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Transposable elements (TEs) are repetitive sequences of DNA that replicate and proliferate throughout genomes. Taken together, all the TEs in a genome form a diverse community of sequences, which can be studied to draw conclusions about genome evolution. TE diversity can be measured using models for ecological community diversity that consider species richness and evenness. Several models predict TE diversity decreasing as genomes expand because of selection against ectopic recombination and/or competition among TEs to garner host replicative machinery and evade host silencing mechanisms. Salamanders have some of the largest vertebrate genomes and highest TE loads. Salamanders of the genus Plethodon, in particular, have genomes that range in size from 20 to 70 Gb. Here, we use Oxford Nanopore sequencing to generate low-coverage genomic sequences for four species of Plethodon that encompass two independent genome expansion events, one in the eastern clade (P. cinereus, 29.3 Gb vs. P. glutinosus, 38.9 Gb) and one in the western clade (P. vehiculum, 46.4 Gb vs P. idahoensis, 67.0 Gb). We classified the TEs in these genomes and found ~52 TE superfamilies, accounting for 27-32% of the genomes. We calculated Simpson’s and Shannon’s diversity indices to quantify overall TE diversity. In both pairwise comparisons, the diversity index values for the smaller and larger genome were almost identical. This result indicates that, when genomes reach extremely large sizes, they maintain high levels of TE diversity at the superfamily level, in contrast to predictions made by previous studies on smaller genomes.

The Cre/loxP system is a powerful tool for generating conditional gene knockout (KO) mice and elucidate gene function in vivo. CD19-Cre and Mb1-iCre transgenic mice are commonly used for generating B cell-specific KO mice and investigate the development, as well as the physiological and pathophysiological roles of B cells. However, the CD19-Cre line low efficiency and the Mb1-iCre line occasional ectopic recombination represent challenges for their use. Thus, we developed a CD19-codon-improved Cre (CD19-iCre) knock-in mouse with the T2A-iCre sequence inserted into the Cd19 locus, just before the stop codon. The CD19-iCre mice were compared with existing models, crossed with the Rosa26-EYFP reporter mice, and their recombination activity in B cells carrying different Cre alleles was assessed. CD19-iCre mice showed more effective Cre recombination in the early B cell developmental stages compared with the CD19-Cre mice. The efficiencies of the CD19-iCre and Mb1-iCre lines were similar; however, the B lineage-specific recombination was more stringent in the CD19-iCre line. Furthermore, the utility value of the CD19-iCre model was superior than that of the CD19-Cre mice regarding deletion efficiency in IL10-floxed mice. Thus, the CD19-iCre line is a valuable tool for highly efficient gene targeting specific to the B cell compartment.

Balancers are rearranged chromosomes used in Drosophila melanogaster to maintain deleterious mutations in stable populations, preserve sets of linked genetic elements and construct complex experimental stocks. Here, we assess the phenotypes associated with breakpoint-induced mutations on commonly used third chromosome balancers and show remarkably few deleterious effects. We demonstrate that a breakpoint in p53 causes loss of radiation-induced apoptosis and a breakpoint in Fucosyltransferase A causes loss of fucosylation in nervous and intestinal tissue—the latter study providing new markers for intestinal cell identity and challenging previous conclusions about the regulation of fucosylation. We also describe thousands of potentially harmful mutations shared among X or third chromosome balancers, or unique to specific balancers, including an Ankyrin 2 mutation present on most TM3 balancers, and reiterate the risks of using balancers as experimental controls. We used long-read sequencing to confirm or refine the positions of two inversions with breakpoints lying in repetitive sequences and provide evidence that one of the inversions, In(2L)Cy, arose by ectopic recombination between foldback transposon insertions and the other, In(3R)C, cleanly separates subtelomeric and telomeric sequences and moves the subtelomeric sequences to an internal chromosome position. In addition, our characterization of In(3R)C shows that balancers may be polymorphic for terminal deletions. Finally, we present evidence that extremely distal mutations on balancers can add to the stability of stocks whose purpose is to maintain homologous chromosomes carrying mutations in distal genes. Overall, these studies add to our understanding of the structure, diversity and effectiveness of balancer chromosomes.

Regulatory T (Treg) cell-specific deletion of a gene of interest is a procedure widely used to study mechanisms controlling Treg development, homeostasis and function. Accordingly, several transgenic mouse lines have been generated that bear the Cre recombinase under control of the Foxp3 promoter either as a random transgene insertion or knocked into the endogenous Foxp3 locus, with the Foxp3YFP-Cre strain of mice being one of the most widely used. In an attempt to generate Treg cells that lacked expression of the insulin receptor (Insr), we crossed Foxp3YFP-Cre mice with Insrfl/fl mice. Using a conventional two-band PCR genotyping method we found that offspring genotypes did not correspond to the expected Mendelian ratios. We therefore developed a quantitative PCR-based genotyping method to investigate possible ectopic recombination outside the Treg lineage. With this method we found that ~50% of the F1 -generation mice showed evidence of ectopic recombination and that ~10% of the F2 -generation mice had germline Cre recombination activity leading to a high frequency of offspring with global Insr deletion. Use of the quantitative PCR genotyping method enabled accurate selection of mice without ectopic recombination and only the desired Treg cell-specific Insr deletion. Our data highlight the need to use genotyping methods that allow for assessment of possible ectopic recombination driven by the Foxp3YFP-Cre allele, particularly when studying genes that are systemically expressed.

Aspergillus flavus is a fungus that produces aflatoxin B1, one of the most carcinogenic secondary metabolites. Understanding the regulation mechanism of aflatoxin biosynthesis in this fungus requires precise methods for genomic integration of mutant alleles. To avoid the disadvantage of DNA integration into the genome by non-homologous or ectopic recombination, we developed a novel strategy for site-specific integration of foreign DNA by using a carboxin-resistant sdh2R allele (His 249 Leu). Our results demonstrated that the transformants were generated with a high efficiency (>96%) of correct integration into the sdh2-lcus of the genome of A. flavus NRRL 3357. The advantage of this method is that introduction of the eGFP expression cassette into the sdh2-locus had little effect on fungal growth and virulence while also being rapid and efficient. This system will be a valuable tool for genetic manipulation in A. flavus. To the best of our knowledge, this is the first report on the efficient site-specific integration at the sdh2-locus in the genome of Aspergillus.

The number and distribution of meiotic crossovers (COs) are highly regulated, reflecting the requirement for COs during the first round of meiotic chromosome segregation. CO control includes CO assurance and CO interference, which promote at least one CO per chromosome bivalent and evenly-spaced COs, respectively. Previous studies revealed a role for the DNA damage response (DDR) clamp and the clamp loader in CO formation by promoting interfering COs and interhomolog recombination, and also by suppressing ectopic recombination. In this study, we use classical tetrad analysis of Saccharomyces cerevisiae to show that a mutant defective in RAD24, which encodes the DDR clamp loader (RAD17 in other organisms), displayed reduced CO frequencies on two shorter chromosomes (III and V), but not on a long chromosome (chromosome VII). The residual COs in the rad24 mutant do not show interference. In contrast to rad24, mutants defective in the ATR kinase homolog Mec1, including a mec1 null and a mec1 kinase-dead mutant, show slight or few defects in CO frequency. On the other hand, mec1 COs show defects in interference, similar to the rad24 mutant. Our results support a model in which the DDR clamp and clamp-loader proteins promote interfering COs by recruiting pro-CO Zip, Mer, and Msh proteins to recombination sites, while the Mec1 kinase regulates CO distribution by a distinct mechanism. Moreover, CO formation and its control are implemented in a chromosome-specific manner, which may reflect a role for chromosome size in regulation.

