Discovery of human inversion polymorphisms by comparative analysis of human and chimpanzee DNA sequence assemblies

With a draft genome-sequence assembly for the chimpanzee available, it is now possible to perform genome-wide analyses to identify, at a submicroscopic level, structural rearrangements that have occurred between chimpanzees and humans. The goal of this study was to investigate chromosomal regions that are inverted between the chimpanzee and human genomes. Using the net alignments for the builds of the human and chimpanzee genome assemblies, we identified a total of 1,576 putative regions of inverted orientation, covering more than 154 mega-bases of DNA. The DNA segments are distributed throughout the genome and range from 23 base pairs to 62 mega-bases in length. For the 66 inversions more than 25 kilobases (kb) in length, 75% were flanked on one or both sides by (often unrelated) segmental duplications. Using PCR and fluorescence in situ hybridization we experimentally validated 23 of 27 (85%) semi-randomly chosen regions; the largest novel inversion confirmed was 4.3 mega-bases at human Chromosome 7p14. Gorilla was used as an out-group to assign ancestral status to the variants. All experimentally validated inversion regions were then assayed against a panel of human samples and three of the 23 (13%) regions were found to be polymorphic in the human genome. These polymorphic inversions include 730 kb (at 7p22), 13 kb (at 7q11), and 1 kb (at 16q24) fragments with a 5%, 30%, and 48% minor allele frequency, respectively. Our results suggest that inversions are an important source of variation in primate genome evolution. The finding of at least three novel inversion polymorphisms in humans indicates this type of structural variation may be a more common feature of our genome than previously realized.     

Characterization of the human lineage-specific pericentric inversion that distinguishes human chromosome 1 from the homologous chromosomes of the great apes

The human and chimpanzee genomes are distinguishable in terms of ten gross karyotypic differences including nine pericentric inversions and a chromosomal fusion. Seven of these large pericentric inversions are chimpanzee-specific whereas two of them, involving human chromosomes 1 and 18, were fixed in the human lineage after the divergence of humans and chimpanzees. We have performed detailed molecular and computational characterization of the breakpoint regions of the human-specific inversion of chromosome 1. FISH analysis and sequence comparisons together revealed that the pericentromeric region of HSA 1 contains numerous segmental duplications that display a high degree of sequence similarity between both chromosomal arms. Detailed analysis of these regions has allowed us to refine the p-arm breakpoint region to a 154.2 kb interval at 1p11.2 and the q-arm breakpoint region to a 562.6 kb interval at 1q21.1. Both breakpoint regions contain human-specific segmental duplications arranged in inverted orientation. We therefore propose that the pericentric inversion of HSA 1 was mediated by intra-chromosomal non-homologous recombination between these highly homologous segmental duplications that had themselves arisen only recently in the human lineage by duplicative transposition.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Justyna M. Szamalek and Violaine Goidts are contributed equally to the paper.     

Assaying chromosomal inversions by single-molecule haplotyping

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.     

STRUCTURE AND POPULATION GENETICS OF THE BREAKPOINTS OF A POLYMORPHIC INVERSION IN DROSOPHILA SUBOBSCURA

Drosophila subobscura is a paleartic species of the obscura group with a rich chromosomal polymorphism. To further our understanding on the origin of inversions and on how they regain variation, we have identified and sequenced the two breakpoints of a polymorphic inversion of D. subobscura—inversion 3 of the O chromosome—in a population sample. The breakpoints could be identified as two rather short fragments (～300 bp and 60 bp long) with no similarity to any known transposable element family or repetitive sequence. The presence of the ～300‐bp fragment at the two breakpoints of inverted chromosomes implies its duplication, an indication of the inversion origin via staggered double‐strand breaks. Present results and previous findings support that the mode of origin of inversions is neither related to the inversion age nor species‐group specific. The breakpoint regions do not consistently exhibit the lower level of variation within and stronger genetic differentiation between arrangements than more internal regions that would be expected, even in moderately small inversions, if gene conversion were greatly restricted at inversion breakpoints. Comparison of the proximal breakpoint region in species of the obscura group shows that this breakpoint lies in a small high‐turnover fragment within a long collinear region (～300 kb).     

