The first division of meiosis is unique in its capacity to halve the ploidy of future gametes. To this end, one key innovation compared with mitosis is the monopolar orientation of the pairs of sister kinetochores required for the proper separation of homologs at meiosis I. How monopolar orientation is imposed is unclear and seems to vary in eukaryotes. Here, we performed a forward genetic screen in Arabidopsis thaliana, specifically designed to identify the molecular components imposing monopolar orientation, based on mutants’ ability to restore fertility in spo11 osd1 haploid plants. We show that monopolar orientation involves all four cohesin subunits (REC8, SCC3, SMC1, and SMC3), the cohesion establishment factors CTF18 and DCC1, the cohesin protectors SGO1/2 and PATRONUS (PANS1), the inner kinetochore protein CENP-C, and the deSUMOylase SUMO PROTEASE RELATED TO FERTILITY 2 (SPF2). The mutants show bipolar orientation of achiasmatic chromosomes; however, monopolar orientation is maintained in the presence of crossovers, despite most of them displaying the splitting of sister kinetochores and reduced levels of cohesin at metaphase I. Taken together, the findings demonstrate that cohesion establishment and maintenance, kinetochore function, and deSUMOylation, together with crossovers, promote monopolar orientation in plants and support a cohesion-driven model of kinetochore orientation at meiosis I that is conserved across kingdoms.

The transfer of apomixis, a clonal mode of reproduction by seeds, to crops has the potential to revolutionize agriculture by enabling the generation of one-line F1 hybrids that propagate clonally by seeds from one generation to the next. However, despite nearly four decades of intensive research, all the attempts to transfer the identified genetic determinants of apomixis from naturally apomictic wild plants to their crop relatives have failed to produce apomictic crops. Engineering of apomixis, mimicking the key features of a natural form of apomixis known as gametophytic diplosporous apomixis, has recently been achieved in rice and further improvements by the introduction of a single "all-in-one" T-DNA construct into calli derived from F1 hybrid seed embryos resulted in high frequency of clonal seeds. The T-DNA encodes a constitutively expressed Cas9 protein guided by sgRNAs designed to knock-out the functions of three genes essential for regulating crucial steps involved in meiosis, thereby eliminating meiosis and creating the Mitosis instead of Meiosis (MiMe) triple mutant. Additionally, the T-DNA contains another gene expression cassette consisting of a parthenogenetic trigger, the BABY BOOM1 (BBM1) transcription factor driven by an egg cell-specific (ECS) promoter. Inactivation of the three MiMe genes converts meiosis into mitosis-apomeiosis-yielding unrecombined and unreduced male and female spores developing into gametophytes. The BBM1 expression triggers parthenogenetic development of an embryo from the diploid egg cell of the female gametophyte. The endosperm develops sexually by the fusion of a diploid sperm cell and the central cell of the female gametophyte, which contains two diploid polar nuclei, resulting in an initially hexaploid endosperm. To date, the "all-in-one" T-DNA method has proved to be the most efficient for achieving high frequency (95-100% clonal seeds) synthetic apomixis in rice. Since the original publication, we have successfully generated synthetic apomictic events in three additional F1 hybrids. Here, we describe the methods for designing T-DNA constructs, analyzing mutations in first-generation (T0) MiMe mutant plants of F1 hybrids, and ascertaining the apomictic nature of the progenies from confirmed MiMe F1 hybrids, which relies on the egg cell-specific accumulation of BBM1.

Crossovers (COs) ensure proper chromosome segregation during meiosis and generate genetic diversity. COs are non-uniformly distributed along chromosomes and almost universally suppressed in centromere-proximal regions, notably creating an important bottleneck for plant breeding. The mechanism of this CO suppression is still not fully understood, but the chromatin state is a contributing factor. Here we identify three factors that actively limit proximal CO in Arabidopsis thaliana: the cohesion establishment factor CTF18, the centromeric cohesin protector SGO2 and the deSUMOylase SPF2. The mutation of these factors allows both the formation of COs in the centromere-proximal region where they were completely absent in the wild type and the enhancement of their frequency where they were rare. COs can be further increased by combining these mutations together or with mutation in the DNA methylase CMT3, suggesting that multiple mechanisms prevent proximal COs in parallel. The identification of the very conserved CTF18, SGO2 and SPF2 as suppressors of centromere-proximal COs highlights the importance of cohesin turnover in this process and opens up new possibilities for plant breeding.

