Project description:Chromosomal rearrangements constitute a significant feature of genome evolution, and inversion polymorphisms in Drosophila have been studied intensely for decades. Population geneticists have long recognized that the sequence features associated with inversion breakpoints would reveal much about the mutational origin, uniqueness, and genealogical history of individual inversion polymorphisms, but the cloning of breakpoint sequences is not trivial. With the aid of a method for rapid recovery of DNA clones spanning rearrangement breakpoints, we recover and examine the DNA sequences spanning the breakpoints of the cosmopolitan inversion In(3L)Payne in Drosophila melanogaster. By examining the sequence diversity associated with six standard and seven inverted chromosomes from natural populations, we find that the inversion is monophyletic in origin, the sequences are genetically isolated from recombination at the breakpoints, and there is no association with features such as transposable elements. The inverted sequences show 17-fold less nucleotide polymorphism, but there are eight fixed differences in the region spanning both breakpoints. This suggests that this inversion is not recently derived. Finally, Northern analysis and transcript mapping find that the distal breakpoint has disrupted three transcripts that are normally expressed in the standard arrangement. Incidentally, the method introduced here can be used to isolate breakpoint sequences of arrangements associated with many human diseases.
Project description:Chromosomal inversions are structural changes that alter gene order but generally not gene content in the affected region. In Drosophila, extensive cytological studies revealed the widespread character of inversion polymorphism, with evidence for its adaptive character. In Drosophila subobscura, polymorphism affects both its four large autosomal elements and its X (A) chromosome. The characterization of eight of these autosomal inversions breakpoints revealed that most of them originated through the staggered-breaks mechanism. Here, we have performed chromosomal walks to identify the breakpoints of two X-chromosome widely distributed inversions -A2 and A1- of D. subobscura. Inversion A2 is considered a warm-adapted arrangement that exhibits parallel latitudinal clines in the species ancestral distribution area and in both American subcontinents, whereas inversion A1 is only present in the Palearctic region where it presents an east-west cline. The duplication detected at the A2 inversion breakpoints is consistent with its origin by the staggered-breaks mechanism. Inversion A1 breakpoints could not be molecularly identified even though they could be narrowly delimited. This result points to chromosome walking limitations when using as a guide the genome of other species. Limitations stem from the rate of evolution by paracentric inversions, which in Drosophila is highest for the X chromosome.
Project description:Cytological studies revealed that the number of chromosomes and their organization varies across species. The increasing availability of whole genome sequences of multiple species across specific phylogenies has confirmed and greatly extended these cytological observations. In the Drosophila genus, the ancestral karyotype consists of five rod-like acrocentric chromosomes (Muller elements A to E) and one dot-like chromosome (element F), each exhibiting a generally conserved gene content. Chromosomal fusions and paracentric inversions are thus the major contributors, respectively, to chromosome number variation among species and to gene order variation within chromosomal element. The subobscura cluster of Drosophila consists in three species that retain the genus ancestral karyotype and differ by a reduced number of fixed inversions. Here, we have used cytological information and the D. guanche genome sequence to identify and molecularly characterize the breakpoints of inversions that became fixed since the D. guanche-D. subobscura split. Our results have led us to propose a modified version of the D. guanche cytological map of its X chromosome, and to establish that (i) most inversions became fixed in the D. subobscura lineage and (ii) the order in which the four X chromosome overlapping inversions occurred and became fixed.
Project description: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 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:<h4>Background</h4>Drosophila subobscura has long been a central model in evolutionary genetics. Presently, its use is hindered by the lack of a reference genome. To bridge this gap, here we used PacBio long-read technology, together with the available wealth of genetic marker information, to assemble and annotate a high-quality nuclear and complete mitochondrial genome for the species. With the obtained assembly, we performed the first synteny analysis of genome structure evolution in the subobscura subgroup.<h4>Results</h4>We generated a highly-contiguous ~ 129 Mb-long nuclear genome, consisting of six pseudochromosomes corresponding to the six chromosomes of a female haploid set, and a complete 15,764 bp-long mitogenome, and provide an account of their numbers and distributions of codifying and repetitive content. All 12 identified paracentric inversion differences in the subobscura subgroup would have originated by chromosomal breakage and repair, with some associated duplications, but no evidence of direct gene disruptions by the breakpoints. Between lineages, inversion fixation rates were 10 times higher in continental D. subobscura than in the two small oceanic-island endemics D. guanche and D. madeirensis. Within D. subobscura, we found contrasting ratios of chromosomal divergence to polymorphism between the A sex chromosome and the autosomes.<h4>Conclusions</h4>We present the first high-quality, long-read sequencing of a D. subobscura genome. Our findings generally support genome structure evolution in this species being driven indirectly, through the inversions' recombination-suppression effects in maintaining sets of adaptive alleles together in the face of gene flow. The resources developed will serve to further establish the subobscura subgroup as model for comparative genomics and evolutionary indicator of global change.
Project description: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 E<sub>1</sub>, E<sub>2</sub>, E<sub>9</sub> and E<sub>3</sub>. 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.
Project description:The adaptive character of inversion polymorphism in Drosophila subobscura is well established. The O(ST) and O(3+4) chromosomal arrangements of this species differ by two overlapping inversions that arose independently on O(3) chromosomes. Nucleotide variation in eight gene regions distributed along inversion O(3) was analyzed in 14 O(ST) and 14 O(3+4) lines. Levels of variation within arrangements were quite similar along the inversion. In addition, we detected (i) extensive genetic differentiation between arrangements in all regions, regardless of their distance to the inversion breakpoints; (ii) strong association between nucleotide variants and chromosomal arrangements; and (iii) high levels of linkage disequilibrium in intralocus and also in interlocus comparisons, extending over distances as great as approximately 4 Mb. These results are not consistent with the higher genetic exchange between chromosomal arrangements expected in the central part of an inversion from double-crossover events. Hence, double crossovers were not produced or, alternatively, recombinant chromosomes were eliminated by natural selection to maintain coadapted gene complexes. If the strong genetic differentiation detected along O(3) extends to other inversions, nucleotide variation would be highly structured not only in D. subobscura, but also in the genome of other species with a rich chromosomal polymorphism.
Project description:Adaptation is defined as an evolutionary process allowing organisms to succeed in certain habitats or conditions. Chromosomal inversions have the potential to be key in the adaptation processes, since they can contribute to the maintenance of favoured combinations of adaptive alleles through reduced recombination between individuals carrying different inversions. We have analysed six genes (Pif1A, Abi, Sqd, Yrt, Atp? and Fmr1), located inside and outside three inversions of the O chromosome in European populations of Drosophila subobscura. Genetic differentiation was significant between inversions despite extensive recombination inside inverted regions, irrespective of gene distance to the inversion breakpoints. Surprisingly, the highest level of genetic differentiation between arrangements was found for the Atp? gene, which is located outside the O1 and O7 inversions. Two derived unrelated arrangements (O3+4+1 and O3+4+7) are nearly fixed for several amino acid substitutions at the Atp? gene that have been described to confer resistance in other species to the cardenolide ouabain, a plant toxin capable of blocking ATPases. Similarities in the Atp? variants, conferring ouabain resistance in both arrangements, may be the result of convergent substitution and be favoured in response to selective pressures presumably related to the presence of plants containing ouabain in the geographic locations where both inversions are present.