In order to understand how DNA sequences of transposable elements (TEs) evolve, extensive simulations were carried out. We first used our previous model, in which the copy number of TEs is mainly controlled by selection against ectopic recombination. It was found that along a simulation run, the shape of phylogeny changes quite much, from monophyletic trees to dimorphic trees with two clusters. Our results demonstrated that the change of the phase is usually slow from a monomorphic phase to a dimorphic phase, accompanied with a growth of an internal branch by accumulation of variation between two types. Then, the phase immediately changes back to a monomorphic phase when one group gets extinct. Under this condition, monomorphic and dimorphic phases arise repeatedly, and it is very difficult to maintain two or more different types of TEs for a long time. Then, how a new subfamily can evolve? To solve this, we developed a new model, in which ectopic recombination is restricted between two types under some condition, for example, accumulation of mutations between them. Under this model, because selection works on the copy number of each types separately, two types can be maintained for a long time. As expected, our simulations demonstrated that a new type arises and persists quite stably, and that it will be recognized as a new subfamily followed by further accumulation of mutations. It is indicated that how ectopic recombination is regulated in a genome is an important factor for the evolution of a new subfamily.

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.

The replicative nature and generally deleterious effects of transposable elements (TEs) give rise to an outstanding question about how TE copy number is stably contained in host populations. Classic theoretical analyses predict that, when the decline in fitness due to each additional TE insertion is greater than linear, or when there is synergistic epistasis, selection against TEs can result in a stable equilibrium of TE copy number. While several mechanisms are predicted to yield synergistic deleterious effects of TEs, we lack empirical investigations of the presence of such epistatic interactions. Purifying selection with synergistic epistasis generates repulsion linkage between deleterious alleles and, accordingly, an underdispersed distribution for the number of deleterious mutations among individuals. We investigated this population genetic signal in an African Drosophila melanogaster population and found evidence for synergistic epistasis among TE insertions, especially those expected to have large fitness impacts. Curiously, even though ectopic recombination has long been predicted to generate nonlinear fitness decline with increased TE copy number, TEs predicted to suffer higher rates of ectopic recombination are not more likely to be underdispersed. On the other hand, underdispersed TE families are more likely to show signatures of deleterious epigenetic effects and stronger ping-pong signals of piRNA amplification, a hypothesized source from which synergism of TE-mediated epigenetic effects arises. Our findings set the stage for investigating the importance of epistatic interactions in the evolutionary dynamics of TEs.

Background Despite progress in genomic analysis of spiders, their chromosome evolution is not satisfactorily understood. Most information on spider chromosomes concerns the most diversified clade, entelegyne araneomorphs. Other clades are far less studied. Our study focused on haplogyne araneomorphs, which are remarkable for their unusual sex chromosome systems and for the co-evolution of sex chromosomes and nucleolus organizer regions (NORs); some haplogynes exhibit holokinetic chromosomes. To trace the karyotype evolution of haplogynes on the family level, we analysed the number and morphology of chromosomes, sex chromosomes, NORs, and meiosis in pholcids, which are among the most diverse haplogyne families. The evolution of spider NORs is largely unknown. Results Our study is based on an extensive set of species representing all major pholcid clades. Pholcids exhibit a low 2n and predominance of biarmed chromosomes, which are typical haplogyne features. Sex chromosomes and NOR patterns of pholcids are diversified. We revealed six sex chromosome systems in pholcids (X0, XY, X1X20, X1X2X30, X1X2Y, and X1X2X3X4Y). The number of NOR loci ranges from one to nine. In some clades, NORs are also found on sex chromosomes. Conclusions The evolution of cytogenetic characters was largely derived from character mapping on a recently published molecular phylogeny of the family. Based on an extensive set of species and mapping of their characters, numerous conclusions regarding the karyotype evolution of pholcids and spiders can be drawn. Our results suggest frequent autosome–autosome and autosome–sex chromosome rearrangements during pholcid evolution. Such events have previously been attributed to the reproductive isolation of species. The peculiar X1X2Y system is probably ancestral for haplogynes. Chromosomes of the X1X2Y system differ considerably in their pattern of evolution. In some pholcid clades, the X1X2Y system has transformed into the X1X20 or XY systems, and subsequently into the X0 system. The X1X2X30 system of Smeringopus pallidus probably arose from the X1X20 system by an X chromosome fission. The X1X2X3X4Y system of Kambiwa probably evolved from the X1X2Y system by integration of a chromosome pair. Nucleolus organizer regions have frequently expanded on sex chromosomes, most probably by ectopic recombination. Our data suggest the involvement of sex chromosome-linked NORs in achiasmatic pairing.

Hybrid sterility maintains reproductive isolation between species by preventing them from exchanging genetic material. The genome sequences of the yeasts Saccharomyces paradoxus and Saccharomyces paradoxus have diverged by about 12%, yet they can mate and form viable diploid hybrids, which can reproduce asexually by mitosis. But these hybrids are sexually sterile: nearly all the gametes they produce by meiosis are inviable because they are aneuploid, lacking at least one essential chromosome. Chromosomes from the different species mis-segregate during hybrid meiosis due to anti-recombination; their sequences are too diverged to efficiently recombine and form crossovers. Previous methods to increase hybrid fertility by enhancing meiotic crossing over have had counteracting detrimental effects on asexual viability, due to increased mutagenesis and ectopic recombination. We were able to increase hybrid fertility 70-fold by suppressing two genes, MSH2 (YOL090W) and SGS1 (YMR190C), specifically during meiosis, whilst maintaining their mitotic expression. This confirms that anti-recombination is the principal cause of hybrid sterility. By overcoming this barrier, we were able to generate viable euploid hybrid gametes containing recombinant genomes from these two highly diverged parent species. We used this unique resource to map intrinsic Bateson-Dobzhansky-Muller incompatibilities contributing to hybrid gamete inviability. The ability to interbreed such diverged species will allow mapping of other intrinsic traits such as hybrid depression or vigour, extrinsic ecological adaptations that differentiate species, or commercially important characteristics.

Background Chromosomal inversions have been pervasive during the evolution of the genus Drosophila, but there is significant variation between lineages in the rate of rearrangement fixation. D. mojavensis, an ecological specialist adapted to a cactophilic niche under extreme desert conditions, is a chromosomally derived species with ten fixed inversions, five of them not present in any other species. Results In order to explore the causes of the rapid chromosomal evolution in D. mojavensis, we identified and characterized all breakpoints of seven inversions fixed in chromosome 2, the most dynamic one. One of the inversions presents unequivocal evidence for its generation by ectopic recombination between transposon copies and another two harbor inverted duplications of non-repetitive DNA at the two breakpoints and were likely generated by staggered single-strand breaks and repair by non-homologous end joining. Four out of 14 breakpoints lay in the intergenic region between preexisting duplicated genes, suggesting an adaptive advantage of separating previously tightly linked duplicates. Four out of 14 breakpoints are associated with transposed genes, suggesting these breakpoints are fragile regions. Finally two inversions contain novel genes at their breakpoints and another three show alterations of genes at breakpoints with potential adaptive significance. Conclusions D. mojavensis chromosomal inversions were generated by multiple mechanisms, an observation that does not provide support for increased mutation rate as explanation for rapid chromosomal evolution. On the other hand, we have found a number of gene alterations at the breakpoints with putative adaptive consequences that directly point to natural selection as the cause of D. mojavensis rapid chromosomal evolution.