Background Meiotic recombination creates genetic diversity through reciprocal exchange of haplotypes between homologous chromosomes. Scalable and robust methods for mapping recombination breakpoints are essential for understanding meiosis and for genetic mapping. Single cell sequencing of gametes offers a direct approach to recombination mapping, yet the effect of technical differences between single-cell sequencing methods for crossover detection remains unclear. Results We benchmark single cell methods for droplet-based chromatin accessibility and RNA sequencing and plate-based whole-genome amplification for mapping meiotic recombination in Arabidopsis thaliana . For this purpose we introduce two novel open-source tools coelsch\_mapping\_pipeline and coelsch for haplotype-aware alignment and per-cell crossover detection, using them to recover known recombination frequencies and quantify the effects of coverage sparsity. We subsequently apply our approach to a panel of 40 recombinant F₁ hybrids derived from crosses of 22 diverse natural accessions, successfully recovering genetic maps for 34 F1s in a single dataset. This analysis reveals substantial variation in recombination rate and identifies a ∼10 Mb pericentric inversion in the accession Zin-9, the largest natural inversion reported in A. thaliana to date. Conclusions These results demonstrate the applicability and scalability of single-cell gamete sequencing for high-throughput mapping of meiotic recombination, and highlight the strengths and limitations of different single-cell modalities. The accompanying open-source tools provide a framework for haplotyping and crossover detection analysis using sparse single-cell sequencing data. Our methodology enables parallel analysis of large numbers of hybrids in a single dataset, removing a major technical barrier to large-scale studies of natural variation in recombination rate. ### Competing Interest Statement The authors declare the following competing interests: Takara provided Takara Shasta Whole Genome Amplification kits free of charge. The company had no role in study design, analysis, or publication decisions. A patent was granted to Institut National de la Recherche Agronomique (INRA) on the use of RECQ4 to manipulate meiotic recombination in crops, with RM listed among the inventors (US10,920,237/EP3149027). A patent was filed by the Max Planck Society on the combined use of RECQ4 and ZYP1 to manipulate recombination in crops, with RM listed among the inventors (EP23179262. 14.06.2023).

F1 hybrids are known for their superior yield and greater yield stability which are due to the phenomenon of hybrid vigor, also called heterosis. In Asian rice, hybrids between compatible lines of the two subspecies indica and japonica benefit from a higher yield heterosis than intra-subspecific hybrids. However, the vast majority of indica/japonica hybrids generally exhibit partial fertility, which currently prevents their large-scale exploitation as F1 cultivars. Recently, synthetic apomixis has emerged as an alternative to male sterility systems to produce F1 hybrids that can be clonally propagated by seeds over generations. The most efficient synthetic apomixis system to date relies on the conversion of meiosis into mitosis (apomeiosis) by the induction of the triple Mitosis instead of Meiosis (MiMe) mutations, followed by the triggering of parthenogenesis by egg cell-specific expression of the BABY BOOM 1 (BBM1) transcription factor. We show here that apomeiosis restores full fertility in primary transformants of two indica/japonica F1 hybrids, normally exhibiting 5.5 and 55% grain filling. We further demonstrate that the restored fertility can be transmitted to the next generation in diploid clonal progeny plants through synthetic apomixis. Our results also indicate that a better tuning of parthenogenesis is likely needed to reach both full fertility and full penetrance of apomixis in hybrid rice. Altogether, these results open new avenues for reproducing distant hybrids with high level of heterosis by clonal seeds in rice and in other crops.

