Mechanistic Insight into Crossing over during Mouse Meiosis.

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2-/- mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution.

Crossover Interference: Shedding Light on the Evolution of Recombination.

Through recombination, genes are freed to evolve more independently of one another, unleashing genetic variance hidden in the linkage disequilibrium that accumulates through selection combined with drift. Yet crossover numbers are evolutionarily constrained, with at least one and not many more than one crossover per bivalent in most taxa. Crossover interference, whereby a crossover reduces the probability of a neighboring crossover, contributes to this homogeneity. The mechanisms by which interference is achieved and crossovers are regulated are a major current subject of inquiry, facilitated by novel methods to visualize crossovers and to pinpoint recombination events. Here, we review patterns of crossover interference and the models built to describe this process. We then discuss the selective forces that have likely shaped interference and the regulation of crossover numbers.

Thermodynamic crossovers in supercritical fluids.

Can liquid-like and gas-like states be distinguished beyond the critical point, where the liquid-gas phase transition no longer exists and conventionally only a single supercritical fluid phase is defined? Recent experiments and simulations report strong evidence of dynamical crossovers above the critical temperature and pressure. Despite using different criteria, many existing theoretical explanations consider a single crossover line separating liquid-like and gas-like states in the supercritical fluid phase. We argue that such a single-line scenario is inconsistent with the supercritical behavior of the Ising model, which has two crossover lines due to its symmetry, violating the universality principle of critical phenomena. To reconcile the inconsistency, we define two thermodynamic crossover lines in supercritical fluids as boundaries of liquid-like, indistinguishable, and gas-like states. Near the critical point, the two crossover lines follow critical scalings with exponents of the Ising universality class, supported by calculations of theoretical models and analyses of experimental data from the standard database. The upper line agrees with crossovers independently estimated from the inelastic X-ray scattering data of supercritical argon, and from the small-angle neutron scattering data of supercritical carbon dioxide. The lower line is verified by the equation of states for the compressibility factor. This work provides a fundamental framework for understanding supercritical physics in general phase transitions.

Diversity in Recombination Hotspot Characteristics and Gene Structure Shape Fine-Scale Recombination Patterns in Plant Genomes.

During the meiosis of many eukaryote species, crossovers tend to occur within narrow regions called recombination hotspots. In plants, it is generally thought that gene regulatory sequences, especially promoters and 5' to 3' untranslated regions, are enriched in hotspots, but this has been characterized in a handful of species only. We also lack a clear description of fine-scale variation in recombination rates within genic regions and little is known about hotspot position and intensity in plants. To address this question, we constructed fine-scale recombination maps from genetic polymorphism data and inferred recombination hotspots in 11 plant species. We detected gradients of recombination in genic regions in most species, yet gradients varied in intensity and shape depending on specific hotspot locations and gene structure. To further characterize recombination gradients, we decomposed them according to gene structure by rank and number of exons. We generalized the previously observed pattern that recombination hotspots are organized around the boundaries of coding sequences, especially 5' promoters. However, our results also provided new insight into the relative importance of the 3' end of genes in some species and the possible location of hotspots away from genic regions in some species. Variation among species seemed driven more by hotspot location among and within genes than by differences in size or intensity among species. Our results shed light on the variation in recombination rates at a very fine scale, revealing the diversity and complexity of genic recombination gradients emerging from the interaction between hotspot location and gene structure.

Multiple Genomic Landscapes of Recombination and Genomic Divergence in Wild Populations of House Mice-The Role of Chromosomal Fusions and Prdm9.

Chromosomal fusions represent one of the most common types of chromosomal rearrangements found in nature. Yet, their role in shaping the genomic landscape of recombination and hence genome evolution remains largely unexplored. Here, we take advantage of wild mice populations with chromosomal fusions to evaluate the effect of this type of structural variant on genomic landscapes of recombination and divergence. To this aim, we combined cytological analysis of meiotic crossovers in primary spermatocytes with inferred analysis of recombination rates based on linkage disequilibrium using single nucleotide polymorphisms. Our results suggest the presence of a combined effect of Robertsonian fusions and Prdm9 allelic background, a gene involved in the formation of meiotic double strand breaks and postzygotic reproductive isolation, in reshaping genomic landscapes of recombination. We detected a chromosomal redistribution of meiotic recombination toward telomeric regions in metacentric chromosomes in mice with Robertsonian fusions when compared to nonfused mice. This repatterning was accompanied by increased levels of crossover interference and reduced levels of estimated recombination rates between populations, together with high levels of genomic divergence. Interestingly, we detected that Prdm9 allelic background was a major determinant of recombination rates at the population level, whereas Robertsonian fusions showed limited effects, restricted to centromeric regions of fused chromosomes. Altogether, our results provide new insights into the effect of Robertsonian fusions and Prdm9 background on meiotic recombination.

