ASYNAPSIS 1 ensures crossover fidelity in polyploid wheat by promoting homologous recombination and suppressing non-homologous recombination

During meiosis, the chromosome axes and synaptonemal complex mediate chromosome pairing and homologous recombination to maintain genomic stability and accurate chromosome segregation. In plants, ASYNAPSIS 1 (ASY1) is a key component of the chromosome axis that promotes inter-homolog recombination, synapsis and crossover formation. Here, the function of ASY1 has been cytologically characterized in a series of hypomorphic wheat mutants. In tetraploid wheat, asy1 hypomorphic mutants experience a reduction in chiasmata (crossovers) in a dosage-specific manner, resulting in failure to maintain crossover (CO) assurance. In mutants with only one functional copy of ASY1, distal chiasmata are maintained at the expense of proximal and interstitial chiasmata, indicating that ASY1 is required to promote chiasma formation away from the chromosome ends. Meiotic prophase I progression is delayed in asy1 hypomorphic mutants and is arrested in asy1 null mutants. In both tetraploid and hexaploid wheat, single asy1 mutants exhibit a high degree of ectopic recombination between multiple chromosomes at metaphase I. To explore the nature of the ectopic recombination, Triticum turgidum asy1b-2 was crossed with wheat-wild relative Aegilops variabilis. Homoeologous chiasmata increased 3.75-fold in Ttasy1b-2/Ae. variabilis compared to wild type/Ae. variabilis, indicating that ASY1 suppresses chiasma formation between divergent, but related chromosomes. These data suggest that ASY1 promotes recombination along the chromosome arms of homologous chromosomes whilst suppressing recombination between non-homologous chromosomes. Therefore, asy1 mutants could be utilized to increase recombination between wheat wild relatives and elite varieties for expediting introgression of important agronomic traits.</p

ASYNAPSIS 1 ensures crossover fidelity in polyploid wheat by promoting homologous recombination and suppressing non-homologous recombination

During meiosis, the chromosome axes and synaptonemal complex mediate chromosome pairing and homologous recombination to maintain genomic stability and accurate chromosome segregation. In plants, ASYNAPSIS 1 (ASY1) is a key component of the chromosome axis that promotes inter-homolog recombination, synapsis and crossover formation. Here, the function of ASY1 has been cytologically characterized in a series of hypomorphic wheat mutants. In tetraploid wheat, asy1 hypomorphic mutants experience a reduction in chiasmata (crossovers) in a dosage-specific manner, resulting in failure to maintain crossover (CO) assurance. In mutants with only one functional copy of ASY1, distal chiasmata are maintained at the expense of proximal and interstitial chiasmata, indicating that ASY1 is required to promote chiasma formation away from the chromosome ends. Meiotic prophase I progression is delayed in asy1 hypomorphic mutants and is arrested in asy1 null mutants. In both tetraploid and hexaploid wheat, single asy1 mutants exhibit a high degree of ectopic recombination between multiple chromosomes at metaphase I. To explore the nature of the ectopic recombination, Triticum turgidum asy1b-2 was crossed with wheat-wild relative Aegilops variabilis. Homoeologous chiasmata increased 3.75-fold in Ttasy1b-2/Ae. variabilis compared to wild type/Ae. variabilis, indicating that ASY1 suppresses chiasma formation between divergent, but related chromosomes. These data suggest that ASY1 promotes recombination along the chromosome arms of homologous chromosomes whilst suppressing recombination between non-homologous chromosomes. Therefore, asy1 mutants could be utilized to increase recombination between wheat wild relatives and elite varieties for expediting introgression of important agronomic traits.</p

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.</p

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 duplicatedArabidopsistransverse filament geneszyp1aandzyp1busing a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of thezyp1null 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 thezyp1mutant compared to wild type. The increase in recombination inzyp1does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% inmsh5/zyp1compared tomsh5. These data suggest that ZYP1 limits the formation of closely spaced Class I COs inArabidopsis. 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

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 duplicatedArabidopsistransverse filament geneszyp1aandzyp1busing a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of thezyp1null 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 thezyp1mutant compared to wild type. The increase in recombination inzyp1does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% inmsh5/zyp1compared tomsh5. These data suggest that ZYP1 limits the formation of closely spaced Class I COs inArabidopsis. 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

MSH2 stimulates interfering and inhibits non-interfering crossovers in response to genetic polymorphism

Meiotic crossovers can be formed through the interfering pathway, in which one crossover prevents another from forming nearby, or by an independent non-interfering pathway. In Arabidopsis, local sequence polymorphism between homologs can stimulate interfering crossovers in a MSH2-dependent manner. To understand how MSH2 regulates crossovers formed by the two pathways, we combined Arabidopsis mutants that elevate non-interfering crossovers with msh2 mutants. We demonstrate that MSH2 blocks non-interfering crossovers at polymorphic loci, which is the opposite effect to interfering crossovers. We also observe MSH2-independent crossover inhibition at highly polymorphic sites. We measure recombination along the chromosome arms in lines differing in patterns of heterozygosity and observe a MSH2-dependent crossover increase at the boundaries between heterozygous and homozygous regions. Here, we show that MSH2 is a master regulator of meiotic DSB repair in Arabidopsis, with antagonistic effects on interfering and non-interfering crossovers, which shapes the crossover landscape in relation to interhomolog polymorphism.</p

