Turning meiosis into mitosis

Abstract

Apomixis, or asexual clonal reproduction through seeds, is of immense interest due to its potential application in agriculture. One key element of apomixis is apomeiosis, a deregulation of meiosis that results in a mitotic-like division. We isolated and characterised a novel gene that is directly involved in controlling entry into the second meiotic division. By combining a mutation in this gene with two others that affect key meiotic processes, we created a genotype called MiMe in which meiosis is totally replaced by mitosis. The obtained plants produce functional diploid gametes that are genetically identical to their mother. The creation of the MiMe genotype and apomeiosis phenotype is an important step towards understanding and engineering apomixis.

Figure 1. Schematic summary of the main results.

During mitosis in diploid cells, chromosomes replicate and sister chromatids segregate to generate daughter cells that are diploid and genetically identical to the initial cell. During meiosis, two rounds of chromosome segregation follow a single round of replication. At division one, homologous chromosomes recombine and are separated. Meiosis II is more similar to mitosis, resulting in equal distribution of sister chromatids. The obtained spores are thus haploid and carry recombined genetic information. In the osd1 mutant (this study), meiosis II is skipped giving rise to diploid spores and gametes with recombined genetic information. The double Atspo11-1/Atrec8 mutant undergoes a mitotic-like division instead of a normal first meiotic division, followed by an unbalanced second division leading to unbalanced spores and sterility . In the triple osd1/Atspo11-1/Atrec8 mutant (MiMe, this study), the presence of the Atspo11-1 and Atrec8 mutations leads to a mitotic-like first meiotic division, and the presence of the osd1 mutation prevents the second meiotic division from occurring. Thus meiosis is replaced by a mitotic-like division. The obtained spores and gametes are genetically identical to the initial cell.

Figure 2. The OSD1 gene and protein.

(A)The OSD1 gene contains three exons and two introns and encodes a protein of 243 amino acids. The positions of the two Ds insertions are indicated by triangles. (B) A multiple sequence alignment of OSD1/UVI4 family members is shown, pointing out the segments of best conservation along OSD1. Particularly high conservation is observed in the central OSD1-motif, which is the only section with clear sequence similarity in moss and club-mosses. The conserved C-terminal 40–60 amino acids are predicted to contribute to alpha-helical structural elements. All available homologues are included for the selected range of species, with the species of origin being indicated by a two letter code preceding each protein identifier. Brassicaceae: Al, Arabidopsis lyrata; At, Arabidopsis thaliana; Br, Brassica rapa. Eurosids I: Gm, Glycine max; Pt, Populus trichocarpa. Poaceae: Os, Oryza sativa; Sb, Sorghum bicolor; Zm, Zea mays. Mosses: Pp, Physcomitrella patens. (C) Unrooted phylogenetic tree inferred from a OSD1/UVI4 family alignment using a neighbour-joining algorithm with pairwise gap removal. The full length sequences are included in the alignment except for Physcomitrella patens (Pp) sequences, for which only the conserved segment is aligned. The fact that only the aligning portions of Pp proteins were used in the reconstruction of the consensus tree topology should be taken into account during Pp-node branch length interpretation. Based on paralogue grouping, it can be hypothesised that gene duplication events occurred repeatedly in the evolution of plant OSD1/UVI4 family. The robustness of the topology is indicated by bootstrap confidence levels in percentage of 1,000 replicates. The analysis was performed with PHYLIPv3.66 (Felsenstein J (2006)) distributed by the author. Department of Genome Sciences, University of Washington, Seattle, United States of America) and PHYLO_WINv2.0 .

Figure 3. osd1 mutants skip meiosis II.

(A and B) Male meiotic products stained with toluidine blue. (A) A wild-type tetrad. (B) A dyad in the osd1-1 mutant. (C–D) Male meiosis in osd1 is indistinguishable from wild type until telophase I (compare to figure 4), but no figures characteristic of a second division were observed. (C) Pachytene. (D) Diakinesis. (E) Metaphase I. (F) Anaphase I. (G) Telophase I. Two nuclei separated by a band of mitochondria are observed. (H) Metaphase I of female meiosis in osd1.

Figure 4. Meiosis in wild type.

(A) Pachytene. Homologous chromosomes are fully synapsed. (B) Diakinesis. Five pairs of homologous chromosomes (bivalent), linked by chiasmata, are observed. (C) Metaphase I. The five bivalent are aligned on the metaphase plate. (D) Anaphase I. The homologous chromosomes are separated. (E) Telophase I. (F) Metaphase II. The pairs of sister chromatids align on the metaphase plates. (G) Anaphase II. The sister chromatids are separated. (H and I) Telophase II. Four haploid spores are formed (tetrad). Scale bar = 10 µm.

Figure 5. Genetic make-up of the osd1 and MiMe diploid gametes.

(A) Triploid offspring of the osd-1 (No-0)/osd1-2 (Ler) × Col-0 crosses were genotyped for several molecular markers. Each line represents one plant. For each marker, plants carrying only the No-0 allele are in green, plants carrying only the Ler allele are in yellow, and plants with both the No-0 and Ler alleles are in red. Col-0 alleles are present in all the plants because it was used as the male or female parent in the cross. The position of each marker (red) and the centromeres (dark blue) are indicated along the chromosomes. (B) Similarly, triploid offspring of the MiMe (patchwork of Col-0 from Atspo11-1/Atrec8 and No-0 from osd1-1) × Le crosses was genotyped for molecular markers that were heterozygous in the MiMe mother plant. All the plants were heterozygous for each marker (Col-0/No-0).

Figure 6. Mitosis-like divisions instead of meiosis in MiMe plants.

(A) Male metaphase I (B) Male anaphase I. The vignette shows a dyad in MiMe. (C) Female metaphase I. (D) Female anaphase I. Scale bar = 10 µm.

Figure 7. Doubling of ploidy at each generation in the MiMe line.

In subsequent generations, MiMe plant ploidy doubled at each generation, from 2n (A) (10 chromosomes), to 4n (B) (20 chromosomes) and 8n (C) (40 chromosomes). The left column shows mitotic metaphase, scale bar = 10 µm. The right column shows the corresponding four-week-old plants, (scale bar = 2 cm) and flowers (scale bar = 1 mm).

Figure 8. Male meiosis in 2n, 4n, and 8n MiMe plants.

MiMe plants underwent a mitosis-like division instead of meiosis in 2n (A), 4n (B), and 8n (C) plants. Meiotic metaphase I, anaphase I, and telophase I are shown for each generation. Scale bar = 10 µm.