Understanding the Genetic Basis of Variation in Meiotic Recombination: Past, Present, and Future.

Meiotic recombination is a fundamental feature of sexually reproducing species. It is often required for proper chromosome segregation and plays important role in adaptation and the maintenance of genetic diversity. The molecular mechanisms of recombination are remarkably conserved across eukaryotes, yet meiotic genes and proteins show substantial variation in their sequence and function, even between closely related species. Furthermore, the rate and distribution of recombination shows a huge diversity within and between chromosomes, individuals, sexes, populations, and species. This variation has implications for many molecular and evolutionary processes, yet how and why this diversity has evolved is not well understood. A key step in understanding trait evolution is to determine its genetic basis-that is, the number, effect sizes, and distribution of loci underpinning variation. In this perspective, I discuss past and current knowledge on the genetic basis of variation in recombination rate and distribution, explore its evolutionary implications, and present open questions for future research.

Non-homologous chromosome pairing: sequence similarity or genetic control?

Polyploids must correctly segregate homologous chromosomes. We propose that this process is dictated not just by sequence similarity, but is also under strong genetic control that may vary between lineages. We also highlight how factors like partner availability and genome structure may influence sequence similarity needed for crossover formation.

FIGL1 prevents aberrant chromosome associations and fragmentation and limits crossovers in polyploid wheat meiosis.

Meiotic crossovers (COs) generate genetic diversity and are crucial for viable gamete production. Plant COs are typically limited to 1-3 per chromosome pair, constraining the development of improved varieties, which in wheat is exacerbated by an extreme distal localisation bias. Advances in wheat genomics and related technologies provide new opportunities to investigate, and possibly modify, recombination in this important crop species. Here, we investigate the disruption of FIGL1 in tetraploid and hexaploid wheat as a potential strategy for modifying CO frequency/position. We analysed figl1 mutants and virus-induced gene silencing lines cytogenetically. Genetic mapping was performed in the hexaploid. FIGL1 prevents abnormal meiotic chromosome associations/fragmentation in both ploidies. It suppresses class II COs in the tetraploid such that CO/chiasma frequency increased 2.1-fold in a figl1 msh5 quadruple mutant compared with a msh5 double mutant. It does not appear to affect class I COs based on HEI10 foci counts in a hexaploid figl1 triple mutant. Genetic mapping in the triple mutant suggested no significant overall increase in total recombination across examined intervals but revealed large increases in specific individual intervals. Notably, the tetraploid figl1 double mutant was sterile but the hexaploid triple mutant was moderately fertile, indicating potential utility for wheat breeding.

ATRX and RECQ5 define distinct homologous recombination subpathways.

Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

HIM-17 regulates the position of recombination events and GSP-1/2 localization to establish short arm identity on bivalents in meiosis.

The position of recombination events established along chromosomes in early prophase I and the chromosome remodeling that takes place in late prophase I are intrinsically linked steps of meiosis that need to be tightly regulated to ensure accurate chromosome segregation and haploid gamete formation. Here, we show that RAD-51 foci, which form at the sites of programmed meiotic DNA double-strand breaks (DSBs), exhibit a biased distribution toward off-centered positions along the chromosomes in wild-type Caenorhabditis elegans, and we identify two meiotic roles for chromatin-associated protein HIM-17 that ensure normal chromosome remodeling in late prophase I. During early prophase I, HIM-17 regulates the distribution of DSB-dependent RAD-51 foci and crossovers on chromosomes, which is critical for the formation of distinct chromosome subdomains (short and long arms of the bivalents) later during chromosome remodeling. During late prophase I, HIM-17 promotes the normal expression and localization of protein phosphatases GSP-1/2 to the surface of the bivalent chromosomes and may promote GSP-1 phosphorylation, thereby antagonizing Aurora B kinase AIR-2 loading on the long arms and preventing premature loss of sister chromatid cohesion. We propose that HIM-17 plays distinct roles at different stages during meiotic progression that converge to promote normal chromosome remodeling and accurate chromosome segregation.

Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation.

Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1-Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ-Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.

Pericentromeric chromatin reorganisation follows the initiation of recombination and coincides with early events of synapsis in cereals.

