Cystain pi absolute p kit

Flow cytometry was performed on sporulating and pre-sporulating spinach leaves mixed with 1 cm² of young leaf tissue from Oryza sativa cv. Kitaake (2C = 867 Mb), which was sufficiently different from the genome size of P. effusa for use as the internal reference. The O. sativa 2C DNA content was determined by calibrating against nuclei from flower buds of Arabidopsis thaliana Col-0 which has a known absolute DNA content of 2C = 314 Mb [62 (link)]. Nuclei extraction and staining with propidium iodide was done using the Cystain PI absolute P kit (Sysmex, Lincolnshire, IL). Flow cytometry was done on a BD FACScan (Becton Dickinson, East Rutherford, NJ). For each measurement, 10,000 nuclei were assessed. Data was analyzed using FlowJo (Ashland, OR).

To measure the genome size of the two model species by flow cytometry, we used the CyStain PI Absolute P kit (Sysmex, Görlitz, Germany) according to the manufacturer's instructions with leaf tissue of the sample and parsley (Petroselinum crispum (Mill.) Fuss, 1C = 2.23 pg; [92 (link)]) or pea (Pisum sativum L. ‘Kleine Rheinländerin’, 1C = 4.42 pg; [93 (link)]) as internal standard. At least 5,000 measurements were gathered in a CyFlow Space (532 nm diode laser; Sysmex) to estimate nuclear DNA content. Two individuals of A. clusiana and three individuals of C. pulla (all from Hochschwab) were measured. One individual of each species was measured twice, one time with parsley as standard, one time with pea as standard. The remaining individuals were measured once, with pea as standard in the case of A. clusiana and with parsley as standard in the case of C. pulla.

Young cultures (2 weeks) were harvested using a vacuum filtration with Whatman #2 papers (GE Healthcare 47 mm), and washed with water for three times. Nuclei isolation was prepared according to Galbraith et al. (1983) (link) with modifications, described as follows. ∼50 mg of 2 weeks old algal filaments were transferred to plastic petri dishes and chopped with a single edge razor blade for 5 min. Then, the chopped algal filaments were transferred to a 1.5 ml Eppendorf tube and immediately put on ice. Then, 0.5 ml of nucleus extraction buffer (CyStain PI Absolute P kit, Sysmex) were added and gently mixed. The samples were filtered through a 20 μm CellTrics filter (Sysmex) into a collection tube cooled over ice. Samples were then centrifuged with 600 × g at 4°C for 10 min and the supernatant decanted. Pellets were washed with 1 ml of nucleus extraction buffer, and centrifuged again with 600 × g at 4°C for 10 min. The washing step was repeated two more times.
Arabidopsis thaliana nuclei were used as control for FC (see below). Three young leaves were taken and transferred into a 1.5 ml Eppendorf tube. Then, 0.5 ml of nucleus extraction buffer was added. The leaves were chopped into very small pieces with spatula for 1 min. The extraction mixture was filtered through a 20 μm CellTrics filter. The remaining steps were the same as described above for the algal samples.

The genome size of N. micrantha was estimated by flow cytometry. About 1 cm² of the young fresh leaf was used for sample preparation using the CyStain^® PI Absolute P kit (Sysmex, Germany) following manufacturer instructions. Solanum lycopersicum L. “Stupicke’ polnı’ rane”’ 1C = 0.98 pg (Doležel et al., 1992 (link)) was used as an internal standard reference. In brief, the nuclei of the standard and the sample were isolated, stained, and analyzed simultaneously. The nuclear DNA content was determined using the CyFlow^® Cube 8 flow cytometer (Sysmex, Germany) for each sample. For both, the standard reference and N. micrantha samples were prepared in replicates, and each was run three times with a minimum of 2,500 nuclei count per sample. The fluorescence values were converted to the DNA content using the following formula:

Genome size was initially estimated experimentally by fluorescence-activated cell sorting (FACs). Approximately 0.5 cm² of fresh tissue from young leaves of Orzya sativa, a P. atlantica female, and a P. integerrima male were used. O. sativa was used as a reference because it is expected to have a comparable genome size to pistachio (430 Mb).
Sysmex CyStain PI Absolute P Kit reagent kit for nuclei extraction and DNA staining of nuclear DNA from plant tissues was used to determine the absolute and relative genome size and ploidy level of a P. atlantica female and P. integerrima male individual. The reagent was created specifically for selected configurations of the Sysmex CyFlow Ploidy Analyser and CyFlow Space flow cytometers. Briefly, the samples were co-chopped in 500 μL nucleic extraction buffer. A razor blade with a single edge was used for chopping each sample in a Petri dish. A 50-μm mesh filter was used to filter the resulting homogenate, staining solution was added, and samples were incubated in a dark −20°C freezer. CellQuest software was used to acquire and analyze data from the flow cytometer.
A computational estimate of genome size and heterozygosity was also calculated. Using Illumina short reads, k-mer counts were generated for each parental tree using Jellyfish (Marcais and Kingsford 2011 (link)) and further analyzed with Genomescope (Vurture et al. 2017 (link)).

DNA content was estimated using flow cytometry as described in Cochetel et al. [47 (link)]. The nuclei extraction was performed using the Cystain PI absolute P kit (Sysmex America Inc., IL, USA). For the internal reference standard, Lycopersicon esculentum cv. Stupické polní tyčkové rané was selected, with a known genome size of 2 C = 1.96 pg; 1 C = 958 Mb [73 (link)]. Young leaves (~ 5 mg) from grape and tomato were finely cut with a razor blade in a Petri dish containing 500 mL of extraction buffer and filtered through a 50-mm filter (CellTrics, Sysmex America Inc., IL, USA). Propidium iodide staining solution (2 mL) was then added to the nuclei suspension [74 (link), 75 (link)]. Measurements were acquired with a Becton Dickinson FACScan (Franklin Lakes, New Jersey) equipped with a 488-nm laser. Raw data were imported and analyzed with the R package flowPloidy v.1.22.0 [76 (link)] (Additional File 1: Table S9). For V. berlandieri, V. riparia, and V. rupestris, estimates from the same species were retrieved from literature [77 (link)].