Nuclei extraction buffer

The genome size of the “Mandevilla 2001” sample, subsequently used for genome sequencing, was determined through flow cytometry of propidium iodide (PI)-stained nuclei, following the procedure described by the CyStain PI Absolute P protocol (Sysmex Partec, Görlitz, Germany). One hundred milligrams of fresh leaf tissue was chopped with a razor blade along with 0.5 ml of Nuclei Extraction Buffer (Sysmex Partec), incubated for 45 min at room temperature and filtered using 30 μm CellTrics (Sysmex Partec). Two milliliters of staining solution (1982 μl of Staining Buffer, 12 μl of PI and 6 μl of RNAse A 3.3 ng/μl) was then added to each filtered sample, and the resulting solution was placed on ice in the dark for 45 min. Analyses were run by setting the following parameters: Nd-YAG green laser: λ = 532 nm; 30 mW, flow rate of 4 μl/s. Raphanus sativus, Glycine max, and Solanum lycopersicum seeds with known 2C DNA content were kindly provided by Prof. Dolezel,¹ adopted as reference standards, and their relative fluorescence was used to estimate the genome size of the 2001 sample. Fluorescence histograms were evaluated using FCS Express 5 Flow software (Sysmex Partec), and c-values were inferred by comparing the sample and standard at G0/G1 peak positions.

Flow cytometric analysis was performed to estimate the size of the P. paniculata genome using CyFlow SL (Sysmex Partec, Japan). Three different organs (leaf, flower and inflorescence stem) from two different individuals were chopped up using a razor in nuclei extraction buffer (Sysmex Partec) and filtered through a 50 μm CellTrics filter (Sysmex Partec). Staining solution containing staining buffer, propidium iodide and RNase (Sysmex Partec) was added to samples. Arabidopsis thaliana leaves were used as an internal standard. The number of nuclei was measured using 488 nm blue lasers. A regression formula of genome size and peak intensities was constructed using A. thaliana, which has a genome size of 125 Mbp. Then, genome size of each P. paniculata sample was estimated from the regression formula.

Ploidy level was evaluated by flow cytometry analysis of stained nuclei by 4′,6-diamidino-2-phenylindole (DAPI), isolated from leaves of 40 walnut progenies in the laboratory of Genomics for Plant Breeding, DAFNAE, University of Padova, Italy. In brief, 35 progenies that -based on SSR data- were predicted to be the result of outcrossing were analyzed to identify any possible polyploid BIII hybrid. Similarly, 5 progenies that -based on SSR data - were predicted to be the result of selfing were analyzed to identify any possible parthenogenesis-derived haploid accession through flow cytometry (CyFlow Ploidy Analyzer, Sysmex, DE). Following the procedure outlined in the CyStain UV Precise protocol, Approximately, 0.5 cm² of young fresh leaves were chopped with 0.5 ml of Nuclei Extraction Buffer (Sysmex Partec) and incubated for 2 minutes at room temperature. Then, each sample was filtered (30 µm CellTrics®, Sysmex) and incubated in 2 ml of staining buffer for 60 sec before analysis (blue fluorescence emission= 435-500 nm; flow rate of 4 µl/sec). Fluorescence histograms were evaluated using Flowing software 2.

Five‐millimetre‐long root tips were harvested from c. 100 seedlings, on DAS5, and placed in 250 ml of cold nuclei Extraction Buffer (Sysmex Partec GmbH, Görlitz, Germany) in a Petri dish on ice. Root tips were chopped with a new razor blade exchanged between every sample. The nuclei suspension was incubated for 2 h in 1 ml of CyStain PI Absolute P (Sysmex Partec GmbH) staining buffer in the dark on ice. The stained homogenate was filtered through a 30 μm CellTrics filters (Sysmex Partec GmbH). Stained particles were excited with 561 nm (110 mW) and 488 nm (100 mW) lasers and emission was detected for propidium iodide‐stained particles (610/20 nm) and forward scattering (488/10 nm). Fluorescence intensity was recorded for 10 000 particles for each sample. facs diva software (BD Lifesciences, Franklin Lakes, NJ, USA) was used for gating the nuclear populations. We analysed six pooled biological samples each for the Col‐0 wild‐type and the aak6 mutant.