Genome stability in radioresistant bacterium Deinococcus radiodurans depends on RecA, the main bacterial recombinase. Without RecA, gross genome rearrangements occur during repair of DNA double-strand breaks. Long repeated (insertion) sequences have been identified as hot spots for ectopic recombination leading to genome rearrangements, and single-strand annealing (SSA) postulated to be the most likely mechanism involved in this process. Here, we have sequenced five isolates of D. radiodurans recA mutant carrying gross genome rearrangements to precisely characterize the rearrangements and to elucidate the underlying repair mechanism. The detected rearrangements consisted of large deletions in chromosome II in all the sequenced recA isolates. The mechanism behind these deletions clearly differs from the classical SSA; it utilized short (4–11 bp) repeats as opposed to insertion sequences or other long repeats. Moreover, it worked over larger linear DNA distances from those previously tested. Our data are most compatible with alternative end-joining, a recombination mechanism that operates in eukaryotes, but is also found in Escherichia coli. Additionally, despite the recA isolates being preselected for different rearrangement patterns, all identified deletions were found to overlap in a 35 kb genomic region. We weigh the evidence for mechanistic vs. adaptive reasons for this phenomenon.

The mitochondria of flowering plants have large and complex genomes whose structure and segregation are modulated by recombination activities. Among unresolved questions is what are the pathways responsible for the late steps of homologous recombination: while the loss of mitochondrial recombination is not viable, a deficiency in RECG1-dependent branch migration has little impact on plant development. Here we present an additional pathway required for the processing of organellar recombination intermediates, the one depending on RADA . RADA is similar in structure and activity to bacterial RadA/Sms, and in vitro it binds to ssDNA and accelerates strand-exchange reactions initiated by RecA. RADA-deficient plants are severely impacted in their development and fertility, correlating with increased mtDNA ectopic recombination and replication of recombination-generated subgenomes. The radA mutation is epistatic to recG1 , indicating that RADA drives the main branch migration pathway of plant mitochondria. In contrast, the double mutation radA recA3 is lethal, revealing the importance of an alternative RECA3-dependent pathway. Interestingly, the radA developmental phenotypes could not be correlated with obvious defects in mitochondrial gene expression. Rather, it seems that it is the activation of genes that repress cell cycle progression that is partially the cause of the stunted growth of radA mutants. SYNOPSIS Arabidopsis RADA is a main branch migration activity in plant mitochondria, whose deficiency leads to mtDNA instability by recombination, and suppression of plant growth by the activation of repressors of cell cycle progression.

During prophase I of meiosis, homologous chromosomes pair, synapse and exchange their genetic material through reciprocal homologous recombination, a phenomenon essential for faithful chromosome segregation. Partial sequence identity between non-homologous and heterologous chromosomes can also lead to recombination (ectopic recombination), a highly deleterious process that rapidly compromises genome integrity. To avoid ectopic exchange, homology recognition must be extended from the narrow position of a crossover-competent double-strand break to the entire chromosome. Here, we review advances on chromosome behaviour during meiotic prophase I in higher plants, by integrating centromere- and telomere dynamics driven by cytoskeletal motor proteins, into the processes of homologue pairing, synapsis and recombination. Centromere–centromere associations and the gathering of telomeres at the onset of meiosis at opposite nuclear poles create a spatially organised and restricted nuclear state in which homologous DNA interactions are favoured but ectopic interactions also occur. The release and dispersion of centromeres from the nuclear periphery increases the motility of chromosome arms, allowing meiosis-specific movements that disrupt ectopic interactions. Subsequent expansion of interstitial synapsis from numerous homologous interactions further corrects ectopic interactions. Movement and organisation of chromosomes, thus, evolved to facilitate the pairing process, and can be modulated by distinct stages of chromatin associations at the nuclear envelope and their collective release., Summary: We review plant meiosis, including chromosome tethering at the nuclear periphery that, we propose, defines chromosome dynamics-facilitating DNA sequence-based pairing of homologues

Mitochondrial and plastid genomes in land plants exhibit some of the slowest rates of sequence evolution observed in any eukaryotic genome, suggesting an exceptional ability to prevent or correct mutations. However, the mechanisms responsible for this extreme fidelity remain unclear. We tested seven candidate genes involved in cytoplasmic DNA replication, recombination, and repair (POLIA, POLIB, MSH1, RECA3, UNG, FPG, and OGG1) for effects on mutation rates in the model angiosperm Arabidopsis thaliana by applying a highly accurate DNA sequencing technique (duplex sequencing) that can detect newly arisen mitochondrial and plastid mutations still at low heteroplasmic frequencies. We find that disrupting MSH1 (but not the other candidate genes) leads to massive increases in the frequency of point mutations and small indels and changes to the mutation spectrum in mitochondrial and plastid DNA. We also used droplet digital PCR to show transmission of de novo heteroplasmies across generations in msh1 mutants, confirming a contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within the mutS mismatch repair family that we find is also present outside of green plants in multiple eukaryotic groups (stramenopiles, alveolates, haptophytes, and cryptomonads), as well as certain bacteria and viruses. MSH1 has previously been shown to limit ectopic recombination in plant cytoplasmic genomes. Our results point to a broader role in recognition and correction of errors in plant mitochondrial and plastid DNA sequence, leading to greatly suppressed mutation rates perhaps via initiation of double-stranded breaks and repair pathways based on faithful homologous recombination.

When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants.

Oriental theileriosis is an economically important tickborne disease of bovines, caused by some members of the Theileria orientalis complex. Currently, 11 distinct operational taxonomic units (OTUs), or genotypes, are recognized based on their major piroplasm surface protein (MPSP) gene sequences. Two of these genotypes (i.e., chitose and ikeda) are recognized as pathogenic in cattle, causing significant disease in countries of the Asia-Pacific region. However, the true extent of genetic variation and associated virulence/pathogenicity within this complex is unknown. Here, we undertook a proof-of-principle study of a small panel of genomic DNAs (n = 13) from blood samples originating from individual cattle known to harbor T. orientalis, in order to assess the performance of a targeted &rdquo, next-generation&rdquo, sequencing-informatic approach to identify genotypes. Five genotypes (chitose, ikeda, buffeli, type 4, and type 5) were defined, multiple genotypes were found within individual samples, with dominant and minor sequence types representing most genotypes. This study indicates that this sequencing-informatic workflow could be useful to assess the nature and extent of genetic variation within and among populations of T. orientalis on a large scale, and to potentially employ panels of distinct gene markers for expanded molecular epidemiological investigations of socioeconomically important protistan pathogens more generally.

In various eukaryotes, supernumerary B chromosomes (Bs) are an optional genomic component that affect their integrity and functioning. In the present study, the impact of Bs on the current changes in the genome of goatgrass, Aegilops speltoides, was addressed. Individual plants from contrasting populations with and without Bs were explored using fluorescence in situ hybridization. In parallel, abundances of the Ty1-copia, Ty3-gypsy, and LINE retrotransposons (TEs), and the species-specific Spelt1 tandem repeat (TR) in vegetative and generative spike tissues were estimated by real-time quantitative PCR. The results revealed: (i) ectopic associations between Bs and the regular A chromosomes, and (ii) cell-specific rearrangements of Bs in both mitosis and microgametogenesis. Further, the copy numbers of TEs and TR varied significantly between (iii) genotypes and (iv) different spike tissues in the same plant(s). Finally, (v) in plants with and without Bs from different populations, genomic abundances and/or copy number dynamics of TEs and TR were similar. These findings indicate that fluctuations in TE and TR copy numbers are associated with DNA damage and repair processes during cell proliferation and differentiation, and ectopic recombination is one of the mechanisms by which Bs play a role in genome changes.

