
==== Front
Plant Cell Physiol
Plant Cell Physiol
pcp
Plant and Cell Physiology
0032-0781
1471-9053
Oxford University Press UK

39107984
10.1093/pcp/pcae074
pcae074
Regular Paper
AcademicSubjects/SCI01180
Wheat Cybrid Plants, OryzaWheat, Regenerated from Wheat–Rice Hybrid Zygotes via in Vitro Fertilization System Possess Wheat–Rice Hybrid Mitochondria
Maryenti Tety Department of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397, Japan
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424, Indonesia

https://orcid.org/0000-0001-9469-3757
Koshimizu Shizuka Bioinformation and DDBJ Center, National Institute of Genetics, Shizuoka 411-8540, Japan
Graduate Institute for Advanced Studies, SOKENDAI, Mishima, Shizuoka 411-8540, Japan

Onda Nonoka Department of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397, Japan

Ishii Takayoshi Arid Land Research Center, Tottori University, Tottori 680-001, Japan

Yano Kentaro Department of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397, Japan
WellGreen-i Co. Ltd., Kanagawa 215-0007, Japan

https://orcid.org/0000-0001-6531-210X
Okamoto Takashi Department of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397, Japan

*Corresponding author: E-mail, okamoto-takashi@tmu.ac.jp
8 2024
07 8 2024
07 8 2024
65 8 13441357
07 3 2024
20 6 2024
06 7 2024
21 6 2024
06 8 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
2024
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Abstract

Hybridization generates biodiversity, and wide hybridization plays a pivotal role in enhancing and broadening the useful attributes of crops. The hybridization barrier between wheat and rice, the two most important cereals, was recently overcome by in vitro production of allopolyploid wheat–rice hybrid zygotes, which can develop and grow into mature plants. In the study, genomic sequences and compositions of the possible hybrid plants were investigated through short- and long-read sequencing analyses and fluorescence in situ hybridization (FISH)-based visualization. The possible hybrid possessed whole wheat nuclear and cytoplasmic DNAs and rice mitochondrial (mt) DNA, along with variable retention rates of rice mtDNA ranging from 11% to 47%. The rice mtDNA retained in the wheat cybrid, termed Oryzawheat, can be transmitted across generations. In addition to mitochondrial hybridization, translocation of rice chromosome 1 into wheat chromosome 6A was detected in a F1 hybrid individual. OryzaWheat can provide a new horizon for utilizing inter-subfamily genetic resources among wheat and rice belonging to different subfamilies, Pooideae and Ehrhartoideae, respectively.

Dryland
Heterogeneous cytoplasm
Mitochondria
Oryza sativa
OryzaWheat
Triticum aestivum
Wide hybridization
Japan Society for the Promotion of Science 10.13039/501100001691 Grant-in-Aid for Scientific Research (C), No. 22K0 Grant-in-Aid for Scientific Research(B), No. 22H02 New Energy and Industrial Technology Development Organization 10.13039/501100001863 Moonshot program, No. 22101490-0 Japan Science and Technology Corporation 10.13039/501100001695 JST-FOREST, No. JPMJFR 2001 JST-Mirai Program, No. 23-231038606 Tottori University 10.13039/501100005695 Joint Research Program of Arid Land Research Cente Japan Society for the Promotion of Science 10.13039/501100001691 Grant-in-Aid for Scientific Research (C), No. 22K0 Grant-in-Aid for Scientific Research(B), No. 22H02 New Energy and Industrial Technology Development Organization 10.13039/501100001863 Moonshot program, No. 22101490-0 Japan Science and Technology Corporation 10.13039/501100001695 JST-FOREST, No. JPMJFR 2001 JST-Mirai Program, No. 23-231038606 Tottori University 10.13039/501100005695 Joint Research Program of Arid Land Research Cente Japan Society for the Promotion of Science 10.13039/501100001691 Grant-in-Aid for Scientific Research (C), No. 22K0 Grant-in-Aid for Scientific Research(B), No. 22H02 New Energy and Industrial Technology Development Organization 10.13039/501100001863 Moonshot program, No. 22101490-0 Japan Science and Technology Corporation 10.13039/501100001695 JST-FOREST, No. JPMJFR 2001 JST-Mirai Program, No. 23-231038606 Tottori University 10.13039/501100005695 Joint Research Program of Arid Land Research Cente Japan Society for the Promotion of Science 10.13039/501100001691 Grant-in-Aid for Scientific Research (C), No. 22K0 Grant-in-Aid for Scientific Research(B), No. 22H02 New Energy and Industrial Technology Development Organization 10.13039/501100001863 Moonshot program, No. 22101490-0 Japan Science and Technology Corporation 10.13039/501100001695 JST-FOREST, No. JPMJFR 2001 JST-Mirai Program, No. 23-231038606 Tottori University 10.13039/501100005695 Joint Research Program of Arid Land Research Cente
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pmcIntroduction

Hybridization plays an important role in the evolution and diversification of angiosperms, as an increase in genetic variation can be achieved through sexual or somatic hybridization between distant species. Sexual hybridization is generally conducted through cross-pollination, and the resulting hybrid zygote, which possesses totipotent potential for embryo formation, can transmit heterogeneous genetic material to the progeny. However, sexual hybridization between intergeneric or inter-subfamily combinations often results in unsuccessful fertilization/embryogenesis owing to pre- and/or post-fertilization barriers (Haig and Westoby 1991, Morgan et al. 2010).

Rice and wheat are significant cereal crops with valuable genetic resources. However, hybridization between wheat and rice has been ineffective due to the fact that wheat and rice belong to different subfamilies, Pooideae and Ehrhartoideae, respectively. A new method of in vitro gamete fusion has facilitated the production of regenerated plants in rice (Uchiumi et al. 2007), wheat (Maryenti et al. 2019) and maize (Kranz and Lörz 1993), and a combination of these three in vitro fertilization systems (IVFs) offers the possibility of artificially generating inter-subfamily zygotes. Recently, we produced allopolyploid wheat–rice hybrid zygotes via IVF with various gamete combinations, and some developed into possible hybrid plants, bypassing hybrid incompatibility (Maryenti et al. 2021). Hybrid zygotes derived from one rice egg cell (Re) and one wheat sperm cell (Ws) ceased at the globular embryo-like structural stage, possibly due to a dysfunction of the nuclear–cytoplasmic relationship between wheat nuclear genome and rice cytoplasmic genome, as the rice nuclear genome was eliminated during the development of ReWs zygotes (Fig. 1A). This hybridization defect was overcome by adding one wheat egg cell to the ReWs zygote, followed by the subsequent culture and regeneration of the resulting ReWsWe zygotes/embryos (Maryenti et al. 2021) (Fig. 1B). Following the success in overcoming hybrid incompatibility through the addition of wheat egg cells, a different combination involving a set of two gametes of rice and wheat (ReRsWeWs), termed the double zygote (DZ), was created (Fig. 1C). These results strongly suggested that ReWsWe and DZ-derived plants possessed a ‘wheat’ nuclear genome and a ‘wheat’ + ‘rice’ cytoplasmic genome (Fig. 1B, C), and that the wheat-like plants derived from these allopolyploid hybrid zygotes will correspond to cytoplasmic hybrid between wheat and rice.

