Meiotic crossovers rearrange existing genetic variation between homologous chromosomes, profoundly affecting genomic diversity. Crossovers are typically constrained to one to three events per chromosome pair, and their distribution is shaped by chromatin accessibility and DNA polymorphisms. Genome-wide crossover maps can be generated in plants by high-throughput short-read sequencing or linked-read sequencing. Here, we use long-read nanopore sequencing technology to develop a crossover mapping pipeline, COmapper, for high-resolution mapping of genome-wide crossovers from pooled DNA of F1 hybrid pollen and F2 recombinant seedlings derived from a cross between Arabidopsis thaliana accessions Col and Ler. We validate the high accuracy of COmapper by applying nanopore long-read sequencing to the pooled DNA of Arabidopsis F2 individuals with crossovers mapped by short-read sequencing. Using the COmapper, we constructed high-resolution genomic maps of crossovers using F1 hybrid pollen and F2 seedlings in wild-type (WT) and crossover-elevated recq4a recq4b mutants, showing results comparable to short-read sequencing. Crossovers were enriched at gene-proximal promoters in WT and increased but reshaped by high polymorphism density in recq4a recq4b. We propose that COmapper will be widely applicable for exploring the effects of genetic, epigenetic, and environmental changes on the crossover patterns across diverse plant species.

Meiotic crossovers rearrange allele combinations and create offspring diversity. Crossovers occur nonrandomly along chromosomes, predominantly in distal euchromatin and less in pericentromeric heterochromatin marked with histone H3 lysine 9 dimethylation (H3K9me2) and the H2A variant H2A.W in Arabidopsis thaliana. Loss of H3K9me2 increases heterochromatic crossovers, but how H2A.W affects crossover formation in pericentromeric regions is unknown. Here, we report that H2A.W is required to restrict heterochromatic crossovers in Arabidopsis. Using meiosis-specific microRNA-induced gene silencing (meiMIGS) and fluorescence-tagged recombination reporters, we show that meiotic knockdown of H2A.W.6, H2A.W.7, and H2A.W.12 (meiMIGS-H2A.W.6/7/12) increases pericentromeric crossovers. High-resolution genomic maps of crossovers show that meiMIGS-H2A.W.6/7/12 enhances heterochromatic crossovers, similar to meiMIGS plants silencing the H3K9me2 pathway. Consistently, genome-wide crossover maps show that the mutants h2a.w.6, h2a.w.7, h2a.w.6 h2a.w.7, and h2a.w.6 h2a.w.7 h2a.w.12, but not h2a.w.12, exhibit a similar increase in heterochromatic crossovers to meiMIGS-H2A.W.6/7/12, demonstrating that H2A.W.6 and H2A.W.7 limit heterochromatic crossovers. Profiling of genome-wide nucleosome density using micrococcal nuclease sequencing reveals that h2a.w mutants with increased heterochromatic crossovers have increased heterochromatin accessibility, with lower H3K9me2 levels during meiosis. Our findings shed light on the role of H2A.W variants as heterochromatin compaction factors that suppress meiotic crossovers within the pericentromeric regions.

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis.

The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a fluorescent crossover reporter in Arabidopsis thaliana, we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers toward the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome, and chromatin immunoprecipitation analysis, we reveal that HSBP is required to repress HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5' untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.

The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a sensitive fluorescent seed crossover reporter in Arabidopsis thaliana we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers towards the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome and chromatin immunoprecipitation analysis, we reveal that HSBP directly represses HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5′ untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.

Meiotic crossovers are tightly restricted in most eukaryotes, despite an excess of initiating DNA double-strand breaks. The majority of plant crossovers are dependent on class I interfering repair, with a minority formed via the class II pathway. Class II repair is limited by anti-recombination pathways; however, similar pathways repressing class I crossovers have not been identified. Here, we performed a forward genetic screen in Arabidopsis using fluorescent crossover reporters to identify mutants with increased or decreased recombination frequency. We identified HIGH CROSSOVER RATE1 (HCR1) as repressing crossovers and encoding PROTEIN PHOSPHATASE X1. Genome-wide analysis showed that hcr1 crossovers are increased in the distal chromosome arms. MLH1 foci significantly increase in hcr1 and crossover interference decreases, demonstrating an effect on class I repair. Consistently, yeast two-hybrid and in planta assays show interaction between HCR1 and class I proteins, including HEI10, PTD, MSH5 and MLH1. We propose that HCR1 plays a major role in opposition to pro-recombination kinases to restrict crossovers in Arabidopsis.

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate genetic dissection of mechanisms that control meiotic recombination.

Meiotic recombination provides genetic diversity in populations and ensures accurate homologous chromosome segregation for genome integrity. During meiosis, recombination processes, from DNA double strand breaks (DSBs) to crossover formation are tightly linked to higher order chromosome structure, including chromatid cohesion, axial element formation, homolog pairing and synapsis. The extensive studies on plant meiosis have revealed the important conserved roles for meiotic proteins in homologous recombination. Recent works have focused on elucidating the mechanistic basis of how meiotic proteins regulate recombination events via protein complex formation and modifications such as phosphorylation, ubiquitination, and SUMOylation. Here, we highlight recent advances on the signaling and modifications of meiotic proteins that mediate the formation of DSBs and crossovers in plants.

Meiotic recombination initiates via DNA double strand breaks (DSBs) generated by SPO11 topoisomerase-like complexes. Recombination frequency varies extensively along eukaryotic chromosomes, with hotspots controlled by chromatin and DNA sequence. To map meiotic DSBs throughout a plant genome, we purified and sequenced Arabidopsis SPO11-1-oligonucleotides. DSB hotspots occurred in gene promoters, terminators and introns, driven by AT-sequence richness, which excludes nucleosomes and allows SPO11-1 access. A strong positive relationship was observed between SPO11-1 DSBs and final crossover levels. Euchromatic marks promote recombination in fungi and mammals, and consistently we observe H3K4me3 enrichment in proximity to DSB hotspots at gene 5’-ends. Repetitive transposons are thought to be recombination-silenced during meiosis, in order to prevent non-allelic interactions and genome instability. Unexpectedly, we found strong DSB hotspots in nucleosome-depleted Helitron/Pogo/Tc1/Mariner DNA transposons, whereas retrotransposons were coldspots. Hotspot transposons are enriched within gene regulatory regions and in proximity to immunity genes, suggesting a role as recombination-enhancers. As transposon mobility in plant genomes is restricted by DNA methylation, we used the met1 DNA methyltransferase mutant to investigate the role of heterochromatin on the DSB landscape. Epigenetic activation of transposon meiotic DSBs occurred in met1 mutants, coincident with reduced nucleosome occupancy, gain of transcription and H3K4me3. Increased met1 SPO11-1 DSBs occurred most strongly within centromeres and Gypsy and CACTA/EnSpm coldspot transposons. Together, our work reveals complex interactions between chromatin and meiotic DSBs within genes and transposons, with significance for the diversity and evolution of plant genomes.