Turning rice meiosis into mitosis

Abstract

Introduction of clonal reproduction through seeds (apomixis) in crops has the potential to revolutionize agriculture by allowing self-propagation of any elite variety, in particular F1 hybrids. In the sexual model plant Arabidopsis thaliana synthetic clonal reproduction through seeds can be artificially implemented by (i) combining three mutations to turn meiosis into mitosis (MiMe) and (ii) crossing the obtained clonal gametes with a line expressing modified CENH3 and whose genome is eliminated in the zygote. Here we show that additional combinations of mutations can turn Arabidopsis meiosis into mitosis and that a combination of three mutations in rice (Oryza sativa) efficiently turns meiosis into mitosis, leading to the production of male and female clonal diploid gametes in this major crop. Successful implementation of the MiMe technology in the phylogenetically distant eudicot Arabidopsis and monocot rice opens doors for its application to any flowering plant and paves the way for introducing apomixis in crop species.

Figure 1

Chromosome spreads of male meiosis in wild-type Arabidopsis and Arabidopsis MiMe genotypes. (A-F) Wild type. (A) Metaphase I with five aligned bivalents. (B) Anaphase I. (C) Telophase I. (D) Metaphase II. (E) Anaphase II. (F) Telophase II. (G-I) prd3 rec8 osd1 triple mutant (n = 17). (J-L) prd1 rec8 osd1 triple mutant (n = 21). (M-O) prd2 rec8 osd1 triple mutant (n = 27). (G, J, M) Metaphase I with 10 aligned univalents. (H, K, N) Anaphase I with segregation of 10 pairs of chromatids. (I, L, O) Telophase. Scale bar = 10 μm.

Figure 2

Phylogram of OSD1 and UVI4 homologues from flowering plants. Consensus tree of OSD1 and UVI4 protein family inferred from a Muscle alignment. Analyses were performed using the MPI Bioinformatic Toolkit, Muscle and Phylip-neighbor with default parameters, enabling boostrapping with 100 replicates. The numbers of forks indicate the number of times the group consisting of the species that are to the right of that fork occurred among the trees, out of 100 trees. AT, Arabidopsis thaliana; AL, Arabidopsis lyrata; BD, Brachypodium distachyon; BR, Brassica rapa (turnip mustard); CL, Citrullus lanatus (water melon); CM, Cucumis melo (muskmelon); CP, Carica papaya (papaya); CRU, Capsella rubella; CS, Citrus sinensis (orange); EG, Eucalyptus grandis (eucalyptus); GM, Glycine max (soybean); GR, Gossypium raimondii (cotton); HV, Hordeum vulgare (barley); LJ, Lotus japonicus; MA, Musa acuminata (banana); MD, Malus domestica (apple); ME, Manihot esculenta (cassava); OS, Oryza sativa (rice); PPE Prunus persica (peach); RC Ricinus communis (castor bean plant); SB, Sorghum bicolor (sorghum); SI, Setaria italica (foxtail millet); SL Solanum lycopersicum (tomato); ST, Solanum tuberosum (potato); TC, Theobroma cacao (cacao); VV, Vitis vinifera (grape vine); ZM, Zea mays (maize).

Figure 3

Male meiotic products in wild-type rice and Ososd1. Fresh anthers squashed in acetocarmine. (A) Tetrads of spores in wild type. (B) Dyad of spores in Ososd1-1 (n = 500).

Figure 4

Chromosome spreads of male meiosis in wild-type rice and Ososd1. (A-F) Wild type. (A) Metaphase I with 12 aligned bivalents. (B) Anaphase I. (C) Telophase I. (D) Metaphase II. (E) Anaphase II. (F) Telophase II. (G-H) Ososd1-1 (n = 62). (G) Metaphase I with 12 aligned bivalents. (H) Anaphase I. (I) Late anaphase I. (J) Telophase I. No second division was observed. Scale bar = 10 μm.

Figure 5

Ploidy of Ososd1-1 pollen grains. The ploidy was determined using staining of isolated nuclei with propidium iodide followed by flow cytometry. (A) Wild-type Nipponbare pollen grains. A single peak is observed, corresponding to haploid nuclei. (B) Wild-type Nipponbare leaf. A single peak, corresponding to diploid nuclei. (C) Ososd1^+/+ pollen grains. A single peak is observed, corresponding to haploid nuclei. (D) Ososd1^−/− pollen grains. A single peak is observed, corresponding to diploid nuclei.

Figure 6

Male meiosis I in rice pair1, Osrec8 and pair1 Osrec8 mutants. (A-B) pair1 (n = 154). (A) Metaphase I with 24 unaligned univalents. (B) Anaphase I with unbalanced segregation of univalents. (C, D) Osrec8 (n = 222). (C) Metaphase I with abnormal chromosomes. (D) Anaphase I with chromosome fragmentation. (E, F) pair1 Osrec8 (n = 154). (E) Metaphase I with 24 aligned univalents. (F) Anaphase I with segregation of 24 pairs of chromatids. Scale bar = 10 μm.

Figure 7

Creating OsMiMe. (A) Crossing scheme. OsMiMe mutant was created first by crossing B01997 plants heterozygous for the Osrec8 mutation with AQUG12 plants heterozygous for the pair1 mutation (step 1). 1/4 of F1 were double heterozygous plants and were in turn crossed with AMBA12 plants heterozygous for the Ososd1 mutation to produce 1/8 of F1 triple heterozygous plants (Osrec8^+/−; pair1^+/−; Ososd1^+/−) (step 2). These plants were selfed (step 3) and the progeny were genotyped to identify 1/64 triple homozygous OsMiMe plants and relevant control segregants. HW, Hwayoung; NB, Nipponbare; He, heterozygous. (B) Fertility in MiMe and related mutants. The histogram represents the percentage of flowers giving a seed by self-fertilization. Numbers of observed flowers and seeds are indicated. Errors bars correspond to the SD for 4-7 plants.

Figure 8

Male meiosis I in OsMiMe. (A) Metaphase I with 24 aligned univalents. (B, C) Anaphase I with segregation of 24 pairs of chromatids. (D) Telophase I. n = 43. Scale bar = 10 μm.

Figure 9

Genotypes of OsOsd1 and OsMiMe diploid parentals and their respective tetraploid progeny siblings. Tetraploid offpring of OsOsd1 and OsMiMe plants (refer to Figure 7 for scheme of creation of plant materials) were genotyped using 10 SNP markers residing on rice chromosomes 7 and 11. Positions of markers (brown) and centromeres (black) are indicated along the chromosomes. Each line represents a plant. For each marker, plants carrying only the Hwayoung allele are in yellow, plants carrying only the Nipponbare allele are in blue, while plants with both the Hwayoung and Nipponbare alleles appear in green. Osd1 and OsMiMe parental plants (top line of upper and lower panels, respectively) are heterozygous at the 10 markers. While the offspring of OsOsd1 exhibit segregation of parental alleles at the 10 markers, all the offspring of OsMiMe are heterozygous at the same 10 markers and exhibit the same genotype like the parental plants.