Chromosomal speciation of humans and chimpanzees revisited: studies of DNA divergence within inverted regions

The human and chimpanzee karyotypes are distinguishable in terms of nine pericentric inversions. According to the recombination suppression model of speciation, these inversions could have promoted the process of parapatric speciation between hominoid populations ancestral to chimpanzees and humans. Were recombination suppression to have occurred in inversion heterozygotes, gene flow would have been reduced, resulting in the accumulation of genetic incompatibilities leading to reproductive isolation and eventual speciation. In an attempt to detect the molecular signature of such events, the sequence divergence of non-coding DNA was compared between humans and chimpanzees. Precise knowledge of the locations of the inversion breakpoints permitted accurate discrimination between inverted and non-inverted regions. Contrary to the predictions of the recombination suppression model, sequence divergence was found to be lower in inverted chromosomal regions as compared to non-inverted regions, albeit with borderline statistical significance. Thus, no signature of recombination suppression resulting from inversion heterozygosity appears to be detectable by analysis of extant human and chimpanzee non-coding DNA. The precise delineation of the inversion breakpoints may nevertheless still prove helpful in identifying potential speciation-relevant genes within the inverted regions.     

Principles of genome evolution in the Drosophila melanogaster species group

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.     

Contrasting Histories of Three Gene Regions Associated with In(3l)payne of Drosophila Melanogaster

In the present report, we studied nucleotide variation in three gene regions of Drosophila melanogaster, spanning >5 kb and showing different degrees of association with the cosmopolitan inversion In(3-L)Payne. The analysis of sequence variation in the regions surrounding the breakpoints and the heat shock 83 (Hsp83) gene locus, located close to the distal breakpoint, revealed the absence of shared polymorphisms and the presence of a number of fixed differences between arrangements, indicating absence of genetic exchange. In contrast, for the esterase-6 gene region, located in the center of the inversion, we observed the presence of shared polymorphisms between arrangements suggesting genetic exchange. In the regions close to the breakpoints, the common St arrangement is 10 times more polymorphic than inverted chromosomes. We propose that the lack of recombination between arrangements in these regions coupled with genetic hitchhiking is the best explanation for the low heterozygosity observed in inverted lines. Using the data for the breakpoints, we estimate that this inversion polymorphism is around 0.36 million yr old. Although it is widely accepted that inversions are examples of balanced polymorphisms, none of the current neutrality tests including our Monte Carlo simulations showed significant departure from neutral expectations.     

The Genetic Content of Chromosomal Inversions across a Wide Latitudinal Gradient

There is increasing evidence regarding the role of chromosomal inversions in relevant biological processes such as local adaptation and speciation. A classic example of the adaptive role of chromosomal polymorphisms is given by the clines of inversion frequencies in Drosophila subobscura, repeatable across continents. Nevertheless, not much is known about the molecular variation associated with these polymorphisms. We characterized the genetic content of ca. 600 individuals from nine European populations following a latitudinal gradient by analysing 19 microsatellite loci from two autosomes (J and U) and the sex chromosome (A), taking into account their chromosomal inversions. Our results clearly demonstrate the molecular genetic uniformity within a given chromosomal inversion across a large latitudinal gradient, particularly from Groningen (Netherlands) in the north to Málaga (Spain) in the south, experiencing highly diverse environmental conditions. This low genetic differentiation within the same gene arrangement across the nine European populations is consistent with the local adaptation hypothesis for th evolutionof chromosomal polymorphisms. We also show the effective role of chromosomal inversions in maintaining different genetic pools within these inverted genomic regions even in the presence of high gene flow. Inversions represent thus an important barrier to gene flux and can help maintain specific allelic combinations with positive effects on fitness. Consistent patterns of microsatellite allele-inversion linkage disequilibrium particularly in loci within inversions were also observed. Finally, we identified areas within inversions presenting clinal variation that might be under selection.     

Interchromosomal exchange of genetic information between gene arrangements on the third chromosome of Drosophila pseudoobscura

During the last 60 years, the inversion polymorphism on the third chromosome of Drosophila pseudoobscura has become a case study of the evolution of linked blocks of genes, isolated from each other by the suppression of recombination in heterozygotes for different inversions. Due to its location within inverted regions in most gene arrangements, the amylase (Amy) gene region can be used to elucidate the molecular pattern of evolution in these inversions. We studied this region in the Tree Line phylad of gene arrangements, with regard to both restriction site polymorphisms (RSP) and nucleotide sequences. The analysis of restriction maps, encompassing 26 kb, corroborates the cytogenetic phylogeny established on the basis of inversion breakpoints. However, we found that the 2.7 kb of nucleotide sequences of the AmyI gene are identical in both Estes Park and Hidalgo arrangements, despite the fact that these inversions arose independently from Tree Line. These contrasting results suggest that a homogenizing force, most likely gene conversion, is able to bring about localized exchanges between otherwise isolated gene arrangements.      

Molecular analysis of recombination in a family with Duchenne muscular dystrophy and a large pericentric X chromosome inversion.

