Molecular characterization of inversion breakpoints in the Drosophila nasuta species group

Chromosomal inversions are fundamental drivers of genome evolution. In the Drosophila genus, inversions have been widely characterized cytologically, and play an important role in local adaptation. Here, we characterize chromosomal inversions in the Drosophila nasuta species group using chromosome-level, reference-quality assemblies of seven species and subspecies in this clade. Reconstruction of ancestral karyotypes allowed us to infer the order in which the 22 identified inversions occurred along the phylogeny. We found a higher rate of inversions on the X chromosome, and heterogeneity in the rate of accumulation across the phylogeny. We molecularly characterize the breakpoints of six autosomal inversions, and found that repeated sequences are associated with inversion breakpoints in four of these inversions, suggesting that ectopic recombination is an important mechanism in generating inversion. Characterization of inversions in this species group provides a foundation for future population genetic and functional studies in this recently diverged species group.

A Nanopore Based Chromosome-Level Assembly Representing Atlantic Cod from the Celtic Sea

Currently available genome assemblies for Atlantic cod (Gadus morhua) have been constructed from fish belonging to the Northeast Arctic Cod (NEAC) population; a migratory population feeding in the Barents Sea. These assemblies have been crucial for the development of genetic markers which have been used to study population differentiation and adaptive evolution in Atlantic cod, pinpointing four discrete islands of genomic divergence located on linkage groups 1, 2, 7 and 12. In this paper, we present a high-quality reference genome from a male Atlantic cod representing a southern population inhabiting the Celtic sea. The genome assembly (gadMor_Celtic) was produced from long-read nanopore data and has a combined contig length of 686 Mb with an N50 of 10 Mb. Integrating contigs with genetic linkage mapping information enabled us to construct 23 chromosome sequences which mapped with high confidence to the latest NEAC population assembly (gadMor3) and allowed us to characterize, to an extent not previously reported large chromosomal inversions on linkage groups 1, 2, 7 and 12. In most cases, inversion breakpoints could be located within single nanopore contigs. Our results suggest the presence of inversions in Celtic cod on linkage groups 6, 11 and 21, although these remain to be confirmed. Further, we identified a specific repetitive element that is relatively enriched at predicted centromeric regions. Our gadMor_Celtic assembly provides a resource representing a ‘southern’ cod population which is complementary to the existing ‘northern’ population based genome assemblies and represents the first step toward developing pan-genomic resources for Atlantic cod.

Fine-Scale Position Effects Shape the Distribution of Inversion Breakpoints in Drosophila melanogaster

Chromosomal inversions are among the primary drivers of genome structure evolution in a wide range of natural populations. Although there is an impressive array of theory and empirical analyses that have identified conditions under which inversions can be positively selected, comparatively little data are available on the fitness impacts of these genome structural rearrangements themselves. Because inversion breakpoints can disrupt functional elements and alter chromatin domains, the precise positioning of an inversion’s breakpoints can strongly affect its fitness. Here, we compared the fine-scale distribution of low-frequency inversion breakpoints with those of high-frequency inversions and inversions that have gone to fixation between Drosophila species. We identified a number of differences among frequency classes that may influence inversion fitness. In particular, breakpoints that are proximal to insulator elements, generate large tandem duplications, and minimize impacts on gene coding spans which are more prevalent in high-frequency and fixed inversions than in rare inversions. The data suggest that natural selection acts to preserve both genes and larger cis-regulatory networks in the occurrence and spread of rearrangements. These factors may act to limit the availability of high-fitness arrangements when suppressed recombination is favorable.

A nanopore based chromosome-level assembly representing Atlantic cod from the Celtic Sea

ABSTRACTCurrently available genome assemblies for Atlantic cod (Gadus morhua) have been constructed using DNA from fish belonging to the Northeast Arctic Cod (NEAC) population; a migratory population feeding in the cold Barents Sea. These assemblies have been crucial for the development of genetic markers which have been used to study population differentiation and adaptive evolution in Atlantic cod, pinpointing four discrete islands of genomic divergence located on linkage groups 1, 2, 7 and 12. In this paper, we present a high-quality reference genome from a male Atlantic cod representing a southern population inhabiting the Celtic sea. Structurally, the genome assembly (gadMor_Celtic) was produced from long-read nanopore data and has a combined contig size of 686 Mb with a N50 of 10 Mb. Integrating contigs with genetic linkage mapping information enabled us to construct 23 chromosome sequences which mapped with high confidence to the latest NEAC population assembly (gadMor3) and allowed us to characterize in detail large chromosomal inversions on linkage groups 1, 2, 7 and 12. In most cases, inversion breakpoints could be located within single nanopore contigs. Our results suggest the presence of inversions in Celtic cod on linkage groups 6, 11 and 21, although these remain to be confirmed. Further, we identified a specific repetitive element that is relatively enriched at predicted centromeric regions. Our gadMor_Celtic assembly provides a resource representing a ‘southern’ cod population which is complementary to the existing ‘northern’ population based genome assemblies and represents the first step towards developing pan-genomic resources for Atlantic cod.