The reciprocal exchange of genetic information between homologous chromosomes during meiotic recombination is essential to secure balanced chromosome segregation and to promote genetic diversity. The chromosomal position and frequency of reciprocal genetic exchange shapes the efficiency of breeding programmes and influences crop improvement under a changing climate. In large genome cereals, such as wheat and barley, crossovers are consistently restricted to subtelomeric chromosomal regions, thus preventing favourable allele combinations being formed within a considerable proportion of the genome, including interstitial and pericentromeric chromatin. Understanding the key elements driving crossover designation is therefore essential to broaden the regions available for crossovers. Here, we followed early meiotic chromatin dynamism in cereals through the visualisation of a homologous barley chromosome arm pair stably transferred into the wheat genetic background. By capturing the dynamics of a single chromosome arm at the same time as detecting the undergoing events of meiotic recombination and synapsis, we showed that subtelomeric chromatin of homologues synchronously transitions to an open chromatin structure during recombination initiation. By contrast, pericentromeric and interstitial regions preserved their closed chromatin organisation and become unpackaged only later, concomitant with initiation of recombinatorial repair and the initial assembly of the synaptonemal complex. Our results raise the possibility that the closed pericentromeric chromatin structure in cereals may influence the fate decision during recombination initiation, as well as the spatial development of synapsis, and may also explain the suppression of crossover events in the proximity of the centromeres.

The Cdk1/Cdk2 homolog CDKA;1 controls the recombination landscape in Arabidopsis.

Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in ArabidopsisArabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner.

The Theory and Applications of Measuring Broad-Range and Chromosome-Wide Recombination Rate from Allele Frequency Decay around a Selected Locus.

Recombination is the exchange of genetic material between homologous chromosomes via physical crossovers. High-throughput sequencing approaches detect crossovers genome wide to produce recombination rate maps but are difficult to scale as they require large numbers of recombinants individually sequenced. We present a simple and scalable pooled-sequencing approach to experimentally infer near chromosome-wide recombination rates by taking advantage of non-Mendelian allele frequency generated from a fitness differential at a locus under selection. As more crossovers decouple the selected locus from distal loci, the distorted allele frequency attenuates distally toward Mendelian and can be used to estimate the genetic distance. Here, we use marker selection to generate distorted allele frequency and theoretically derive the mathematical relationships between allele frequency attenuation, genetic distance, and recombination rate in marker-selected pools. We implemented nonlinear curve-fitting methods that robustly estimate the allele frequency decay from batch sequencing of pooled individuals and derive chromosome-wide genetic distance and recombination rates. Empirically, we show that marker-selected pools closely recapitulate genetic distances inferred from scoring recombinants. Using this method, we generated novel recombination rate maps of three wild-derived strains of Drosophila melanogaster, which strongly correlate with previous measurements. Moreover, we show that this approach can be extended to estimate chromosome-wide crossover interference with reciprocal marker selection and discuss how it can be applied in the absence of visible markers. Altogether, we find that our method is a simple and cost-effective approach to generate chromosome-wide recombination rate maps requiring only one or two libraries.

Crossover Interference, Crossover Maturation, and Human Aneuploidy.

A striking feature of human female sexual reproduction is the high level of gametes that exhibit an aberrant number of chromosomes (aneuploidy). A high baseline observed in women of prime reproductive age is followed by a dramatic increase in older women. Proper chromosome segregation requires one or more DNA crossovers (COs) between homologous maternal and paternal chromosomes, in combination with cohesion between sister chromatid arms. In human females, CO designations occur normally, according to the dictates of CO interference, giving early CO-fated intermediates. However, ≈25% of these intermediates fail to mature to final CO products. This effect explains the high baseline of aneuploidy and is predicted to synergize with age-dependent cohesion loss to explain the maternal age effect. Here, modern advances in the understanding of crossing over and CO interference are reviewed, the implications of human female CO maturation inefficiency are further discussed, and areas of interest for future studies are suggested.

Unleashing meiotic crossovers in hybrid plants.

Meiotic crossovers shuffle parental genetic information, providing novel combinations of alleles on which natural or artificial selection can act. However, crossover events are relatively rare, typically one to three exchange points per chromosome pair. Recent work has identified three pathways limiting meiotic crossovers in Arabidopsis thaliana that rely on the activity of FANCM [Crismani W, et al. (2012) Science 336:1588-1590], RECQ4 [Séguéla-Arnaud M, et al. (2015) Proc Natl Acad Sci USA 112:4713-4718], and FIGL1 [Girard C, et al. (2015) PLoS Genet 11:e1005369]. Here we analyzed recombination in plants in which one, two, or all three of these pathways were disrupted in both pure line and hybrid contexts. The greatest effect was observed when combining recq4 and figl1 mutations, which increased the hybrid genetic map length from 389 to 3,037 cM. This corresponds to an unprecedented 7.8-fold increase in crossover frequency. Disrupting the three pathways did not further increase recombination, suggesting that some upper limit had been reached. The increase in crossovers is not uniform along chromosomes and rises from centromere to telomere. Finally, although in wild type recombination is much higher in male meiosis than in female meiosis (490 cM vs. 290 cM), female recombination is higher than male recombination in recq4 figl1 (3,200 cM vs. 2,720 cM), suggesting that the factors that make wild-type female meiosis less recombinogenic than male wild-type meiosis do not apply in the mutant context. The massive increase in recombination observed in recq4 figl1 hybrids opens the possibility of manipulating recombination to enhance plant breeding efficiency.

