Comparative Analysis of orf182 Widespread in Chinese Wild Rice Species for Hybrid Rice Breeding

Comparative Analysis of orf182 Widespread in Chinese Wild Rice Species for Hybrid Rice Breeding | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Analysis of orf182 Widespread in Chinese Wild Rice Species for Hybrid Rice Breeding Weixiong Long, Jie Wang, Lihua Luo, Lujian Zhou, Wei Chen, Laiyang Luo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7649942/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The utilization of hybrid rice has successfully addressed food security challenges in China. However, the predominant use of wild-abortive (WA) cytoplasmic male sterility (CMS) systems in hybrid rice cultivation raises significant safety concerns. In this study, we have elucidated the distribution patterns of orf182 , a novel CMS gene identified in our previous work, in Chinese common wild rice. Mitochondrial sequencing and assembly of 11 representative wild rice species harboring orf182 revealed 16 novel genes. A total of 469 mitochondrial genes were classified into 36 gene families, with nine families containing single-copy homologous genes, indicating significant gene duplication in mitochondria. We observed a strong positive correlation between mitogenome size and the quantity and size of repetitive sequences. Collinearity analysis revealed extensive mitochondrial variation and large-scale inversions in Guangdong wild rice. Comparative genome analysis uncovered inversions, translocations, and several variations surrounding orf182 , with a 71 bp repeat sequence mediating the formation of the orf182 - nad6 chimeric gene. Gene copy number analysis (GCNV) revealed variable orf182 gene copy counts (1, 2, and 3) in wild rice species. Additionally, successful transformation of orf182 from various sources into sterile lines was achieved. These findings provide valuable resources for advancing hybrid rice development in China, thus contributing to enhanced food security. Oryza rufipogon orf182 wild rice CMS structural variation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Mitochondria, often termed the powerhouses of eukaryotic cells, are semi-autonomous organelles believed to have originated from prokaryotic ancestors via endosymbiosis. They harbor their own genetic material, which is indispensable for mitochondrial and cellular functions [1]. The mitochondrial genome (mitogenome or mtDNA) is typically maternally inherited and follows evolutionary trajectories distinct from those of nuclear genomes. Outbreeding-mediated reshuffling of nuclear and cytoplasmic genomes occurs widely and frequently, potentially reconfiguring the functional and structural interplay between these genetic systems. Rearrangements, mutations, and inheritance patterns in mitochondrial genomes can provoke evolutionary conflicts that significantly influence plant diversification, domestication, and hybridization [2, 3]. Nevertheless, the evolutionary mechanisms shaping cytoplasmic genomes—especially among wild relatives of crops—remain inadequately explored. Structural variation (SV) is considerably more prevalent in mitochondrial genomes than in chloroplast genomes, where SVs are rare and occur at low frequency [4], whereas mitochondrial genomes exhibit extensive SVs driven by frequent repeat-mediated recombination[5]. In contrast, plant mitochondrial genomes undergo extensive recombination mediated by repetitive sequences, leading to pronounced structural diversity [6] and can disrupt gene expression through the reshuffling of regulatory elements or the creation of chimeric open reading frames (ORFs). Many such chimeric ORFs have been associated with cytoplasmic male sterility (CMS), a phenotype characterized by aberrant floral development and abortion of male gametophytes [7, 8]. Recombination events are often facilitated by short repeats (SRs; 50–500 bp) and micro-repeats (MRs; <50 bp), which promote asymmetric and largely irreversible recombination, yielding recombinant molecules present at low (sub-stoichiometric) levels [9, 10]. These molecules, termed sublimons , can rapidly amplify under certain conditions through a mechanism known as sub-stoichiometric shifting (SSS), thereby altering the mitochondrial genotype (mitotype) and driving mtDNA evolution [11]. Collectively, recombination processes mediated by SRs, MRs, intracellular gene transfer (IGT), and horizontal gene transfer (HGT) generate diverse SVs—including rearrangements, duplications, insertions, and deletions [5, 10, 12]. Although SVs within gene regions are generally deleterious and selected against, those in intergenic regions may persist, contributing to the high sequence divergence observed in mitochondrial intergenic spacers [13-15]. Wild rice ( Oryza rufipogon ), the direct progenitor of cultivated rice ( Oryza sativa ), was domesticated approximately 8,000–10,000 years ago. It represents a critical genetic resource for improving stress tolerance, yield, and grain quality in modern rice breeding [16]. While the nuclear genome of O. rufipogon has been extensively studied, providing key insights into rice evolution and domestication [17, 18], research on its mitochondrial genome remains limited. Early studies using restriction fragment length polymorphism (RFLP) analysis revealed mtDNA variation and distinct mitotypes among wild and cultivated rice populations [19]. Furthermore, several CMS types—including the widely used WA-, BT-, HL-, and D1-CMS systems—have been identified in cultivated rice, many tracing back to O. rufipogon or closely related accessions [7, 20-22]. However, only a few complete mitogenomes of O. rufipogon have been reported to date, and a comprehensive comparative analysis is still lacking. As a result, the extent of mitochondrial genomic diversity and its evolutionary relationship with cultivated rice remain poorly understood. In this study, we employed D1-CMS-type wild rice as a model to investigate cytoplasmic genome evolution. We constructed a pan-mitogenome based on high-quality long-read assemblies from 11 representative accessions of common wild rice. Our comprehensive analyses revealed extensive mitochondrial rearrangements and the presence of chimeric ORFs. We generated genome-wide mitochondrial