Cystain pi absolute p

Genome sizes were measured for 134 individuals. Due to a lack of material, we could not measure the genome sizes of C. neapolitanus and C. siculus. Genome sizes for C. bertiscensis were partially taken from Raca et al. [16 (link)].
Genome size was determined using propidium iodide (PI) as a stain in flow cytometry with a Cyflow Space (Sysmex Partec) flow cytometer, following essentially the procedure described in Jakob et al. [6 (link)]. We mainly used rye (Secale cereale; 16.01 pg/2C) or pea (Pisum sativum; 9.09 pg/2C) as internal size standards and the buffer CyStain PI Absolute P (Sysmex Partec). Genome size measurements aimed at identifying diploids and polyploids. To link the genome sizes with the molecular data, we used silica-gel-dried leaves from the same individual used for DNA extraction whenever possible. Initial tests showed that fresh and silica-gel-dried materials arrived at the same genome size estimations in Crocus. However, the quality of data obtained is slightly lower for dried leaves. A detailed overview of the material measured and standards used is given in Supplementary Table S3.

Measuring of relative DNA amount of nuclei occurred by flow cytometry (Facs calibur, Becton Dickinson, BD) with a red fluorescence laser as basis for detection of ploidy level. For each probe, leaf material was chopped with razor blades in 500 μl nuclei extraction buffer (CyStain PI absolute P, Sysmex) and stained with the corresponding staining buffer, containing 5 % polyvinylpyrrolidone 25 (Serva) and 0.6 % propidium iodide (Serva). Immediately after staining, the nuclei suspension was filtered using a 5 ml polystyrene round-button tube with a cell-strainer cap (BD). For reference, radish was measured in separate sample after five samples of balm.

Flow cytometric analysis of putative polyploid shoots was performed according to [52 (link)], after the removal of leaf trichomes as described by [53 (link)] by using Ploidy Analyzer PA I (Sysmex Partec, Germany). The leaflets were chopped with a sharp razor blade while submerged in the nuclei extraction buffer from CyStain™PI Absolute P (Sysmex Partec GmbH, Görlitz, Germany). The homogenate was filtered through a 30 μm nylon mesh to remove debris and then stained with RNAse A and propidium iodide (PI) according to the manufacturers’ instructions. To assess ploidy levels of the putative polyploids, Allium cepa was used as an internal standard.

For nuclei isolation, roughly 0.5 cm² of fresh leaf tissue was chopped together with equivalent amounts of leaf tissue of one of the internal reference standards, Raphanus sativus var. ‘Voran’ (Gatersleben genebank accession number: RA 34; 1.11 pg/2C) or Lycopersicon esculentum var. ‘Stupicke Rane’ (Gatersleben genebank accession number: LYC 418; 1.96 pg/2C), in a petri dish using the reagent kit ‘CyStain PI Absolute P’ (Sysmex) following the manufacturer’s instructions. The resulting nuclei suspension was filtered through a 50-μm filter mesh (CellTrics, Sysmex) and measured either on a CyFlow Space (Sysmex) or on a BD Influx cell sorter (BD Biosciences). The absolute DNA content (pg/2C) was calculated based on the values of the G1 peak means and the corresponding genome size (Mbp/1C), according to Dolêzel et al. (2003 (link)).

For nuclei isolation, approximately 0.5 cm² of fresh leaf tissue was chopped together with equivalent amounts of leaf tissue of the internal reference standard, Glycine max (L.) Merr. convar. max var. max, Sorte Cina 5202 (Gatersleben GeneBank accession number: SOJA 392; 2.21 pg/2C), in a petri dish using the reagent kit “CyStain PI Absolute P” (Sysmex-Partec) following the manufacturer’s instructions. The resulting nuclei suspension was filtered through a 50-μm CellTrics filter (Sysmex-Partec) and measured on a BD Influx cell sorter (BD Biosciences). Six independent measurements were performed for each genotype. The absolute DNA content (pg/2C) was calculated based on the values of the G1 peak means and the corresponding genome size (Mbp/1C), according to Dolezel et al. (2003) (link).

To isolate nuclei, approximately 0.5 cm² of fresh leaf tissue from M. fragrans and the internal reference standard, Lycopersicon esculentum Mill. convar. infiniens Lehm. var. flammatum Lehm., Stupicke Rane, Genebank accession number LYC 418, were chopped together in a petri dish using the reagent kit ‘CyStain PI Absolute P’ (Sysmex-Partec) following the manufacturer’s instructions. The nuclei suspension was filtered through a 50-μm CellTrics filter (Sysmex-Partec) and measured on a CyFlow Space flow cytometer (Partec-Sysmex). At least five independent measurements were performed of each of the four individual plants. The absolute DNA content (pg/2C) was calculated based on the values of the G1 peak means and converted to the corresponding genome size (Mbp/1C) according to Dolezel et al. (2007 (link)).

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.