Synthetic apomixis with normal hybrid rice seed production

Heterosis has long been exploited in the hybrid seed industry, which contributes to high and stable yields of modern agriculture (Huang et al., 2016Huang X. Yang S. Gong J. Zhao Q. Feng Q. Zhan Q. Zhao Y. Li W. Cheng B. Xia J. et al.Genomic architecture of heterosis for yield traits in rice.Nature. 2016; 537: 629-633Crossref PubMed Scopus (261) Google Scholar). However, heterosis phenotypes of hybrid plants are segregated in its offspring. Apomixis allows instant fixation and propagation though seeds with heterozygous genotypes, showing great potential in plant breeding and agricultural practice (Ye and Cui, 2019Ye J. Cui X. Clonal propagation of hybrid seeds.Mol. Plant. 2019; 12: 141-142Abstract Full Text Full Text PDF Google Scholar). Apomixis naturally occurs in hundreds of plant species, but it is absent in major crop species (Underwood and Mercier, 2022Underwood C.J. Mercier R. Engineering apomixis: clonal seeds approaching the fields.Annu. Rev. Plant Biol. 2022; 73: 201-225Crossref PubMed Scopus (11) Google Scholar). Recently, synthetic apomixis has been engineered in rice by combining Mitosis instead of Meiosis (MiMe) with a mutation of MATRILINEAL or ectopic expression of BABY BOOM1 (BBM1), enabling clonal reproduction of F1 hybrids through seeds and stable transmission of heterotic phenotypes over generations (Khanday et al., 2019Khanday I. Skinner D. Yang B. Mercier R. Sundaresan V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.Nature. 2019; 565: 91-95Crossref PubMed Scopus (200) Google Scholar; Wang et al., 2019Wang C. Liu Q. Shen Y. Hua Y. Wang J. Lin J. Wu M. Sun T. Cheng Z. Mercier R. Wang K. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes.Nat. Biotechnol. 2019; 37: 283-286Crossref PubMed Scopus (157) Google Scholar; Liu et al., 2022Liu C. He Z. Zhang Y. Hu F. Li M. Liu Q. Huang Y. Wang J. Zhang W. Wang C. Wang K. Synthetic apomixis enables stable transgenerational transmission of heterotic phenotypes in hybrid rice.Plant Commun. 2022; 100470: 100470Abstract Full Text Full Text PDF Scopus (2) Google Scholar). However, the fertility of both two strategies was significantly reduced compared with that of the wild type, which hinders the application of both strategies in agriculture. In this study, we established synthetic apomixis with a high fertility that is almost comparable to normal hybrid rice. BBM belongs to the AP2 family of transcription factors and plays important roles in embryo development (Boutilier et al., 2002Boutilier K. Offringa R. Sharma V.K. Kieft H. Ouellet T. Zhang L.M. Hattori J. Liu C.M. van Lammeren A.A.M. Miki B.L.A. et al.Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth.Plant Cell. 2002; 14: 1737-1749Crossref PubMed Scopus (695) Google Scholar). Several studies revealed that ectopic expression of the PsBBM-like gene in egg cells could induce parthenogenesis in sexual pearl millet, maize, and rice (Conner et al., 2015Conner J.A. Mookkan M. Huo H. Chae K. Ozias-Akins P. A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant.Proc. Natl. Acad. Sci. USA. 2015; 112: 11205-11210Crossref PubMed Scopus (116) Google Scholar, Conner et al., 2017Conner J.A. Podio M. Ozias-Akins P. Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes.Plant Reprod. 2017; 30: 41-52Crossref PubMed Scopus (54) Google Scholar). In addition, it is further found that parthenogenesis can also be induced when rice endogenous BBM1 was ectopically expressed in egg cells (Khanday et al., 2019Khanday I. Skinner D. Yang B. Mercier R. Sundaresan V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.Nature. 2019; 565: 91-95Crossref PubMed Scopus (200) Google Scholar). Besides BBM1, three BBM-like genes, namely BBM2, BBM3, and BBM4, were also identified with varying expression patterns in rice (Anderson et al., 2017Anderson S.N. Johnson C.S. Chesnut J. Jones D.S. Khanday I. Woodhouse M. Li C.X. Conrad L.J. Russell S.D. Sundaresan V. The zygotic transition is initiated in unicellular plant zygotes with asymmetric activation of parental genomes.Dev. Cell. 2017; 43: 349-358Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar; Khanday et al., 2019Khanday I. Skinner D. Yang B. Mercier R. Sundaresan V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.Nature. 