Multidimensional Mortality Selection: Why Individual Dimensions of Frailty Don't Act Like Frailty.

Theoretical models of mortality selection have great utility in explaining otherwise puzzling phenomena. The most famous example may be the Black-White mortality crossover: at old ages, Blacks outlive Whites, presumably because few frail Blacks survive to old ages while some frail Whites do. Yet theoretical models of unidimensional heterogeneity, or frailty, do not speak to the most common empirical situation for mortality researchers: the case in which some important population heterogeneity is observed and some is not. I show that, when one dimension of heterogeneity is observed and another is unobserved, neither the observed nor the unobserved dimension need behave as classic frailty models predict. For example, in a multidimensional model, mortality selection can increase the proportion of survivors who are disadvantaged, or "frail," and can lead Black survivors to be more frail than Whites, along some dimensions of disadvantage. Transferring theoretical results about unidimensional heterogeneity to settings with both observed and unobserved heterogeneity produces misleading inferences about mortality disparities. The unusually flexible behavior of individual dimensions of multidimensional heterogeneity creates previously unrecognized challenges for empirically testing selection models of disparities, such as models of mortality crossovers.

The genetic architecture of genome-wide recombination rate variation in allopolyploid wheat revealed by nested association mapping.

Recombination affects the fate of alleles in populations by imposing constraints on the reshuffling of genetic information. Understanding the genetic basis of these constraints is critical for manipulating the recombination process to improve the resolution of genetic mapping, and reducing the negative effects of linkage drag and deleterious genetic load in breeding. Using sequence-based genotyping of a wheat nested association mapping (NAM) population of 2,100 recombinant inbred lines created by crossing 29 diverse lines, we mapped QTL affecting the distribution and frequency of 102 000 crossovers (CO). Genome-wide recombination rate variation was mostly defined by rare alleles with small effects together explaining up to 48.6% of variation. Most QTL were additive and showed predominantly trans-acting effects. The QTL affecting the proximal COs also acted additively without increasing the frequency of distal COs. We showed that the regions with decreased recombination carry more single nucleotide polymorphisms (SNPs) with possible deleterious effects than the regions with a high recombination rate. Therefore, our study offers insights into the genetic basis of recombination rate variation in wheat and its effect on the distribution of deleterious SNPs across the genome. The identified trans-acting additive QTL can be utilized to manipulate CO frequency and distribution in the large polyploid wheat genome opening the possibility to improve the efficiency of gene pyramiding and reducing the deleterious genetic load in the low-recombining pericentromeric regions of chromosomes.

Engineering meiotic recombination pathways in rice.

In the last 15 years, outstanding progress has been made in understanding the function of meiotic genes in the model dicot and monocot plants Arabidopsis and rice (Oryza sativa L.), respectively. This knowledge allowed to modulate meiotic recombination in Arabidopsis and, more recently, in rice. For instance, the overall frequency of crossovers (COs) has been stimulated 2.3- and 3.2-fold through the inactivation of the rice FANCM and RECQ4 DNA helicases, respectively, two genes involved in the repair of DNA double-strand breaks (DSBs) as noncrossovers (NCOs) of the Class II crossover pathway. Differently, the programmed induction of DSBs and COs at desired sites is currently explored by guiding the SPO11-1 topoisomerase-like transesterase, initiating meiotic recombination in all eukaryotes, to specific target regions of the rice genome. Furthermore, the inactivation of 3 meiosis-specific genes, namely PAIR1, OsREC8 and OsOSD1, in the Mitosis instead of Meiosis (MiMe) mutant turned rice meiosis into mitosis, thereby abolishing recombination and achieving the first component of apomixis, apomeiosis. The successful translation of Arabidopsis results into a crop further allowed the implementation of two breakthrough strategies that triggered parthenogenesis from the MiMe unreduced clonal egg cell and completed the second component of diplosporous apomixis. Here, we review the most recent advances in and future prospects of the manipulation of meiotic recombination in rice and potentially other major crops, all essential for global food security.