification in variable, less stable environments).

Our findings provide a platform to test hypotheses about the potential evolutionary roles of TE activity and TE-gene associations, particularly in groups with high species, ecological, and functional diversities.