ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis

Significance The synaptonemal complex (SC) is a meiosis-specific proteinaceous ultrastructure required to ensure cross-over (CO) formation in the majority of sexually reproducing eukaryotes. It is composed of two lateral elements adjoined by transverse filaments. Even though the general structure of the SC is conserved throughout kingdoms, phenotypic differences between mutants perpetuate the enigmatic role of the SC. Here, we have used genetic and cytogenetic approaches to show that the transverse filament protein, ZYP1, acts on multiple pathways of meiotic recombination in Arabidopsis. ZYP1 is required for CO assurance, thus ensuring that every chromosome pair receives at least one CO. ZYP1 limits the number of COs and mediates CO interference, the phenomenon that reduces the probability of multiple COs forming close together.

and transformants were selected on 50 µM phosphinothricin. Mutations were confirmed by sequencing amplicons (Eurofins) and homozygous plants carrying frameshift mutations were used for analysis. T-DNA insertions were confirmed by PCR as previously described (2).
zyp1a-2 and zyp1a-3 were genotyped by CAPS as the mutation abolished a HindIII site and zyp1b-2 was genotyped by amplicon sequencing.

Super-resolution microscopy and analysis
Image capture and reconstruction for SIM was performed using a Zeiss ELYRA PS1 microscope (John Innes Centre) and OMX microscope (Applied Precision) at the University of Dundee with Imaris software (Oxford Instruments). Images were imported into NIS-elements software (Nikon) for measuring distances between ASY1-labelled axial bridges. Three independent cells and 18 counts were recorded. The distance between lateral elements/axes was measured for a maximum intensity projection of the SMC3 channel using ImageJ and R.
For each cell (wild type, n = 2; zyp1a-2/zyp1b-1, n = 3), random rectangular regions 50 pixels wide were manually annotated with perpendicular lines bisecting the two juxtaposed axes and repeated 20 times. The plot profile of fluorescence intensity was then exported and analysed in R. For each line an interpolating spline anchored was fitted (spline function -stats package).
Fitted splines were used to predict a denser array of values (predict -stats package). The turning points corresponding to the centre of each lateral element were identified (findpeakspracma package), and the distance between the centre of the axes was calculated.

Chiasmata counts
Metaphase I chromosome spread preparations were prepared from ethanol: acetic acid (3:1) fixed material. Chiasma counts were based on the shape of bivalents and fluorescence in situ hybridization was performed with the 5S and 45S rDNA probes (3).
Pollen FTL and viability analysis zyp1a-3/zyp1b-1/I3bc qrt1-2/I5ab qrt1-2 crosses were sown, following which F1 and F2 generations were allowed to self. F3 plants were then genotyped to identify zyp1a-3/zyp1b-1 mutants and segregated wild-type controls. These were screened to identify qrt1-2 plants with the correct hemizygous fluorescence patterns. For the I3bc cross, 3 mutants and 1 wild type plant were recovered with the correct pattern (CYR/+++). For I5ab no plants were recovered to analyse all three intervals (RYC/+++) but 1 wild type plant and 1 mutant plant with appropriate I5b fluorescence patterns was recovered (+YC/+++). Fluorescent tetrad analysis was carried out as previously described (4 Seed scoring was automated in a method adapted from (7). An ImageJ macro was used to detect and measure the mean RFP and GFP fluorescence intensity of every seed in each field of view for all images per plant. A rolling ball background subtraction was applied to all channels (subtract background: radius of 50 pixels for 'brightfield' channel seeds appeared as dark objects against a light background). The 'brightfield' image was then converted to binary (Make Binary), the binary mask was eroded to reduce contacts between adjacent seeds (Erode) and the image was then watershed to split groups (Watershed). Following this, seed outlines were detected (filtering for size and circularity) and added to the ROI manager counted, and map distance determined as previously described (6,7). This process enabled rapid and accurate measurements of >1000 seeds per individual.

Statistical analysis
All statistical analyses (excluding pollen quartet analysis) were performed using R.