A novel allele of ASY3 is associated with greater meiotic stability in autotetraploid Arabidopsis lyrata

In this study we performed a genotype-phenotype association analysis of meiotic stability in 10 autotetraploid Arabidopsis lyrata and A. lyrata/A. arenosa hybrid populations collected from the Wachau region and East Austrian Forealps. The aim was to determine the effect of eight meiosis genes under extreme selection upon adaptation to whole genome duplication. Individual plants were genotyped by high-throughput sequencing of the eight meiosis genes (ASY1, ASY3, PDS5b, PRD3, REC8, SMC3, ZYP1a/b) implicated in synaptonemal complex formation and phenotyped by assessing meiotic metaphase I chromosome configurations. Our results reveal that meiotic stability varied greatly (20–100%) between individual tetraploid plants and associated with segregation of a novel ASYNAPSIS3 (ASY3) allele derived from A. lyrata. The ASY3 allele that associates with meiotic stability possesses a putative in-frame tandem duplication (TD) of a serine-rich region upstream of the coiled-coil domain that appears to have arisen at sites of DNA microhomology. The frequency of multivalents observed in plants homozygous for the ASY3 TD haplotype was significantly lower than in plants heterozygous for ASY3 TD/ND (non-duplicated) haplotypes. The chiasma distribution was significantly altered in the stable plants compared to the unstable plants with a shift from proximal and interstitial to predominantly distal locations. The number of HEI10 foci at pachytene that mark class I crossovers was significantly reduced in a plant homozygous for ASY3 TD compared to a plant heterozygous for ASY3 ND/TD. Fifty-eight alleles of the 8 meiosis genes were identified from the 10 populations analysed, demonstrating dynamic population variability at these loci. Widespread chimerism between alleles originating from A. lyrata/A. arenosa and diploid/tetraploids indicates that this group of rapidly evolving genes may provide precise adaptive control over meiotic recombination in the tetraploids, the very process that gave rise to them

MSH2 stimulates interfering and inhibits non-interfering crossovers in response to genetic polymorphism

Meiotic crossovers can be formed through the interfering pathway, in which one crossover prevents another from forming nearby, or by an independent non-interfering pathway. In Arabidopsis, local sequence polymorphism between homologs can stimulate interfering crossovers in a MSH2-dependent manner. To understand how MSH2 regulates crossovers formed by the two pathways, we combined Arabidopsis mutants that elevate non-interfering crossovers with msh2 mutants. We demonstrate that MSH2 blocks non-interfering crossovers at polymorphic loci, which is the opposite effect to interfering crossovers. We also observe MSH2-independent crossover inhibition at highly polymorphic sites. We measure recombination along the chromosome arms in lines differing in patterns of heterozygosity and observe a MSH2-dependent crossover increase at the boundaries between heterozygous and homozygous regions. Here, we show that MSH2 is a master regulator of meiotic DSB repair in Arabidopsis, with antagonistic effects on interfering and non-interfering crossovers, which shapes the crossover landscape in relation to interhomolog polymorphism.</p

Distal Bias of Meiotic Crossovers in Hexaploid Bread Wheat Reflects Spatio-Temporal Asymmetry of the Meiotic Program

Meiotic recombination generates genetic variation and provides physical links between homologous chromosomes (crossovers) essential for accurate segregation. In cereals the distribution of crossovers, cytologically evident as chiasmata, is biased toward the distal regions of chromosomes. This creates a bottleneck for plant breeders in the development of varieties with improved agronomic traits, as genes situated in the interstitial and centromere proximal regions of chromosomes rarely recombine. Recent advances in wheat genomics and genome engineering combined with well-developed wheat cytogenetics offer new opportunities to manipulate recombination and unlock genetic variation. As a basis for these investigations we have carried out a detailed analysis of meiotic progression in hexaploid wheat (Triticum aestivum) using immunolocalization of chromosome axis, synaptonemal complex and recombination proteins. 5-Bromo-2′-deoxyuridine (BrdU) labeling was used to determine the chronology of key events in relation to DNA replication. Axis morphogenesis, synapsis and recombination initiation were found to be spatio-temporally coordinated, beginning in the gene-dense distal chromosomal regions and later occurring in the interstitial/proximal regions. Moreover, meiotic progression in the distal regions was coordinated with the conserved chromatin cycles that are a feature of meiosis. This mirroring of the chiasma bias was also evident in the distribution of the gene-associated histone marks, H3K4me3 and H3K27me3; the repeat-associated mark, H3K27me1; and H3K9me3. We believe that this study provides a cytogenetic framework for functional studies and ongoing initiatives to manipulate recombination in the wheat genome

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.</p