It has been demonstrated in animal studies that, in animals heterozygous for pericentric chromosomal inversions, loop formation is greatly reduced during meiosis. This results in absence of recombination within the inverted segment, with recombination seen only outside the inversion. A recent study in yeast has shown that telomeres, rather than centromeres, lead in chromosome movement just prior to meiosis and may be involved in promoting recombination. We studied by cytogenetic analysis and DNA polymorphisms the nature of meiotic recombination in a three-generation family with a large pericentric X chromosome inversion, inv(X)(p21.1q26), in which Duchenne muscular dystrophy (DMD) was cosegregating with the inversion. On DNA analysis there was no evidence of meiotic recombination between the inverted and normal X chromosomes in the inverted segment. Recombination was seen at the telomeric regions, Xp22 and Xq27-28. No deletion or point mutation was found on analysis of the DMD gene. On the basis of the FISH results, we believe that the X inversion is the mutation responsible for DMD in this family. Our results indicate that (1) pericentric X chromosome inversions result in reduction of recombination between the normal and inverted X chromosomes; (2) meiotic X chromosome pairing in these individuals is likely initiated at the telomeres; and (3) in this family DMD is caused by the pericentric inversion.     

Chromosomal inversions associated with environmental adaptation in honeybees

Chromosomal inversions can facilitate local adaptation in the presence of gene flow by suppressing recombination between well‐adapted native haplotypes and poorly adapted migrant haplotypes. East African mountain populations of the honeybee Apis mellifera are highly divergent from neighbouring lowland populations at two extended regions in the genome, despite high similarity in the rest of the genome, suggesting that these genomic regions harbour inversions governing local adaptation. Here, we utilize a new highly contiguous assembly of the honeybee genome to characterize these regions. Using whole‐genome sequencing data from 55 highland and lowland bees, we find that the highland haplotypes at both regions are present at high frequencies in three independent highland populations but extremely rare elsewhere. The boundaries of both divergent regions are characterized by regions of high homology with each other positioned in opposite orientations and contain highly repetitive, long inverted repeats with homology to transposable elements. These regions are likely to represent inversion breakpoints that participate in nonallelic homologous recombination. Using long‐read data, we confirm that the lowland samples are contiguous across breakpoint regions. We do not find evidence for disruption of functional sequence by these breakpoints, which suggests that the inversions are likely maintained due to their allelic content conferring local adaptation in highland environments. Finally, we identify a third divergent genomic region, which contains highly divergent segregating haplotypes that also may contain inversion variants under selection. The results add to a growing body of evidence indicating the importance of chromosomal inversions in local adaptation.     

Mapping regions within cosmopolitan inversion In(3R)Payne associated with natural variation in body size in Drosophila melanogaster

Associations between genotypes for inversions and quantitative traits have been reported in several organisms, but little has been done to localize regions within inversions controlling variation in these traits. Here, we use an association mapping technique to identify genomic regions controlling variation in wing size within the cosmopolitan inversion In(3R)Payne in Drosophila melanogaster. Previous studies have shown that this inversion strongly influences variation in wing size, a trait highly correlated with body size. We found three alleles from two separate regions within In(3R)Payne with significant additive effects on wing size after the additional effect of the inversion itself had been taken into account. There were also several alleles with significant genotype-by-inversion interaction effects on wing size. None of the alleles tested had a significant additive effect on development time, suggesting different genes control these traits and that clinal patterns in them have therefore arisen independently. The presence of multiple regions within In(3R)Payne controlling size is consistent with the idea that inversions persist in populations because they contain multiple sets of locally adapted alleles, but more work needs to be done to test if they are indeed coadapted.     

Chromosomal Inversions between Human and Chimpanzee Lineages Caused by Retrotransposons

The long interspersed element-1 (LINE-1 or L1) and Alu elements are the most abundant mobile elements comprising 21% and 11% of the human genome, respectively. Since the divergence of human and chimpanzee lineages, these elements have vigorously created chromosomal rearrangements causing genomic difference between humans and chimpanzees by either increasing or decreasing the size of genome. Here, we report an exotic mechanism, retrotransposon recombination-mediated inversion (RRMI), that usually does not alter the amount of genomic material present. Through the comparison of the human and chimpanzee draft genome sequences, we identified 252 inversions whose respective inversion junctions can clearly be characterized. Our results suggest that L1 and Alu elements cause chromosomal inversions by either forming a secondary structure or providing a fragile site for double-strand breaks. The detailed analysis of the inversion breakpoints showed that L1 and Alu elements are responsible for at least 44% of the 252 inversion loci between human and chimpanzee lineages, including 49 RRMI loci. Among them, three RRMI loci inverted exonic regions in known genes, which implicates this mechanism in generating the genomic and phenotypic differences between human and chimpanzee lineages. This study is the first comprehensive analysis of mobile element bases inversion breakpoints between human and chimpanzee lineages, and highlights their role in primate genome evolution.     