In eukaryotes, chromosome segregation relies on attachment to the spindle, ensured by the kinetochore. The outer kinetochore attaches to the microtubules and is named after three sub-complexes KNL1C, MIS12C, and NDC80C (KMN). While the KMN complex comprises ten proteins in humans S. cerevisiae, its conservation in more distant eukaryotes is unclear. Here, we aimed to define the KMN complex in the plant Arabidopsis using affinity purification and identified thirteen KMN proteins. Seven were previously known to have a conserved function (atMIS12, atNNF1, atNDC80, atSPC24, atSPC25, atNUF2, and atKNL1) and six were uncharacterized. These six proteins show remote similarity to yeast/human KMN-associated proteins, whose homologs have not yet been characterized in plants. We named them atDSN1, atCSM1, atNSL1.1/.2, and atZWINT1.1/.2. We confirmed kinetochore localization for atDSN1, atCSM1, atNSL1.1, and atZWINT1.1 in planta. In addition, atDSN1, atCSM1, and ZWINT1.1/.2 are essential, further supporting their kinetochore function. AlphaFold3 predicts an alike3D organization of the KMN complex in plants and mammals. We conclude that the KMN complex is globally conserved with a matching composition and similar organization in distant eukaryotes, with some local variations, suggesting its presence in the common ancestors of all living eukaryotes.

Sexual dysmorphism in the number and distribution of meiotic crossovers is seen across species but is poorly understood. Here, we disrupt multiple anti-crossover pathways in hermaphrodite Arabidopsis and analyze thousands of female and male progeny genomes. The greatest crossover increase is seen in zyp1 recq4 mutants, with a 12-fold rise in females and 4.5-fold in males. Additional manipulation of crossover regulators does not further increase crossovers but shifts the balance between crossover pathways, suggesting competition for a shared, limited precursor pool. While wild-type crossover patterns differ between sexes, mutant crossover landscapes converge on a unique distinct profile, which we term Crossover Potential (COP). COP can be accurately predicted using only sequence and chromatin features. We propose that COP reflects the density of eligible recombination precursors, which is determined by genomic features and is thus identical across sexes, with sexual dimorphism resulting solely from differential regulation of their maturation into crossovers.

During meiosis, each chromosome pair experiences at least one crossover (CO), which directs their balanced segregation in addition to shuffling genetic information. COs tend to be away from each other, a phenomenon known as CO interference. The main biochemical pathway for CO formation, which is conserved in distant eukaryotes, involves the ZMM proteins together with the MLH1–MLH3 complex (MutLγ). Here, we aim to clarify the role of MutLγ in CO formation in Arabidopsis thaliana. We show that AtMutLγ is partially dispensable for ZMM-dependent CO formation. HEI10 large foci—that mark CO sites in wild-type—form at a normal level in mlh1 and mlh3 mutants, but are inefficiently maturated into COs. Mutating the MUS81 nuclease in either mlh1 or mlh3 leads to chromosome fragmentation, which is suppressed by further mutating the zmm msh5. This suggests that in the absence of MutLγ, recombination intermediates produced by ZMMs are resolved by MUS81, which does not ensure CO formation. Finally, CO interference is marginally affected in mlh1, which is compatible with a random sub-sampling of normally patterned CO sites. We conclude that AtMutLγ imposes designated recombination intermediates to be resolved exclusively as COs, supporting the view that MutLγ asymmetrically resolves double-Holliday junctions, yielding COs.

The first division of meiosis is unique in its capacity to halve the ploidy of the future gametes. To this end, one key innovation compared to mitosis is the monopolar orientation of the pairs of sister kinetochores required for the proper separation of homologs at meiosis I. How monopolar orientation is imposed is unclear and seems to vary in eukaryotes. Here we performed a forward genetic screen in Arabidopsis thaliana , specifically designed to identify the molecular components imposing monopolar orientation, based on mutants’ ability to restore fertility in spo11 osd1 haploid plants. We show that monopolar orientation involves all four cohesin subunits (REC8, SCC3, SMC1, SMC3), the cohesion establishment factors CTF18 and DCC1, and the cohesin protectors SGO1/2 and PANS1, the inner kinetochore protein CENP-C, and the desumoylase SPF2. The mutants show bipolar orientation of achiasmatic chromosomes, but monopolar orientation is maintained in the presence of crossovers despite splitting of sister kinetochores at metaphase I and reduced levels of cohesin. Taken together, the findings demonstrate that cohesion establishment and protection, kinetochore function, and deSUMOylation, together with crossovers, enforce monopolar orientation in plants and support a cohesion-driven model of kinetochore orientation at meiosis I that is conserved across kingdoms. ### Competing Interest Statement The authors have declared no competing interest.