Fig. 1 Schematic representation of the effects of gamete combination on the developmental profiles of wheat–rice hybrid zygotes (A–C) and mature plants regenerated from the hybrid zygotes (D). (A) Hybrid zygotes derived from one rice egg (Re) and one wheat sperm (Ws) cease their development at the globular embryo-like structure stage after a selective elimination of rice chromosomes. Cells of the resulting embryos possess wheat nuclear genome and rice cytoplasmic genome, which possibly trigger nuclear–cytoplasmic dysfunction between wheat and rice. (B) Hybrid incompatibility of ReWs zygote presented in panel (A) can be overcome by additional fusion of one wheat egg cell (We) to the wheat–rice hybrid zygote. During the development of the resulting ReWsWe hybrid zygotes, rice chromosomes are eliminated and cells of the ReWsWe embryos possess a ‘wheat’ nuclear genome and a ‘rice + wheat’ cytoplasm genome. The ReWsWe embryos proliferate and regenerate into mature plants. (C) Fusion of wheat and rice gamete sets, wheat egg cell (We), wheat sperm cell (Ws), rice egg cell (Re) and rice sperm cell (Rs) results in fusion products between wheat and rice zygote. The wheat–rice DZ also develops and regenerates into mature plant via elimination of rice chromosomes. (D) Regenerated plants DZ-1A, -2A, -3B and -5B from DZ and ReWsWe-1A, -2A and -3A from ReWsWe zygotes.

Cytoplasmic hybridization in angiosperms has been intensively investigated in mitochondria and mitochondrial DNA, as active fusion and fission of mitochondria occur between two genetically different mitochondria that are colocalized within a cell. The mitochondrial hybridization has been reported in hybrid plants produced by somatic cell fusion and subsequent culture of the hybrid cells (Rothenberg and Hanson 1988, Temple et al. 1992, Sanchez-Puerta et al. 2015) and in Pelargonium, whose mitochondria are transmitted through a biparental manner (Matsushima et al. 2008, Apitz et al. 2013). In addition, mitochondrial hybridization across species boundaries can be achieved through natural horizontal transfer (Hao et al. 2010, Mower et al. 2010) and grafting system (Gurdon et al. 2016). As eukaryotic organisms have evolved through endo symbiosis, the expression of cytoplasm functions is achieved through nucleus–cytoplasm (CN) relationship, and cytoplasmic variation is tightly linked with phenotypic expression through NC relationship (Sloan et al. 2018, Postel and Touzet 2020, Nakamura 2023). The cytoplasmic hybridization increases the CN relationship and combination, and the variations in CN relationship/combination are relevant from both agricultural and evolutionary perspectives (Levings 1990, Bock et al. 2014), as the proper functioning of metabolism, cellular homeostasis and environmental sensing highly rely on plasmotypic activity (Johnston 2018). Moreover, impacts of plasmotypic variation are particularly pronounced under conditions of fluctuating and stressful environments, because plasmotype serves as a reservoir of variations that are predominantly exposed under certain conditions (Flood et al. 2020). These suggest that distant hybridization of cytoplasm among major crops will be highly important to broaden the variety of CN combination and subsequent physiological and agricultural traits of crops. Therefore, addressing the hybridization status of the possible cytoplasmic hybrid wheats derived ReWsWe and DZ zygotes is crucial, as cytoplasmic hybridization between wheat and rice has not been created due to reproductive barriers and an inability in somatic hybridization between wheat and rice. In the present study, genomic sequences and compositions of the possible cytoplasmic hybrids were investigated through short- and long-read sequencing analyses and fluorescence in situ hybridization (FISH)-based visualization.

Results

Genome composition of possible wheat–rice hybrids

We first conducted short-read sequencing of genome DNA (gDNA) isolated from a possible wheat–rice hybrid plant, line DZ-1A (Fig. 1D), and estimated the genomic origin of the sequence reads from the DZ-1A plant. For mitochondrial genomes, the reads estimated as ‘wheat origin’ were found to be derived from whole regions of the wheat mitochondrial genome (Fig. 2A, lower panel; Supplementary Fig. S1A). Notably, in addition to wheat, the reads estimated as ‘rice origin’ were found to be derived from eight major regions on the rice mitochondrial genome (Fig. 2A, upper panel), and the DNA regions unmapped by ‘rice origin’ reads on the rice mitochondrial genome did not correspond to genome region where reads estimated as ‘common between wheat and rice genome’ were mapped (Supplementary Fig. S1B). These suggest that partial regions from rice mitochondrial genome were retained in DZ-1A plant. The coverage of the eight regions from which the rice origin reads were derived was calculated based on raw read counts (≥5) (Supplementary Table S1) and estimated to encompass 47% of the rice mitochondrial genome. In the case of wheat and rice nuclear genomes, the reads estimated as wheat origin were found to be derived from whole regions of the wheat nuclear genome, whereas apparent mapping peak from rice-origin reads was not detected in the rice nuclear genome and coverage of the leads on rice genome was less than that on wheat genome (Fig. 2B, Supplementary Fig. S1C, D). These findings are consistent with the massive elimination of rice chromosomes during the development of DZ zygotes (Maryenti et al. 2021), and strongly suggested that DZ zygote-derived plant possessed a ‘wheat’ nuclear genome and a ‘wheat’ + ‘rice’ cytoplasmic genome. For plastid genomes, we found that the reads estimated for wheat origin were derived from whole regions of the wheat plastid genome (Supplementary Fig. S2A, lower panel). In addition to wheat, reads estimated as rice origin were detected in two major regions of the rice plastid genome (Supplementary Fig. S2A, upper panel). However, although genomic PCR using specific primers set for rice mitochondrial DNA regions, which are putatively retained in DZ-1A plant, resulted in detection of amplified DNA bands specifically derived from rice mitochondria (Supplementary Fig. S2B, left panel), the rice plastid genome region could not be assessed via genomic PCR even if we tried to set primers possibly specific to rice plastid genome (Supplementary Fig. S2B, right panel). This is because, in contrast to the relatively low sequence similarity in mtDNAs between rice and wheat (Supplementary Fig. S2C), the sequence similarity of the plastid DNA between rice and wheat was extremely high (Supplementary Fig. S2D), making it difficult to specifically detect any possible rice plastid DNA regions.