In various eukaryotes, supernumerary B chromosomes (Bs) are an optional genomic component that affect their integrity and functioning. In the present study, the impact of Bs on the current changes in the genome of goatgrass, Aegilops speltoides, was addressed. Individual plants from contrasting populations with and without Bs were explored using fluorescence in situ hybridization. In parallel, abundances of the Ty1-copia, Ty3-gypsy, and LINE retrotransposons (TEs), and the species-specific Spelt1 tandem repeat (TR) in vegetative and generative spike tissues were estimated by real-time quantitative PCR. The results revealed: (i) ectopic associations between Bs and the regular A chromosomes, and (ii) cell-specific rearrangements of Bs in both mitosis and microgametogenesis. Further, the copy numbers of TEs and TR varied significantly between (iii) genotypes and (iv) different spike tissues in the same plant(s). Finally, (v) in plants with and without Bs from different populations, genomic abundances and/or copy number dynamics of TEs and TR were similar. These findings indicate that fluctuations in TE and TR copy numbers are associated with DNA damage and repair processes during cell proliferation and differentiation, and ectopic recombination is one of the mechanisms by which Bs play a role in genome changes.

Malaria remains one of the most prevalent and deadly infectious diseases of the developing world, causing approximately 228 million clinical cases and nearly half a million deaths annually. The disease is caused by protozoan parasites of the genus Plasmodium, and of the five species capable of infecting humans, infections with P. falciparum are the most severe. In addition to the parasites that infect people, there are hundreds of additional species that infect birds, reptiles, and other mammals, each exquisitely evolved to meet the specific challenges inherent to survival within their respective hosts. By comparing the unique strategies that each species has evolved, key insights into host-parasite interactions can be gained, including discoveries regarding the pathogenesis of human disease. Here, we describe the surprising observation that closely related parasites with different hosts have evolved remarkably different methods for repairing their genomes. This observation has important implications for the ability of parasites to maintain chronic infections and for the development of host immunity., The protozoan parasites that cause malaria infect a wide variety of vertebrate hosts, including birds, reptiles, and mammals, and the evolutionary pressures inherent to the host-parasite relationship have profoundly shaped the genomes of both host and parasite. Here, we report that these selective pressures have resulted in unexpected alterations to one of the most basic aspects of eukaryotic biology, the maintenance of genome integrity through DNA repair. Malaria parasites that infect humans continuously generate genetic diversity within their antigen-encoding gene families through frequent ectopic recombination between gene family members, a process that is a crucial feature of the persistence of malaria globally. The continuous generation of antigen diversity ensures that different parasite isolates are antigenically distinct, thus preventing extensive cross-reactive immunity and enabling parasites to maintain stable transmission within human populations. However, the molecular basis of the recombination between gene family members is not well understood. Through computational analyses of the antigen-encoding, multicopy gene families of different Plasmodium species, we report the unexpected observation that malaria parasites that infect rodents do not display the same degree of antigen diversity as observed in Plasmodium falciparum and appear to undergo significantly less ectopic recombination. Using comparative genomics, we also identify key molecular components of the diversification process, thus shedding new light on how malaria parasites balance the maintenance of genome integrity with the requirement for continuous genetic diversification.

Retrotransposable elements are widely distributed and diverse in eukaryotes. Their copy number increases through reverse-transcription-mediated propagation, while they can be lost through recombinational processes, generating genomic rearrangements. We previously identified extensive structurally uniform retrotransposon groups in which no member contains the gag, pol, or env internal domains. Because of the lack of protein-coding capacity, these groups are non-autonomous in replication, even if transcriptionally active. The Cassandra element belongs to the non-autonomous group called terminal-repeat retrotransposons in miniature (TRIM). It carries 5S RNA sequences with conserved RNA polymerase (pol) III promoters and terminators in its long terminal repeats (LTRs). Here, we identified multiple extended tandem arrays of Cassandra retrotransposons within different plant species, including ferns. At least 12 copies of repeated LTRs (as the tandem unit) and internal domain (as a spacer), giving a pattern that resembles the cellular 5S rRNA genes, were identified. A cytogenetic analysis revealed the specific chromosomal pattern of the Cassandra retrotransposon with prominent clustering at and around 5S rDNA loci. The secondary structure of the Cassandra retroelement RNA is predicted to form super-loops, in which the two LTRs are complementary to each other and can initiate local recombination, leading to the tandem arrays of Cassandra elements. The array structures are conserved for Cassandra retroelements of different species. We speculate that recombination events similar to those of 5S rRNA genes may explain the wide variation in Cassandra copy number. Likewise, the organization of 5S rRNA gene sequences is very variable in flowering plants, part of what is taken for 5S gene copy variation may be variation in Cassandra number. The role of the Cassandra 5S sequences remains to be established.

The nucleolus organizer regions (NORs) demonstrate differences in genomic dispersion and transcriptional activity among all organisms. I postulate that such differences stem from distinct genomic structures and their interactions from chromosome observations using fluorescence in situ hybridization and silver nitrate staining methods. Examples in primates and Australian bulldog ants indicate that chromosomal features indeed play a significant role in determining the properties of NORs. In primates, rDNA arrays that are located on the short arm of acrocentrics frequently form reciprocal associations (“affinity”), but they lack such associations (“non-affinity”) with other repeat arrays—a binary molecular effect. These “rules” of affinity vs. non-affinity are extrapolated from the chromosomal configurations of meiotic prophase. In bulldog ants, genomic dispersions of rDNA loci expand much more widely following an increase in the number of acrocentric chromosomes formed by centric fission. Affinity appears to be a significantly greater force: associations likely form among rDNA and heterochromatin arrays of acrocentrics—thus, more acrocentrics bring about more rDNA loci. The specific interactions among NOR-related genome structures remain unclear and require further investigation. Here, I propose that there are limited and non-limited genomic dispersion systems that result from genomic affinity rules, inducing specific chromosomal configurations that are related to NORs.

Retrotransposon Hot Spot (RHS) is the most abundant gene family in Trypanosoma cruzi, with unknown function in this parasite. The aim of this work was to shed light on the organization and expression of RHS in T. cruzi. The diversity of the RHS protein family in T. cruzi was demonstrated by phylogenetic and recombination analyses. Transcribed sequences carrying the RHS domain were classified into ten distinct groups of monophyletic origin. We identified numerous recombination events among the RHS and traced the origins of the donors and target sequences. The transcribed RHS genes have a mosaic structure that may contain fragments of different RHS inserted in the target sequence. About 30% of RHS sequences are located in the subtelomere, a region very susceptible to recombination. The evolution of the RHS family has been marked by many events, including gene duplication by unequal mitotic crossing-over, homologous, as well as ectopic recombination, and gene conversion. The expression of RHS was analyzed by immunofluorescence and immunoblotting using anti-RHS antibodies. RHS proteins are evenly distributed in the nuclear region of T. cruzi replicative forms (amastigote and epimastigote), suggesting that they could be involved in the control of the chromatin structure and gene expression, as has been proposed for T. brucei.

In Drosophila, large variations in rearrangement rate have been reported among different lineages and among Muller’s elements. Nevertheless, the mechanisms that are involved in the generation of inversions, their increase in frequency, as well as their impact on the genome are not completely understood. This is in part due to the lack of comparative studies on species distantly related to Drosophila melanogaster. Therefore, we sequenced and assembled the genomes of two species of the virilis phylad (Drosophila novamexicana [15010-1031.00] and Drosophila americana [SF12]), which are diverging from D. melanogaster for more than 40 Myr. Based on these data, we identified the precise location of six novel inversion breakpoints. A molecular characterization provided clear evidence that DAIBAM (a miniature inverted–repeat transposable element) was involved in the generation of eight out of the nine inversions identified. In contrast to what has been previously reported for D. melanogaster and close relatives, ectopic recombination is thus the prevalent mechanism of generating inversions in species of the virilis phylad. Using pool-sequencing data for three populations of D. americana, we also show that common polymorphic inversions create a high degree of genetic differentiation between populations for chromosomes X, 4, and 5 over large physical distances. We did not find statistically significant differences in expression levels between D. americana (SF12) and D. novamexicana (15010-1031.00) strains for the three genes surveyed (CG9588, Fig 4, and fab1) flanking three inversion breakpoints. This article is a result of the project Norte-01-0145-FEDER-000008—Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). N.P. and M.R. are funded by the Emmy Noether Programme of the Deutsche Forschungsgemeinschaft (Grant Number: PO 1648/3-1 to N.P.). We would like to thank the Transcriptome Analysis Lab (TAL) (University Medical Center Göttingen, UMG) in Göttingen for the Illumina sequencing.