variation maps from diverse Oryza accessions for population-level genomic studies. The potential role of chimeric ORFs—classified into distinct phylogenetic clades based on mitotype-specific markers (MSS)—in CMS was evaluated using cytoplasmic substitution lines. Furthermore, we examined recombination dynamics around the mitochondrial gene orf182 to elucidate its structural complexity and explore the mechanisms underlying rice mtDNA evolution. Finally, we characterized structural variations across the mitochondrial genome to uncover general architectural features and evolutionary constraints. Results Phylogenetic tree constructed by Mitotype-specific sequence (MSS) Previous studies identified the orf182 gene exclusively in 18 Oryza rufipogon accessions from China, distributed among 17 species across nine genomic types, based on a screen of 446 wild rice samples [22]. To further elucidate the evolutionary origin and distribution of this gene, we collected an additional 320 common wild rice accessions from three provinces in China: Guangdong, Jiangxi, and Guangxi. Among these, 76 accessions (23.75%) exhibiting the D1-type cytoplasmic male sterility (CMS) phenotype were selected for subsequent genetic and genomic analyses (Table S1). From a total of 57 mitochondrial structural sequence (MSS) profiles obtained from these accessions [23], 18 representative samples showing pronounced polymorphisms were used to reconstruct a phylogenetic tree, aiming to clarify the evolutionary relationships within the D1-type wild rice group (Fig. 1, Tables S2, S3). Furthermore, 11 representative wild rice accessions spanning nine distinct D1-type phylogenetic clades were subjected to high-quality mitochondrial genome assembly using a hybrid sequencing strategy combining long-read and short-read technologies. These high-resolution assemblies facilitated detailed comparative and evolutionary analyses of the mitochondrial genome, with particular emphasis on structural variation, recombination events, and the potential functional implications of CMS-associated open reading frames such as orf182 . Based on three Oryza rufipogon accessions carrying orf182 fromdifferent geographical origins and phylogenetic tree branch, we successfully converted Mingxiangsi into male sterile lines after only five backcross generations(Fig. 1B). These results demonstrates that the D1-type CMS system can be readily introduced into diverse genetic backgrounds, highlighting its efficiency and broad applicability in hybrid breeding programs. General features of the Oryza rufipogon mitochondrial genome A total of 69 Gb of raw Illumina reads (150 bp in length) from 11 samples, along with 506 Mb of Nanopore PromethION data from 9 wild rice varieties and 572 Mb of PacBio long reads (average length ~10 kb), were generated for mitochondrial genome assembly (Table S4). The assembled mitogenomes of the 11 wild rice accessions ranged in size from 438,632 bp to 718,357 bp. Functional annotations and physical locations of genes across these mitochondrial genomes are summarized in Fig. 2 and Fig. S1. In the JX2 mitogenome, 77 genes were annotated, including 46 protein-coding genes, 28 tRNAs, and 3 rRNAs. These encode 37 distinct proteins, with atp4 , atp6 , atp8 , atp9 , cob-like , orf182 , and orfX present in two copies each, and nad6 in three copies. The protein-coding genes were classified into 11 functional categories: ATP synthases (5 genes), cytochrome C biogenesis (4 genes), ubiquinol cytochrome c reductase (1 gene), cytochrome c oxidase (3 genes), maturases (1 gene), NADH dehydrogenase (9 genes), cytoplasmic male sterility-related (1 gene), open reading frames (1 gene), large ribosomal subunit proteins (3 genes), and small ribosomal subunit proteins (8 genes). On average, seven chimeric ORFs derived from fragments of known and conserved protein-coding genes (excluding tRNAs and rRNAs) were identified in each mitogenome. Notably, nad6 (from the nad family) and rpl2 and rpl16 (from the rpl family) frequently participated in the formation of prominent chimeric ORFs across all wild rice accessions examined (Table S5). To assess structural variation (SV) and its functional implications, we performed gene copy number analysis. A core set of 37 protein-coding genes was conserved across all 11 mitochondrial genomes. Among these, nad6 exhibited the highest copy number. The accession GX34 showed the most extensive gene loss, affecting atp4 , atp8 , rps7 , cox3 , nad5 , nad7 , rps13 , ccmFc , rpl5 , and cox1 . Additionally, genes such as cob-like and orfX were lost in two other accessions. Gene duplication was widespread: cox1 was replicated in nearly all wild rice mitogenomes, and nad6 duplication occurred in all 11 accessions (Fig. S2). Repeat sequence variation and correlations between genome size and repeat sequences Mitogenome repeats were classified into class I (repeats ≤ 100 bp), class II (100–500 bp), class III (500–2000 bp), and IV (> 2000 bp) based on repeat length. The number of class I repeat sequences ranged from 239 (GX44 and GX51) to 724 (GX4), account for 1.53-1.68% of mitochondrial genome size (Fig. 3A; Table S6). Among the samples, the number of class I repeats in GX4 samples was significantly higher than samples from wild rice in same province and other wild rice, while the number of repeats longer than 100 bp was not significantly increased (t-test, P-value = 0.11) (Table S6). Meanwhile the total repeat number (864) of GX4 was higher than that of other sample (total repeat number: 316–490) (Fig. 3), and the total repeat length show the similar pattern, while the mitochondrial size of GX4 greater than other contributed by the class IV type repeat. These were followed by repeats in class II, which accounted for approximately 15% of the total repeats, with 56–90 in number. There were most least repeats length located in class III and only accounted for 0.92%. In contrast, a total of 12–38 repeats in class IV were identified, with lengths ranging from 114.38 to 574.38kb, contributing 26.04–79.96% of the mitogenome size. Despite the relatively low number of repeats in class IV, they were the major contributors to the total mitogenome size (Fig. 3B). For all of the 11 wild rice samples, mitogenomes size show significantly high