Dynamics Of DNA Replication During Premeiosis And Early Meiosis ...

Identification, isolation and flow cytometric analysis of meiocytes during premeiosis and early meiosis in wheat

To study the progression of replication during early meiosis in wheat, one anther per floret was carefully checked to determine the meiotic stage using a light microscopy. Since all the anthers in the same flower are synchronised, the two remaining anthers were stored in 100% ethanol: acetic acid (3∶1, v/v) at 4°C. The identification, selection and isolation of anthers was carried out until a total of 150 anthers were accumulated in each meiosis stage of prophase I (leptotene, zygotene, pachytene, diplotene and diakinesis) and metaphase I, either in the presence or in the absence of the Ph1 locus. Each sample was then separated in three aliquots of 50 anthers each with the aim of having three independent replicates of each stage of meiosis, either in the presence or in the absence of the Ph1 locus for three independent experiments. In addition, three different flow cytometric measurements were taken from each sample in each experiment to account for equipment deviations.

Flow cytometric determination of the nuclear DNA content in a wheat anther sample, either in the presence or in the absence of the Ph1 locus, was distributed in a histogram with two peaks corresponding to G0/G1 phases (un-replicated cells; 2C DNA content) and G2/M phases (replicated cells; 4C DNA content), respectively (Figures 1 and 2). As expected, most of the cells in each meiosis stage were identified in the 2C peak (G0/G1) for all the samples analysed (Figures 1 and 2). The small peak (4C) corresponded to those cells that had already finished replication and cells going under active replication were detected between the 2C and 4C peaks (Figures 1 and 2).

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Figure 1. Flow cytometric analysis of DNA replication in wheat anthers during early meiosis in the presence of the Ph1 locus.

a) Flow cytometric histograms of the nuclear DNA content of isolated anther nuclei in each stage of meiosis, which was determined by the number and organisation of centromeres (green) and telomeres (red) using fluorescence in situ hybridisation. The percentage of cells in G0/G1, S and G2/M are in brackets. G0/G1 and G2/M phases are shown in red, S phase is shown in green and total histogram (measured as total nuclei) is shown in blue. b) Progression of the percentage of cells in G0/G1, S and G2/M phases during early meiosis.

https://doi.org/10.1371/journal.pone.0107714.g001

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Figure 2. Flow cytometric analysis of DNA replication in wheat anthers during early meiosis in the absence of the Ph1 locus.

a) Flow cytometric histograms of the nuclear DNA content of isolated anther nuclei in each stage of meiosis, which was determined by the number and organisation of centromeres (green) and telomeres (red) using fluorescence in situ hybridisation. The percentage of cells in G0/G1, S and G2/M are in brackets. G0/G1 and G2/M phases are shown in red, S phase is shown in green and total histogram (measured as total nuclei) is shown in blue. b) Progression of the percentage of cells in G0/G1, S and G2/M phases during early meiosis.

https://doi.org/10.1371/journal.pone.0107714.g002

The amount of DNA during the cell cycle in somatic tissues was always measured by flow cytometry at the beginning and at the end of each experiment for both wheat lines (Ph1+ and Ph1-), to have a basal reference of unreplicated cells, replicated cells and somatic replication of each wheat line and, in addition, to monitor instrument or staining variations (Figures 1 and 2). As expected, most of the cells were in G0/G1 stage and no significant differences were found in wheat somatic tissue in the presence and in the absence of the Ph1 locus in any case (Table 1). The replication value obtained for the somatic tissue (3.1±0.2 and 2.6±0.6 in Ph1+ and Ph1- wheat lines, respectively) was always at least five times lower than the minimum replication value obtained in early meiosis (15.5±1.2 and 13.5±0.1 in diakinesis in Ph1+ and Ph1- wheat lines, respectively) (Tables 2 and 3).

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Table 1. Flow cytometric determination of the percentage distributions of wheat somatic nuclei in each phase of the cell cycle in the presence and in the absence of the Ph1 locus (Ph1+ and Ph1-, respectively).

https://doi.org/10.1371/journal.pone.0107714.t001

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Table 2. Flow cytometric determination of the percentage distributions of nuclei from wheat anthers in each phase of the cell cycle during meiosis in the presence of the Ph1 locus.

https://doi.org/10.1371/journal.pone.0107714.t002

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Table 3. Flow cytometric determination of the percentage distributions of nuclei from wheat anthers in each phase of the cell cycle during meiosis in the absence of the Ph1 locus.

https://doi.org/10.1371/journal.pone.0107714.t003