Transposable elements (TEs) are genomic parasites that impose fitness costs on their hosts by producing deleterious mutations and disrupting gametogenesis. Host genomes avoid these costs by regulating TE activity, particularly in germline cells where new insertions are heritable and TEs are exceptionally active. However, the capacity of different TE-associated fitness costs to select for repression in the host, and the role of selection in the evolution of TE regulation more generally, remain controversial. In this study, we use forward, individual-based simulations to examine the evolution of small-RNA-mediated TE regulation, a conserved mechanism for TE repression that is employed by both prokaryotes and eukaryotes. To design and parameterize a biologically realistic model, we drew on an extensive survey of empirical studies of the transposition and regulation ofP-element DNA transposons inDrosophila melanogaster. We observed that even under conservative assumptions, where small-RNA-mediated regulation reduces transposition only, repression evolves rapidly and adaptively after the genome is invaded by a new TE. We further show that the spread of repressor alleles is greatly enhanced by two additional TE-imposed fitness costs: dysgenic sterility and ectopic recombination. Finally, we demonstrate that the mutation rate to repression (i.e., the size of the mutational target) is a critical parameter that influences both the evolutionary trajectory of host repression and the associated proliferation of TEs after invasion. Our findings suggest that adaptive evolution of TE regulation may be stronger and more prevalent than previously appreciated, and provide a framework for evaluating empirical data.

Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, ‘safe’ repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. Here we show that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes. Relocalization of heterochromatic double-strand breaks to the nuclear periphery in Drosophila cells occurs via directed motions driven by nuclear actin filaments and myosins activated by the Smc5/6 complex.

Transposable elements (TE) can constitute a large fraction of plant genomes, yet our understanding of their evolution and fitness effect is still limited. Here we tested several models of evolution that make specific predictions about differences in TE abundance between selfing and outcrossing taxa, and between small and large populations. We estimated TE abundance in multiple populations of North American Arabidopsis lyrata differing in mating system and long-term size, using transposon insertion display on several TE families. Selfing populations had higher TE copy numbers per individual and higher TE allele frequencies, supporting models which assume that selection against TEs acts predominantly against heterozygotes via the process of ectopic recombination. In outcrossing populations differing in long-term size, the data supported neither a model of density-regulated transposition nor a model of direct deleterious effect. Instead, the population structure of TEs revealed that outcrossing populations tended to split into western and eastern groups - as previously detected using microsatellite markers - whereas selfing populations from west and east were less differentiated. This, too, agrees with the model of ectopic recombination. Overall, our results suggest that TE elements are nearly neutral except for their deleterious potential to disturb meiosis in the heterozygous state.

The genome of Plasmodium falciparum, the causative agent of malaria in Africa, has been extensively studied since it was first fully sequenced in 2002. However, many open questions remain, including understanding the chromosomal context of molecular evolutionary changes (e.g., relationship between chromosome map and phylogenetic conservation, patterns of gene duplication, and patterns of selection). Here, we present PhyloChromoMap, a method that generates a phylogenomic map of chromosomes from a custom-built bioinformatics pipeline. Using P. falciparum 3D7 as a model, we analyze 2,116 genes with homologs in up to 941 diverse eukaryotic, bacterial and archaeal lineages. We estimate the level of conservation along chromosomes based on conservation across clades, and identify “young” regions (i.e., those with recent or fast evolving genes) that are enriched in subtelomeric regions as compared with internal regions. We also demonstrate that patterns of molecular evolution for paralogous genes differ significantly depending on their location as younger paralogs tend to be found in subtelomeric regions whereas older paralogs are enriched in internal regions. Combining these observations with analyses of synteny, we demonstrate that subtelomeric regions are actively shuffled among chromosome ends, which is consistent with the hypothesis that these regions are prone to ectopic recombination. We also assess patterns of selection by comparing dN/dS ratios of gene family members in subtelomeric versus internal regions, and we include the important antigenic gene family var. These analyses illustrate the highly dynamic nature of the karyotype of P. falciparum, and provide a method for exploring genome dynamics in other lineages.

Plant genomes contain numerous transposable elements (TEs), and many hypotheses on the evolutionary drivers that restrict TE activity have been postulated. Few models, however, have focused on the evolutionary epigenomic interaction between the plant host and its TE. The host genome recruits epigenetic factors, such as methylation, to silence TEs but methylation can spread beyond the TE sequence and influence the expression of nearby host genes. In this study, we investigated this epigenetic trade-off between TE and proximal host gene silencing by studying the epigenomic regulation of repressing long terminal repeat (LTR) retrotransposons (RTs) in Oryza sativa. Results showed significant evidence of methylation spreading originating from the LTR-RT sequences, and the extent of spreading was dependent on five factors: 1) LTR-RT family, 2) time since the LTR-RT insertion, 3) recombination rate of the LTR-RT region, 4) level of LTR-RT sequence methylation, and 5) chromosomal location. Methylation spreading had negative effects by reducing host gene expression, but only on host genes with LTR-RT inserted in its introns. Our results also suggested high levels of LTR-RT methylation might have a role in suppressing TE-mediated deleterious ectopic recombination. In the end, despite the methylation spreading, no strong epigenetic trade-off was detected and majority of LTR-RT may have only minor epigenetic effects on nearby host genes.

In the yeast Saccharomyces cerevisiae, the genes encoding the metallothionein protein Cup1 are located in a tandem array on chromosome VIII. Using a diploid strain that is heterozygous for an insertion of a selectable marker (URA3) within this tandem array, and heterozygous for markers flanking the array, we measured interhomolog recombination and intra/sister chromatid exchange in the CUP1 locus. The rate of intra/sister chromatid recombination exceeded the rate of interhomolog recombination by >10-fold. Loss of the Rad51 and Rad52 proteins, required for most interhomolog recombination, led to a relatively small reduction of recombination in the CUP1 array. Although interhomolog mitotic recombination in the CUP1 locus is elevated relative to the average genomic region, we found that interhomolog meiotic recombination in the array is reduced compared to most regions. Lastly, we showed that high levels of copper (previously shown to elevate CUP1 transcription) lead to a substantial elevation in rate of both interhomolog and intra/sister chromatid recombination in the CUP1 array; recombination events that delete the URA3 insertion from the CUP1 array occur at a rate of >10−3/division in unselected cells. This rate is almost three orders of magnitude higher than observed for mitotic recombination events involving single-copy genes. In summary, our study shows that some of the basic properties of recombination differ considerably between single-copy and tandemly-repeated genes.

Transposable elements (TEs) are virtually ubiquitous components of genomes, yet they often impose significant fitness consequences on their hosts. In addition to producing specific deleterious mutations by insertional inactivation, TEs also impose general fitness costs by inducing DNA damage and participating in ectopic recombination. These latter fitness costs are often assumed to be dosage-dependent, with stronger effects occurring in the presence of higher TE copy numbers. We test this assumption in Drosophila melanogaster by considering the relationship between the copy number of two active DNA transposons, P-element and hobo element, and the incidence of hybrid dysgenesis, a sterility syndrome associated with transposon activity in the germline. By harnessing a subset of the Drosophila Genetic Reference Panel (DGRP), a group of fully-sequenced D. melanogaster strains, we describe quantitative and structural variation in P-elements and hobo elements among wild-derived genomes and associate these factors with hybrid dysgenesis. We find that the incidence of hybrid dysgenesis is associated with both P-element and hobo element copy number in a dosage-dependent manner. However, the relationship is weak for both TEs, suggesting that dosage alone explains only a small part of TE-associated fitness costs.

