Susan J. Armstrong

Using a positional candidate-gene approach, we show that semi-sterile desynaptic8 mutants are associated with deleterious variants of the barley homolog of XRCC2 (X-Ray Repair Cross Complementing 2). In barley XRCC2 mutants, the initial meiotic progression is normal, albeit with a small delay in initiation, with completion of synapsis. However, the absence of HvXRCC2 subsequently leads to a dramatic reduction in the number of crossovers, chromosome mis-segregation, and infertility, suggesting that HvXRCC2 plays a major role in recombination. This mutant phenotype is congruent with that reported in mammalian studies but contrasts with the XRCC2 mutant in Arabidopsis which is fertile, exhibits normal chromosome pairing and correct chromosome segregation, and is associated with an increased rate of crossovers. This observation indicates that the XRCC2 mutant phenotype in Arabidopsis is not representative of all plants and that XRCC2 is not a good candidate for the modulation of recombination in barley.

Using a positional candidate-gene approach we show that semi-sterile desynaptic8 mutants are associated with deletions in or complete knockout of the barley homolog of XRCC2 ( X-Ray Repair Cross Complementing 2 ). In barley XRCC2 mutants, the initial meiotic progression is normal, albeit with a small delay in initiation, with completion of synapsis. However, the absence of HvXRCC2 subsequently leads to a dramatic reduction in the number of crossovers, chromosome mis-segregation, and infertility, suggesting that HvXRCC2 plays a major role in recombination. This mutant phenotype is congruent with that reported in mammalian studies but contrasts with the XRCC2 mutant in Arabidopsis which is fertile, exhibits normal chromosome pairing and correct chromosome segregation, and is associated with an increased rate of crossovers. This indicates that the XRCC2 mutant phenotype in Arabidopsis is not representative of all plants and that XRCC2 is not a good candidate for the modulation of recombination in barley. Highlight: The mutants of the barley homolog of XRCC2 exhibit delays in replication leading to defective meiosis, altered RAD51 orthologue behaviour, and significant reduction in the number of crossovers as in canonical mammalian XRCC2 mutants but unlike those in Arabidopsis.

Meiosis generates genetic variation through homologous recombination (HR) that is harnessed during breeding. HR occurs in the context of meiotic chromosome axes and the synaptonemal complex. To study the role of axis remodelling in crossover (CO) formation in a crop species, we characterized mutants of the axis-associated protein ASY1 and the axis-remodelling protein PCH2 in Brassica rapa. asy1 plants form meiotic chromosome axes that fail to synapse. CO formation is almost abolished, and residual chiasmata are proportionally enriched in terminal chromosome regions, particularly in the nucleolar organizing region (NOR)-carrying chromosome arm. pch2 plants show impaired ASY1 loading and remodelling, consequently achieving only partial synapsis, which leads to reduced CO formation and loss of the obligatory CO. PCH2-independent chiasmata are proportionally enriched towards distal chromosome regions. Similarly, in Arabidopsis pch2, COs are increased towards telomeric regions at the expense of (peri-) centromeric COs compared with the wild type. Taken together, in B. rapa, axis formation and remodelling are critical for meiotic fidelity including synapsis and CO formation, and in asy1 and pch2 CO distributions are altered. While asy1 plants are sterile, pch2 plants are semi-sterile and thus PCH2 could be an interesting target for breeding programmes.

Summary: Ensuring faithful homologous recombination in allopolyploids is essential to maintain optimal fertility of the species. Variation in the ability to control aberrant pairing between homoeologous chromosomes in Brassica napus has been identified. The current study exploited the extremes of such variation to identify genetic factors that differentiate newly resynthesised B. napus, which is inherently unstable, and established B. napus, which has adapted to largely control homoeologous recombination. A segregating B. napus mapping population was analysed utilising both cytogenetic observations and high‐throughput genotyping to quantify the levels of homoeologous recombination. Three quantitative trait loci (QTL) were identified that contributed to the control of homoeologous recombination in the important oilseed crop B. napus. One major QTL on BnaA9 contributed between 32 and 58% of the observed variation. This study is the first to assess homoeologous recombination and map associated QTLs resulting from deviations in normal pairing in allotetraploid B. napus. The identified QTL regions suggest candidate meiotic genes that could be manipulated in order to control this important trait and further allow the development of molecular markers to utilise this trait to exploit homoeologous recombination in a crop.