2019; 565: 91-95Crossref PubMed Scopus (200) Google Scholar). As all BBM proteins shared high similarity with BBM1 (Supplemental Figure 1), we wondered whether those genes can also be used to trigger parthenogenesis in rice, so we ectopically expressed each of them by using the Arabidopsis promoter pDD45 (Figure 1A ), which has been shown to induce egg-cell-specific expression in rice (Khanday et al., 2019Khanday I. Skinner D. Yang B. Mercier R. Sundaresan V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.Nature. 2019; 565: 91-95Crossref PubMed Scopus (200) Google Scholar). The elite inter-subspecific hybrid rice Chunyou84 (CY84) was selected for Agrobacterium-mediated genetic transformation. A total of 10, 7, and 8 independent ectopically expressed plants were generated for BBM2, BBM3, and BBM4, respectively (referred to as EE–BBM2, EE–BBM3, and EE–BBM4 hereafter). These transgenic plants showed normal vegetative growth and plant morphology. We further investigated the seed setting and found that all EE–BBM3 lines exhibited a dramatically decreased seed-setting rate (range: 10.3%–55%) compared with that of CY84 (79%, range: 73.4%–83.3%), whereas the EE–BBM2 lines (range: 49%–85.7%) and EE–BBM4 plants (range: 21.1%–82.6%) showed varied but relatively high seed-setting rates (Figure 1B and 1C and Supplemental Figures 2 and 3). To determine whether those ectopic expression plants gained the ability to induce parthenogenesis, we harvested the seeds and genotyped their self-pollinated progeny by using 12 insertion/deletion markers (one per chromosome; Supplemental Table 1; Supplemental Figure 4) to identify probable haploid or doubled haploid offspring, which theoretically would be homozygous at all markers. No plants homozygous at all markers were found in the 140 progenies from the wild-type CY84. Similarly, no homozygous individuals were identified in the 966 offspring of EE–BBM2 and 691 offspring of EE–BBM3 plants, respectively (Figure 1B). In contrast, 13 individuals from 411 EE–BBM4 progeny were found to be homozygous at all 12 markers. To further verify the genotype of those identified plants, the whole genomes of 4 plants were resequenced with next-generation sequencing technology. By analyzing >581 737 SNP markers that differed between two parents (16A and C84), we found that all four plants were fully homozygous at all chromosomes and recombinant compared with the parental genome (Figure 1D), indicating that those plants were each derived from a single gamete. We further determined the ploidy level of the homozygous plants using flow cytometry and found that all of them were haploids (Figure 1E). All haploid plants exhibited reduced plant height, decreased panicle length and glume size, and the complete loss of fertility (Figure 1F). These results demonstrate that egg-cell expression of BBM4 is able to trigger parthenogenesis and induce haploids in hybrid rice. Next, to explore whether ectopic BBM4 expression could be applied to engineer synthetic apomixis, we designed a single construct, sgMiMe_pDD45:BBM4, that combined MiMe (simultaneous editing of the REC8, PAIR1, and OSD1 genes) with egg-cell expression of BBM4 (Figure 1G) and introduced it into CY84 by genetic transformation. Out of 26 transgenic plants, six were found to contain co-mutations in PAIR1, REC8, and OSD1 (Supplemental Figure 5), as well as the ectopic expression of BBM4. Those plants (named Fix2, for the Fixation of hybrids2) were grown for further phenotype analysis. During the vegetative stage, the Fix2 plants displayed normal morphology (Figure 1H). Unexpectedly, at the mature stage, all Fix2 plants showed a high seed-setting rate (range: 80.9%–86.1%), which was comparable to that of CY84 (84.4%, range: 82.1%–86.6%; Figure 1I and Supplemental Figure 6). The results suggest that the reduced fertility of EE–BBM4 might be rescued by introducing MiMe in hybrid rice. To investigate whether Fix2 plants with a high seed-setting rate can produce clonal seeds, the offspring of three plants were selected for further analysis (Figure 1I). In the progeny plants, we conducted flow cytometry to determine the ploidy level of the progeny of Fix2 plants (Figure 1J). Out of 156, 152, and 169 progeny individuals from the Fix2 plants, two, two, and four were, respectively, identified to be diploid plants, while the remaining plants were all identified as tetraploid plants. To examine whether the hybrid vigor, especially the high fertility, can be inherited to the next generation, we planted the diploid offspring and