seeds from four different Cypriot populations (ARI00-845, ARI00-853, ARI00-856
and ARI00-876; Table 1) were
acquired from the Agricultural Research Institute of Cyprus (ARI). All seeds
were germinated in 10-inch pots containing peat, in a growth room under a 16/8
light/dark cycle (200 ?einstein/m-2/sec-1) at 25°C.
Plants were transferred in a glasshouse and regularly watered till anthesis.
Pollen size and viability
grains were sampled from up to ten plants per accession. Mature (but undehisced)
anthers were excised from the primary floret of sampled spikelets from each
plant, and pollen grains were released from the anther with a needle. Pollen
stainability, as a measure of pollen viability, was assessed by staining mature
pollen in a 1% aceto-carmine solution. The AxioCam MRc Zeiss system (Zeiss,
Jena, Germany) was used for imaging. Regularly shaped and darkly stained pollen
grains were considered to be viable. Image J (https://imagej.nih.gov/ij/) was
employed to examine pollen grains’ number and size. More than 500 pollen grains
were analyzed from each genotype.
following procedures were completed at 4oC or on ice. A two-step
procedure using Otto I 0.1 M citric acid monohydrate, 0.5 % (v/v) Tween 20
(Sigma-Aldrich, St. Louis,MO, USA) and Otto II buffers 0.4M Na2HPO4.12H2O,
0.05 mg/mL Propidium Iodide (Sigma-Aldrich, St. Louis, MO, USA), 0.05 mg/mL
RNase (Takara, Otsu Shiga Japan) was used for both pollen and leave tissues. In
order to isolate pollen grains, we placed mature anthers in Otto I buffer and
vortexed them vigorously for 10 sec. The homogenate was then passed through a
pre-filter to remove large contaminants, while allowing pollen grains to pass.
The flow through, with the collected pollen,
was loaded to a bursting filter (30 ?m) placed on a 1.5 mL Eppendorf tube and pollen
grains were moderately pressed against the filter for a few sec, using a
rounded end rod, as previously described (Kron and Husband 2012). Nuclei were
rinsed with 0.5 mL Otto I buffer.
one cm2 of excised leaf tissue was placed in a plastic 60-mm petri
dish settled on ice with 0.5 mL of pre-chilled Otto I buffer. Tissues were
chopped using a sterilized hand razor blade for 2–3 min. The homogenate was
further filtered through a 30 ?m filter to retain debris. Homogenates (0.2 mL) were
added to a labeled tube containing one mL of Otto II buffer, and incubated in
dark for 20 min prior to analysis.
levels were estimated using an Accuri C6 flow cytometer (Accuri Cytometers,
Inc., Ann Arbor, MI, USA), following a standard protocol (Galbraith 2009). Analysis
was based on light-scatter and fluorescence signals emitted from a 20-mW laser
illumination at 488 nm. Precision of the instrument was validated using 6-peak
fluorescent bead mixtures (Spherotech), as suggested by the manufacturer (CFlow
User Guide, Accuri). Threshold levels were set (80,000 on FSC-H and 1,000 FL-2)
to eradicate unrelated debris from being detected. Fluidics were set on slow
and measurements were collected to a total count of 10,000 nuclei. In order to discriminate
among 2n gametes and aggregates (two
or more adhering nuclei), 2C events with a markedly lower FL-H/FL-A comparative
to that of single 2C nuclei were removed (Kreiner et al. 2017). The regions
of nuclei were located diagonally using a FL3-A/FL2A plot and values
representing peak positions and variances were linearly estimated across the lowest
peaks in order to export. The flow histograms showed sharp peaks, with CVs
typically <5%, for both the pollen and leave nuclei. Replicates were highly reproducible with little systematic errors. Pollen Mother Cells analysis Pollen Mother Cells (PMCs) were examined from florets that were excised from panicles collected at the booting stage (the flag leaf was 3 to 7 cm above the base of the last true leaf). Immature panicles were fixed for 48 h in Carnoy's buffer (6:3:1, alcohol:chloroform:acetic acid v/v/v) and subsequently stored in 70% (v/v) ethanol at 40 C. The young spikelets were hydrolyzed in 1N HCl solution at 60°C for 10 min. Anthers were isolated and squashed in one drop of 1% aceto-carmine solution. Chromosome configurations at meiosis were studied from individual plants (population) that contained large pollen grains.