correlations (P-value < 0.001) with total repeat number and length (Fig. S3, Fig. 3c, d). All the count of the four repeat categories showed significant correlation with variations in genome size (R2 adj ranged from 0.63 to 0.99, P-value < 0.003) (Fig. S3). While the Class II repeat size show low correlations with the mitochondrial genome size (Fig. 3d). In addition, several mitogenomes may have an overrepresentation of repeat content (Table S6). For example, GX46 (total repeat length: 466 kb) had a similar genome size to GX51 and GX44 (438 kb), but the total repeat length was about 1.46-fold of GX51 (131 kb). Identified the structural variation in D1-wild rice mitogenomes Previous studies have indicated that repeat sequences can mediate rearrangements of varying extent in the mitochondrial genome. By aligning the 11 assembled mitogenomes to the Nipponbare reference genome, we identified syntenic regions accounting for 12% to 30% of each genome (Table S7). Structural variations across assemblies included one to three inversions, two to six translocations, 27 to 68 insertions, and 32 to 61 deletions (Fig. S4). Wild rice accessions with D1-CMS exhibited the highest abundance of SVs (Fig. S5). Notably, although GX34 ranked fourth lowest in terms of SV quantity, it displayed the greatest total SV length. This apparent discrepancy is largely attributable to the absence of large-scale translocations in GX34, resulting in a notably low minimal SV length. Mitogenomes of wild rice from Guangdong province appeared relatively conserved, a pattern that may reflect either geographic sampling concentration or limited underlying genetic differentiation. In contrast, the two Jiangxi accessions exhibited markedly distinct profiles in both the number and size of SVs (Fig. S5). Whole-genome alignment using Mauve further characterized large-scale structural rearrangements, revealing 17 locally collinear blocks (LCBs) with variable ordering across accessions (Fig. S6). Three major rearrangement events were detected in Jiangxi Dongxiang wild rice, whereas only five LCBs were identified in GX4 and GX34. These results suggest that the mitochondrial genomes of Guangxi D1-CMS wild rice have lost numerous conserved genomic modules during evolution, consistent with previous reports of extensive structural variation in accessions from this region. Pan-mitochondrial genome of 11 selected D1 CMS wild rice and Nipponbare To further investigate mitochondrial genomic diversity across different origins, we selected 11 D1-CMS wild rice accessions representing distinct groups, along with the previously published Nipponbare mitochondrial genome, for pan-genome construction and comparative analysis. The gene accumulation curve (Fig. 5C) indicated an open pan-genome, suggesting that new genes are likely to be identified as more mitochondrial genomes from rice are sequenced. Comparative analysis of the eleven D1-CMS wild rice mitogenomes revealed no private genes among these accessions. Mitochondrial genomes from Guangdong accessions were generally the most conserved. However, one accession, GD4, exhibited a reduced number of core genes and a higher proportion of variable genes within the pan-genome. In contrast, accessions from Guangxi showed greater variability in pan-genome composition. Among these, GX34 contained fewer variable genes, implying a more stable evolutionary history and possible adaptation to a narrower ecological niche. Further comparison between Jiangxi accessions indicated that JX5 possessed more variable genes than JX2. Notably, private genes were exclusively identified in the Nipponbare reference genome, highlighting substantial divergence in mitochondrial gene content between the reference and the D1-CMS wild rice lineages examined in this study (Fig. 5A). We also quantified the proportional distribution of different genomic components. Core gene families accounted for the largest share (44.23%), followed by private (30.77%) and variable (25%) gene families. In terms of copy number, core genes constituted 66.55% of all genes, while private genes represented only 2.89%. Discussion In this study, we identified the chimeric mitochondrial gene orf182 as a unique genetic element present exclusively in a subset of Chinese wild rice accessions. This finding offers significant insights into the evolutionary dynamics of plant mitochondrial genomes, the origin of cytoplasmic male sterility (CMS)-associated genes, and the potential utility of orf182 in hybrid rice breeding. The restricted distribution of orf182 suggests that it likely originated from a localized mitochondrial recombination event. This observation supports the hypothesis that CMS-related genes often emerge independently in different populations through rapid and extensive mitochondrial restructuring. Similar to orf79 and orfH79 , orf182 exemplifies the high structural plasticity of plant mitochondrial genomes, wherein recurrent gene fusion events give rise to chimeric open reading frames. The occurrence of orf182 solely in Chinese wild rice further indicates that CMS genes are not universally conserved but rather represent region-specific evolutionary innovations. Its discovery highlights the distinctive genetic value of Chinese wild rice germplasm within the global rice gene pool. As a naturally occurring CMS-inducing gene, orf182 provides direct evidence of functional diversity preserved in wild populations. Among the collected Chinese Oryza rufipogon accessions, 23.75% were found to carry orf182 . Based on these findings, we propose that the mitochondrial genome harboring orf182 has not evolved through conservative mechanisms but rather via dynamic structural rearrangements. Mitochondrial genome structural plasticity and chimeric gene formation Our results demonstrate that large-scale structural variations (LSVs) are prevalent in the mitochondrial genomes of wild rice, including among accessions from the same geographic region. Accessions such as GD4 and GX4 exhibit pronounced structural divergence compared to others. Notably, the physical scale of SVs did not correlate with their quantity; for instance, GX34 displayed the largest SV size but ranked among the lowest in terms of SV number. Substantial variation in mitochondrial genome size was