High Resolution Analysis of Meiotic Chromosome Structure and Behaviour in Barley (Hordeum vulgare L.)

Reciprocal crossing over and independent assortment of chromosomes during meiosis generate most of the genetic variation in sexually reproducing organisms. In barley, crossovers are confined primarily to distal regions of the chromosomes, which means that a substantial proportion of the genes of this crop rarely, if ever, engage in recombination events. There is potentially much to be gained by redistributing crossovers to more proximal regions, but our ability to achieve this is dependent upon a far better understanding of meiosis in this species. This study explores the meiotic process by describing with unprecedented resolution the early behaviour of chromosomal domains, the progression of synapsis and the structure of the synaptonemal complex (SC). Using a combination of molecular cytogenetics and advanced fluorescence imaging, we show for the first time in this species that non-homologous centromeres are coupled prior to synapsis. We demonstrate that at early meiotic prophase the loading of the SC-associated structural protein ASY1, the cluster of telomeres, and distal synaptic initiation sites occupy the same polarised region of the nucleus. Through the use of advanced 3D image analysis, we show that synapsis is driven predominantly from the telomeres, and that new synaptic initiation sites arise during zygotene. In addition, we identified two different SC configurations through the use of super-resolution 3D structured illumination microscopy (3D-SIM)

SC structures and features revealed by 3D-SIM.

<p>(A & B) Cross-sectional views of the variant SC structure in the <i>yz</i> (A) and <i>xy</i> planes (B) showing ASY1 (green) and ZYP1 (orange). Average dimensions of the various SC components (the number of measurements taken and the standard deviation shown in parenthesis). (C & D) An SC shown in the <i>xz</i> (C) and <i>yz</i> (D) planes, with two cross-sectional views (E & F) 2µm apart (red arrows) showing a change in SC conformation. (G) Series of 3 images showing a single <i>z</i> section from a limited region of a pachytene nucleus, with DAPI (grey) in the upper pane, two SCs with different conformations in the centre pane and a merged image of the upper two panes. (H & I) Twisting of the LEs during pachytene as revealed by detecting ASY1 protein (green). A left-handed twist (H) and a right-handed twist (I) are shown, together with an interpretive diagram generated in Imaris. All images have been captured by 3D-SIM from pachytene nuclei embedded in polyacrylamide. The <i>xyz</i> angles shown in each image relate to the orientation of the captured image.</p

Proposed 3D model of the variant SC structure.

<p>Proposed 3D model of the variant SC structure based upon published information about ZYP1 and ASY1 proteins and the 3D-SIM images described in this study. Chromatin loops (blue) are shown to attach in the vicinity of the ASY1 protein (green). The C-terminus of the ZYP1 protein (red) is the epitope for its antibody. The precise location of the LEs is currently unknown, so they have been excluded from the diagram.</p

Analysis of synaptic progression during barley prophase I.

<p>(A & E) Deconvolved maximum projection of a zygotene nucleus embedded in polyacrylamide and captured using CLSM, showing ASY1 cores (green) and ZYP1 cores (red). (B) Zygotene nucleus shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039539#pone-0039539-g002" target="_blank">Figure 2A</a> processed using Imaris with each of the seven synapsing bivalents isolated, showing generated surfaces for ASY1 (green) and ZYP1 (red). (C) A single bivalent extracted from the reconstruction in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039539#pone-0039539-g002" target="_blank">Figure 2B</a>, containing an example of a ZYP1 focus present on a single AE (white box). (D) Detail of an interlock isolated from the nucleus shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039539#pone-0039539-g002" target="_blank">Figure 2A</a>. For ease of interpretation, one of the bivalents has been re-coloured to show ASY1 in grey and ZYP1 in black. (E) Zygotene nucleus containing a telomere cluster delimited by darker staining DAPI (blue) and emanating Zyp1 cores identified by the white arrow. (F) Stacked bar graph showing the cumulative lengths of ZYP1 and ASY1 fragments constituting each bivalent isolated from 9 nuclei. Bivalents from the same nucleus are grouped together and ordered by descending length. Bivalent complements are ordered by ascending average percentage synapsis shown below the groups.</p

Centromere and telomere behaviour during prophase I.

