Differentiated function and localisation of SPO11-1 and PRD3 on the chromosome axis during meiotic DSB formation in Arabidopsis thaliana.

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.

Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination.

During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural Arabidopsis thaliana accessions, we identified two major recombination quantitative trait loci (rQTLs) that explain 56.9% of crossover variation in Col×Ler F2 populations. We mapped rQTL1 to semidominant polymorphisms in HEI10, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null hei10 mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional HEI10 copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest HEI10-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in Arabidopsis recombination. Our work reveals that HEI10 naturally limits Arabidopsis crossovers and has the potential to influence the response to selection.

Crossover-active regions of the wheat genome are distinguished by DMC1, the chromosome axis, H3K27me3, and signatures of adaptation.

The hexaploid bread wheat genome comprises over 16 gigabases of sequence across 21 chromosomes. Meiotic crossovers are highly polarized along the chromosomes, with elevation in the gene-dense distal regions and suppression in the Gypsy retrotransposon-dense centromere-proximal regions. We profiled the genomic landscapes of the meiotic recombinase DMC1 and the chromosome axis protein ASY1 in wheat and investigated their relationships with crossovers, chromatin state, and genetic diversity. DMC1 and ASY1 chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed strong co-enrichment in the distal, crossover-active regions of the wheat chromosomes. Distal ChIP-seq enrichment is consistent with spatiotemporally biased cytological immunolocalization of DMC1 and ASY1 close to the telomeres during meiotic prophase I. DMC1 and ASY1 ChIP-seq peaks show significant overlap with genes and transposable elements in the Mariner and Mutator superfamilies. However, DMC1 and ASY1 ChIP-seq peaks were detected along the length of each chromosome, including in low-crossover regions. At the fine scale, crossover elevation at DMC1 and ASY1 peaks and genes correlates with enrichment of the Polycomb histone modification H3K27me3. This indicates a role for facultative heterochromatin, coincident with high DMC1 and ASY1, in promoting crossovers in wheat and is reflected in distalized H3K27me3 enrichment observed via ChIP-seq and immunocytology. Genes with elevated crossover rates and high DMC1 and ASY1 ChIP-seq signals are overrepresented for defense-response and immunity annotations, have higher sequence polymorphism, and exhibit signatures of selection. Our findings are consistent with meiotic recombination promoting genetic diversity, shaping host-pathogen co-evolution, and accelerating adaptation by increasing the efficiency of selection.

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.

ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis.

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.

Identification, characterization, and rescue of CRISPR/Cas9 generated wheat SPO11-1 mutants.

Increasing crop yields through plant breeding is time consuming and laborious, with the generation of novel combinations of alleles being limited by chromosomal linkage blocks and linkage-drag. Meiotic recombination is essential to create novel genetic variation via the reshuffling of parental alleles. The exchange of genetic information between homologous chromosomes occurs at crossover (CO) sites but CO frequency is often low and unevenly distributed. This bias creates the problem of linkage-drag in recombination 'cold' regions, where undesirable variation remains linked to useful traits. In plants, programmed meiosis-specific DNA double-strand breaks, catalysed by the SPO11 complex, initiate the recombination pathway, although only ~5% result in the formation of COs. To study the role of SPO11-1 in wheat meiosis, and as a prelude to manipulation, we used CRISPR/Cas9 to generate edits in all three SPO11-1 homoeologues of hexaploid wheat. Characterization of progeny lines shows plants deficient in all six SPO11-1 copies fail to undergo chromosome synapsis, lack COs and are sterile. In contrast, lines carrying a single copy of any one of the three wild-type homoeologues are phenotypically indistinguishable from unedited plants both in terms of vegetative growth and fertility. However, cytogenetic analysis of the edited plants suggests that homoeologues differ in their ability to generate COs and in the dynamics of synapsis. In addition, we show that the transformation of wheat mutants carrying six edited copies of SPO11-1 with the TaSPO11-1B gene, restores synapsis, CO formation, and fertility and hence opens a route to modifying recombination in this agronomically important crop.

Interacting Genomic Landscapes of REC8-Cohesin, Chromatin, and Meiotic Recombination in Arabidopsis.

Meiosis recombines genetic variation and influences eukaryote genome evolution. During meiosis, DNA double-strand breaks (DSBs) enter interhomolog repair to yield crossovers and noncrossovers. DSB repair occurs as replicated sister chromatids are connected to a polymerized axis. Cohesin rings containing the REC8 kleisin subunit bind sister chromatids and anchor chromosomes to the axis. Here, we report the genomic landscape of REC8 using chromatin immunoprecipitation sequencing (ChIP-seq) in Arabidopsis (Arabidopsis thaliana). REC8 associates with regions of high nucleosome occupancy in multiple chromatin states, including histone methylation at H3K4 (expressed genes), H3K27 (silent genes), and H3K9 (silent transposons). REC8 enrichment is associated with suppression of meiotic DSBs and crossovers at the chromosome and fine scales. As REC8 enrichment is greatest in transposon-dense heterochromatin, we repeated ChIP-seq in kyp suvh5 suvh6 H3K9me2 mutants. Surprisingly, REC8 enrichment is maintained in kyp suvh5 suvh6 heterochromatin and no defects in centromeric cohesion were observed. REC8 occupancy within genes anti-correlates with transcription and is reduced in COPIA transposons that reactivate expression in kyp suvh5 suvh6 Abnormal axis structures form in rec8 that recruit DSB-associated protein foci and undergo synapsis, which is followed by chromosome fragmentation. Therefore, REC8 occupancy correlates with multiple chromatin states and is required to organize meiotic chromosome architecture and interhomolog recombination.