observed across the wild rice panel (Fig. 3 ). The assembly and annotation of 11 mitochondrial genomes provide new insights into this variability. None of the wild rice accessions shared a common presence–absence variation (PAV) region associated with orf182 , indicating that this gene did not originate from a recent insertion event. Instead, analysis of repetitive sequences revealed that some orf182 chimeric genes likely arose from duplication events, while others were generated through recombination mediated by a 71-bp repetitive element, which appears to have undergone multiple recombination cycles (Fig. 6 ). CMS gene diversity and implications for domestication and hybrid breeding Dynamic structural changes, including gene duplications, have facilitated the emergence of new gene copies in the mitochondrial genome. Nucleotide alignment of orf182 sequences identified several SNP sites, which were categorized into three haplotypes (Fig. S7 ). All detected SNPs were synonymous, suggesting neutral evolution at the sequence level. CMS genes such as orf182 may have historically functioned as genetic barriers that contributed to reproductive isolation, thereby influencing domestication pathways and exhibiting geographic specificity. The D1-CMS system, characterized by orf182 , represents a promising resource for the development of novel hybrid rice breeding systems. Material and methods Plant materials Oryza rufipogon collected from three province, Guangdong(GD), Guangxi(GX) and Jiangxi (JX), were used in the present research. The Guangdong common wild rice materials were generously contributed and identified by professor Yonggen Lu (South China Agricultural University), Guangxi wild rice were obtained from Researcher Yuntao Liang (Guangxi Academy of Agricultural Sciences), while Jiangxi wild rice were kindly provided by the Dongxiang Wild Rice Resource Nursery and identified by Hongwei Xie. Oryza spontanea (N104625) was provided by international rice research institute, Philippines. The voucher specimens (refer to Table S1) are stored in the herbarium of Jiangxi Academy of Agricultural Sciences, relevant data are stored in the Germplasm Resource Database of the Jiangxi Academy of Agricultural Sciences (currently an internal website, and are available from the corresponding author upon reasonable request). All the wild rice and O. spontanea were grown in the Wild Rice Germplasm Resource Nursery of the Jiangxi Academy of Agricultural Sciences (28°56′20″N, 115°39′59″E). Construction of maximum likelihood tree based on the Mitotype-specific sequences (MSSs) To selected the representative D1-CMS relatives among the 76 common wild rice collected from worldwide. The previous developed 20 pairs of evenly distribution D1-CMS mitochondrial specific sequences were used (Table S2). The 0/1 matrix was generated by MTTs gel results (Table S3). The phylogenetic tree was constructed using the maximum likelihood method in IQ-TREE software v1.6.12 [24], and the resulting maximum likelihood tree was visualized using ITOL software [25]. Samples, DNA extraction and Mitochondrial genome sequencing A total of 11 representative wild rice were selected based on the phylogenetic tree constructed by MSSs in this study (Table 1). Fresh leaves from the wild rice species were collected from a local filed experiment field of Jiangxi Academy of Agricultural Science, Nanchang, China. High-molecular-weight genomic DNA was extracted from 1-month-old leaves following the standard CTAB method. The mitochondrial genome of 11 wild rice was sequenced using the Illimina, Pacbio and Nanopore methods. Paired-end libraries with insert sizes of ∼300 bp were prepared following Illumina’s standard genomic DNA library preparation procedure. The qualified Illumina pair-end library would be used for Illumina NovaSeq 2500 sequencing (150 bp × 2, Shanghai BIOZERON Co., Ltd., Shanghai, China). Mitochondrial genome assembly and annotation Raw sequencing reads were trimmed using Btrim software to filter out low-quality bases and sequences. The high quality reads were assembled into bp using SOAP de novo v2.04 ( http://soap.genomics.org.cn/) and Celera Assembler 8.0. Gaps in the SSPACE scaffolds were filled using GapCloser v.1.12 [26] with default parameters. Finally, redundant sequences were manually removed to obtain the final mitochondrial genome. The denovo annotation strategy and homology-based comparison were conducted for Genome annotation. We utilized the protein sequences of the reference genome for homology alignment prediction. First, we rapidly aligned the protein sequences to each genome sequence, filtered out the poor alignment results, removed redundancies, and then using Genewise for precise alignment. The AUGUSTUS software (http://bioinf.uni-greifswald.de/augustus/) was performed to de novo gene prediction for plant mitochondrial genomes. Finally, we integrated the gene sets using the EVidenceModeler v1.1.1 software [27] to obtain the comprehensive annotation file. The output genebank format file was manually confirmed, and the mitochondrial circular map was drawn using Organellar Genome DRAW (MPI-MP CHLOROBOX - OGDRAW (mpg.de)). Analysis of repeated sequences BlastN was used to determine whether there were intragenomic duplications of large fragments or interspersed repeats in the 11 wild rice mitogenomes. Microsatellite identification tool was used to detect simple sequence repeats. The repeats of 1, 2, 3, 4, 5, and 6 bases with 8, 4, 4, 3, 3, and 3 repeats numbers, repeatively, were identified in this analysis. The tandem repeats with > 7p repeats units were detected using Tandem Repeats Finder v4.10 [28]. The direct and inverted repeats were identified using REPuter software (BiBiServ - Temporariliy not available (uni-bielefeld.de)) with the minimal repeat size set to 20 bp. Collinear analysis and comparative analysis among the D1-CMS wild rice The mitogenome of Nipponbare was characterized by Notsu [29] were used for comparision. Collinear analysis between the two mitogenomes were conducted in Mummer [30]. Collinear regions were identified using the nucmer module with an identity threshold of 95% under the many-to-many alignment mode. The resulting collinearity maps were visualized with Plotsr implemented in the SYRI