Field cress (Lepidium campestreL.), despite its potential as a sustainable alternative oilseed plant, has been underutilized, and no prior attempts to characterize the genome at the genetic or molecular cytogenetic level have been conducted. Genetic maps are the foundation for anchoring and orienting annotated genome assemblies and positional cloning of candidate genes. Our principal goal was to construct a genetic map using integrated approaches of genetic, comparative and cytogenetic map analyses. In total, 503 F₂interspecific hybrid individuals were genotyped using 7,624 single nucleotide polymorphism markers. Comparative analysis demonstrated that ~57% of the sequenced loci inL. campestrewere congruent withArabidopsis thaliana(L.) genome and suggested a novel karyotype, which predates the ancestral crucifer karyotype. Aceto-orcein chromosome staining and fluorescencein situhybridization (FISH) analyses confirmed thatL. campestre,L. heterophyllumBenth. and their hybrids had a chromosome number of 2n = 2x = 16. Flow cytometric analysis revealed that both species possess 2C roughly 0.4 picogram DNA. Integrating linkage and comparative maps with cytogenetic map analyses assigned two linkage groups to their particular chromosomes. Future work could incorporate FISH utilizingA. thalianamapped BAC clones to allow the chromosomes of field cress to be identified reliably.

The heavy selection pressure due to intensive breeding of Brassica napus has created a narrow gene pool, limiting the ability to produce improved varieties through crosses between B. napus cultivars. One mechanism that has contributed to the adaptation of important agronomic traits in the allotetraploid B. napus has been chromosomal rearrangements resulting from homoeologous recombination between the constituent A and C diploid genomes. Determining the rate and distribution of such events in natural B. napus will assist efforts to understand and potentially manipulate this phenomenon. The Brassica high-density 60K SNP array, which provides genome-wide coverage for assessment of recombination events, was used to assay 254 individuals derived from 11 diverse cultivated spring type B. napus. These analyses identified reciprocal allele gain and loss between the A and C genomes and allowed visualization of de novo homoeologous recombination events across the B. napus genome. The events ranged from loss/gain of 0.09 Mb to entire chromosomes, with almost 5% aneuploidy observed across all gametes. There was a bias toward sub-telomeric exchanges leading to genome homogenization at chromosome termini. The A genome replaced the C genome in 66% of events, and also featured more dominantly in gain of whole chromosomes. These analyses indicate de novo homoeologous recombination is a continuous source of variation in established Brassica napus and the rate of observed events appears to vary with genetic background. The Brassica 60K SNP array will be a useful tool in further study and manipulation of this phenomenon.

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. In the reproductive cells of many eukaryotes, a process called meiosis generates haploid gametes. During meiosis, homologous parental chromosomes (homologs) recombine forming crossovers (CO) that provide genetic variation. CO formation generates physical links called chiasmata, which are essential for accurate homolog segregation. CO control designates a sub-set of recombination precursors that will mature to form at least one chiasma between each homolog pair. Recombination is accompanied by extensive chromosome reorganization. Formation of a proteinaceous axis organizes the pairs of sister chromatids of each homolog into conjoined linear looped chromatin arrays. Pairs of homologs then align and synapse becoming closely associated along their length by a protein structure, the synaptonemal complex (SC). The SC is disassembled at the end of prophase I and recombination is completed. We have investigated the link between recombination and chromosome remodelling by analysing the role of a protein, PCH2, which we show is required for remodelling of the chromosome axis during SC formation. In wild type, immunolocalization reveals depletion of the axis-associated signal of the axis component, ASY1, along synapsed regions of the chromosomes. In the absence of PCH2, the ASY1 signal is not depleted from the chromosome axis and the SC does not form normally. Although this defect in chromosome remodelling has no obvious effect on CO designation, CO maturation is perturbed such that the formation of at least one CO per homolog pair no longer occurs.

