High-frequency synthetic apomixis in hybrid rice

Abstract

Introducing asexual reproduction through seeds - apomixis - into crop species could revolutionize agriculture by allowing F1 hybrids with enhanced yield and stability to be clonally propagated. Engineering synthetic apomixis has proven feasible in inbred rice through the inactivation of three genes (MiMe), which results in the conversion of meiosis into mitosis in a line ectopically expressing the BABYBOOM1 (BBM1) parthenogenetic trigger in egg cells. However, only 10-30% of the seeds are clonal. Here, we show that synthetic apomixis can be achieved in an F1 hybrid of rice by inducing MiMe mutations and egg cell expression of BBM1 in a single step. We generate hybrid plants that produce more than 95% of clonal seeds across multiple generations. Clonal apomictic plants maintain the phenotype of the F1 hybrid along successive generations. Our results demonstrate that there is no barrier to almost fully penetrant synthetic apomixis in an important crop species, rendering it compatible with use in agriculture.

Fig. 1. Ploidy and genotype of progeny plants of transformation events harboring the T314 and T315 apomixis-inducing T-DNA constructs.

A Schematic representation of the T-DNA constructs used to induce the triple MiMe mutation and the triggering of parthenogenesis, resulting in synthetic apomixis. Upper: T313 sgRNA MiMe T-DNA Middle: T314 sgRNA MiMe_pAtECS:BBM1 T-DNA Lower: T315 sgRNA_pOsECS:BBM1 T-DNA: LB and RB: left and right borders of the T-DNA; p35S: promoter of the Cauliflower Mosaic Virus (CaMV); 35S: polyadenylation signal of CaMV; hpt cat int: hygromycin phosphotransferase II with castor bean catalase intron; ZmUbi: promoter, first intron and first exon of the maize Ubiquitin 1 gene; “Os”Cas9: rice-optimized Cas9 coding sequence; NLS: nucleus localization signal fused to Cas9; OSD1, OSD1/2, PAIR1 and REC8: Four cassettes including sgRNAs (20 bp crRNA specific to the target gene + 82 bp tracR RNA) driven by the Pol III U3 promoter targeting OsOSD1, PAIR1, and OsREC8; EC1.2: egg cell-specific promoter from Arabidopsis^,; OsECA1: egg cell-specific promoter from rice^,. B Principle for formation of tetraploid and diploid clonal progenies in MiMe and MiMe + BBM1 plants, respectively. C Representative flow cytometry histograms of DAPI-marked nuclei suspensions isolated from young leaf blade of a diploid (upper) and tetraploid (lower) progeny plants. D Upper: Genealogy of the plants selected for whole-genome sequencing including 1 F and D24 parents (two plants each); heterozygous F1 hybrid BRS-CIRAD 302 (two plants), six F2 sexual progenies; T314 15.1 and 37.7 primary transformants (T0); three T1 progeny plants of each of the sequenced T0 plants; three T2 progenies of each of the sequenced T1 plants (i.e., nine T2 plants per event). Lower: Graphical representation of genotypes of the 12 rice chromosomes established from whole-genome sequences of homozygous parents, heterozygous F1 hybrid, F2 progeny plants, T0 events T314 15.1 and 37.7 and their T1 and T2 progenies. Changes in color along F2 progeny chromosomes mark heterozygous-to-homozygous transitions resulting from meiotic crossovers.

Fig. 2. Phenotype, panicle fertility, and grain quality of progeny plants of selected apomictic events harboring the T314 and T315 T-DNA constructs.

A Phenotypes of plants grown under controlled greenhouse conditions. Left: Five F2 progeny plants derived from the self-fertilization of BRS-CIRAD 302 compared to a BRS-CIRAD 302 F1 plant. Right: Three T1 progenies from T314 15.1 event compared to a BRS-CIRAD 302 F1 plant. B Phenotypes of T2 progenies grown under controlled greenhouse conditions: 5–6 T2 progeny plants of a T1 plant of events T314 15.1, T314 37.7, T315 5.4, and T315 8.1 are compared to a BRS-CIRAD 302 F1 plant. Senescent leaves of the plants have been removed for photographing. C Panicles of the BRS-CIRAD 302 F1 hybrid and of T314 15.1 T2 plants. The master panicles of five distinct plants have been pooled for photographing. D Distribution of seed filling rate among BRS-CIRAD 302 F1 plants, and T2 progeny plants of T314 events (15.1 and 37.7) and T315 events (5.4 and 8.1 events). Average panicle fertilities of the apomictic lines represent 75.8%, 60.7, 79.9%, and 74.7%, respectively, of that of control plants (100%) (dotted red line). Significance of the differences are based on Duncan’s test, dof = 4, confidence interval of 95%. E Husked and dehulled seeds of 1 F and D24 parents, F1 and F2 generations and apomictic lines. Upper: 1 F and D24 parents, F1 hybrid seeds harvested on 1 F parent, F2 seeds harvested on the F1 hybrid. Lower: T3 seeds harvested from apomictic plants in the four selected apomictic lines. F Starch and amylose content of F2 seeds and T3 seeds harvested from BRS-CIRAD 302 F1 plants and T2 apomictic plants, respectively. Different letters indicate significant differences (α-risk = 0.05) in a Kruskal–Wallis test: Event T314 37.7 exhibits a significantly lower starch content than F2 seeds and seeds of event T315 8.1 and 5.4. Source data are provided as a Source Data file.