software[31]. The genome sizes, GC contents, base compositions, start and stop codons, gene counts, and intron numbers of 11wild rice mitogenomes were compared to assess their variations and similarities. The copy number of protein coding genes among the mitogenome were identified. Rearrangement event identification in D1-type wild rice mitogenome Mauve[32] genome alignments serve as a powerful tool for identify conserved genomic regions and structural rearrangements across different samples. In this study, we conducted a series of Mauve alignments under default parameters to compare mitochondrial genomes of GD, GX and JX, representing three distinct province region of China. To infer the rearrangement rate between eleven wild rice, multiple alignment of all pairwise combinations of the mitogenomes were conducted using Mauve (version 20150226) to identify locally collinear blocks (LCBs) in each mitogenome with default parameters. Structural variation between the wild rice mitochondrial genome and cultivate rice To construct the D1-type wild rice mitochondrial SV maps, we divided the SVs into four categories-insertions (INSs), deletions (DELs), inversions (INVs) and duplications (DUPs). A whole genome alignment by using mummer combined with SYRI software based on the NIP reference mitochondrial genome. Construction of mitochondrial genome variant maps The SNP and INDEL calling of 11 D1-wild rice accessions was also conducted using NIP (BA000029.3) as a reference mitochondrial genome. Raw data of 11 wild rice were trimmed by Trimmomatic v0.39 [33]. Clean data was mapped to the reference genome using BWA[34]. Samtools [35] was used to convert the SAM into BAM format file. Picards (Picard Tools - By Broad Institute) was used to remove the duplicated reads. Variation identification and filtering were conducted by using GATK 4.1[36]. Finally, all SNP and INDELs with MAF large than 0.05 and max-missing rate less than 0.1 was kept for subsequent analysis. Analyses of the protein-coding-gene based Pan-mitochondrial genome The all-to-all comparison of the peptide sequence of protein-coding genes was carried out using BLASTP with an E-value of e-10, and followed by clustering of the blast results using OrthoFinder [37] with default parameters. The clusters were categorized into three groups: the core genome shared by all 12 genomes, the dispensable genome containing genes present in 2 to 11 genomes, and specific gene families unique to only one genome. Declarations Data Availability The mitochondrial genome sequences of long reads and short reads supporting in this study have been submitted to the National Center for Biotechnology Information (NCBI) with BioProject ID: PRJNA1338361 and PRJNA1338456, the material that support the findings in this study are available from the corresponding author, Hongwei Xie, upon reasonable request. Acknowledgements We appreciate professor Yonggen Lu, and Researcher Yuntao Liang for kindly providing the wild rice materials. Funding This work was supported by Major Talent Development Program of the Jiangxi Academy of Agricultural Sciences (JXSNKYJCRC202501), Jiangxi Province Science Foundation for Youths (20224BAB215008), Major Discipline Academic and Technical Leaders Training Program of Jiangxi Province (20213BCJL22044), China Agriculture Research System (CARS-01-08). Author information Authors and Affiliations Jiangxi Super-Rice Research and Development Center, Jiangxi Provincial Key Laboratory of Rice Germplasm Innovation and Breeding, National Engineering Research Center for Rice, Jiangxi Academy of Agricultural Sciences, Nanchang, 330200, China Weixiong Long, Jie Wang, Lihua Luo, Lujian Zhou, Wei Chen, Laiyang Luo, Weibiao Xu, Yonghui Li, Longan Yan, Yaohui Cai & Hongwei Xie Contributions Weixiong Long, Longan Yan, Yaohui Cai, and Hongwei Xie conceived and designed the project. Jie wang, Lihua Luo, Lujian Zhou and Wei Chen were responsible for the management of the wild rice materials. Laiyang Luo, Weibiao Xu, and Yonghui Li helped in the preparation of the manuscript. Weixiong Long and Hongwei Xie wrote and supervised the manuscript. All authors read and approved the manuscript. Corresponding authors Correspondence to Hongwei Xie. Ethics declarations Ethics approval and consent to participate Oryza rufipogon seedings collected from Guangdong province in China and were identified by professor Yonggen Lu, while the Guangxi wild rice ( Oryza rufipogon ) seedlings were collected from Guangxi Academy of Agricultural Sciences and were identified by researcher Yuntao Liang, the Jiangxi common wild rice were collected from Dongxiang, Jiangxi, China and were identified by researcher Hongwei Xie. The methods involved in the study were carried out in compliance with local and national regulations. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. References Milani L, Ghiselli F: Mitochondrial activity in gametes and transmission of viable mtDNA . Biol Direct 2015, 10 :22. Eyre-Walker A, Gaut RL, Hilton H, Feldman DL, Gaut BS: Investigation of the bottleneck leading to the domestication of maize . Proc Natl Acad Sci U S A 1998, 95 (8):4441-4446. 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Supplementary Files TableS1Alistofwildricespecieswhichcontainorf182inthisstudy.xlsx TableS2Theinformationoforf182andMSSmarkersusedinthisstudy.xlsx TableS3Thegenotypegenerateby18MSSmarker.xlsx TableS4Shortandlongreadsandtotalsize.xlsx TableS5chimericgene.xlsx TableS6wildricemitochondrialgenomerepeat.csv TableS7syri.xlsx FigS1genomeassemble.tif figS2generepeat.tif FigS3Repeatcount.tif figS4syrisummary.tif figS5.SV.tif FigS6mauve.tif FigS7orf182snp.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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A. the phylogenetic tree constructed by 20 MSTs, the sample in different clade\u003c/p\u003e\n\u003cp\u003emasked by colors were selected for sequencing. B. Three wild rice (\u003cem\u003eO. rufipogon\u003c/em\u003e) accessions collected from separate provinces in China representing different D1 type variations, served as the cytoplasm sources for breeding male-sterile lines. MXS: Mingxiangsi (WA type restorer line).