The molecular genealogy of sequential overlapping inversions implies both homologous chromosomes of a heterokaryotype in an inversion origin

Cytological and molecular studies have revealed that inversion chromosomal polymorphism is widespread across taxa and that inversions are among the most common structural changes fixed between species. Two major mechanisms have been proposed for the origin of inversions considering that breaks occur at either repetitive or non-homologous sequences. While inversions originating through the first mechanism might have a multiple origin, those originating through the latter mechanism would have a unique origin. Variation at regions flanking inversion breakpoints can be informative on the origin and history of inversions given the reduced recombination in heterokaryotypes. Here, we have analyzed nucleotide variation at a fragment flanking the most centromere-proximal shared breakpoint of several sequential overlapping inversions of the E chromosome of Drosophila subobscura —inversions E1, E2, E9 and E3. The molecular genealogy inferred from variation at this shared fragment does not exhibit the branching pattern expected according to the sequential origin of inversions. The detected discordance between the molecular and cytological genealogies has led us to consider a novel possibility for the origin of an inversion, and more specifically that one of these inversions originated on a heterokaryotype for chromosomal arrangements. Based on this premise, we propose three new models for inversions origin.

Fine-Mapping Complex Inversion Breakpoints and Investigating Somatic Pairing in the Anopheles gambiae Species Complex Using Proximity-Ligation Sequencing

Chromosomal inversions are fundamental drivers of genome evolution. In the main Afrotropical malaria vector species, belonging to the Anopheles gambiae species complex, inversions play an important role in local adaptation and have a rich history of cytological study. Despite the importance and ubiquity of some chromosomal inversions across the species complex, inversion breakpoints are often challenging to map molecularly due to the presence of large repetitive regions. Here, we develop an approach that uses Hi-C sequencing data to molecularly fine-map the breakpoints of inversions. We demonstrate that this approach is robust and likely to be widely applicable for both identification and fine-mapping inversion breakpoints in species whose inversions have heretofore been challenging to characterize. We apply our method to interrogate the previously unknown inversion breakpoints of 2Rbc and 2Rd in An. coluzzii. We found that inversion breakpoints occur in large repetitive regions, and, strikingly, among three inversions analyzed, two breakpoints appear to be reused in two separate inversions. These breakpoint-adjacent regions are strongly enriched for the presence of a 30 bp satellite repeat sequence. Because low frequency inversion breakpoints are not correlated with genomic regions containing this satellite, we suggest that interrupting this particular repeat may result in arrangements with higher relative fitness. Additionally, we use heterozygous individuals to quantitatively investigate the impacts of somatic pairing in the regions immediately surrounding inversion breakpoints. Finally, we discuss important considerations for possible applications of this approach for inversion breakpoint identification in a range of organisms.

Fine-scale position effects shape the distribution of inversion breakpoints in Drosophila melanogaster

Chromosomal inversions are among the primary drivers of genome structure evolution in a wide range of natural populations. While there is an impressive array of theory and empirical analyses that has identified conditions under which inversions can be positively selected, comparatively little data is available on the fitness impacts of these genome structural rearrangements themselves. Because inversion breakpoints can interrupt functional elements and alter chromatin domains, each rearrangement may in itself have strong effects on fitness. Here, we compared the fine-scale distribution of low frequency inversion breakpoints with those of high frequency inversions and inversions that have fixed between Drosophila species. We identified important differences that may influence inversion fitness. In particular, proximity to insulator elements, large tandem duplications adjacent to the breakpoints, and minimal impacts on gene coding spans are more prevalent in high frequency and fixed inversions than in rare inversions. The data suggest that natural selection acts both to preserve both genes and larger cis-regulatory networks in the occurrence and spread of rearrangements. These factors may act to limit the availability of high fitness arrangements when suppressed recombination is favorable.

Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR

ABSTRACTDespite the interest in characterizing all genomic variation, the presence of large repeats at the breakpoints of many structural variants hinders their analysis. This is especially problematic in the case of inversions, since they are balanced changes without gain or loss of DNA. Here we tested novel linkage-based droplet digital PCR (ddPCR) assays on 20 inversions ranging from 3.1 to 742 kb and flanked by long inverted repeats (IRs) of up to 134 kb. Among these, we validated 13 inversions predicted by different genome-wide techniques. In addition, we have generated new experimental human population information across 95 African, European and East-Asian individuals for 16 of them, including four already known inversions for which there were no high-throughput methods to determine directly the orientation, like the well-characterized 17q21 inversion. Through comparison with previous data, independent replicates and both inversion breakpoints, we have demonstrated that the technique is highly accurate and reproducible. Most of the studied inversions are frequent and widespread across continents, showing a negative correlation with genetic length. Moreover, all except two show clear signs of being recurrent, and the new data allowed us to define more clearly the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, thanks to the generated genotypes, we have been able to check inversion functional effects in multiple tissues, validating gene expression differences reported before for two inversions and finding new candidate associations. Our work therefore provides a tool to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications.

Fine-mapping complex inversion breakpoints and investigating somatic pairing in the Anopheles gambiae species complex using proximity-ligation sequencing

Chromosomal inversions are fundamental drivers of genome evolution. In the main afro-tropical malaria vector species, belonging to the Anopheles gambiae species complex, inversions play an important role in local adaptation and have a rich history of cytological study. Despite the importance and ubiquity of some chromosomal inversions across the species complex, inversion breakpoints are often challenging to map molecularly due to the presence of large repetitive regions. Here, we develop an approach that uses Hi-C sequencing data to molecularly fine-map the breakpoints of inversions 2Rbc and 2Rd in A. coluzzii. We found that inversion breakpoints occur in large repetitive regions, and strikingly among three inversions analyzed, two breakpoints appear to be reused in two separate inversions. Additionally, we use heterozygous individuals to quantitatively investigate somatic pairing disruption in the regions immediately surrounding inversion breakpoints, and we find that pairing disruption is undetectable beyond approximately 250 Kb from the inversion breakpoints.