Meiotic crossovers (COs) shape genetic diversity by mixing homologous chromosomes at each generation. CO distribution is a highly regulated process. CO assurance forces the occurrence of at least one obligatory CO per chromosome pair, CO homeostasis smoothes out the number of COs when faced with variation in precursor number and CO interference keeps multiple COs away from each other along a chromosome. In several organisms, it has been shown that cytoskeleton forces are transduced to the meiotic nucleus via KASH- and SUN-domain proteins, to promote chromosome synapsis and recombination. Here we show that the Arabidopsis kinesin AtPSS1 plays a major role in chromosome synapsis and regulation of CO distribution. In Atpss1 meiotic cells, chromosome axes and DNA double strand breaks (DSBs) appear to form normally but only a variable portion of the genome synapses and is competent for CO formation. Some chromosomes fail to form the obligatory CO, while there is an increased CO density in competent regions. However, the total number of COs per cell is unaffected. We further show that the kinesin motor domain of AtPSS1 is required for its meiotic function, and that AtPSS1 interacts directly with WIP1 and WIP2, two KASH-domain proteins. Finally, meiocytes missing AtPSS1 and/or SUN proteins show similar meiotic defects suggesting that AtPSS1 and SUNs act in the same pathway. This suggests that forces produced by the AtPSS1 kinesin and transduced by WIPs/SUNs, are required to authorize complete synapsis and regulate maturation of recombination intermediates into COs. We suggest that a form of homeostasis applies, which maintains the total number of COs per cell even if only a part of the genome is competent for CO formation. In species that reproduce sexually, diploid individuals have two copies of each chromosome, inherited from their father and mother. During a special cell division called meiosis, these two sets of chromosomes are mixed by homologous recombination to give genetically unique chromosomes that will be transmitted to the next generation. Homologous recombination processes are highly controlled in terms of number and localization of events within and among chromosomes. Disruption of this control (a lack of or improper positioning of homologous recombination events) causes deleterious chromosome associations in the offspring. Using the model plant Arabidopsis thaliana we reveal here that the AtPSS1 gene is required for proper localization of these homologous recombination events along the genome. We also show that AtPSS1, which belongs to a family of proteins able to move along the cytoskeleton, is likely part of a module that allows cytoplasmic forces to be transmitted through the nucleus envelope to promote chromosome movements during homologous recombination progression.

Much of meiosis research is focussed on Arabidopsis thaliana, largely due to the significant advantages it brings, having a small sequenced genome with comparatively little repetitive DNA, the ease of forward and reverse genetics, and a short life cycle. On the other hand, due the small genome size using Arabidopsis may be problematic for generating sufficient meiotic material for other types of analysis e.g., proteomics using prophase meiocytes and cytological analysis of the synaptonemal complex at the subcellular level. One solution is to use closely related species with larger genomes, in this case the Brassicas. This chapter contains methods for spreading of Brassica oleracea meiocytes for the analysis of the synaptonemal complex by silver staining and immunolocalization with gold-coupled antibodies using transmission electron microscopy.

During meiosis, homologous chromosomes recognize each other, align, and exchange genetic information. This process requires the action of RecA-related proteins Rad51 and Dmc1 to catalyze DNA strand exchanges. The Mnd1–Hop2 complex has been shown to assist in Dmc1-dependent processes. Furthermore, higher eukaryotes possess additional RecA-related proteins, like XRCC3, which are involved in meiotic recombination. However, little is known about the functional interplay between these proteins during meiosis. We investigated the functional relationship between AtMND1, AtDMC1, AtRAD51, and AtXRCC3 during meiosis in Arabidopsis thaliana. We demonstrate the localization of AtMND1 to meiotic chromosomes, even in the absence of recombination, and show that AtMND1 loading depends exclusively on AHP2, the Arabidopsis Hop2 homolog. We provide evidence of genetic interaction between AtMND1, AtDMC1, AtRAD51, and AtXRCC3. In vitro assays suggest that this functional link is due to direct interaction of the AtMND1–AHP2 complex with AtRAD51 and AtDMC1. We show that AtDMC1 foci accumulate in the Atmnd1 mutant, but are reduced in number in Atrad51 and Atxrcc3 mutants. This study provides the first insights into the functional differences of AtRAD51 and AtXRCC3 during meiosis, demonstrating that AtXRCC3 is dispensable for AtDMC1 focus formation in an Atmnd1 mutant background, whereas AtRAD51 is not. These results clarify the functional interactions between key players in the strand exchange processes during meiotic recombination. Furthermore, they highlight a direct interaction between MND1 and RAD51 and show a functional divergence between RAD51 and XRCC3. During meiosis, two rounds of chromosome segregation follow a single round of chromosome duplication, leading to the production of haploid gametes. It is during this specialised cell division that genetic traits are recombined, achieved by mutual exchange of DNA sequences of homologous chromosomes. Recombination events must be well controlled to ensure that: (i) they occur between homologous chromosomes rather than sister chromatids and (ii) they occur between true homologous sequences and not duplicated sequences present elsewhere in the genome. At one of the early steps of homologous recombination, a single-strand DNA molecule recognizes and invades the targeted homologous sequence. Many proteins, which are conserved throughout the kingdoms, are involved in this crucial step of DNA homology search. In this study, we analyze the role of a series of such proteins and their complex functional relationships in the model plant A. thaliana, shedding new light on this highly regulated step of meiotic recombination.