\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7649942/v1/03400fac52716593673c6ebf.png"},{"id":95232452,"identity":"e0628220-b92f-443d-a87c-dc6fec7905c0","added_by":"auto","created_at":"2025-11-05 16:40:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4043999,"visible":true,"origin":"","legend":"\u003cp\u003eThe features of the wild rice mitochondrial genome. A-K. GD4, GD5, GD7, GD9, GX4, GX34, GX44, GX46, GX51, JX2 and JX5, the outward-facing text on the out circular represents forward-oriented genes, while the inward-facing text denotes reverse-oriented genes. labels in red indicate the chimeric gene \u003cem\u003eorf182\u003c/em\u003e/\u003cem\u003enad6\u003c/em\u003e. The inner circular illustrates segments derived from full length mitochondrial genome duplication.\u003c/p\u003e","description":"","filename":"Figure2mitogenomelargerepeat.png","url":"https://assets-eu.researchsquare.com/files/rs-7649942/v1/48c5a81b1ad007fcae8cf09f.png"},{"id":95232485,"identity":"4ec7739f-5c4c-4502-8a5b-834b2000b458","added_by":"auto","created_at":"2025-11-05 16:40:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":311924,"visible":true,"origin":"","legend":"\u003cp\u003eStructural variation between D1-type wild rice and Nipponbare. The red vertical tags on the mitochondrial genome represent \u003cem\u003eorf182\u003c/em\u003e. Gray lines between two genomes indicate collinearity, while yellow denotes inversions, green represents translocations, and blue corresponds to duplications.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7649942/v1/fa7fbb5b8bba220239103854.png"},{"id":95232678,"identity":"80534361-b178-4f89-a803-2918301aef11","added_by":"auto","created_at":"2025-11-05 16:40:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":673563,"visible":true,"origin":"","legend":"\u003cp\u003eRepeat content in D1-type wild rice mitochondrial genome. A-B.Total number and length of different repeat types in 11 wild rice accessions mitochondrial genomes. class I: \u0026lt;100bp, class II:(100bp, 500bp), class III: (500bp, 2000bp), class IV: \u0026gt;2000bp. C-F: The correlation between genome size and different repeat type number (left), total repeat length. R indicates the correlation coefficient, and the p-value was determined by a two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"fig4repeat.png","url":"https://assets-eu.researchsquare.com/files/rs-7649942/v1/8d885d73afee44dc596e57ac.png"},{"id":95232573,"identity":"6cec626c-fc94-4f27-a78d-7aa29f0c1d00","added_by":"auto","created_at":"2025-11-05 16:40:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":551774,"visible":true,"origin":"","legend":"\u003cp\u003ePan-mitochondrial genome of the 11 D1-type wild rice and Nipponbare. A.\u003c/p\u003e\n\u003cp\u003eThe annotated protein-coding genes, and composition of core (red), dispensable (green), and private (blue) genes in each individual accession. B. Core gene cluster and pan-mitochondrial genome of D1-type wild rice. C. Modeling of pangenome and core-genome sizes when incorporating additional genomes into clustering and composition of the rice pan mitochondrial genome (pie chart). D. The orthogroup gene count per sample. E. the number and proportion of core, dispensable and private gene families in the pan mitochondrial genome.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7649942/v1/74e28e7c14605e2115b1c47e.png"},{"id":95232667,"identity":"180b63b1-77a5-4a49-adb3-c3373155b9c1","added_by":"auto","created_at":"2025-11-05 16:40:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":299706,"visible":true,"origin":"","legend":"\u003cp\u003eThe evolutionary trajectory of \u003cem\u003eorf182\u003c/em\u003e in rice. Green indicates 71 bp repeat sequence shared by mitochondrial and nuclear genome. empty rectangular area means unknown region, blue rectangular shows the sequence derived from \u003cem\u003eSorghum biocolor\u003c/em\u003e mitochondrial genome. blue rectangular with black vertical line (SNP) and white region (InDel) denotes sequences variation compared to \u003cem\u003eSorghum biocolor \u003c/em\u003esequence, yellow rectangular stands for \u003cem\u003enad6\u003c/em\u003e gene, the hairpin arrow indicates a large inverted repeat region.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7649942/v1/eb00620f2fe4b82ba3ce545d.png"},{"id":100370345,"identity":"de7a09bb-bb36-4f17-8651-5b5028ba9fb9","added_by":"auto","created_at":"2026-01-16 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16:40:33","extension":"jpg","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":7739951,"visible":true,"origin":"","legend":"","description":"","filename":"FigS7orf182snp.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7649942/v1/755035957aa0c5280380257f.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Analysis of orf182 Widespread in Chinese Wild Rice Species for Hybrid Rice Breeding","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMitochondria, often termed the powerhouses of eukaryotic cells, are semi-autonomous organelles believed to have originated from prokaryotic ancestors via endosymbiosis. They harbor their own genetic material, which is indispensable for mitochondrial and cellular functions [1]. The mitochondrial genome (mitogenome or mtDNA) is typically maternally inherited and follows evolutionary trajectories distinct from those of nuclear genomes. Outbreeding-mediated reshuffling of nuclear and cytoplasmic genomes occurs widely and frequently, potentially reconfiguring the functional and structural interplay between these genetic systems. Rearrangements, mutations, and inheritance patterns in mitochondrial genomes can provoke evolutionary conflicts that significantly influence plant diversification, domestication, and hybridization [2, 3]. Nevertheless, the evolutionary mechanisms shaping cytoplasmic genomes\u0026mdash;especially among wild relatives of crops\u0026mdash;remain inadequately explored.