Fig. 2 Genome composition of DZ-1A wheat–rice hybrids. (A) Mapping pattern of the short-read sequences from DZ-1A (F1) genome DNA onto rice (upper panel) and wheat (bottom panel) mitochondrial genomes. Only reads for which an origin could be estimated were mapped to each genome. Eight DNA regions of rice mitochondrial genome were numbered as regions I–VIII. (B) Mapping pattern of the short-read sequences of DZ-1A genome onto rice (upper panel) and wheat (bottom panel) nuclear genomes. Only reads for which an origin could be estimated were mapped to each genome. The log of raw read count is the ordinary logarithm.

We further conducted genome sequencing for an additional six possible wheat–rice hybrid lines, DZ-2A, DZ-3B, DZ-5B, ReWsWe-1A, ReWsWe-2A, ReWsWe-3A (Supplementary Figs. S3 and S4). Except for DZ-3B, the reads were found to be derived from several regions of the rice mitochondrial genome, with a coverage rate of 11% in DZ-2A, 46% in DZ-5B, 32% in ReWsWe-1A, 39% in ReWsWe-2A, and 16% in ReWsWe-3A (Table 1, Supplementary Figs. S3 and S4, Supplementary Tables S2 to S7). Although the proportions of retained rice mtDNA varied in the hybrid lines, ranging from 11% to 47% (Supplementary Tables S1 to S7), several regions of the rice mitochondrial genome appeared to be preferentially retained in the wheat–rice hybrid genome (Fig. 2A, Supplementary Figs. S3 and S4). In the case of wheat and rice nuclear genomes, the reads estimated as wheat origin were found to be derived from whole wheat nuclear genome in the six possible hybrid lines, whereas apparent mapping peak from rice-origin reads was not detected in the rice nuclear genome (Table 1, Supplementary Figs. S3 and S4). Judging from the genome composition of these possible wheat–rice hybrids that possess ‘wheat’ nuclear genome and ‘rice (Oryza sativa) + wheat’ mitochondrial genome, we term these plants as Oryzawheat.

Table 1 OryzaWheat hybrid plants (F1) prepared in the study

		Mitochondrial genome	a Nuclear genome			
Plant lines	Gamete combination	Wheat	Rice	Wheat	Rice	Fertility	Number of harvested seeds	
DZ-1A	Wheat egg, wheat sperm, rice egg, rice sperm	Full	47%	Full	None	Fertile	91	
DZ-2A	Wheat egg, wheat sperm, rice egg, rice sperm	Full	11%	Full	None	Infertile	–	
DZ-3B	Wheat egg, wheat sperm, rice egg, rice sperm	Full	None	Full	F1: Ch. 1 (partial) F2: None	Fertile	64	
DZ-5B	Wheat egg, wheat sperm, rice egg, rice sperm	Full	46%	Full	None	Fertile	107	
ReWsWe-1A	Wheat egg, wheat sperm, rice egg	Full	32%	Full	None	Fertile	44	
ReWsWe-2A	Wheat egg, wheat sperm, rice egg	Full	39%	Full	None	Fertile	22	
ReWsWe-3A	Wheat egg, wheat sperm, rice egg	Full	16%	Full	None	Fertile	15	
a Degree or percentage of genome coverage, where sequencing reads estimated as ‘wheat origin’ or ‘wheat origin’ were mapped. Full: whole-genome regions were mapped by sequence reads. None: apparent mapping peak from mapped reads was not detected. %: coverage percentage of the mapped regions.

Transmission of rice mtDNA in Oryzawheat progenies

Six out of seven Oryzawheat lines used in the study were fertile and normally set the seeds (Table 1). To address the transmission of the genomic regions derived from rice mtDNA, two seeds (F2) harvested from the DZ 1A zygote-derived plant were germinated and grown into plants (F2-1 and F2-2), and gDNA isolated from these plants was analyzed by PCR using specific primer sets (Supplementary Table S8) for several regions that were detected as the rice origin reads on the rice mitochondrial genome (regions I–VIII in the panel of Figs. 2A, 3A, Supplementary Table S9). Among the seven tested regions for rice mtDNA (regions I–VIII in the panel of Fig. 3A), PCR bands from six regions (regions I–V, VII, and VIII) were detected in the F2-1 progeny, although an amplification band was not detected in one region (region VI in Fig. 3B, upper panels). These results suggest that the inserted rice mtDNA region was partially eliminated across one generation. In contrast, all inserted regions were detected in the F2-2 progeny (Fig. 3B, lower panels). We further examined the transmission situation in two F3 progenies obtained from the F2-1 plant using genomic PCR. In both F3 progenies (F3-1-1 and F3-1-2), amplified DNA bands corresponding to regions V, VI and VII were not detected, although the DNA bands for the remaining four regions were amplified (Fig. 3C).

Fig. 3 Transmission of rice mtDNA into progenies of DZ-1A (A–C) and ReWsWe-2A (D–F) wheat–rice hybrids. (A–C) Genomic PCR using genome DNA from DZ-1A F1 hybrid (A), two F2 progenies derived from the DZ-1A F1 hybrid (B), or two F3 progenies from the F2-1 DZ-1A progeny (C) with specific primers for the regions I–VIII on the rice mitochondrial genome (Fig. 1B). (D–F) Genomic PCR using genome DNA from ReWsWe-2A F1 hybrid (D), two F2 progenies form the ReWsWe-2A F1 hybrid (E), or two F3 progenies form the F2-1 ReWsWe-2A progeny (F) with specific primers for the regions I–VIII on the rice mitochondrial genome.

