One-line hybrid rice with high-efficiency synthetic apomixis and near-normal fertility

Abstract

High-frequency clonal seeds and near-normal fertility were obtained by engineering synthetic apomixis in hybrid rice. The one-line strategy, with the advantage of unnecessary seed production, is the final stage for the hybrid rice development and can be achieved through the fixation of heterosis via artificially inducing apomixis. Recently, synthetic apomixis has been generated in rice by combining MiMe (Mitosis instead of Meiosis) with either the ectopic expression of BABY BOOM (BBM1 or BBM4) or mutation of MATRILINEAL (MTL), resulting in over 95.00% of clonal seeds. However, the frequency of clonal seeds was only 29.20% when AtDD45 promoter was used to drive BBM1. In addition, achieving both a high frequency of clonal seeds and near-normal fertility simultaneously had been elusive in earlier strategies. In this study, using AtDD45 promoter to drive BBM1 expression in combination with the MiMe mutant resulted in the apomixis frequency as high as 98.70%. Even more, employing fusion promoters (AtMYB98_AtDD1_OsECA1-like1) to drive WUS expression in combination with pAtDD45:BBM1 and MiMe could produce clonal seeds at rates of up to 98.21%, the highest seed setting rate reached to 83.67%. Multiple-embryos were observed in clonal lines at a frequency ranging from 3.37% to 60.99%. Transmission of the high frequency of apomixis through skipped generations (atavism) was identified in two clonal lines, even though it remained stable in the majority of clonal lines. These findings significantly advance the pursuit of fixed heterosis in rice through synthetic apomixis, edging closer to its agricultural application.

Keywords: Apomixis; Atavism; Multiple-embryos; One-line hybrid rice.

Fig. 1

Ploidy, genotype, and phenotype of progeny plants of transformations in hybrid rice. a Schematic diagram of the T-DNA structure sgMiMe (p24MiMe) targeting OsOSD1, PAIR1, and OsREC8 (MiMe). pZmUBIQUITIN1, Zea mays UBIQUITIN 1 promoter; Cas9, Cas9 gene; Nos, nopaline synthase terminator. b Schematic diagram of the T-DNA construct sgMiMe_pAtDD45:BBM1 (p63C) containing the MiMe and pAtDD45:BBM1 gene expression cassettes. pAtDD45, egg cell-specific from Arabidopsis; BBM1, BBM1 gene. c Schematic diagram of the T-DNA construct ‘sgMiMe’_pAtDD45:BBM1 (p94C). Compared to p63C, p94C was created by altering the gRNAs of three MiMe genes to increase editing efficiency. d Schematic diagram of the T-DNA construct ‘sgMiMe’_pAtMYB98_pAtDD1_pOsECA1-like1:WUS_pAtDD45:BBM1 (p95C). pAtMYB98, synergid cell-specific from Arabidopsis; pAtDD1, antipodal cell-specific from Arabidopsis; pOsECA1-like1, egg cell-specific from rice; WUS, WUS gene from Arabidopsis. PinII, the terminator of proteinase inhibitor II. e Plant morphology of line G23-6 and line G23-18 (p63C) were compared to wild-type. The sexual tetraploid plant displayed high sterility. Scale bars, 20 cm. f Comparison of the T₁ seeds between line G23-6 (p63C) and wild-type. The seed of tetraploid showed a large size with awns. Scale bars, 2 mm. g Representative ploidy analysis of a tetraploid and diploid according to flow cytometry. h The frequency of clonal seeds and seed-setting of p95C events. In the T₁ clonal lines of p95C, a high seed-setting rate was observed, but the clonal seed rate was low. Conversely, there was a high frequency of clonal seeds with a low seed-setting rate. Details were provided in Table 2. i Plants morphology of line HW14 (p94C) and line CL3 (p95C) were compared to wild-type. Scale bars, 20 cm. j Comparison of the panicles between T₁ diploid clonal plants (p94C and p95C) and wild-type. Scale bars, 5 cm. k Genotyping by pinpoint sequencing of wild-type, F₂ sexual progenies, T₁ clonal lines of p94C and p63C. Compared to wild-type, the site similarity of F₂ progeny plants ranged from 43.62% to 56.44% resulting from meiotic crossovers, while diploid clonal plants maintained a site similarity of more than 95.00% due to the absence of meiotic division

Fig. 2

Ploidy, phenotype, and embryo sac observation of multiple-embryos. a, b Discovery of multiple-embryos. In terms of morphology, the multiple-embryos displayed a single seed with two or three plumule axes. Scale bars, 2 mm. c, d Morphology of multiple-embryos. At 15 d after germination, the morphology of multiple-embryos showed two or three seedlings. Scale bars, 2 cm. e Discovery of multiple-embryos in p63C transgenic plants (without MiMe mutation). Germination of 6 d-old twin-embryos. Scale bars, 2 mm. f Flow cytometry DNA histograms for ploidy determination of multiple-embryos (2n/n). One was a sexual diploid showing a 2n peak, and the other was an asexual haploid showing a 1n peak. g Characterization of haploids. Differences in height between wild-type and haploid seedlings derived from p63C transformants (without MiMe mutation) at 6 d after germination. Scale bars, 2 mm. h Comparative analysis of the morphology of diploid and haploid floral organs. Scale bars, 1 mm. i Comparison of the morphology across different ploidy (2n/n) in multiple-embryos. Two seedlings were planted separately because the asexual haploid had limited growth potential. Scale bars, 20 cm. j Comparison of the main spikes in multiple-embryos (2n/n). Compared to the sexual diploid, the panicle of the asexual haploid showed infertile seeds. Scale bars, 5 cm. k Ploidy analysis of the multiple-embryos (2n/4n) according to flow cytometry. One of the multiple-embryos was diploid (asexual), showing a 2n peak; and the other was tetraploid (sexual), showing a 4n peak. l Comparison of the morphology across different ploidy (2n/2n, 4n/2n, and 4n/4n) in twin-embryos. Scale bars, 20 cm. m Comparison of the morphology across different ploidy (4n/2n/2n and 2n/2n/2n) in triple-embryos. Scale bars, 20 cm. n Representative embryos at 48 h and 72 h after flowering (HAF) from wild-type (I, II) and multiple-embryos (III, IV). Compared to wild-type, multiple-seedlings showed two embryos formed at the micropyle. Ge globular embryos, Pe pyriform embryos. Scale bars, 2 mm

Fig. 3

The phenomenon of atavism. a Frequency of clonal seeds in T₁, T₂, T₃ and T₄ plants of line G23-18 (p63C). b Comparison of frequency of clonal seeds from T₁ to T₅ plants in line G23-18 (p63C). In the T₁, T₂, T₃, T_4, and T₅ generations, the average frequency of diploid clonal plants was 96.55%, 15.18%, 96.75%, 13.54%, and 17.56%, respectively. Scale bars, 20 cm. Details were provided in Table S3. c Seed-setting rate in T₁ to T₅ clonal plants of line G23-18 (p63C). In the T₁, T₂, T₃, T₄ and T₅ generations, the average seed-setting rate was 51.35%, 56.92%, 49.64%, 58.82%, and 55.88%, respectively