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStructural variation (SV) is considerably more prevalent in mitochondrial genomes than in chloroplast genomes, where SVs are rare and occur at low frequency [4], whereas mitochondrial genomes exhibit extensive SVs driven by frequent repeat-mediated recombination[5]. In contrast, plant mitochondrial genomes undergo extensive recombination mediated by repetitive sequences, leading to pronounced structural diversity [6] and can disrupt gene expression through the reshuffling of regulatory elements or the creation of chimeric open reading frames (ORFs). Many such chimeric ORFs have been associated with cytoplasmic male sterility (CMS), a phenotype characterized by aberrant floral development and abortion of male gametophytes [7, 8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRecombination events are often facilitated by short repeats (SRs; 50\u0026ndash;500 bp) and micro-repeats (MRs; \u0026lt;50 bp), which promote asymmetric and largely irreversible recombination, yielding recombinant molecules present at low (sub-stoichiometric) levels [9, 10]. These molecules, termed \u003cem\u003esublimons\u003c/em\u003e, can rapidly amplify under certain conditions through a mechanism known as sub-stoichiometric shifting (SSS), thereby altering the mitochondrial genotype (mitotype) and driving mtDNA evolution [11].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, recombination processes mediated by SRs, MRs, intracellular gene transfer (IGT), and horizontal gene transfer (HGT) generate diverse SVs\u0026mdash;including rearrangements, duplications, insertions, and deletions [5, 10, 12]. Although SVs within gene regions are generally deleterious and selected against, those in intergenic regions may persist, contributing to the high sequence divergence observed in mitochondrial intergenic spacers [13-15].\u003c/p\u003e\n\u003cp\u003eWild rice (\u003cem\u003eOryza rufipogon\u003c/em\u003e), the direct progenitor of cultivated rice (\u003cem\u003eOryza sativa\u003c/em\u003e), was domesticated approximately 8,000\u0026ndash;10,000 years ago. It represents a critical genetic resource for improving stress tolerance, yield, and grain quality in modern rice breeding [16]. While the nuclear genome of \u003cem\u003eO. rufipogon\u003c/em\u003e has been extensively studied, providing key insights into rice evolution and domestication [17, 18], \u0026nbsp;research on its mitochondrial genome remains limited. Early studies using restriction fragment length polymorphism (RFLP) analysis revealed mtDNA variation and distinct mitotypes among wild and cultivated rice populations [19]. Furthermore, several CMS types\u0026mdash;including the widely used WA-, BT-, HL-, and D1-CMS systems\u0026mdash;have been identified in cultivated rice, many tracing back to \u003cem\u003eO. rufipogon\u003c/em\u003e or closely related accessions [7, 20-22]. However, only a few complete mitogenomes of \u003cem\u003eO. rufipogon\u003c/em\u003e have been reported to date, and a comprehensive comparative analysis is still lacking. As a result, the extent of mitochondrial genomic diversity and its evolutionary relationship with cultivated rice remain poorly understood.\u003c/p\u003e\n\u003cp\u003eIn this study, we employed D1-CMS-type wild rice as a model to investigate cytoplasmic genome evolution. We constructed a pan-mitogenome based on high-quality long-read assemblies from 11 representative accessions of common wild rice. Our comprehensive analyses revealed extensive mitochondrial rearrangements and the presence of chimeric ORFs. We generated genome-wide mitochondrial variation maps from diverse \u003cem\u003eOryza\u003c/em\u003e accessions for population-level genomic studies. The potential role of chimeric ORFs\u0026mdash;classified into distinct phylogenetic clades based on mitotype-specific markers (MSS)\u0026mdash;in CMS was evaluated using cytoplasmic substitution lines. Furthermore, we examined recombination dynamics around the mitochondrial gene \u003cem\u003eorf182\u003c/em\u003e to elucidate its structural complexity and explore the mechanisms underlying rice mtDNA evolution. Finally, we characterized structural variations across the mitochondrial genome to uncover general architectural features and evolutionary constraints.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree constructed by Mitotype-specific sequence (MSS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies identified the \u003cem\u003eorf182\u003c/em\u003e gene exclusively in 18 \u003cem\u003eOryza rufipogon\u003c/em\u003e accessions from China, distributed among 17 species across nine genomic types, based on a screen of 446 wild rice samples [22]. To further elucidate the evolutionary origin and distribution of this gene, we collected an additional 320 common wild rice accessions from three provinces in China: Guangdong, Jiangxi, and Guangxi. Among these, 76 accessions (23.75%) exhibiting the D1-type cytoplasmic male sterility (CMS) phenotype were selected for subsequent genetic and genomic analyses (Table S1).\u003c/p\u003e\n\u003cp\u003eFrom a total of 57 mitochondrial structural sequence (MSS) profiles obtained from these accessions [23], 18 representative samples showing pronounced polymorphisms were used to reconstruct a phylogenetic tree, aiming to clarify the evolutionary relationships within the D1-type wild rice group (Fig. 1, Tables S2, S3). Furthermore, 11 representative wild rice accessions spanning nine distinct D1-type phylogenetic clades were subjected to high-quality mitochondrial genome assembly using a hybrid sequencing strategy combining long-read and short-read technologies. These high-resolution assemblies facilitated detailed comparative and evolutionary analyses of the mitochondrial genome, with particular emphasis on structural variation, recombination events, and the potential functional implications of CMS-associated open reading frames such as \u003cem\u003eorf182\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eBased on three \u003cem\u003eOryza rufipogon\u003c/em\u003e accessions carrying \u003cem\u003eorf182\u0026nbsp;\u003c/em\u003efromdifferent geographical origins and phylogenetic tree branch, we successfully converted Mingxiangsi into male sterile lines after only five backcross generations(Fig. 1B). These results demonstrates that the D1-type CMS system can be readily introduced into diverse genetic backgrounds, highlighting its efficiency and broad applicability in hybrid breeding programs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral features of the \u003cem\u003eOryza rufipogon\u003c/em\u003e mitochondrial genome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 69 Gb of raw Illumina reads (150 bp in length) from 11 samples, along with 506 Mb of Nanopore PromethION data from 9 wild rice varieties and 572 Mb of PacBio long reads (average length ~10 kb), were generated for mitochondrial genome assembly (Table S4). The assembled mitogenomes of the 11 wild rice accessions ranged in size from 438,632 bp to 718,357 bp. Functional annotations and physical locations of genes across these mitochondrial genomes are summarized in Fig. 2 and Fig. S1.