The transmission of the genomic regions derived from rice mitochondrial genome DNA was also examined in F2 progenies (F2-1 and F2-2) derived from F1 of the ReWsWe-2A plant and in two F3 progenies (F3-1-1 and F3-1-2) from the F2-1 ReWsWe-2A plant. Among the six tested regions (regions I–V, VII, and VIII in Fig. 3D), PCR products from three regions (regions I, II, and VIII) were amplified in the F2-1 individual, although the amplified DNA band was not detected in the remaining three regions (Fig. 3E). In the case of the F2-2 ReWsWe-2A plant, four regions (regions I, II, VII, and VIII) were detectable, and the amplified bands of the remaining two regions were absent. When two F3 progenies (F3-1-1 and F3-1-2) obtained from the F2-1 plant were examined, both showed amplification profiles identical to those of the F2 generation (F2-1) (Fig. 3F). These suggest that the rice mitochondrial genome in Oryzawheat were maintained and transmitted to their progenies (F2 and F3), although the retention profiles of F2 and F3 Oryzawheat displayed a possible loss of insertional regions.

Insertion and spatial profiles of rice mtDNA in DZ plants

Long read sequencing of DZ-1A plant gDNA was performed to examine how the rice mtDNA regions were retained in the F2 generation of DZ-1A plant cells. We identified 77 sequence reads that showed a full-length match to the rice mtDNA sequence, and their mapping profiles are presented in Fig. 4A. Importantly, when the mapped regions obtained from long sequencing reads were merged with those obtained from short sequencing reads (Fig. 2A, upper panel), all six mapped regions from the long sequencing reads overlapped with those from the short sequencing reads (Fig. 4B). In addition to the 77 reads, we detected additional 125 reads that possessed a perfectly matched region with a length of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \ge $\end{document}2,000 bp to the rice mtDNA sequence and a non-matched (soft-clipped) region at the 5′ or 3′ end. Fig. 4C shows the mapping profiles of these 125 soft-clipped reads along with the 77 reads, and the regions mapped by these mixed reads also overlapped with those mapped by short sequencing reads (Fig. 4D). These results indicate that rice mtDNAs were retained as relatively long DNA regions with at least 2,000-bp length in DZ 1A-derived hybrid plants.

Fig. 4 Mapping profile of sequence reads from long-read sequencing of DZ-1A genome (A, C), comparison of the mapping profiles between long- and short-read sequencing results (B, D) and putative insertion sites of rice mtDNA regions in wheat mitochondrial genome (E). (A) Seventy-seven sequence reads from long-read sequencing of DZ-1A (F2) genome, which showed full-length match to rice mtDNA sequence, were mapped onto rice mitochondria genome. (B) The mapping profile of the panel (A) was merged with that of sequence reads from short-read sequencing of DZ-1A (F1) genome. (C) In addition to the 77 reads showing full-length match to rice mtDNA sequence, additional 125 reads, which possessed perfect match region with ≥2,000-bp length to rice mtDNA sequence and non-matched (soft-clipped) region at 5′ or 3′ region were mapped onto rice mitochondrial genome. (D) The mapping profile of the panel (C) was merged with that of sequence reads from short-read sequencing of DZ-1A (F1) genome. (E) Five putative hotspot sites, wherein the rice mitochondrial genome tends to be inserted in the wheat mitochondrial genome, are indicated by black triangles. Putatively inserted DNA regions from rice mitochondrial genome were indicated in parentheses after each possible hotspot. The log of raw read count in panels of A–D is the ordinary logarithm.

We next conducted BLAST searches of these 125 soft-clipped reads. Among the 125 reads, the soft-clipped regions of 119 sequences perfectly matched the DNA sequence of the wheat mitochondrial genome (Supplementary Table S10). In addition, the insertion site was estimated using the sequence information of the clipped sites (connected sites between the rice mitochondrial sequence and wheat mitochondrial sequence) (Supplementary Table S10). Based on this information, it was suggested that the wheat mitochondrial genome consisted of several sites where exogenous rice mtDNA regions were very likely to be inserted (Fig. 4E).

In situ localization profile of wheat and rice mitochondria/mitochondrial nucleoids was addressed by FISH analyses using specific probes for rice and wheat mtDNA. To verify the accuracy of the FISH probes for rice and wheat mitochondria, root cells from rice and wheat were used (Fig. 5C, D). Rice and wheat mitochondrial signals were specifically detected in the cytoplasmic region of root cells of DZ-1A plants (Fig. 5A). In addition to mitochondria labeled with probes for rice or wheat mitochondria, respectively, mitochondria with merged signals were also detected (Fig. 5A). These results suggest the possibility that fusion between rice and wheat mitochondria and subsequent recombination between rice and wheat mtDNAs occurred in hybrid cells. Moreover, the detection of the wheat–rice merged signal via FISH analyses is consistent with the results from genome sequencing, showing the possible insertion of rice mtDNA regions into the wheat mitochondrial genome. Similar FISH results were obtained when root cells of ReWsWe-1A plant was examined (Fig. 5B).

Fig. 5 FISH analyses for rice and wheat mitochondrial genomes in cells of DZ-1A and ReWsWe-2A hybrid plants. FISH analyses using specific fluorescent probes for rice and wheat mitochondrial genomic DNA were conducted on root cells of DZ-1A hybrid plants (A), ReWsWe-2A hybrid plants (B), rice plants (C) and wheat plants (D). Red indicates FISH probes targeting wheat mitochondria-specific sequences. Green indicates FISH probes targeting mitochondria-specific sequences in rice. Yellow indicates a fusion point resulting in a merged fluorescence signal of wheat and rice mitochondria. Gray indicates DNA counterstaining with DAPI. Bars, 10 µm.

Retention of rice chromosome 1 in DZ-3B

On estimating the genomic origin of the sequence reads from the DZ-3B plant (Supplementary Fig. S2c), the reads estimated as ‘wheat origin’ were found to be derived from whole regions of the wheat mitochondrial genome (Fig. 6A, lower panels). However, apparent mapping peak from rice origin reads was not detected in DZ-3B plant and coverage of the leads on rice genome was less than that on wheat genome (Fig. 6A, upper panels, Supplementary Table S6). In the case of nuclear genomes, the reads estimated as wheat origin were found to be derived from whole regions of the wheat nuclear genome although mapping rate on latter half of chromosome 6A was highly reduced (panel Ch 6A in Fig. 6B, lower panels). Upon searching for the sequence reads estimated to originate from the rice nuclear genome, sequence reads of rice origin were mapped to most regions of rice chromosome 1 (panel Ch 1 in Fig. 6B, upper panels, Table 1). To verify the presence of rice nuclear DNA corresponding to most of the chromosome 1 regions in the DZ-3B genome, genomic PCR was conducted using SSR markers specific to rice chromosome 1. Among the seven SSR primer sets on rice chromosome 1, six primer sets (#1–#6) and one primer set (#7) were positioned in the putatively retained and unretained regions, respectively (Fig. 6C, Supplementary Table S3). When PCR was performed using DZ-3B gDNA as a template, amplified DNA bands were detectable only when six primer sets (#1–#6) were used (Fig. 6D), indicating that most regions of rice chromosome 1 were retained in the DZ-3B plant. However, when genomic PCR was conducted on 12 F2 progenies, no amplified band was detected (Fig. 6E). These provide a possibility that the retained rice chromosome 1 may be rearranged and translocated to wheat chromosome 6A and these rice–wheat translocated chromosome can be maintained in DZ-3B plants with chimera, and that the rice–wheat translocated chromosome in the DZ-3B F1 plant was not transmitted to F2 progenies due to the chimerism.

