A molecular perspective on a complex polymorphic inversion system with cytological evidence of multiply reused breakpoints.
ABSTRACT: Genome sequence comparison across the Drosophila genus revealed that some fixed inversion breakpoints had been multiply reused at this long timescale. Cytological studies of Drosophila inversion polymorphism had previously shown that, also at this shorter timescale, some breakpoints had been multiply reused. The paucity of molecularly characterized polymorphic inversion breakpoints has so far precluded contrasting whether cytologically shared breakpoints of these relatively young inversions are actually reused at the molecular level. The E chromosome of Drosophila subobscura stands out because it presents several inversion complexes. This is the case of the E1+2+9+3 arrangement that originated from the ancestral Est arrangement through the sequential accumulation of four inversions (E1, E2, E9 and E3) sharing some breakpoints. We recently identified the breakpoints of inversions E1 and E2, which allowed establishing reuse at the molecular level of the cytologically shared breakpoint of these inversions. Here, we identified and sequenced the breakpoints of inversions E9 and E3, because they share breakpoints at sections 58D and 64C with those of inversions E1 and E2. This has allowed establishing that E9 and E3 originated through the staggered-break mechanism. Most importantly, sequence comparison has revealed the multiple reuse at the molecular level of the proximal breakpoint (section 58D), which would have been used at least by inversions E2, E9 and E3. In contrast, the distal breakpoint (section 64C) might have been only reused once by inversions E1 and E2, because the distal E3 breakpoint is displaced >70 kb from the other breakpoint limits.
Project description:Chromosomal inversions are fundamental drivers of genome evolution. In the main Afrotropical malaria vector species, belonging to the <i>Anopheles gambiae</i> 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 <i>An. coluzzii</i> 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.
Project description:Inversions are an important form of structural variation, but they are difficult to characterize, as their breakpoints often fall within inverted repeats. We have developed a method called 'haplotype fusion' in which an inversion breakpoint is genotyped by performing fusion PCR on single molecules of human genomic DNA. Fusing single-copy sequences bracketing an inversion breakpoint generates orientation-specific PCR products, exemplified by a genotyping assay for the int22 hemophilia A inversion on Xq28. Furthermore, we demonstrated that inversion events with breakpoints embedded within long (>100 kb) inverted repeats can be genotyped by haplotype-fusion PCR followed by bead-based single-molecule haplotyping on repeat-specific markers bracketing the inversion breakpoint. We illustrate this method by genotyping a Yp paracentric inversion sponsored by >300-kb-long inverted repeats. The generality of our methods to survey for, and genotype chromosomal inversions should help our understanding of the contribution of inversions to genomic variation, inherited diseases and cancer.
Project description:Chromosomal polymorphism is widespread in the Drosophila genus, with extensive evidence supporting its adaptive character in diverse species. Moreover, inversions are the major contributors to the genus chromosomal evolution. The molecular characterization of a reduced number of polymorphic inversion breakpoints in Drosophila melanogaster and Drosophila subobscura supports that their inversions would have mostly originated through a mechanism that generates duplications -staggered double-strand breaks- and has thus the potential to contribute to their adaptive character. There is also evidence for inversion breakpoint reuse at different time scales. Here, we have characterized the breakpoints of two inversions of D. subobscura -O4 and O8- involved in complex arrangements that are frequent in the warm parts of the species distribution area. The duplications detected at their breakpoints are consistent with their origin through the staggered-break mechanism, which further supports it as the prevalent mechanism in D. subobscura. The comparative analysis of inversions breakpoint regions across the Drosophila genus has revealed several genes affected by multiple disruptions due not only to inversions but also to single-gene transpositions and duplications.
Project description:Chromosomal inversions can contribute to the adaptation of organisms to their environment by capturing particular advantageous allelic combinations of a set of genes included in the inverted fragment and also by advantageous functional changes due to the inversion process itself that might affect not only the expression of flanking genes but also their dose and structure. Of the two mechanisms originating inversions -ectopic recombination, and staggered double-strand breaks and subsequent repair- only the latter confers the inversion the potential to have dosage effects and/or to generate advantageous chimeric genes. In Drosophila subobscura, there is ample evidence for the adaptive character of its chromosomal polymorphism, with an important contribution of some warm-climate arrangements such as E1+2+9+12. Here, we have characterized the breakpoints of inversion E12 and established that it originated through the staggered-break mechanism like four of the five inversions of D. subobscura previously studied. This mechanism that also predominates in the D. melanogaster lineage might be prevalent in the Sophophora subgenus and contribute to the adaptive character of the polymorphic and fixed inversions of its species. Finally, we have shown that the D. subobscura inversion breakpoint regions have generally been disrupted by additional structural changes occurred at different time scales.
Project description:Chromosomal rearrangements (CRs) play important roles in karyotype diversity and speciation. While many CR breakpoints have been characterized at the sequence level in yeast, insects, and primates, little is known about the structure of evolutionary CR breakpoints in plant genomes, which are much more dynamic in genome size and sequence organization. Here, we report identification of breakpoints of a translocation between chromosome arms 4L and 5L of Triticeae, which is fixed in several species, including diploid wheat and rye, by comparative mapping and analysis of the draft genome and chromosome survey sequences of the Triticeae species. The wheat translocation joined the ends of breakpoints downstream of a WD40 gene on 4AL and a gene of the PMEI family on 5AL. A basic helix-loop-helix transcription factor gene in 5AL junction was significantly restructured. Rye and wheat share the same position for the 4L breakpoint, but the 5L breakpoint positions are not identical, although very close in these two species, indicating the recurrence of 4L/5L translocations in the Triticeae. Although barley does not carry the translocation, collinearity across the breakpoints was violated by putative inversions and/or transpositions. Alignment with model grass genomes indicated that the translocation breakpoints coincided with ancient inversion junctions in the Triticeae ancestor. Our results show that the 4L/5L translocation breakpoints represent two CR hotspots reused during Triticeae evolution, and support breakpoint reuse as a widespread mechanism in all eukaryotes. The mechanisms of the recurrent translocation and its role in Triticeae evolution are also discussed.