<p>(A) Leptotene nucleus with 28 rendered telomere signals (red), 7 rendered centromere signals (pink) and diffuse, polarised ASY1 signals (green). (B) Leptotene nucleus with a cluster of 7 rendered telomere signals (red), 7 rendered centromere signals (pink) and linearising elements of ASY1 (green) in the same hemisphere as the telomeres. (C) FISH to an embedded leptotene nucleus with a Rabl orientation of 19 rendered telomere signals (red) and 5 rendered centromere signals (pink), and 2 separate single-locus BAC DH053N18 signals (green). (D) FISH to an embedded leptotene nucleus with a bouquet orientation of 7 rendered telomere signals (red) and 7 rendered centromere signals (pink), and 2 separate single-locus BAC DH053N18 signals (green). (E) FISH to a squashed pachytene nucleus showing centromeres (red) and a single BAC signal (green). (F) Enlargement of red box shown in E, showing the close proximity of the BAC (green) to the centromere (red). All images are deconvolved maximum projections of nuclei, and are counterstained with DAPI (blue). (A–B) were imaged by CLSM, and (C–G) by wide-field fluorescence microscopy. (A–D) For ease of counting, the positions of centromeric and telomeric FISH signals have been marked by rendered spears using Imaris, an un-rendered nucleus is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039539#pone.0039539.s001" target="_blank">Figure S1A</a>.</p

High resolution imaging of barley prophase I nuclei.

<p>(A) Diagram illustrating the nomenclature of the various planes of view of the SC, according to Moses (1968). Note that oblique section is defined as any plane of section at an angle to the axis that is not perpendicular. (B) Zygotene nucleus showing ASY1 (green) and ZYP1 (orange) cores, and chromatin (blue). (C–E) Enlargement of the region delimited by the red box in (B) showing the standard SC structure in frontal view (C), cross-sectional view (D) and oblique view (E) where a single ZYP1 structure is visible. (F–H) Enlargement of the region delimited by the yellow box in (B) showing the different SC structure. Frontal view (F), lateral view (G) and oblique view (H) where the two ZYP1 structures are visible (orange arrows) are also shown. (I) Pachytene nucleus showing ASY1 (green) and ZYP1 (orange) cores, and chromatin counterstained with DAPI (blue). (J–L) Enlargement of the synapsed region delimited by the green box in (I) showing the usual SC structure in frontal view (J), cross-sectional view (K) and lateral view (L). (M–O) Enlargement of the synapsed region delimited by the pink box in (I) showing the different SC structure in frontal view (J), cross-sectional view (N) and lateral view (O). All images are maximum projections of nuclei embedded in polyacrylamide and captured by 3D-SIM. The <i>xyz</i> angles shown in each image relate to the orientation of the captured image.</p

Density of ZYP1 in synapsing bivalents.

<p>Plot of the density of ZYP1 fragments in along the synapsing bivalent (grey circles), and the number of ZYP1 sites along the bivalent (excluding distal synapsis) (black diamonds) against percentage synapsis of 45 zygotene bivalents. P-values calculated using a 2 tailed t-test on the slope coefficient in a simple regression.</p

Quantitative high resolution mapping of HvMLH3 foci in barley pachytene nuclei reveals a strong distal bias and weak interference

In barley (Hordeum vulgare L.), chiasmata (the physical sites of genetic crossovers) are skewed towards the distal ends of chromosomes, effectively consigning a large proportion of genes to recombination coldspots. This has the effect of limiting potential genetic variability, and of reducing the efficiency of map-based cloning and breeding approaches for this crop. Shifting the sites of recombination to more proximal chromosome regions by forward and reverse genetic means may be profitable in terms of realizing the genetic potential of the species, but is predicated upon a better understanding of the mechanisms governing the sites of these events, and upon the ability to recognize real changes in recombination patterns. The barley MutL Homologue (HvMLH3), a marker for class I interfering crossovers, has been isolated and a specific antibody has been raised. Immunolocalization of HvMLH3 along with the synaptonemal complex transverse filament protein ZYP1, used in conjunction with fluorescence in situ hybridization (FISH) tagging of specific barley chromosomes, has enabled access to the physical recombination landscape of the barley cultivars Morex and Bowman. Consistent distal localization of HvMLH3 foci throughout the genome, and similar patterns of HvMLH3 foci within bivalents 2H and 3H have been observed. A difference in total numbers of HvMLH3 foci between these two cultivars has been quantified, which is interpreted as representing genotypic variation in class I crossover frequency. Discrepancies between the frequencies of HvMLH3 foci and crossover frequencies derived from linkage analysis point to the existence of at least two crossover pathways in barley. It is also shown that interference of HvMLH3 foci is relatively weak compared with other plant species