Fig. 6 Genomic composition of DZ-3B wheat–rice hybrids. (A) Mapping pattern of sequence reads from short-read sequencing of DZ-3B (F1) genome DNA onto rice (upper panel) and wheat (bottom panel) mitochondrial genomes. (B) Mapping pattern of sequence reads from short-read sequencing of DZ-3B genome onto rice (upper panel) and wheat (bottom panel) nuclear genomes. (C) Positions of SSR markers on rice chromosome 1 are presented on mapping profiles of sequence reads from DZ-3B and DZ-1A on rice chromosome 1, as presented in Fig. 2B and panel (B), respectively. (D) Genomic PCR of DZ-3B F1, DZ-1A F1, rice and wheat plants using specific primer sets for SSR markers indicated in (C). (E) Genomic PCR for 12 F2 progenies from DZ-3B F1 plant using specific primer sets for the SSR markers. CP3: Genomic PCR for wheat CP3 gene was conducted as internal control for wheat and hybrid plants. M: Molecular markers for DNA bands for 100, 200 and 300 bp. The log of raw read count in panels of A and B is the ordinary logarithm.

Discussion

The IVF system, filling a gap between sexual crossing and somatic hybridization

The primary challenges in sexual hybridization through cross-pollination between distant species are the presence of reproductive barriers. In addition, as the cytoplasmic organelles are maternally transmitted into the zygote through sexual reproduction, hybridization of the cytoplasmic genome hardly occurs. Somatic hybridization offers distinct advantages compared to sexual hybridization. This process entails the fusion of somatic protoplasts originating from distinct species, resulting in the creation of hybrid somatic cells containing both nuclear and cytoplasmic genomes from the respective species. However, it is crucial to highlight that the isolation and culture of protoplasts pose challenges in most plant species. In addition, in the case of a majority of monocotyledonous plants, including vital crops such as rice, maize and wheat, the regeneration of plants from protoplasts remains either unattainable or inefficient (Davey et al. 2005, Eeckhaut et al. 2013). IVF system using isolated gametes provides a solution to these limitations in sexual and somatic hybridizations, and serves as a bridge between sexual crossing and somatic hybridization. The present study showed that IVF system has the capacity to overcome the reproductive barriers between wheat and rice, resulting in the hybridization of cytoplasmic organelles across subfamilies (Fig. 7). This indicates that the IVF system introduces a new hybridization procedure that combines the advantages of sexual and somatic hybridization, as totipotent hybrid zygotes possessing a hetero-plastome/nucleome can be produced by the optimized combination of inter-subfamily gamete fusion.

Fig. 7 Diagrammatic illustration of production of Oryzawheat by in vitro fertilization system and composition of nucleus and mitochondria in Oryzawheat cells. Following the electrical fusion of wheat and rice gametes with suitable combinations, allopolyploid zygotes produced by the electrical fusion of wheat and rice gametes with suitable combinations develop and grow into Oryzawheat plants through the elimination of rice chromosomes. OryzaWheat cells are estimated to possess wheat nucleus, wheat mitochondria, rice mitochondria and mixed mitochondria.

OryzaWheat, wheat hybrids possessing rice mitochondrial genome

The variations in the plasmotype are important from both agricultural and evolutionary perspectives (Levings 1990, Bock et al. 2014), as the proper functioning of metabolism, cellular homeostasis and environmental sensing highly rely on plasmotypic activity (Johnston 2018), and an increase in cytoplasmic variation through hybridization enhances the nucleotype–plasmotype combination. In addition, it has been suggested that plasmotypic variation exerts a more significant impact under fluctuating and stressful environmental conditions and that the plasmotype serves as a reservoir of variations that are predominantly exposed under certain conditions (Flood et al. 2020). In the context of cytoplasmic variation in wheat, extensive crossbreeding and several subsequent rounds of backcrossing were necessary as the cytoplasmic genome in wheat is maternally inherited. For instance, phenotypic effects of Aegilops plasmotype in wheat have been extensively studied using cytoplasmic substitution wheat lines, indicating that the cytoplasm of Aegilops provides multiple phenotypes, including male sterility, panicle emergence delay, and parthenogenesis induction (Tsunewaki 1993, Tsunewaki et al. 2002). However, the nucleotype–plasmotype combination in wheat has been limited within the same subspecies or genera due to crossbreeding limitations. In this study, we observed that two types of wheat–rice hybrid zygotes, DZ and ReWsWe zygotes, developed into mature plants possessing whole wheat nuclear and cytoplasmic DNAs and rice mtDNA, along with variable retention rates of rice mtDNA ranging from 11% to 47%. The emergence of the first hybrid wheat possessing a heterogeneous cytoplasm across subfamilies offers new possibilities for utilizing inter-subfamily genetic resources of cytoplasmic organelles between wheat (Pooideae) and rice (Ehrhartoideae). Understanding how rice and wheat mitochondria coexist in the cells of Oryzawheat is crucial, as it is closely linked to the artificial production of hybrids and/or cybrids across distantly related plant species.

