Following the Formation of Synaptonemal Complex Formation in Wheat and Barley by High-Resolution Microscopy.

Wheat and barley have large genomes of 15 Gb and 5.1 Gb, respectively, which is much larger than the human genome (3.3 Gb). The release of their respective genomes has been a tremendous advance the understanding of the genome organization and the ability for deeper functional analysis in particular meiosis. Meiosis is the cell division required during sexual reproduction. One major event of meiosis is called recombination, or the formation of crossing over, a tight link between homologous chromosomes, ensuring gene exchange and faithful chromosome segregation. Recombination is a major driver of genetic diversity but in these large genome crops, the vast majority of these events is constrained at the end of their chromosomes. It is estimated that in barley, about 30% of the genes are located within the poor recombining centromeric regions, making important traits, such as resistance to pest and disease for example, difficult to access. Increasing recombination in these crops has the potential to speed up breeding program and requires a good understand of the meiotic mechanism. However, most research on recombination in plant has been carried in Arabidopsis thaliana which despite many of the advantages it brings for plant research, has a small genome and more spread out of recombination compare to barley or wheat. Advance in microscopy and cytological procedures have emerged in the last few years, allowing to follow meiotic events in these crops. This protocol provides the steps required for cytological preparation of barley and wheat pollen mother cells for light microscopy, highlighting some of the differences between the two cereals.

Observation of Extensive Chromosome Axis Remodeling during the "Diffuse-Phase" of Meiosis in Large Genome Cereals.

The production of balanced fertile haploid gametes requires the faithful separation of paired (synapsed) chromosomes toward the end of meiotic prophase I (desynapsis). This involves the timely dissolution of the synaptonemal complex during the pachytene-diplotene transition, a stage traditionally referred to as the "diffuse stage." In species with large genomes such as, barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) we know most about the early stages of meiotic prophase I. There, synapsis initiates at the telomeric ends of chromosomes and progresses toward the centromeric regions through the ordered assembly of the synaptonemal complex (SC). Synapsis is impacted by recombination (crossing over, CO) which locally modifies the extent of chromatin compaction and extension. CO is uneven along the chromosomes, occurring mainly toward the telomeric regions resulting in a highly skewed distribution of recombination events. However, we know very little about the process of desynapsis which occurs during the "diffuse stage," where the synapsed and recombined chromosomes faithfully desynapse and separate into daughter cells. Here, using 3D-SIM super-resolution immuno-cytology combined with the use of antibodies directed against two crucial SC proteins, ASY1 and ZYP1, we followed the whole of meiosis I (i.e., both synapsis and desynapsis) in both barley and wheat. We showed that synapsis forms a characteristic tri-partite SC structure in zygotene (more clearly seen in barley). Toward the end of meiosis I, as the SC starts to disassemble, we show that extensive chromosome axis remodeling results in the formation of characteristic "tinsel-like" structures in both wheat and barley. By using a mutant (des10) that is severely compromised in polymerization of ZYP1during synapsis, we show that tinsel structure formation during SC dissolution is not dependant on full synapsis and may relate instead to changes in expansion stress. Our observations highlight a potentially new role for ASYNAPSIS1 (ASY1) in desynapsis, in addition to chromosome synapsis and cohesion.