Derived alleles of two axis proteins affect meiotic traits in autotetraploid Arabidopsis arenosa.

Polyploidy, which results from whole genome duplication (WGD), has shaped the long-term evolution of eukaryotic genomes in all kingdoms. Polyploidy is also implicated in adaptation, domestication, and speciation. Yet when WGD newly occurs, the resulting neopolyploids face numerous challenges. A particularly pernicious problem is the segregation of multiple chromosome copies in meiosis. Evolution can overcome this challenge, likely through modification of chromosome pairing and recombination to prevent deleterious multivalent chromosome associations, but the molecular basis of this remains mysterious. We study mechanisms underlying evolutionary stabilization of polyploid meiosis using Arabidopsis arenosa, a relative of A. thaliana with natural diploid and meiotically stable autotetraploid populations. Here we investigate the effects of ancestral (diploid) versus derived (tetraploid) alleles of two genes, ASY1 and ASY3, that were among several meiosis genes under selection in the tetraploid lineage. These genes encode interacting proteins critical for formation of meiotic chromosome axes, long linear multiprotein structures that form along sister chromatids in meiosis and are essential for recombination, chromosome segregation, and fertility. We show that derived alleles of both genes are associated with changes in meiosis, including reduced formation of multichromosome associations, reduced axis length, and a tendency to more rod-shaped bivalents in metaphase I. Thus, we conclude that ASY1 and ASY3 are components of a larger multigenic solution to polyploid meiosis in which individual genes have subtle effects. Our results are relevant for understanding polyploid evolution and more generally for understanding how meiotic traits can evolve when faced with challenges.

Factors underlying restricted crossover localization in barley meiosis.

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.

Unravelling mechanisms that govern meiotic crossover formation in wheat.

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.

The Role of Topoisomerase II in DNA Repair and Recombination in Arabidopsis thaliana.

DNA entanglements and supercoiling arise frequently during normal DNA metabolism. DNA topoisomerases are highly conserved enzymes that resolve the topological problems that these structures create. Topoisomerase II (TOPII) releases topological stress in DNA by removing DNA supercoils through breaking the two DNA strands, passing a DNA duplex through the break and religating the broken strands. TOPII performs key DNA metabolic roles essential for DNA replication, chromosome condensation, heterochromatin metabolism, telomere disentanglement, centromere decatenation, transmission of crossover (CO) interference, interlock resolution and chromosome segregation in several model organisms. In this study, we reveal the endogenous role of Arabidopsis thaliana TOPII in normal root growth and cell cycle, and mitotic DNA repair via homologous recombination. Additionally, we show that the protein is required for meiotic DSB repair progression, but not for CO formation. We propose that TOPII might promote mitotic HR DNA repair by relieving stress needed for HR strand invasion and D-loop formation.

Identification of ASYNAPTIC4, a Component of the Meiotic Chromosome Axis.

During the leptotene stage of prophase I of meiosis, chromatids become organized into a linear looped array via a protein axis that forms along the loop bases. Establishment of the axis is essential for the subsequent synapsis of the homologous chromosome pairs and the progression of recombination to form genetic crossovers. Here, we describe ASYNAPTIC4 (ASY4), a meiotic axis protein in Arabidopsis (Arabidopsis thaliana). ASY4 is a small coiled-coil protein that exhibits limited sequence similarity with the carboxyl-terminal region of the axis protein ASY3. We used enhanced yellow fluorescent protein-tagged ASY4 to show that ASY4 localizes to the chromosome axis throughout prophase I. Bimolecular fluorescence complementation revealed that ASY4 interacts with ASY1 and ASY3, and yeast two-hybrid analysis confirmed a direct interaction between ASY4 and ASY3. Mutants lacking full-length ASY4 exhibited defective axis formation and were unable to complete synapsis. Although the initiation of recombination appeared to be unaffected in the asy4 mutant, the number of crossovers was reduced significantly, and crossovers tended to group in the distal parts of the chromosomes. We conclude that ASY4 is required for normal axis and crossover formation. Furthermore, our data suggest that ASY3/ASY4 are the functional homologs of the mammalian SYCP2/SYCP3 axial components.

Understanding and Manipulating Meiotic Recombination in Plants.

