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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., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Wheat is a major cereal crop that possesses a large allopolyploid genome formed through hybridisation of tetraploid and diploid progenitors. During meiosis, crossovers (COs) are constrained in number to 1-3 per chromosome pair that are predominantly located towards the chromosome ends. This reduces the probability of advantageous traits recombining onto the same chromosome, thus limiting breeding. Therefore, understanding the underlying factors controlling meiotic recombination may provide strategies to unlock the genetic potential in wheat. In this mini-review, we will discuss the factors associated with restricted CO formation in wheat, such as timing of meiotic events, chromatin organisation, pre-meiotic DNA replication and dosage of CO genes, as a means to modulate recombination., (© 2022 The Author(s).)

FANCM suppresses crossovers in plants by unwinding recombination intermediates. In wheat, crossovers are skewed toward the chromosome ends, thus limiting generation of novel allelic combinations. Here, we observe that FANCM maintains the obligate crossover in tetraploid and hexaploid wheat, thus ensuring that every chromosome pair exhibits at least one crossover, by localizing class I crossover protein HEI10 at pachytene. FANCM also suppresses class II crossovers that increased 2.6-fold in fancm msh5 quadruple mutants. These data are consistent with a role for FANCM in second-end capture of class I designated crossover sites, whilst FANCM is also required to promote formation of non-crossovers. In hexaploid wheat, genetic mapping reveals that crossovers increase by 31% in fancm compared to wild type, indicating that fancm could be an effective tool to accelerate breeding. Crossover rate differences in fancm correlate with wild type crossover distributions, suggesting that chromatin may influence the recombination landscape in similar ways in both wild type and fancm., (© 2022. The Author(s).)

The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5 These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

In most eukaryotes, a set of conserved proteins that are collectively termed ZMM proteins (named for molecular zipper 1 [ZIP1], ZIP2, ZIP3, and ZIP4, MutS homologue 4 [MSH4] and MSH5, meiotic recombination 3, and sporulation 16 [SPO16] in yeast [ Saccharomyces cerevisiae ]) are essential for the formation of the majority of meiotic crossovers (COs). Recent reports indicated that ZIP2 acts together with SPO16 and ZIP4 to control CO formation through recognizing and stabilizing early recombination intermediates in budding yeast. However, whether this mechanism is conserved in plants is not clear. Here, we characterized the functions of SHORTAGE OF CHIASMATA 1 (OsSHOC1; ZIP2 ortholog) and PARTING DANCERS (OsPTD; SPO16 ortholog) and their interactions with other ZMM proteins in rice ( Oryza sativa ). We demonstrated that disruption of OsSHOC1 caused a reduction of CO numbers to ∼83% of wild-type CO numbers, whereas synapsis and early meiotic recombination steps were not affected. Furthermore, OsSHOC1 interacts with OsPTD, which is responsible for the same set of CO formations as OsSHOC1. In addition, OsSHOC1 and OsPTD are required for the normal loading of other ZMM proteins, and conversely, the localizations of OsSHOC1 and OsPTD were also affected by the absence of OsZIP4 and human enhancer of invasion 10 in rice (OsHEI10). OsSHOC1 interacts with OsZIP4 and OsMSH5, and OsPTD interacts with OsHEI10. Furthermore, bimolecular fluorescence complementation and yeast-three hybrid assays demonstrated that OsSHOC1, OsPTD, OsHEI10, and OsZIP4 were able to form various combinations of heterotrimers. Moreover, statistical and genetic analysis indicated that OsSHOC1 and OsPTD are epistatic to OsHEI10 and OsZIP4 in meiotic CO formation. Taken together, we propose that OsSHOC1, OsPTD, OsHEI10, and OsZIP4 form multiple protein complexes that have conserved functions in promoting class I CO formation., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Background: Sequence exchange between homologous chromosomes through crossing over and gene conversion is highly conserved among eukaryotes, contributing to genome stability and genetic diversity. A lack of recombination limits breeding efforts in crops; therefore, increasing recombination rates can reduce linkage drag and generate new genetic combinations., Results: We use computational analysis of 13 recombinant inbred mapping populations to assess crossover and gene conversion frequency in the hexaploid genome of wheat (Triticum aestivum). We observe that high-frequency crossover sites are shared between populations and that closely related parents lead to populations with more similar crossover patterns. We demonstrate that gene conversion is more prevalent and covers more of the genome in wheat than in other plants, making it a critical process in the generation of new haplotypes, particularly in centromeric regions where crossovers are rare. We identify quantitative trait loci for altered gene conversion and crossover frequency and confirm functionality for a novel RecQ helicase gene that belongs to an ancient clade that is missing in some plant lineages including Arabidopsis., Conclusions: This is the first gene to be demonstrated to be involved in gene conversion in wheat. Harnessing the RecQ helicase has the potential to break linkage drag utilizing widespread gene conversions.