Retention of rice mtDNA and its transmission

Mitochondria are dynamics organelles that regularly undergo fusion and fission (Arimura et al. 2004, Sheahan et al. 2005). The possible active fusion between rice and wheat mitochondria in DZ and ReWsWe zygotes (Fig. 5A, B), followed by possible recombination across their mtDNAs, will be the driving force for the emergence of Oryzawheat where significant portions of rice mtDNA, ranging from 11% to 47% are retained as relatively large DNA regions. Although the retention proportions of rice mtDNA varied, several regions of the rice mitochondrial genome appeared to be preferentially retained in the Oryzawheat genome (Fig. 2A, Supplementary Figs S2 and S3). Chimerism of mtDNA is often caused by a recombinant pathway, and recombination can affect the structure, repeat content and length of the cybrid mitochondrial genome (Albert et al. 1996, Greiner et al. 2015, Garcia et al. 2019). In Arabidopsis and several cultivars of the same or related species, short repeats are reportedly involved in mitochondrial genomic rearrangements (Grabau et al. 1992, Kanazawa et al. 1992, Unseld et al. 1997), which are known as hot spots for recombination and have been identified in the mitochondrial genomes of numerous plant species (Hanlon and Grabau 1997, Scotti et al. 2004). In the present study, putative hot spots for recombination, wherein the exogenous rice mitochondrial genome tended to be inserted, were identified in the wheat mitochondrial genome (Fig. 4E). The resulting chimeric mitogenomes in these two economically important crops can enhance the diversity of nucleotype–plasmotype combinations in wheat. In addition, mixing mtDNA can lead to modifications in gene organization and the formation of chimeric genes that play a role in evolution (Gualberto and Newton 2017) and yield dramatic changes in phenotypes (Mujeeb-Kazi and Rodriguez 1984). The basic and agricultural phenotypic traits of Oryzawheat are currently being examined in our laboratories and experimental fields.

The rice mitochondrial genome in Oryzawheat were maintained and transmitted to their progenies (F2 and F3), although the retention profiles of F2 and F3 Oryzawheat displayed a possible loss of insertional regions. A progressive loss in rice mitochondrial retention was detected in the F2/F3 generation of DZ (regions V, VI and VII) and ReWsWe (regions III, V and VII) (Fig. 3B, C, E, F). Whereas, regions I, II and VIII of the inserted rice mtDNA were detected in all Oryzawheat progenies (Fig. 3). Thus, these regions were stably transmitted across generations in Oryzawheat. The differential transmission of the mitochondrial genome may be explained by the high level of complexity in terms of mitotype diversity, stoichiometry and the origins of the individual mtDNA molecules (Woloszynska 2009). Investigating these factors may help unravel the intricate and dynamic nature of mitochondrial genome retention and inheritance in the Oryzawheat.

When mitochondria were visualized using FISH with specific probes for rice or wheat mtDNAs, three distinct types of mitochondria, were observed in the cells of the Oryzawheat plants (Fig. 5A, B). In addition to wheat and merged mitochondria, mitochondria, possibly containing rice mtDNA as major DNA components, co-existed in cells of Oryzawheat, although long DNA regions from rice mitochondrial genome are considered to be inserted in wheat mitochondrial DNA. Such rice-like mitochondrial may be produced via active fission and genome recombination/rearrangement of merged mitochondria. As the rice-like mitochondria were abundantly detected in cells of Oryzawheat, they may be amplified and transmitted to progeny through somatic and reproductive cell division, similar to wheat and merged mitochondria. A comprehensive understanding of how rice mitochondria are formed and retained in Oryzawheat cells is essential because the mechanisms governing the stability of alien cytoplasm are closely linked to regulating the stability of introduced cytoplasm in plants of interest, thereby promoting greater variation in cytoplasmic organelles.

Wheat hybrid possessing rice chromosome 1

The Triticeae tribe comprises several plant species, including wheat, barley, rye and approximately 300 wild species. Artificial crossings between wheat and alien species in Triticeae have produced several amphidiploids and alien chromosome addition lines of wheat through repetitive backcrossing (Dewey 1984, Kishii 2019). Wild species exist in a range of environments and their alien chromosomes offer diverse traits and incorporating these chromosomes into the wheat genome is a common practice in breeding programs, as they provide disease/insect resistance (Friebe et al. 1996) and biological nitrification inhibition (Friebe et al. 1996, Subbarao et al. 2021) properties. These strongly indicate that addition/introgression of alien chromosomes can significantly increase genetic variation and that crossbreeding wheat with plants from Poaceae family beyond Triticeae tribe can enhance genetic diversity. However, attempts to hybridize wheat with distant Poaceae plants, such as rice, maize and pearl millet, have been futile due to reproductive barriers or the complete elimination of alien chromosomes (Ishii et al. 2016).

One of the possible wheat–rice hybrids observed in the study possessed a full wheat genome along with an additional rice chromosome 1, and this possible hybrid (DZ-3B) is the first report of a wheat–rice hybrid across the subfamily between Pooideae (wheat) and Ehrhartoideae (rice). However, the stability of the rice chromosome appeared to diminish over generations, from F1 to F2 (Fig. 6c-E). Judging from mapping profile of sequence reads on wheat chromosome 6A (Fig. 6B, lower panels) and roughly estimated number of mapped reads on rice chromosome 1 (Fig. 6B, upper panels), it can be estimated that rice chromosome 1 was translocated to approximate middle position of wheat chromosome 6A, and that the DZ-3B plant was in chimeric situation having intact wheat 6A chromosome and rice–wheat translocated chromosome. These chromosomal situation in DZ-3B plant will be a reason for non-transmission of the translocated chromosome to progenies. In the case of wheat–rice hybridization via IVF system, rice chromosomes are generally eliminated during the early zygotic developmental stage (Maryenti et al. 2021). Therefore, it can be postulated that, in the cells of early embryo derived from the DZ-3B zygote, the rice chromosome 1 was not eliminated due to the translocation into the wheat chromosome 6A. In fact, chromosome breakage, incomplete chromosome elimination, chimerism or aneuploids and translocation of chromosomes are found during chromosome elimination process in plant and animal hybrid cells (Gernand et al. 2005, Ishii et al. 2010, Karimi-Ashtiyani et al. 2015, Tan et al. 2015, Wada et al. 2017, Marimuthu et al. 2021). In addition, inter-subfamily wide hybridization such as oat-maize and oat-pearl millet combinations generates stable inter-subfamily distant chromosomes in hybrid cells, and genes are expressed from chromosomes of both species in these inter-subfamily hybrids (Riera-Lizarazu et al. 1996, Ishii et al. 2013, 2015, Ishii 2017). Toward wide hybridization between wheat and rice, elucidating the molecular mechanisms involved in the elimination of rice chromosomes in IVF-derived wheat–rice hybrid cells is crucial. Manipulating and regulating the elimination process can enhance the efficiency of translocating rice chromosomes into wheat chromosomes. For subsequent experiments, we intend to conduct transcriptome analyses for wheat–rice hybrid embryos before or after the elimination of the rice chromosome. Alternatively, considering that one F1 individual possessed a rice chromosome among the seven produced wheat–rice hybrid lines, increasing the number of produced wheat–rice hybrid lines may result in the production of wheat plants possessing rice chromosomes that can be transmitted across generations.