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.

Naturally occurring differences in CENH3 affect chromosome segregation in zygotic mitosis of hybrids.

The point of attachment of spindle microtubules to metaphase chromosomes is known as the centromere. Plant and animal centromeres are epigenetically specified by a centromere-specific variant of Histone H3, CENH3 (a.k.a. CENP-A). Unlike canonical histones that are invariant, CENH3 proteins are accumulating substitutions at an accelerated rate. This diversification of CENH3 is a conundrum since its role as the key determinant of centromere identity remains a constant across species. Here, we ask whether naturally occurring divergence in CENH3 has functional consequences. We performed functional complementation assays on cenh3-1, a null mutation in Arabidopsis thaliana, using untagged CENH3s from increasingly distant relatives. Contrary to previous results using GFP-tagged CENH3, we find that the essential functions of CENH3 are conserved across a broad evolutionary landscape. CENH3 from a species as distant as the monocot Zea mays can functionally replace A. thaliana CENH3. Plants expressing variant CENH3s that are fertile when selfed show dramatic segregation errors when crossed to a wild-type individual. The progeny of this cross include hybrid diploids, aneuploids with novel genetic rearrangements and haploids that inherit only the genome of the wild-type parent. Importantly, it is always chromosomes from the plant expressing the divergent CENH3 that missegregate. Using chimeras, we show that it is divergence in the fast-evolving N-terminal tail of CENH3 that is causing segregation errors and genome elimination. Furthermore, we analyzed N-terminal tail sequences from plant CENH3s and discovered a modular pattern of sequence conservation. From this we hypothesize that while the essential functions of CENH3 are largely conserved, the N-terminal tail is evolving to adapt to lineage-specific centromeric constraints. Our results demonstrate that this lineage-specific evolution of CENH3 causes inviability and sterility of progeny in crosses, at the same time producing karyotypic variation. Thus, CENH3 evolution can contribute to postzygotic reproductive barriers.

Arabidopsis PCH2 Mediates Meiotic Chromosome Remodeling and Maturation of Crossovers.

Meiotic chromosomes are organized into linear looped chromatin arrays by a protein axis localized along the loop-bases. Programmed remodelling of the axis occurs during prophase I of meiosis. Structured illumination microscopy (SIM) has revealed dynamic changes in the chromosome axis in Arabidopsis thaliana and Brassica oleracea. We show that the axis associated protein ASY1 is depleted during zygotene concomitant with synaptonemal complex (SC) formation. Study of an Atpch2 mutant demonstrates this requires the conserved AAA+ ATPase, PCH2, which localizes to the sites of axis remodelling. Loss of PCH2 leads to a failure to deplete ASY1 from the axes and compromizes SC polymerisation. Immunolocalization of recombination proteins in Atpch2 indicates that recombination initiation and CO designation during early prophase I occur normally. Evidence suggests that CO interference is initially functional in the mutant but there is a defect in CO maturation following designation. This leads to a reduction in COs and a failure to form COs between some homologous chromosome pairs leading to univalent chromosomes at metaphase I. Genetic analysis reveals that CO distribution is also affected in some chromosome regions. Together these data indicate that the axis remodelling defect in Atpch2 disrupts normal patterned formation of COs.

The synaptonemal complex protein ZYP1 is required for imposition of meiotic crossovers in barley.

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.

A spontaneous mutation in MutL-Homolog 3 (HvMLH3) affects synapsis and crossover resolution in the barley desynaptic mutant des10.

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.

Retinoblastoma protein is essential for early meiotic events in Arabidopsis.

We have analysed the role of RBR (retinoblastoma related), the Arabidopsis homologue of the tumour suppressor Retinoblastoma protein (pRb), during meiosis. We characterise the rbr-2 mutation, which causes a loss of RBR in male meiocytes. The rbr-2 plants exhibit strongly reduced fertility, while vegetative growth is generally unaffected. The reduced fertility is due to a meiotic defect that results in reduced chiasma formation and subsequent errors in chromosome disjunction. Immunolocalisation studies in wild-type meiocytes reveal that RBR is recruited as foci to the chromosomes during early prophase I in a DNA double-strand-break-dependent manner. In the absence of RBR, expression of several meiotic genes is reduced. The localisation of the recombinases AtRAD51 and AtDMC1 is normal. However, localisation of the MutS homologue AtMSH4 is compromised. Additionally, polymerisation of the synaptonemal complex protein AtZYP1 is abnormal. Together, these data indicate that loss of RBR during meiosis results in a reduction of crossover formation and an associated failure in chromosome synapsis. Our results indicate that RBR has an important role in meiosis affecting different aspects of this complex process.

Spatiotemporal asymmetry of the meiotic program underlies the predominantly distal distribution of meiotic crossovers in barley.

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.

Inter-homolog crossing-over and synapsis in Arabidopsis meiosis are dependent on the chromosome axis protein AtASY3.

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.