Repairing double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination and chromosome rearrangements. Recent studies in Drosophila cells revealed that faithful homologous recombination repair of heterochromatic DSBs relies on the relocalization of DSBs to the nuclear periphery before Rad51 recruitment. Here we summarize the exciting progress in understanding this pathway, including conserved responses in mammalian cells and surprising similarities with mechanisms available in yeast to deal with DSBs in other sites that are difficult to repair, including other repeated sequences. We will also point out some of the most important open questions in the field and emerging evidence suggesting that deregulating these pathways might have dramatic consequences for human health.

Meiosis is a specialized cell division, essential in most reproducing organisms to halve the number of chromosomes, thereby enabling the restoration of ploidy levels during fertilization. A key step of meiosis is homologous recombination, which promotes homologous pairing and generates crossovers (COs) to connect homologous chromosomes until their separation at anaphase I. These CO sites, seen cytologically as chiasmata, represent a reciprocal exchange of genetic information between two homologous nonsister chromatids. This gene reshuffling during meiosis has a significant influence on evolution and also plays an essential role in plant breeding, because a successful breeding program depends on the ability to bring the desired combinations of alleles on chromosomes. However, the number and distribution of COs during meiosis is highly constrained. There is at least one CO per chromosome pair to ensure accurate segregation of homologs, but in most organisms, the CO number rarely exceeds three regardless of chromosome size. Moreover, their positions are not random on chromosomes but exhibit regional preference. Thus, genes in recombination-poor regions tend to be inherited together, hindering the generation of novel allelic combinations that could be exploited by breeding programs. Recently, much progress has been made in understanding meiotic recombination. In particular, many genes involved in the process in Arabidopsis (Arabidopsis thaliana) have been identified and analyzed. With the coming challenges of food security and climate change, and our enhanced knowledge of how COs are formed, the interest and needs in manipulating CO formation are greater than ever before. In this review, we focus on advances in understanding meiotic recombination and then summarize the attempts to manipulate CO formation. Last, we pay special attention to the meiotic recombination in polyploidy, which is a common genomic feature for many crop plants.

Homologous chromosomes exchange genetic information through recombination during meiosis, a process that increases genetic diversity, and is fundamental to sexual reproduction. In an attempt to shed light on the dynamics of mammalian recombination and its implications for genome organization, we have studied the recombination characteristics of 112 individuals belonging to 28 different species in the family Bovidae. In particular, we analyzed the distribution of RAD51 and MLH1 foci during the meiotic prophase I that serve, respectively, as proxies for double-strand breaks (DSBs) which form in early stages of meiosis and for crossovers. In addition, synaptonemal complex length and meiotic DNA loop size were estimated to explore how genome organization determines DSBs and crossover patterns. We show that although the number of meiotic DSBs per cell and recombination rates observed vary between individuals of the same species, these are correlated with diploid number as well as with synaptonemal complex and DNA loop sizes. Our results illustrate that genome packaging, DSB frequencies, and crossover rates tend to be correlated, while meiotic chromosomal axis length and DNA loop size are inversely correlated in mammals. Moreover, axis length, DSB frequency, and crossover frequencies all covary, suggesting that these correlations are established in the early stages of meiosis.

During meiosis, homologous recombination repairs programmed DNA double-stranded breaks. Meiotic recombination physically links the homologous chromosomes (“homologs”), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1’s activity is regulated by its accessory factors including the heterodimeric protein Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister joint molecule intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1. Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filament lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements., Author summary During meiosis, two rounds of division follow a single round of DNA replication to create the gametes for biparental reproduction. The first round of division requires that the homologous chromosomes become physically linked to one another to create the tension that is necessary for their segregation. This linkage is achieved through DNA recombination between the two homologous chromosomes, followed by resolution of the recombination intermediate into a crossover. Central to this process is the meiosis-specific recombinase Dmc1, and its accessory factors, which provide important regulatory functions to ensure that recombination is accurate, efficient, and occurs predominantly between homologous chromosomes, and not sister chromatids. To gain insight into the regulation of Dmc1 by its accessory factors, we mutated Dmc1 such that it was no longer dependent on its accessory factor Mei5-Sae3. Our analysis reveals that Dmc1 accessory factors Mei5-Sae3 and Rad51 have independent roles in stabilizing Dmc1 filaments. Furthermore, we find that although Rad51 is required for promoting recombination between homologous chromosomes, Mei5-Sae3 is not. Lastly, we show that our Dmc1 mutant forms abnormally long filaments, and high levels of aberrant recombination intermediates and products. These findings suggest that filaments are actively maintained at short lengths to prevent deleterious genome rearrangements.

LTR retrotransposons constitute a significant part of plant genomes and their evolutionary dynamics play an important role in genome size changes. Current methods of LTR retrotransposon age estimation are based only on LTR (long terminal repeat) divergence. This has prompted us to analyze sequence similarity of LTRs in 25,144 LTR retrotransposons from fifteen plant species as well as formation of solo LTRs. We found that approximately one fourth of nested retrotransposons showed a higher LTR divergence than the pre-existing retrotransposons into which they had been inserted. Moreover, LTR similarity was correlated with LTR length. We propose that gene conversion can contribute to this phenomenon. Gene conversion prediction in LTRs showed potential converted regions in 25% of LTR pairs. Gene conversion was higher in species with smaller genomes while the proportion of solo LTRs did not change with genome size in analyzed species. The negative correlation between the extent of gene conversion and the abundance of solo LTRs suggests interference between gene conversion and ectopic recombination. Since such phenomena limit the traditional methods of LTR retrotransposon age estimation, we recommend an improved approach based on the exclusion of regions affected by gene conversion.

Expansions of CNG microsatellite tracts are responsible for several neurodegenerative diseases, including myotonic dystrophy type 1, Huntington disease, and spinocerebellar ataxia type 8. Here we show that expanded (CNG)n repeats are susceptible not only to expansions and contractions, but are prone to DNA double strand breaks following replication stress. We describe a general strategy for the construction of clonal cell lines containing CNG repeats of various lengths, in which the microsatellites are integrated using the yeast FLP recombinase at a single ectopic recombination acceptor site in the HeLa genome. We illustrate two types of (CTG/CAG) cell lines, one of which contains dual fluorescent marker genes flanking the (CTG/CAG) repeat, and one which does not. We show that long CNG repeats are prone to DNA double strand breaks (DSBs) upon exposure of these cell lines to prolonged replication stress.

Background Large palindromes (inverted repeats) make up substantial proportions of mammalian sex chromosomes, often contain genes, and have high rates of structural variation arising via ectopic recombination. As a result, they underlie many genomic disorders. Maintenance of the palindromic structure by gene conversion between the arms has been documented, but over longer time periods, palindromes are remarkably labile. Mechanisms of origin and loss of palindromes have, however, received little attention. Results Here, we use fiber-FISH, 10x Genomics Linked-Read sequencing, and breakpoint PCR sequencing to characterize the structural variation of the P8 palindrome on the human Y chromosome, which contains two copies of the VCY (Variable Charge Y) gene. We find a deletion of almost an entire arm of the palindrome, leading to death of the palindrome, a size increase by recruitment of adjacent sequence, and other complex changes including the formation of an entire new palindrome nearby. Together, these changes are found in ~ 1% of men, and we can assign likely molecular mechanisms to these mutational events. As a result, healthy men can have 1–4 copies of VCY. Conclusions Gross changes, especially duplications, in palindrome structure can be relatively frequent and facilitate the evolution of sex chromosomes in humans, and potentially also in other mammalian species.