IVF system, method for producing a variety of hybrids among Poaceae

Typical genomic composition of oryzawheat was illustrated in Fig. 7. Beyond the wheat–rice combination, this IVF system enables the creation of novel inter-subfamily genome combinations, as egg and sperm cells can be isolated from important crop plants belonging to the family Poaceae, such as rice (Uchiumi et al. 2007), maize (Kranz and Lörz 1993), wheat (Maryenti et al. 2019), barley (Holm et al. 1994), setaria (Toda et al. 2022) and Brachypodium (Matsumura and Okamoto 2016). Importantly, hybrid plants produced through in vitro fusion of gametes, which are isolated from plant species within same Poaceae family, are free from legislative concerns related to genetically modified organisms (Jones 2015). This opens up the possibility of cultivating these hybrid plants in open fields immediately after their production. We are currently working on producing hybrid zygotes with other plant combinations, such as wheat and maize, in our laboratories, with the aim of creating Poaceae plants across subfamilies without the hindrance of reproductive barriers.

Materials and Methods

Plant materials

The wheat plants (Triticum aestivum L. cv. Fielder) and wheat–rice hybrid plants (F1, F2 and F3 generations) used in this study were grown in an environmental chamber with cycles of 23°C light (16 h)/20°C dark (8 h) and a photosynthetic photon flux density of 100–150 µmol photons/m2/s. Wheat–rice hybrid plants (F1) were prepared by electrical fusion as previously described (Maryenti et al. 2021). F2 and F3 hybrid plants (seeds) were obtained by self-pollination of F1 and F2 wheat–rice hybrid plants, respectively. Oryza sativa L. cv. Nipponbare plants were grown in an environmental chamber at 26°C under light (13 h)/dark (11 h) conditions.

DNA extraction, library preparation and Illumina sequencing

Young leaf tissues of the F1 hybrid lines were harvested and stored at −80°C until DNA extraction. gDNA was isolated from the leaves of wheat plants, rice plants and seven hybrid plants (ReWsWe-1A,-2A,-3A and DZ-1A,-2A,-3B,-5B) as presented in Fig. 1D (Maryenti et al. 2021) using the Nucleo Spin II Kit (Macherey-Nagel, Duren, Germany) following the manufacturer’s instructions. The isolated gDNA was stored at −20°C until use.

DNA libraries were constructed from 25 ng/µl gDNA using the Nextera DNA Flex Library Preparation Kit (Illumina, California, USA). The quality and quantity of the amplified DNA libraries were determined using an Agilent 2100 BioAnalyzer with a high-Sensitivity DNA chip (Agilent Technologies, California, USA) and NanoDrop UV/Vis 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, USA), respectively. Pooled libraries were sequenced on an Illumina HiSeqX_Ten platform (Illumina, San Diego, USA) with 150-bp paired-end reads at Macrogen-Japan (Tokyo, Japan). Sequencing data were deposited in DNA Data Bank of Japan as ID DRA016401.

DNA extraction, HiFi library preparation and PacBio sequencing

gDNA was extracted from the young leaves of F2 of DZ-1A plants using the NucleoBond HMW Spin II DNA Kit (Macherey-Nagel, Duren, Germany), following the manufacturer’s instructions. Using the gDNA, the DNA library was generated according to the protocol titled ‘Preparing HiFi SMRTbell Libraries using SMRTbell Express Template Prep Kit 2.0’ from PacBio. Sequencing of the pooled libraries was performed on the Sequel II system with v8.0 software and sequencing chemistry 2.0. Hifi reads were generated using circular consensus sequencing. Library generation and HiFi read generation were performed at Rhelixa (Tokyo, Japan). Sequencing data were deposited in DNA Data Bank of Japan as ID DRA016401.

Processing and analyses of data obtained from short- and long-read-based genome sequencing for wheat–rice hybrids

The obtained short-read sequences were evaluated using FastQC (v0.11.8) (Simon 2010), and adapter- and low-quality sequences were trimmed using Cutadapt (v2.10) (Martin 2011). Processed reads were mapped to both rice and wheat reference sequences using bowtie2 (v2.3.5.1) (Langmead and Salzberg 2012) with paired-end option. The genomic origin (rice or wheat) of each sequence read was estimated using HomeoRoq (Akama et al. 2014). HomeoRoq determines the origin species based on mismatches between the read and each template genome; thus, species with a low number of mismatches were estimated as the genomic ‘origin’ of the reads. If the number of mismatches was equal between the rice and wheat genomes, the reads were treated as ‘common’ sequence reads (origin could not be determined).

The obtained long (HiFi) reads were mapped to both rice and wheat reference sequences using pbmm2 (github.com/PacificBiosciences/pbmm2) with ‘—sort’ option. The genomic origin of each read was estimated using Eagle-RC (Kuo et al. 2018), which determines the species of origin based on mismatches between the read and each template genome, similar to HomeoRoq. The reads estimated for ‘rice origin’ were BLASTN searched (v2.10.1+) (Altschul et al. 1997, Camacho et al. 2009) for the rice and wheat reference sequences and selected showing ‘query coverage = 100’ against the rice reference sequence. Around regions in the rice reference sequence to which these ‘full-length rice origin reads’ were mapped, chimeric reads of rice and wheat were searched following the threshold; at least 2,000 bp must be mapped to rice without mismatch, and either the 3ʹ or 5ʹ end should be soft-clipped and mapped.

Raw read counts were calculated using featureCounts (Liao et al. 2013) with the ‘-O’ option every 1 Mb and 1 kb for the nuclear and organelle genomes, respectively. The values of the retained regions on the hybrids were calculated based on the depth of the raw read counts (≥5) (Supplementary Tables S1–S7). The following data sets were used as the reference sequences: nuclear genome sequences: RAP-DB Os-Nipponbare-Reference-IRGSP-1.0 (Kawahara et al. 2013, Sakai et al. 2013) and IWGSC RefSeq v1.0 [International Wheat Genome Sequencing Consortium (IWGSC) 2018] for rice and wheat, respectively; mitochondrial and plastid genomes: DQ167400.1 and AY522330.1 for rice and MH051716.1 and MH051715.1 for wheat. The organelle genomes with accession numbers were obtained from the NCBI database.