Project description:Apparently balanced chromosomal inversions may lead to disruption of developmentally important genes at the breakpoints of the inversion, causing congenital malformations. Characterization of such inversions may therefore lead to new insights in human development. Here, we report on a de novo inversion of chromosome 7 (p15.2q36.3) in a patient with postaxial polysyndactyly. The breakpoints do not disrupt likely candidate genes for the limb phenotype observed in the patient. However, on the p-arm the breakpoint separates the HOXA cluster from a gene desert containing several conserved noncoding elements, suggesting that a disruption of a cis-regulatory circuit of the HOXA cluster could be the underlying cause of the phenotype in this patient.
Project description:That closely related species often differ by chromosomal inversions was discovered by Sturtevant and Plunkett in 1926. Our knowledge of how these inversions originate is still very limited, although a prevailing view is that they are facilitated by ectopic recombination events between inverted repetitive sequences. The availability of genome sequences of related species now allows us to study in detail the mechanisms that generate interspecific inversions. We have analyzed the breakpoint regions of the 29 inversions that differentiate the chromosomes of Drosophila melanogaster and two closely related species, D. simulans and D. yakuba, and reconstructed the molecular events that underlie their origin. Experimental and computational analysis revealed that the breakpoint regions of 59% of the inversions (17/29) are associated with inverted duplications of genes or other nonrepetitive sequences. In only two cases do we find evidence for inverted repetitive sequences in inversion breakpoints. We propose that the presence of inverted duplications associated with inversion breakpoint regions is the result of staggered breaks, either isochromatid or chromatid, and that this, rather than ectopic exchange between inverted repetitive sequences, is the prevalent mechanism for the generation of inversions in the melanogaster species group. Outgroup analysis also revealed evidence for widespread breakpoint recycling. Lastly, we have found that expression domains in D. melanogaster may be disrupted in D. yakuba, bringing into question their potential adaptive significance.
Project description:Genomic structural variants, including translocations, inversions, insertions, deletions, and duplications, are challenging to be reliably detected by traditional genomic technologies. In particular, balanced translocations and inversions can neither be identified by microarrays since they do not alter chromosome copy numbers, nor by short-read sequencing because of the unmappability of short reads against repetitive genomic regions. The precise localization of breakpoints is vital for exploring genetic causes in patients with balanced translocations or inversions. Long-read sequencing techniques may detect these structural variants in a more direct, efficient, and accurate manner. Here, we performed whole-genome, long-read sequencing using the Oxford Nanopore GridION sequencer to detect breakpoints in six balanced chromosome translocation carriers and one inversion carrier. The results showed that all the breakpoints were consistent with the karyotype results with only ~10× coverage. Polymerase chain reaction (PCR) and Sanger sequencing confirmed 8 out of 14 breakpoints; however, other breakpoint loci were slightly missed since they were either in highly repetitive regions or pericentromeric regions. Some of the breakpoints interrupted normal gene structure, and in other cases, micro-deletions/insertions were found just next to the breakpoints. We also detected haplotypes around the breakpoint regions. Our results suggest that long-read, whole-genome sequencing is an ideal strategy for precisely localizing translocation breakpoints and providing haplotype information, which is essential for medical genetics and preimplantation genetic testing.
Project description:Chromosomal inversions are usually portrayed as simple two-breakpoint rearrangements changing gene order but not gene number or structure. However, increasing evidence suggests that inversion breakpoints may often have a complex structure and entail gene duplications with potential functional consequences. Here, we used a combination of different techniques to investigate the breakpoint structure and the functional consequences of a complex rearrangement fixed in Drosophila buzzatii and comprising two tandemly arranged inversions sharing the middle breakpoint: 2m and 2n. By comparing the sequence in the breakpoint regions between D. buzzatii (inverted chromosome) and D. mojavensis (noninverted chromosome), we corroborate the breakpoint reuse at the molecular level and infer that inversion 2m was associated with a duplication of a ~13 kb segment and likely generated by staggered breaks plus repair by nonhomologous end joining. The duplicated segment contained the gene CG4673, involved in nuclear transport, and its two nested genes CG5071 and CG5079. Interestingly, we found that other than the inversion and the associated duplication, both breakpoints suffered additional rearrangements, that is, the proximal breakpoint experienced a microinversion event associated at both ends with a 121-bp long duplication that contains a promoter. As a consequence of all these different rearrangements, CG5079 has been lost from the genome, CG5071 is now a single copy nonnested gene, and CG4673 has a transcript ~9 kb shorter and seems to have acquired a more complex gene regulation. Our results illustrate the complex effects of chromosomal rearrangements and highlight the need of complementing genomic approaches with detailed sequence-level and functional analyses of breakpoint regions if we are to fully understand genome structure, function, and evolutionary dynamics.
Project description:Anopheles arabiensis, one of the two most potent malaria vectors of the gambiae complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion 2Rd' of A. arabiensis. A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different A. arabiensis strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element Odysseus. The presence of Odysseus at the junction of the naturally occuring inversion 2Rd' suggests that the inversion may be the result of the transposable element's activity. Characteristics of Odysseus' terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of Odysseus.