Speciation is a central mechanism of biological diversification. While speciation is well studied in plants and animals, in comparison, relatively little is known about speciation in fungi. One fungal model is the Cryptococcus genus, which is best known for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex that causes over 200,000 new infections in humans annually. The closest non-human pathogenic relatives are the sibling species, Cryptococcus amylolentus and Tsuchiyaea wingfieldii. However, because relatively few isolates of each species are available, it is unclear whether they represent divergent lineages of the same species or different biological species. The recent isolation of an additional strain, preliminarily identified as T. wingfieldii, prompted us to reexamine this group as it may inform about the evolutionary processes underlying the diversification of both non-pathogenic and pathogenic Cryptococcus lineages. Using genomic data, we reappraised the phylogenetic relationship of the four available strains and confirmed the genetic separation of C. amylolentus and T. wingfieldii (now Cryptococcus wingfieldii), and revealed an additional cryptic species, for which the name Cryptococcus floricola is proposed. Comparison of full-length chromosome assemblies revealed approximately 6% pairwise sequence divergence between the three species, and identified significant genomic changes, including inversions as well as a reciprocal translocation that involved inter-centromeric ectopic recombination, which together likely impose significant barriers to genetic exchange. Using genetic crosses, we show that while C. wingfieldii cannot interbreed with any of the other strains, C. floricola can undergo sexual reproduction with C. amylolentus. However, most of the spores resulting from this cross were inviable, and many were sterile, indicating that the two species are genetically isolated through intrinsic post-zygotic barriers and possibly due to niche differentiation. Genome sequencing and analysis of the progeny demonstrated decreased recombination frequency during meiosis in heterospecific crosses compared to C. amylolentus conspecific crosses. This study advances our understanding of speciation in fungi and highlights the power of genomics in assisting our ability to correctly identify and discriminate fungal species.Author SummaryThe idea of species as discrete natural units seems rather intuitive for most people, just as cells are the basic units of life. However, when observing variation across a species range, boundaries can become blurred making it less than obvious when different populations evolve into separate species. Additionally, separate species can still interbreed, such as lions breeding with tigers to produce a liger or a tigon (depending on the paternal and maternal species of origin), but the resulting offspring is usually inviable or sterile, which in turn is evidence that the parents involved are distinct species. Therefore, what species are and how they originate is still an open question in evolutionary biology. While recent advances have been made in the fields of animal and plant speciation, many other important components of biological diversity, such as fungi, are still understudied. Genome sequencing is now providing new tools to address the genetic mechanisms that drive divergence and reproductive isolation between populations, including genetic incompatibilities, sequence divergence, and chromosomal rearrangements. Here we focus on the Cryptococcus amylolentus species complex, a non-pathogenic fungal lineage closely related to the human pathogenic Cryptococcus neoformans/Cryptococcus gattii complex. Using genetic and genomic analysis we reexamined the species boundaries of four available isolates within the C. amylolentus complex and revealed three genetically isolated species. The genomes of these species are ~6% divergent and exhibit chromosome rearrangements, including translocations and small-scale inversions. Although two of the species (C. amylolentus and newly described C. floricola) are still able to interbreed, the resulting hybrid progeny were mostly inviable, and many were sterile, indicating that barriers to reproduction have already been established. Our results will foster additional studies addressing the transitions between non-pathogenic and pathogenic Cryptococcus lineages.

Repairing DNA double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. InDrosophilaKc cells, accurate homologous recombination (HR) repair of heterochromatic DSBs relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins’ ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic DSBs in mouse cells, and their defects lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. InDrosophilapolytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic DSBs relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response in this structure. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types.Impact StatementHeterochromatin relies on dedicated pathways for ‘safe’ recombinational repair. In mouse and fly cultured cells, DNA repair requires the movement of repair sites away from the heterochromatin ‘domain’vianuclear actin filaments and myosins. Here, we explore the importance of these pathways inDrosophilasalivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary for repair and stability of heterochromatic sequences. This deepens our understanding of repair mechanisms in polytenes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage or rDNA breaks, providing a new understanding of the importance of the nucleoskeleton in genome stability. We expect these discoveries to shed light on tumorigenic processes, including therapy-induced cancer relapses.

Crossover (CO) recombination is essential for chromosome segregation during meiosis I. The number and distribution of COs are tightly regulated during meiosis. CO control includes CO assurance and CO interference, which guarantee at least one CO per a bivalent and evenly-spaced CO distribution, respectively. Previous studies showed the role of DNA damage response (DDR) clamp and its loader in efficient formation of meiotic COs by promoting the recruitment of a pro-CO protein Zip3 and interhomolog recombination, and also by suppressing ectopic recombination. In this study, by classical tetrad analysis ofSaccharomyces cerevisiae, we showed that a mutant defective in theRAD24 gene(RAD17in other organisms), which encodes the DDR clamp loader, displayed reduced CO frequencies on two shorter chromosomes (IIIandV) but not on a long chromosome (chromosomeVII). The residual COs in therad24mutant do not show interference. In contrast to therad24mutant, mutants defective in the ATR kinase homolog Mec1/Esr1, including amec1null and amec1kinase-dead mutant, show little or no defect in CO frequency. On the other hand,mec1COs show defects in interference, similar to therad24mutant. Moreover, CO formation and its control are implemented in a chromosome-specific manner, which may reflect a role for chromosome size in regulation.

To better understand the structure and variability of the 45S rDNA cistron and its evolutionary dynamics in grasshoppers, we performed a detailed analysis combining classical and molecular cytogenetic data with whole-genome sequencing in Abracris flavolienata, which shows extraordinary variability in the chromosomal distribution for this element. We found astonishing variability in the number and size of rDNA clusters at intra- and inter-population levels. Interestingly, FISH using distinct parts of 45S rDNA cistron (18S rDNA, 28S rDNA, and ITS1) as probes revealed a distinct number of clusters, suggesting independent mobility and amplification of the 45S rDNA components. This hypothesis is consistent with the higher genomic coverage of almost the entire cistron of 45S rDNA observed in A. flavolineata compared to other grasshoppers, besides coverage variability along the 45S rDNA cistron in the species. In addition, these differences in coverage for distinct components of the 45S rDNA cistron indicate emergence of pseudogenes evidenced by existence of truncated sequences, demonstrating the rDNA dynamics in the species. Although the chromosomal distribution of 18S rDNA was highly variable, the chromosomes 1, 3, 6, and 9 harbored rDNA clusters in all individuals with the occurrence of NOR activity in pair 9, suggesting ancestry or selective pressures to prevent pseudogenization of rDNA sequences in this chromosome pair. Additionally, small NORs and cryptic rDNA loci were observed. Finally, there was no evidence of enrichment and association of transposable elements, at least, inside or nearby rDNA cistron. These findings broaden our knowledge of rDNA dynamics, revealing an independent movement and amplification of segments of 45S rDNA cistron, which in A. flavolineata could be attributed to ectopic recombination. Fil: Ferretti, Ana B. S. M.. Universidade Estadual Paulista Julio de Mesquita Filho. Faculdade de Engenharia.; Brasil Fil: Ruiz Ruano, Francisco J.. Uppsala University; Suecia. Universidad de Granada; España Fil: Milani, Diogo. Universidade Estadual Paulista Julio de Mesquita Filho. Faculdade de Engenharia.; Brasil Fil: Loreto, Vilma. Universidade Federal de Pernambuco; Brasil Fil: Marti, Dardo Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Ramos, Érica Daniela. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Martins, Cesar. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Cabral de Mello, Diogo C.. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil

African great apes have large constitutive heterochromatin (C-band) blocks in subtelomeric regions of the majority of their chromosomes, but humans lack these. Additionally, the chimpanzee meiotic cell division process demonstrates unique partial terminal associations in the first meiotic prophase (pachytene). These are likely formed as a result of interaction among subtelomeric C-band blocks. We thus conducted an extensive study to define the features in the subtelomeric heterochromatic regions of chimpanzee chromosomes undergoing mitotic metaphase and meiotic cell division. Molecular cytogenetic analyses with probes of both subterminal satellite DNA (a main component of C-band) and rDNA demonstrated principles of interaction among DNA arrays. The results suggest that homologous and ectopic recombination through persistent subtelomeric associations (post-bouquet association observed in 32% of spermatocytes in the pachytene stage) appears to create variability in heterochromatin patterns and simultaneously restrain subtelomeric genome polymorphisms. That is, the meeting of non-homologous chromosome termini sets the stage for ectopic pairing which, in turn, is the mechanism for generating variability and genomic dispersion of subtelomeric C-band blocks through a system of concerted evolution. Comparison between the present study and previous reports indicated that the chromosomal distribution rate of sutelomeric regions seems to have antagonistic correlation with arm numbers holding subterminal satellite blocks in humans, chimpanzees, and gorillas. That is, the increase of subterminal satellite blocks probably reduces genomic diversity in the subtelomeric regions. The acquisition vs. loss of the subtelomeric C-band blocks is postulated as the underlying engine of this chromosomal differentiation yielded by meiotic chromosomal interaction.

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.

Inducible cyclization recombinase (Cre) transgenic mouse strains are powerful tools for cell lineage tracing and tissue-specific knockout experiments. However, low efficiency or leaky expression can be important pitfalls. Here, we compared the efficiency and specificity of two commonly used cholangiocyte-specific Cre drivers, the Opn-iCreERT2 and Ck19-CreERT drivers, using a tdTomato reporter strain. We found that Opn-iCreERT2 triggered recombination of the tdTomato reporter in 99.9% of all cholangiocytes while Ck19-CreERT only had 32% recombination efficiency after tamoxifen injection. In the absence of tamoxifen, recombination was also induced in 2% of cholangiocytes for the Opn-iCreERT2 driver and in 13% for the Ck19-CreERT driver. For both drivers, Cre recombination was highly specific for cholangiocytes since recombination was rare in other liver cell types. Toxic liver injury ectopically activated Opn-iCreERT2 but not Ck19-CreERT expression in hepatocytes. However, ectopic recombination in hepatocytes could be avoided by applying a three-day long wash-out period between tamoxifen treatment and toxin injection. Therefore, the Opn-iCreERT2 driver is best suited for the generation of mutant bile ducts, while the Ck19-CreERT driver has near absolute specificity for bile duct cells and is therefore favorable for lineage tracing experiments.

The accuracy of most DNA processes depends on chromatin integrity and dynamics. Our analyses in the yeast Saccharomyces cerevisiae show that an absence of Swr1 (the catalytic and scaffold subunit of the chromatin-remodeling complex SWR) leads to the formation of long-duration Rad52, but not RPA, foci and to an increase in intramolecular recombination. These phenotypes are further increased by MMS, zeocin, and ionizing radiation, but not by double-strand breaks, HU, or transcription/replication collisions, suggesting that they are associated with specific DNA lesions. Importantly, these phenotypes can be specifically suppressed by mutations in: (1) chromatin-anchorage internal nuclear membrane components (mps3D75-150 and src1D); (2) actin and actin regulators (act1-157, act1-159, crn1D, and cdc42-6); or (3) the SWR subunit Swc5 and the SWR substrate Htz1. However, they are not suppressed by global disruption of actin filaments or by the absence of Csm4 (a component of the external nuclear membrane that forms a bridging complex with Mps3, thus connecting the actin cytoskeleton with chromatin). Moreover, swr1D-induced Rad52 foci and intramolecular recombination are not associated with tethering recombinogenic DNA lesions to the nuclear periphery. In conclusion, the absence of Swr1 impairs efficient recombinational repair of specific DNA lesions by mechanisms that are influenced by SWR subunits, including actin, and nuclear envelope components. We suggest that these recombinational phenotypes might be associated with a pathological effect on homologous recombination of actin-containing complexes., M.M.-P. was recipient of a predoctoral training grant (FPU; Formación de Personal Universitario) from the Spanish government. Research was funded by grants to F.P. from the Spanish government (BFU2012-38171 and BFU2015-63689-P).

The Plasmodium falciparum var multigene family encodes the cytoadhesive, variant antigen PfEMP1. P. falciparum antigenic variation and cytoadhesion specificity are controlled by epigenetic switching between the single, or few, simultaneously expressed var genes. Most var genes are maintained in perinuclear clusters of heterochromatic telomeres. The active var gene(s) occupy a single, perinuclear var expression site. It is unresolved whether the var expression site forms in situ at a telomeric cluster or whether it is an extant compartment to which single chromosomes travel, thus controlling var switching. Here we show that transcription of a var gene did not require decreased colocalisation with clusters of telomeres, supporting var expression site formation in situ. However following recombination within adjacent subtelomeric sequences, the same var gene was persistently activated and did colocalise less with telomeric clusters. Thus, participation in stable, heterochromatic, telomere clusters and var switching are independent but are both affected by subtelomeric sequences. The var expression site colocalised with the euchromatic mark H3K27ac to a greater extent than it did with heterochromatic H3K9me3. H3K27ac was enriched within the active var gene promoter even when the var gene was transiently repressed in mature parasites and thus H3K27ac may contribute to var gene epigenetic memory.

1022 I. 1022 II. 1023 III. 1023 IV. 1025 V. 1026 1027 References 1027 SUMMARY: Meiosis is fundamental to sexual reproduction and creates genetic variation in progeny. During meiosis paired homologous chromosomes undergo recombination, which can result in reciprocal crossovers. This process can recombine independently arising mutations onto the same chromosome. Recombination locations are highly variable between meioses, although total crossover numbers are tightly regulated. In addition to the effect of meiosis on genetic variation, sequence polymorphisms between homologous chromosomes can feedback onto the recombination pathways. Here we review the major crossover pathways in plants and some of the known homeostatic mechanisms that act during meiotic recombination. We then examine how sequence polymorphisms between homologous chromosomes, that is, heterozygosity, can influence meiotic recombination pathways in cis and trans. Finally, we provide a brief perspective on the relevance of these interconnections for natural selection and adaptation in plants.

Meiotic recombination in mammals has been shown to largely cluster into hotspots, which are targeted by the chromatin modifier PRDM9. The canid family, including wolves and dogs, has undergone a series of disrupting mutations in this gene, rendering PRDM9 inactive. Given the importance of PRDM9, it is of great interest to learn how its absence in the dog genome affects patterns of recombination placement. We have used genotypes from domestic dog pedigrees to generate sex-specific genetic maps of recombination in this species. On a broad scale, we find that placement of recombination events in dogs is consistent with that in mice and apes, in that the majority of recombination occurs toward the telomeres in males, while female crossing over is more frequent and evenly spread along chromosomes. It has been previously suggested that dog recombination is more uniform in distribution than that of humans; however, we found that recombination in dogs is less uniform than in humans. We examined the distribution of recombination within the genome, and found that recombination is elevated immediately upstream of the transcription start site and around CpG islands, in agreement with previous studies, but that this effect is stronger in male dogs. We also found evidence for positive crossover interference influencing the spacing between recombination events in dogs, as has been observed in other species including humans and mice. Overall our data suggests that dogs have similar broad scale properties of recombination to humans, while fine scale recombination is similar to other species lacking PRDM9.