Genomic PCR for mitochondrial and nuclear genome of hybrid plants

gDNA of the DZ-1A, ReWsWe-2A and hybrids in the F1 generation, wheat plants and rice plants were prepared as described above. As for the gDNA of wheat–rice hybrid lines at F2 generation, gDNAs were isolated from the young leaves using PrepMan Ultra Sample Preparation Reagent (Thermo Fisher Scientific, Warrington, UK) according to the manufacturer’s protocol. PCR primers (Supplementary Table S8) were set for possible rice mitochondrial regions whose sequences were verified by analyzing the mapping data from short-read sequencing using the IGV software (Robinson et al. 2011) (Supplementary Table S9). After the primer sequences were preliminarily determined using primer3 software, the specificity of the primer sequences was confirmed by BLAST search. For the PCR of mitochondrial genome targets, 0.5 μl of gDNA (10 ng/μl) was used as the template in a 20 μl PCR reaction with 0.3 μM of primers using KOD-FX DNA polymerase (Toyobo, Osaka, Japan) as follows: an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 68°C for 2 min with a final extension at 4°C for 7 min.

For genomic PCR detection of rice chromosome 1 in hybrid plants, rice simple sequence repeat (SSR) markers on chromosome 1 were used. A set of 50 SSR markers that were evenly distributed across all 12 rice chromosomes was obtained from the Gramene marker database (https://archive.gramene.org/markers/microsat/50_ssr.html) developed from Oryza sativa L. Nipponbare genome sequencing. Among the 50 SSR markers, those on rice chromosome 1 (seven SSR marker sets) were selected. Detailed information about the primers for the seven SSR markers and the N-terminal region of the wheat TaCP3 gene, which acts as an internal control, used in the study are presented in Supplementary Table S11. The touchdown PCR method was used for SSR marker detection under the following conditions: initial denaturation at 95°C for 5 min, followed by 13 cycles of denaturation at 95°C for 30 s, annealing at 70°C for 1 min (−1°C/cycle), and extension at 72°C for 30 s and another 27 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s with a final extension at 72°C for 1 min.

FISH of mtDNA

Seeds from F1 hybrid plants, wheat seeds and rice seeds were germinated on moist filter paper in a Petri dish at 25°C. Healthy root tips showing creamy white hue from the primary roots, approximately 0.5–1 cm in length, were excised. The collected root tips were fixed in a fixative solution containing 3:1 (v/v) ethanol:glacial acetic acid, kept at room temperature for 3 d, and stored at 4°C until use. The fixed root tips were then soaked in distilled water for 10 min and then treated with enzymatic solutions consisting of 2% (w/v) of pectolyase Y23 (Kyowa Kasei, Osaka, Japan), 2% (w/v) of cytohelicase from Helix pomatia (Sigma-Aldrich, MO, USA), and 2% (w/v) of cellulase ‘ONOZUKA’ R-10 (Yakult, Tokyo, Japan) at 37°C for 1 h. Thereafter, the enzymatic solution was replaced with 10 mM citrate buffer for 10 min, and the root tips were transferred on slide glass. Ten microliters of 60% acetic acid was added to the root tips, and 100 µl of fixative fluid containing 3:1 (v/v) ethanol:glacial acetic acid was added to the root tips to spread the cells on the slide grass, which was kept at room temperature until dry. The slides with cells were fixed with a post-fixative solution containing 4% formaldehyde solution in PBS for 5 min at room temperature, washed with 1× ice-cold PBS for 5 min and dehydrated sequentially in 70, 90 and 99.5% ethanol at room temperature for 2 min each.

MyTags FISH probe sets (Arbor Biosciences, MI, USA) were used to detect rice and wheat mtDNA. Species-specific sequences were detected in the rice and wheat mitochondrial genomic sequences (rice: BA000029.3 and wheat: AP008982), and specific probe sets for these genomes were designed at 6766 and 6877 positions for rice and wheat, respectively. MyTags FISH immortal probe sets were generated according to the protocols provided by Arbor Biosciences. Specific probe sets were labeled with Alexafuluor-488 for rice and with rhodamine for wheat. Labeled probes were generated according to the manufacturer’s protocol. Rice and wheat mitochondrial probes were resuspended in hybridization solution (10 μl) containing 50% formamide, 10% dextran sulfate and fragmented salmon sperm DNA (2 μg/ml) in 2× saline sodium citrate (SSC). The mixture solution was then applied to the slides, covered with a 22 × 22 mm cover slip, sealed with paper bond glue (Kokuyo, Osaka, Japan), and hybridized at 37°C for 24–36 h. Slides were then washed sequentially at room temperature with 0.1% Triton X-100 in 2 × SSC for 5 min and 2× SSC for 5 min. The slides were dehydrated sequentially in 70, 90 and 99.5% ethanol at room temperature and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) containing 1 ng/μl 4ʹ,6-diamidino-2-phenylindole (DAPI). Fluorescence imaging was performed using an Olympus BX61 microscope equipped with a Cool SNAP HQ2 CCD camera (Photometrics). All images were acquired in grayscale using MetaMorph software (Molecular Devices, CA, USA), pseudo-colored, and merged using Adobe Photoshop 2023 (Adobe, San Jose, CA, USA).

Supplementary Material

pcae074_Supp

Acknowledgments

We thank Mrs. T. Mochizuki (Tokyo Metropolitan University) for isolating the rice egg cells and the National Bio-Resource Project of the MEXT, Japan for analytical methods and platform for genome analyses. Computations were partially performed on the NIG supercomputer at the ROIS National Institute of Genetics.

Supplementary Data

Supplementary data are available at PCP online.

Data Availability

Sequencing data were deposited in DNA Data Bank of Japan as ID DRA016401.

Funding

The JSPS KAKENHI [Grant-in-Aid for Scientific Research(B), grant no. 22H02315 to T.O., and Grant-in-Aid for Scientific Research (C), grant no. 22K05572 to T.I. in part]; the NEDO Moonshot Program (grant no. 22101490–0 to T.O. and T.I.); the JST-Mirai Program (grant no. 23–231038606 to T.O. and T.I.); the JST-FOREST (grant no. JPMJFR 2001 to T.I.); the Joint Research Program of Arid Land Research Center, Tottori University (grant no. 04B2020 and PT#2402 to T.O.).

Author Contributions

T.M., T.I. and T.O. came up with the study idea; T.M., S.K., K.Y., T.I. and T.O. designed the experiments; T.M. performed genomic sequencing, PCR and FISH analysis; S.K. and K.Y. performed bioinformatic analysis; T.I. and N.O. conducted FISH and genomic PCR, respectively. T.O. supervised the project; T.M., S.K., T.I. and T.O. conceived the project and wrote the article.

Disclosures

The authors declare that they have no competing interests.
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