Meiotic crossovers (COs) are needed to produce genetically balanced gametes. In mammals, CO formation is mediated by a conserved set of pro-CO proteins via mechanisms that remain unclear. Here, we characterize a mammalian pro-CO factor HEIP1. In mouse HEIP1 is essential for crossover and fertility of both sexes. HEIP1 promotes crossover by orchestrating the recruitment of other pro-CO proteins, including the MutSγ complex (MSH4-MSH5) and E3 ligases (HEI10, RNF212, and RNF212B), that are required to mature CO sites and recruit the CO-specific resolution complex MutLγ. Moreover, HEIP1 directly interacts with HEI10, suggesting a direct role in controlling the recruitment of pro-CO E3 ligases. During early stages of meiotic prophase I, HEIP1 interacts with the chromosome axes, independently of recombination, before relocalizing to the central region of the synaptonemal complex. We propose that HEIP1 is a conserved master regulator of CO proteins that controls different CO maturation steps.

Crossovers (COs) ensure proper chromosome segregation during meiosis and generate genetic diversity. COs are non-uniformly distributed along chromosomes and almost universally suppressed in centromere-proximal regions, notably creating a significant bottleneck for plant breeding. The mechanism of this CO suppression is elusive, but chromatin state is a contributing factor. Here, we identify three factors that actively limit proximal CO in Arabidopsis, the cohesion establishment factor CTF18, the centromeric cohesin protector SGO2, and the deSUMOylase SPF2. The mutation of these factors allows both the formation of CO in centromere-proximal region where they were completely absent in the wild-type, and enhance their frequency where they were rare. COs can be further increased by combining these mutations together or with mutation in the DNA methylase CMT3 , suggesting that multiple mechanism prevent proximal CO in parallel. The identification of the very conserved CTF18, SGO2 and SPF2 as suppressors of centromere-proximal CO highlights the importance of cohesin turnover in this process and opens up new possibilities for plant breeding. ### Competing Interest Statement The authors declare the following competing interests: A patent was filed by the Max Planck Society on the use of CTF18, SGO2 and SPF2 to manipulate meiotic recombination in plants, with RM, RSG, JBF, SD, and QL listed as inventors (EP25184806.5. 24.06.2025).

Summary In most eukaryotes, balanced chromosome segregation at meiosis requires crossovers, but female Bombyx mori lack these structures. Instead, the synaptonemal complex is repurposed to compensate for this absence of crossovers, a remarkable example of exaptation.

During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1’s role during meiotic recombination in mammals remains unknown. Here, we decipher the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1ΔN alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates. Successful meiotic homologous recombination relies on the tight regulation of RAD51 and DMC1 strand exchange proteins. This study shows that the conserved FIGNL1-FIRRM complex plays a critical role for fertility by restraining uncontrolled assembly of RAD51 and DMC1 filament during meiosis.

Chromosome-level genome sequences of 69 diverse Arabidopsis thaliana strains reveal a quasi-fixed genome structure worldwide, in which large rearrangement is limited almost exclusively to the centromeric regions. Pan-genome analysis uncovered substantial diversity in gene content that, together with the genome assemblies, will fuel future genetic research.