investigated their phenotype. The diploid offspring of Fix2 also exhibited similar plant, panicle, and grain morphology to that of the hybrid rice CY84 (Figure 1K and Supplemental Figure 7). Particularly, the seed-setting rate of diploid offspring of Fix2 (range: 80.9%–82%) was also comparable to that of CY84 (81.9%, range: 78.8%–88.1%), indicating that the high fertility of Fix2 can be inherited to the next generation (Figure 1L). Finally, to test whether the heterozygosity was fixed, we performed whole-genome sequencing of three diploid offspring of Fix2 plants with an average of 15-fold coverage. Bioinformatic analysis using >1 620 954 SNPs showed that the diploid progeny plants were heterozygous at whole genome and genetically identical to CY84 (Figure 1M), suggesting that the heterozygous genotype was fully fixed in those diploid clonal plants. Additionally, the foreign elements of sgMiMe_pDD45:BBM4 were also retained in the clonal Fix2 plants (Supplemental Figure 8). Taken together, the results imply that synthetic apomixis with high fertility can be established by combining MiMe with ectopic expression of BBM4 in hybrid rice. Seed production is one of the most important traits for hybrid rice cultivation. However, in previously established MATRILINEAL-dependent or BBM1-dependent synthetic apomictic strategies, the fertility in both is severely decreased, leading to a significant yield loss and therefore prohibiting the immediate application of both strategies (Khanday et al., 2019Khanday I. Skinner D. Yang B. Mercier R. Sundaresan V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.Nature. 2019; 565: 91-95Crossref PubMed Scopus (200) Google Scholar; Wang et al., 2019Wang C. Liu Q. Shen Y. Hua Y. Wang J. Lin J. Wu M. Sun T. Cheng Z. Mercier R. Wang K. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes.Nat. Biotechnol. 2019; 37: 283-286Crossref PubMed Scopus (157) Google Scholar). In this study, we revealed that egg-cell expression of BBM4 can also trigger parthenogenesis and the production of haploid offspring in hybrid rice. However, the fertility of those EE–BBM4 plants were also affected to different degrees. When egg-cell expression of BBM4 was combined with the mutations of MiMe, the synthetic apomixis was successfully induced, and clonal offspring with fixed genotype can be obtained. Strikingly, the fertility of the apomictic plant was not obviously affected, resulting in a high seed-setting rate almost comparable to that of the normal hybrid. Moreover, the high fertility can be stably inherited to the next generation. As the mutations of MiMe lead to the doubling of the ploidy of the gametes, the rescued fertility of apomictic plant implies that the diploid egg cells might be more likely to survive than the haploid egg cells when parthenogenesis occurs in hybrid rice. Of course, the BBM4-dependent synthetic apomictic strategy is still not fully penetrated, and only a small percentage of the offspring were identified as clonal diploid plants. In the future, more egg-cell-specific promoters, especially rice native promoters, can be chosen to drive the expression of BBM4 and test their ability in inducing parthenogenesis and synthetic apomixis. In addition, it has been shown revealed that BBM4 displays different expression patterns from BBM1, implying at least partially divergent roles between them during early embryogenesis (Anderson et al., 2017Anderson S.N. Johnson C.S. Chesnut J. Jones D.S. Khanday I. Woodhouse M. Li C.X. Conrad L.J. Russell S.D. Sundaresan V. The zygotic transition is initiated in unicellular plant zygotes with asymmetric activation of parental genomes.Dev. Cell. 2017; 43: 349-358Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar; Khanday et al., 2019Khanday I. Skinner D. Yang B. Mercier R. Sundaresan V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.Nature. 2019; 565: 91-95Crossref PubMed Scopus (200) Google Scholar). Thus, co-expression of both BBM1 and BBM4 in egg cells may also be used to elevate the frequency of clonal seeds and promote the application of synthetic apomixis in agriculture. This work was supported by the National Natural Science Foundation of China (32188102, 32025028, and U20A2030), the National Key Research and Development Program of China (2022YFF1003304), the Central Public-interest Scientific Institution Basal Research Fund (Y2022QC20), and the Hainan Yazhou Bay Seed Laboratory (B21HJ0215).