FIGURE 4.

Meiotic anomalies associated with FDR and SDR in WK21/Nip F₁. (A) Schematic diagram of chromosomal separations following normal, FDR, and SDR meiotic events. The three different chromosomal separation pathways following normal, FDR, and SDR events during meiotic division are based on De Storme and Geelen (2013). In the normal situation, bivalent homologous chromosomes separate after recombination at the end of meiosis I, with the sister chromatids remaining attached at the beginning of meiosis II and then separating. In FDR, homologous chromosomes fail to separate at the end of meiosis I, resulting in homologous chromosomes in the gametes. SDR bypasses meiosis II, and sister chromatids are distributed into gametes. FDR and SDR lead to centromeric regions (shown as knobs) that are respectively heterozygous or homozygous between homologous chromosomes. Red and blue are used to indicate the parental origin of chromosomal regions. (B) Genetic zygosities of centromeric regions of the 12 chromosomes of 12 regenerated plants and detection of FDR and SDR. Markers in centromeric regions (two of each chromosome) used in this analysis are detailed in Supplementary Figure 1 and Supplementary Table 3. In the table, the zygosities of the 12 centromeres are indicated by “N” for homozygous centromeres from O. sativa (Nip), “W” for homozygous centromeres for O. glaberrima (WK21), or “H” for heterozygous centromeres. (C) Immunohistochemical detection of normal and anomalous gametes during meiosis I in WK21/Nip F₁. Using anti-OsCenH3 antibody, centromeric regions were observed in chromosomes at diplotene in meiosis I in PMCs from WK21/Nip F₁. Left: detection of paired signals (red spots) from centromeres at diplotene in a PMC, implying normal bivalent chromosomes (white portions). Right: non-aligned, dispersed centromeric signals, indicative of univalent chromosomes. (D) Immunohistochemical detection of normal and anomalous gametes during meiosis II of WK21/Nip F₁. Using anti-α-tubulin mouse antibody, spindle fiber formation (green zone) was observed at anaphase II in PMCs from WK21/Nip F₁. During normal anaphase II, sister chromatids (white zone) prepared to move toward opposite poles of the cell to generate haploid gametes. Left: normal division, showing movement of sister chromatids to the poles via the spindle fibers in both compartments as monitored using α-tubulin antibody. Right: unequal division in a PMC. In the upper compartment, no α-tubulin was observed, and sister chromatids were unable to separate and move to the poles; in contrast, the movement of sister chromatids along spindle fibers was apparent in the lower compartment.