Although meiosis is evolutionarily conserved, many of the underlying mechanisms show species-specific differences. These are poorly understood in large genome plant species such as barley (Hordeum vulgare) where meiotic recombination is very heavily skewed to the ends of chromosomes. The characterization of mutant lines can help elucidate how recombination is controlled. We used a combination of genetic segregation analysis, cytogenetics, immunocytology and 3D imaging to genetically map and characterize the barley meiotic mutant DESYNAPTIC 10 (des10). We identified a spontaneous exonic deletion in the orthologue of MutL-Homolog 3 (HvMlh3) as the causal lesion. Compared with wild-type, des10 mutants exhibit reduced recombination and fewer chiasmata, resulting in the loss of obligate crossovers and leading to chromosome mis-segregation. Using 3D structured illumination microscopy (3D-SIM), we observed that normal synapsis progression was also disrupted in des10, a phenotype that was not evident with standard confocal microscopy and that has not been reported with Mlh3 knockout mutants in Arabidopsis. Our data provide new insights on the interplay between synapsis and recombination in barley and highlight the need for detailed studies of meiosis in nonmodel species. This study also confirms the importance of early stages of prophase I for the control of recombination in large genome cereals., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

In many cereal crops, meiotic crossovers predominantly occur toward the ends of chromosomes and 30 to 50% of genes rarely recombine. This limits the exploitation of genetic variation by plant breeding. Previous reports demonstrate that chiasma frequency can be manipulated in plants by depletion of the synaptonemal complex protein ZIPPER1 (ZYP1) but conflict as to the direction of change, with fewer chiasmata reported in Arabidopsis thaliana and more crossovers reported for rice (Oryza sativa). Here, we use RNA interference (RNAi) to reduce the amount of ZYP1 in barley (Hordeum vulgare) to only 2 to 17% of normal zygotene levels. In the ZYP1(RNAi) lines, fewer than half of the chromosome pairs formed bivalents at metaphase and many univalents were observed, leading to chromosome nondisjunction and semisterility. The number of chiasmata per cell was reduced from 14 in control plants to three to four in the ZYP1-depleted lines, although the localization of residual chiasmata was not affected. DNA double-strand break formation appeared normal, but the recombination pathway was defective at later stages. A meiotic time course revealed a 12-h delay in prophase I progression to the first labeled tetrads. Barley ZYP1 appears to function similarly to ZIP1/ZYP1 in yeast and Arabidopsis, with an opposite effect on crossover number to ZEP1 in rice, another member of the Poaceae.

Meiotic recombination results in the formation of cytological structures known as chiasmata at the sites of genetic crossovers (COs). The formation of at least one chiasma/CO between homologous chromosome pairs is essential for accurate chromosome segregation at the first meiotic division as well as for generating genetic variation. Although DNA double-strand breaks, which initiate recombination, are widely distributed along the chromosomes, this is not necessarily reflected in the chiasma distribution. In many species there is a tendency for chiasmata to be distributed in favored regions along the chromosomes, whereas in others, such as barley and some other grasses, chiasma localization is extremely pronounced. Localization of chiasma to the distal regions of barley chromosomes restricts the genetic variation available to breeders. Studies reviewed herein are beginning to provide an explanation for chiasma localization in barley. Moreover, they suggest a potential route to manipulating chiasma distribution that could be of value to plant breeders.

