Cyflow ploidy analyzer

Four lines from the genus Brassica were used, including a red-green Chinese cabbage line (B. rapa cv. Asia), a red cabbage line (B. oleracea cv. Rubea), a green resynthesized rapeseed line (allopolyploid B. napus cv. Rs306), and a red resynthesized rapeseed line (allopolyploid B. napus cv. Rs035). The Rs035 line was developed by crossing the inbred Chinese cabbage line B. rapa cv. Asia with the cabbage line B. oleracea cv. Rubea. The embryos were rescued 18–20 days after crossing and were cultured on Murashige and Skoog (MS) medium. The chromosomes of the developed plants were diploidized using a 4-hr 0.1% colchicine root treatment. Successfully crossed hybrid plants were identified using the conserved ortholog set (COS) marker COS1078 [73 (link)]. The genotypes of the allopolyploid plants with diploid chromosomes were confirmed using a CyFlow ploidy analyzer and 4′,6-diamidino-2-phenylindole (DAPI) solution (Sysmex, Norderstedt, Germany).

The F₁ hybrids were identified by hybrid identification, ploidy identification, and flesh color-specific marker analysis; the segregation ratios of red-fleshed and white-fleshed loquats were calculated; and the inheritance of tetraploid flesh color was explored. SSR markers were used to identify true and false hybrids. The SSR markers were derived from loquat genome data (Additional file 4: Table S2). For each cross combination, three SSR markers on different chromosomes were used. When two or more male parent-specific bands appeared in the F₁ hybrids, they were considered to be true hybrids. The PCR amplification and electrophoresis of SSRs were run according to the literature with slight modifications [42 , 81 (link)].
The ploidy of hybrids was identified by flow cytometry [82 (link)]. Fresh young plant tissue (1 g) was placed in a culture dish, and 1 ml of nuclear extract was added after chopping. The liquid was filtered into a 2 mL centrifuge tube through a 30 μm filter membrane and centrifuged at 1000 r/min for 5 min. The supernatant was discarded, 500 μL of cell nucleus extract was added to prepare a cell nucleus suspension, 50 μL of DAPI (5 μg/mL) was added, and the cells were stained for 3–4 min in the dark and then tested on a CyFlow^® Ploidy Analyzer (Sysmex-Partec GmbH).

Flow cytometric analyses were performed on leaves of in vitro regenerated plants. Samples were prepared as previously described [24 (link)] using Galbraith’s nuclear isolation buffer [40 (link)] supplemented with 2% (w/v) polyvinylpyrrolidone (PVP-10), propidium iodide (PI; 50 μg/mL), and ribonuclease A (50 μg/mL). Solanum lycopersicum cv. Stupicke (2C = 1.96 pg/2C) [41 (link)] was used as an internal standard. For each sample, PI fluorescence was measured in at least 5000 nuclei, using a CyFlow Ploidy Analyzer flow cytometer (Sysmex Partec, Kobe, Japan). Histograms were analyzed using CyView 1.6 software. The coefficient of variation (CV) of the G0/G1 peak of Salix ranged from 3.16% to 6.68%. Nuclear DNA content was calculated based on the linear relationship between the ratios of the 2C peak positions of Salix/Solanum on a histogram of fluorescence intensities. Genome size was estimated for five control plants (leaves collected in the natural habitat) and 350 in vitro-derived plants.

Young leaves of 15 A. mucosa individuals were collected and used for genome size measurements by flow cytometry. Approximately 1 cm² of young leaf tissue was minced with fresh leaf material from an internal standard (Petroselinum crispum (Mill.), Apiaceae, 2C = 4.50 pg). Nuclear suspensions were prepared according to Doležel, Greilhuber & Suda (2007) (link), adding 1.5 mL of WPB (woody plant buffer) (Loureiro et al., 2007 (link)) to the nucleus extract, and then the nuclear suspension was filtered through a 30 μm nylon mesh. The nuclear suspension was stained with 50 μL of 1 mg/mL propidium iodide, incubated for at least 10 min and analyzed by flow cytometry. At least 5,000 nuclei from three replicates were analyzed for each sample using a CyFlow Ploidy Analyzer (Sysmex, Kobe, Japan) cytometer. Each set of histograms from the flow cytometry analyses was analyzed using Flowing Software v2.5.1 by Perttu Terho (Turku Center for Biotechnology, University of Turku, Turku, Finland).

To analyze the ploidy levels of S. bicolor and S. kaptarae, flow cytometry (FC) was conducted using silica-gel-dried leaf material. Samples (~1 cm²) were reduced to small pieces using Tissue Lyzer II (Qiagen; 30 Hz, time 3 s). A total of 200 µL of 1% PVP Otto I extraction buffer were added, and samples were gently inverted for 1 min. Samples were filtered through CellTrics ^® filter (30 µm mesh, Sysmex Partec GmbH, Görlitz, Germany) into a flow cytometry sample tube. A total of 800 µL of DAPI-containing Otto II buffer [81 ] were then added to strain the DNA. FC analyses were carried out on a CyFlow Ploidy Analyzer (Sysmex, Norderstedt, Germany) and the Software CUBE16 v.1.6 (Sysmex, Norderstedt, Germany) was used to analyze the results and compute DNA content. Gain was set at 530 nm for an optimal differentiation between DNA content. Three different diploid S. caprea samples were used as external standard.

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.