Background Centromeres load kinetochore complexes onto chromosomes, which mediate spindle attachment and allow segregation during cell division. Although centromeres perform a conserved cellular function, their underlying DNA sequences are highly divergent within and between species. Despite variability in DNA sequence, centromeres are also universally suppressed for meiotic crossover recombination, across eukaryotes. However, the genetic and epigenetic factors responsible for suppression of centromeric crossovers remain to be completely defined. Results To explore the centromere-proximal recombination landscape, we mapped 14,397 crossovers against fully assembled Arabidopsis thaliana genomes. A. thaliana centromeres comprise megabase-scale satellite repeat arrays that load nucleosomes containing the CENH3 histone variant. Each chromosome possesses a structurally polymorphic 3-4 megabase region where crossovers were absent, that includes the satellite arrays, flanked by 1-2 megabase low-recombination zones. The recombination-suppressed regions are enriched for Gypsy/Ty3 retrotransposons, and additionally contain expressed genes with high genetic diversity that initiate meiotic recombination, yet do not crossover. We mapped crossovers at high-resolution in proximity to CEN3 , which resolved punctate centromere-proximal hotspots that overlapped gene islands embedded in heterochromatin. Centromeres are densely DNA methylated and the recombination landscape was remodelled in DNA methylation mutants. We observed that the centromeric low-recombining zones decreased and increased crossovers in CG ( met1 ) and non-CG ( cmt3 ) mutants, respectively, whereas the core non recombining zones remained suppressed. Conclusion Our work relates the genetic and epigenetic organisation of the A. thaliana centromeres and flanking pericentromeric heterochromatin to the zones of crossover suppression that surround the CENH3-occupied satellite repeat arrays.

AbstractModern plant breeding, such as genomic selection and gene editing, is based on the knowledge of the genetic architecture of desired traits. Quantitative trait loci (QTL) analysis, which combines high throughput phenotyping and genotyping of segregating populations, is a powerful tool to identify these genetic determinants and to decipher the underlying mechanisms. However, meiotic recombination, which shuffles genetic information between generations, is limited: Typically only one to two exchange points, called crossovers, occur between a pair of homologous chromosomes. Here we test the effect on QTL analysis of boosting recombination, by mutating the anti-crossover factors RECQ4 and FIGL1 in Arabidopsis thaliana full hybrids and lines in which a single chromosome is hybrid. We show that increasing recombination ~6-fold empowers the detection and resolution of QTLs, reaching the gene scale with only a few hundred plants. Further, enhanced recombination unmasks some secondary QTLs undetected under normal recombination. These results show the benefits of enhanced recombination to decipher the genetic bases of traits.

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.

SUMMARYGenetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a research model. Here, we describe the whole‐genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high‐confidence mutations per HEM line, over three independent mutations altering protein sequences are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental, immune and physiological traits confirmed the efficacy of the tool for identifying both null, knockdown and gain‐of‐function alleles. The possibility of conducting subtle repeated phenotyping and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm‐browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.

Heterosis boosts crop yield; however, harnessing additional progressive heterosis in polyploids is challenging for breeders. We bioengineered a 'mitosis instead of meiosis' (MiMe) system that generates unreduced, clonal gametes in three hybrid tomato genotypes and used it to establish polyploid genome design. Through the hybridization of MiMe hybrids, we generated '4-haplotype' plants that encompassed the complete genetics of their four inbred grandparents, providing a blueprint for exploiting polyploidy in crops. An approach to generate unreduced, clonal gametes in hybrid tomato genotypes enables polyploid genome design through controlled combination of four predefined genome haplotypes, thereby establishing a framework for exploiting progressive heterosis in crops.

Arabidopsis thaliana was the first plant for which a high-quality genome sequence became available. The publication of the first reference genome sequence almost 25 years ago was already accompanied by genome-wide data on sequence polymorphisms in another accession, or naturally occurring strain. Since then, inventories of genome-wide diversity have been generated at increasingly precise levels. High-density genotype data for A. thaliana , including those from the 1001 Genomes Project, were key to demonstrating the enormous power of GWAS in inbred populations of wild plants, and the comparison of intraspecific polymorphism with interspecific divergence has illuminated many aspects of plant genome evolution. Over the past decade, an increasing number of nearly complete genome sequences have been published for many more accessions. Here, we highlight the diversity of a curated collection of previously published and so far unpublished genome sequences assembled using different types of long reads, including PacBio Continuous Long Reads (CLR), PacBio High Fidelity (HiFi) reads, and Oxford Nanopore Technologies (ONT) reads. This 1001 Genomes Plus (1001G+) resource is being made available at http://1001genomes.org. We invite colleagues with yet unpublished genome assemblies from A. thaliana accessions to contribute to this effort. ### Competing Interest Statement D.W. holds equity in Computomics, which advises plant breeders. D.W. also consults for KWS SE, a globally active plant breeder and seed producer. J.F. is an employee of Tropic TI, Lda. All other authors declare no competing interests.