Replication protein A (RPA) is a conserved heterotrimeric protein complex comprising RPA1, RPA2, and RPA3 subunits involved in multiple DNA metabolism pathways attributable to its single-stranded DNA binding property. Unlike other species possessing a single RPA2 gene, rice (Oryza sativa) possesses three RPA2 paralogs, but their functions remain unclear. In this study, we identified RPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (rpa2c) exhibited reduced bivalent formation, leading to chromosome nondisjunction. In rpa2c, chiasma frequency is reduced by ~78% compared with the wild type and is accompanied by loss of the obligate chiasma. The residual ~22% chiasmata fit a Poisson distribution, suggesting loss of crossover control. RPA2c colocalized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of RPA2c to the chromosomes. In addition, RPA2c partially colocalized with MER3 during late leptotene, thus indicating that RPA2c is required for class I crossover formation at a late stage of homologous recombination. Furthermore, we identified RPA1c, an RPA1 subunit with nearly overlapping distribution to RPA2c, required for ~79% of chiasmata formation. Our results demonstrate that an RPA complex comprising RPA2c and RPA1c is required to promote meiotic crossovers in rice.

Meiosis involves reciprocal exchange of genetic information between homologous chromosomes to generate new allelic combinations. In cereals, the distribution of genetic crossovers, cytologically visible as chiasmata, is skewed toward the distal regions of the chromosomes. However, many genes are known to lie within interstitial/proximal regions of low recombination, creating a limitation for breeders. We investigated the factors underlying the pattern of chiasma formation in barley (Hordeum vulgare) and show that chiasma distribution reflects polarization in the spatiotemporal initiation of recombination, chromosome pairing, and synapsis. Consequently, meiotic progression in distal chromosomal regions occurs in coordination with the chromatin cycles that are a conserved feature of the meiotic program. Recombination initiation in interstitial and proximal regions occurs later than distal events, is not coordinated with the cycles, and rarely progresses to form chiasmata. Early recombination initiation is spatially associated with early replicating, euchromatic DNA, which is predominately found in distal regions. We demonstrate that a modest temperature shift is sufficient to alter meiotic progression in relation to the chromosome cycles. The polarization of the meiotic processes is reduced and is accompanied by a shift in chiasma distribution with an increase in interstitial and proximal chiasmata, suggesting a potential route to modify recombination in cereals.

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation., Competing Interests: The authors have declared that no competing interests exist.

MSH5, a meiosis-specific member of the MutS-homologue family of genes, is required for normal levels of recombination in budding yeast, mouse and Caenorhabditis elegans. In this paper we report the identification and characterization of the Arabidopsis homologue of MSH5 (AtMSH5). Transcripts of AtMSH5 are specific to reproductive tissues, and immunofluorescence studies indicate that expression of the protein is abundant during prophase I of meiosis. In a T-DNA tagged insertional mutant (Atmsh5-1), recombination is reduced to about 13% of wild-type levels. The residual chiasmata are randomly distributed between cells and chromosomes. These data provide further evidence for at least two pathways of meiotic recombination in Arabidopsis and indicate that AtMSH5 protein is required for the formation of class I interference-sensitive crossovers. Localization of AtMSH5 to meiotic chromosomes occurs at leptotene and is dependent on DNA double-strand break formation and strand exchange. Localization of AtMSH5 to the chromatin at mid-prophase I is dependent on expression of AtMSH4. At late zygotene/early pachytene a proportion of AtMSH5 foci co-localize with AtMLH1 which marks crossover-designated sites. Chromosome synapsis appears to proceed normally, without significant delay, in Atmsh5-1 but the pachytene stage is extended by several hours, indicative of the operation of a surveillance system that monitors the progression of prophase I.