Investigation of inversion polymorphisms in the human genome using principal components analysis

Despite the significant advances made over the last few years in mapping inversions with the advent of paired-end sequencing approaches, our understanding of the prevalence and spectrum of inversions in the human genome has lagged behind other types of structural variants, mainly due to the lack of a cost-efficient method applicable to large-scale samples. We propose a novel method based on principal components analysis (PCA) to characterize inversion polymorphisms using high-density SNP genotype data. Our method applies to non-recurrent inversions for which recombination between the inverted and non-inverted segments in inversion heterozygotes is suppressed due to the loss of unbalanced gametes. Inside such an inversion region, an effect similar to population substructure is thus created: two distinct "populations" of inversion homozygotes of different orientations and their 1:1 admixture, namely the inversion heterozygotes. This kind of substructure can be readily detected by performing PCA locally in the inversion regions. Using simulations, we demonstrated that the proposed method can be used to detect and genotype inversion polymorphisms using unphased genotype data. We applied our method to the phase III HapMap data and inferred the inversion genotypes of known inversion polymorphisms at 8p23.1 and 17q21.31. These inversion genotypes were validated by comparing with literature results and by checking Mendelian consistency using the family data whenever available. Based on the PCA-approach, we also performed a preliminary genome-wide scan for inversions using the HapMap data, which resulted in 2040 candidate inversions, 169 of which overlapped with previously reported inversions. Our method can be readily applied to the abundant SNP data, and is expected to play an important role in developing human genome maps of inversions and exploring associations between inversions and susceptibility of diseases.     

Principles of Genome Evolution in the Drosophila melanogaster Species Group

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.     

Pervasive gene conversion in chromosomal inversion heterozygotes

Chromosomal inversions shape recombination landscapes, and species differing by inversions may exhibit reduced gene flow in these regions of the genome. Though single crossovers within inversions are not usually recovered from inversion heterozygotes, the recombination barrier imposed by inversions is nuanced by noncrossover gene conversion. Here, we provide a genomewide empirical analysis of gene conversion rates both within species and in species hybrids. We estimate that gene conversion occurs at a rate of 1 × 10^–5 to 2.5 × 10^–5 converted sites per bp per generation in experimental crosses within Drosophila pseudoobscura and between D. pseudoobscura and its naturally hybridizing sister species D. persimilis. This analysis is the first direct empirical assessment of gene conversion rates within inversions of a species hybrid. Our data show that gene conversion rates in interspecies hybrids are at least as high as within‐species estimates of gene conversion rates, and gene conversion occurs regularly within and around inverted regions of species hybrids, even near inversion breakpoints. We also found that several gene conversion events appeared to be mitotic rather than meiotic in origin. Finally, we observed that gene conversion rates are higher in regions of lower local sequence divergence, yet our observed gene conversion rates in more divergent inverted regions were at least as high as in less divergent collinear regions. Given our observed high rates of gene conversion despite the sequence differentiation between species, especially in inverted regions, gene conversion has the potential to reduce the efficacy of inversions as barriers to recombination over evolutionary time.     

Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system

During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Therefore, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild‐type and in the non‐homologous end‐joining (NHEJ) mutant ku70‐1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild‐type, most junctions are formed by microhomology‐mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg‐cell‐specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will.     

Inferring the demographic history of Drosophila subobscura from nucleotide variation at regions not affected by chromosomal inversions

Drosophila subobscura presents a rich and complex chromosomal inversion polymorphism. It can thus be considered a model system (i) to study the mechanisms originating inversions and how inversions affect the levels and patterns of variation in the inverted regions and (ii) to study adaptation at both the single‐gene and chromosomal inversion levels. It is therefore important to infer its demographic history as previous information indicated that its nucleotide variation is not at mutation–drift equilibrium. For that purpose, we sequenced 16 noncoding regions distributed across those parts of the J chromosome not affected by inversions in the studied population and possibly either by other selective events. The pattern of variation detected in these 16 regions is similar to that previously reported within different chromosomal arrangements, suggesting that the latter results would, thus, mainly reflect recent demographic events rather than the partial selective sweep imposed by the origin and frequency increase of inversions. Among the simple demographic models considered in our Approximate Bayesian Computation analysis of variation at the 16 regions, the model best supported by the data implies a population size expansion soon after the penultimate glacial period. This model constitutes a better null model, and it is therefore an important resource for subsequent studies aiming among others to uncover selective events across the species genome. Our results also highlight the importance of introducing the possibility of multiple hits in the coalescent simulations with an outgroup.