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.

. * For correspondence (fax +34 91 394 4844; e-mail pradillo@bio.ucm.es). † E. Sá nchez-Morá n and J.L. Santos acted as joint senior authors. SUMMARYThe eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, nonhomologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

Meiotic recombination is the fundamental process that produces balanced gametes and generates diversity within species. For successful meiosis, crossovers must form between homologous chromosomes. This condition is more difficult to fulfill in allopolyploid species, which have more than two sets of related chromosomes (homoeologs). Here, we investigated the formation, progression, and completion of several key hallmarks of meiosis in Brassica napus (AACC), a young polyphyletic allotetraploid crop species with closely related homoeologous chromosomes. Altogether, our results demonstrate a precocious and efficient sorting of homologous versus homoeologous chromosomes during early prophase I in two representative B. napus accessions that otherwise show a genotypic difference in the progression of homologous recombination. More strikingly, our detailed comparison of meiosis in near isogenic allohaploid and euploid plants showed that the mechanism(s) promoting efficient chromosome sorting in euploids is adjusted to promote crossover formation between homoeologs in allohaploids. This suggests that, in contrast to other polyploid species, chromosome sorting is context dependent in B. napus.

The structural features and evolutionary state of the sex chromosomes of the XX/XY species of Rumex are unknown. Here, we report a study of the meiotic behaviour of the XY bivalent in Rumex acetosella and R. suffruticosus, a new species which we describe cytogenetically for the first time in this paper, and also that of the XY(1)Y(2) trivalent of R. acetosa by both conventional cytogenetic techniques and analysis of synaptonemal complex formation. Fluorescent in situ hybridization with satellite DNA and rDNA sequences as probes was used to analyse the degree of cytogenetic differentiation between the X and Y chromosomes in order to depict their evolutionary stage in the three species. Contrasting with the advanced state of genetic differentiation between the X and the Y chromosomes in R. acetosa, we have found that R. acetosella and R. suffruticosus represent an early stage of genetic differentiation between sex chromosomes. Our findings further demonstrate the usefulness of the genus Rumex as a model for analysing the evolution of sex chromosomes in plants, since within this genus it is now possible to study the different levels of genetic differentiation between the sex chromosomes and to analyse their evolutionary history from their origin.

Triticum aestivum is an allohexaploid wheat (AABBDD) that shows diploid-like behaviour at metaphase-I. This behaviour is influenced by the action of several loci, Ph1 and Ph2 being the main loci involved.To study the effect of these two loci on chromosome pairing in T. aestivum we have analysed the synaptic pattern in fully traced spread nuclei at mid-and late-zygotene, and at pachytene, of three different genotypes of cv Chinese Spring: standard line, ph1b and ph2b mutants. The analysis of the synaptic progression showed that only a few nuclei accomplish synapsis in the ph2b genotype, whereas most nuclei completed synapsis in the standard and ph1b genotypes. This result indicates that the Ph2 locus affects synaptic progression. The number of synaptonemal complex (SC) bivalents and of the different SC multivalent associations were determined in each nucleus. The mean number of lateral elements involved in SC multivalent associations (LEm) at midzygotene was relatively high and showed similar values in the three genotypes. These values decreased progressively between mid-zygotene and pachytene in the genotypes with the Ph1 locus because of the transformation of multivalents into bivalents. In the ph1b genotype, this value only decreased between late-zygotene and pachytene. Therefore, multivalent correction was more efficient in the presence than in the absence of the Ph1 locus. It is concluded that the Ph1 and Ph2 loci bring about diploidization of allohexaploid wheat via a different mechanism.