Meiotic crossovers/chiasmata, that are required to ensure chromosome disjunction, arise via the class I interference-dependent pathway or via the class II interference-free pathway. The proportions of these two classes vary considerably between different organisms. In Arabidopsis, about 85% of chiasmata are eliminated in Atmsh4 mutants, denoting that these are class I events. In budding and fission yeasts Msh4-independent crossovers arise largely or entirely via a Mus81-dependent pathway. To investigate the origins of the 15% residual (AtMSH4-independent) chiasmata in Arabidopsis we conducted a cytological and molecular analysis of AtMUS81 meiotic expression and function. Although AtMUS81 functions in somatic DNA repair and recombination, it is more highly expressed in reproductive tissues. The protein is abundantly present in early prophase I meiocytes, where it co-localizes, in a double-strand break-dependent manner, with the recombination protein AtRAD51. Despite this, an Atmus81 mutant shows normal growth and has no obvious defects in reproductive development that would indicate meiotic impairment. A cytological analysis confirmed that meiosis was apparently normal in this mutant and its mean chiasma frequency was similar to that of wild-type plants. However, an Atmsh4/Atmus81 double mutant revealed a significantly reduced mean chiasma frequency (0.85 per cell), compared with an Atmsh4 single mutant (1.25 per cell), from which we conclude that AtMUS81 accounts for some, but not all, of the 15% AtMSH4-independent residual crossovers. It is possible that other genes are responsible for these residual chiasmata. Alternatively the AtMUS81 pathway coexists with an alternative parallel pathway that can perform the same functions.

The duplicated Arabidopsis genes ZYP1a/ZYP1b encode closely related proteins with structural similarity to the synaptonemal complex (SC) transverse filament proteins from other species. Immunolocalization detects ZYP1 foci at late leptotene, which lengthen until at pachytene fluorescent signals extending the entire length of the fully synapsed homologs are observed. Analysis of zyp1a and zyp1b T-DNA insertion mutants indicates that the proteins are functionally redundant. The SC is not formed in the absence of ZYP1 and prophase I progression is significantly delayed suggesting the existence of an intraprophase I surveillance mechanism. Recombination is only slightly reduced in the absence of ZYP1 such that the chiasma frequency at metaphase I is approximately 80% of wild type. Moreover cytological analysis indicates that chiasma distribution within zyp1 bivalents is indistinguishable from wild type, providing evidence that the SC is not required for the imposition of interference. Importantly in the absence of ZYP1, recombination occurs between both homologous and nonhomologous chromosomes suggesting the protein is required to ensure the fidelity of meiotic chromosome associations.

MSH4, a meiosis-specific member of the MutS-homolog family of genes, is required for normal levels of recombination and fertility in budding yeast, mouse, and Caenorhabditis elegans. In this paper, we report the identification and characterization of the Arabidopsis homolog of MSH4 (AtMSH4). We demonstrate that AtMSH4 expression can only be detected in floral tissues, consistent with a role in reproduction. Immunofluorescence studies indicate that its expression is limited to early meiotic prophase I, preceding the synapsis of homologous chromosomes. A T-DNA insertional mutant (Atmsh4) exhibited normal vegetative growth but a severe reduction in fertility, consistent with a meiotic defect; this was confirmed by cytological analysis of meiosis. RNAi-induced down-regulation of the MSH4 gene resulted in a similar fertility and meiotic phenotype. We demonstrate that prophase I chromosome synapsis is delayed and may be incomplete in Atmsh4, and metaphase I chiasma frequency is greatly reduced to approximately 15% of wild type, leading to univalence and nondisjunction. We show that these residual chiasmata are randomly distributed among cells and chromosomes. These features of chiasma frequency and distribution in Atmsh4 show close parallels to MSH4-independent crossovers in budding yeast that have been proposed to originate by a separate pathway. Furthermore, the characteristics of the MSH4-independent chiasmata in the Atmsh4 mutant closely parallel those of second-pathway crossovers that have been postulated from Arabidopsis crossover analysis and mathematical modeling. Taken together, this evidence strongly indicates that Arabidopsis possesses two crossover pathways.