\u003c/p\u003e\n\u003cp\u003eIn the JX2 mitogenome, 77 genes were annotated, including 46 protein-coding genes, 28 tRNAs, and 3 rRNAs. These encode 37 distinct proteins, with \u003cem\u003eatp4\u003c/em\u003e, \u003cem\u003eatp6\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003eatp9\u003c/em\u003e, \u003cem\u003ecob-like\u003c/em\u003e, \u003cem\u003eorf182\u003c/em\u003e, and \u003cem\u003eorfX\u003c/em\u003e present in two copies each, and \u003cem\u003enad6\u003c/em\u003e in three copies. The protein-coding genes were classified into 11 functional categories: ATP synthases (5 genes), cytochrome C biogenesis (4 genes), ubiquinol cytochrome c reductase (1 gene), cytochrome c oxidase (3 genes), maturases (1 gene), NADH dehydrogenase (9 genes), cytoplasmic male sterility-related (1 gene), open reading frames (1 gene), large ribosomal subunit proteins (3 genes), and small ribosomal subunit proteins (8 genes).\u003c/p\u003e\n\u003cp\u003eOn average, seven chimeric ORFs derived from fragments of known and conserved protein-coding genes (excluding tRNAs and rRNAs) were identified in each mitogenome. Notably, \u003cem\u003enad6\u003c/em\u003e (from the \u003cem\u003enad\u003c/em\u003e family) and \u003cem\u003erpl2\u003c/em\u003e and \u003cem\u003erpl16\u003c/em\u003e (from the \u003cem\u003erpl\u003c/em\u003e family) frequently participated in the formation of prominent chimeric ORFs across all wild rice accessions examined (Table S5).\u003c/p\u003e\n\u003cp\u003eTo assess structural variation (SV) and its functional implications, we performed gene copy number analysis. A core set of 37 protein-coding genes was conserved across all 11 mitochondrial genomes. Among these, \u003cem\u003enad6\u003c/em\u003e exhibited the highest copy number. The accession GX34 showed the most extensive gene loss, affecting \u003cem\u003eatp4\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003erps7\u003c/em\u003e, \u003cem\u003ecox3\u003c/em\u003e, \u003cem\u003enad5\u003c/em\u003e, \u003cem\u003enad7\u003c/em\u003e, \u003cem\u003erps13\u003c/em\u003e, \u003cem\u003eccmFc\u003c/em\u003e, \u003cem\u003erpl5\u003c/em\u003e, and \u003cem\u003ecox1\u003c/em\u003e. Additionally, genes such as \u003cem\u003ecob-like\u003c/em\u003e and \u003cem\u003eorfX\u003c/em\u003e were lost in two other accessions. Gene duplication was widespread: \u003cem\u003ecox1\u003c/em\u003e was replicated in nearly all wild rice mitogenomes, and \u003cem\u003enad6\u003c/em\u003e duplication occurred in all 11 accessions (Fig. S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepeat sequence variation and\u0026nbsp;correlations between\u0026nbsp;genome size and\u0026nbsp;repeat sequences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitogenome repeats were classified into class I (repeats ≤ 100 bp), class II (100–500 bp), class III (500–2000 bp), and IV (\u0026gt; 2000 bp) based on repeat length. The number of class I repeat sequences ranged from 239 (GX44 and GX51) to 724 (GX4), account for 1.53-1.68% of mitochondrial genome size (Fig. 3A; Table S6). Among the samples, the number of class I repeats in GX4 samples was significantly higher than samples from wild rice in same province and other wild rice, while the number of repeats longer than 100 bp was not significantly increased (t-test, P-value = 0.11) (Table S6). Meanwhile the total repeat number (864) of GX4 was higher than that of other sample (total repeat number: 316–490) (Fig. 3), and the total repeat length show the similar pattern, while the mitochondrial size of GX4 greater than other contributed by the class IV type repeat. These were followed by repeats in class II, which accounted for approximately 15% of the total repeats, with 56–90 in number. There were most least repeats length located in class III and only accounted for 0.92%. In contrast, a total of 12–38 repeats in class IV were identified, with lengths ranging from 114.38 to 574.38kb, contributing 26.04–79.96% of the mitogenome size. Despite the relatively low number of repeats in class IV, they were the major contributors to the total mitogenome size (Fig. 3B).\u003c/p\u003e\n\u003cp\u003eFor all of the 11 wild rice samples, mitogenomes size show significantly high correlations (P-value \u0026lt; 0.001) with total repeat number and length (Fig. S3, Fig. 3c, d). All the count of the four repeat categories showed significant correlation with variations in genome size (R2 adj ranged from 0.63 to 0.99, P-value \u0026lt; 0.003) (Fig. S3). While the Class II repeat size show low correlations with the mitochondrial genome size (Fig. 3d). In addition, several mitogenomes may have an overrepresentation of repeat content (Table S6). For example, GX46 (total repeat length: 466 kb) had a similar genome size to GX51 and GX44 (438 kb), but the total repeat length was about 1.46-fold of GX51 (131 kb).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentified the structural variation in D1-wild rice mitogenomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have indicated that repeat sequences can mediate rearrangements of varying extent in the mitochondrial genome. By aligning the 11 assembled mitogenomes to the Nipponbare reference genome, we identified syntenic regions accounting for 12% to 30% of each genome (Table S7). Structural variations across assemblies included one to three inversions, two to six translocations, 27 to 68 insertions, and 32 to 61 deletions (Fig. S4). Wild rice accessions with D1-CMS exhibited the highest abundance of SVs (Fig. S5). Notably, although GX34 ranked fourth lowest in terms of SV quantity, it displayed the greatest total SV length. This apparent discrepancy is largely attributable to the absence of large-scale translocations in GX34, resulting in a notably low minimal SV length.\u003c/p\u003e\n\u003cp\u003eMitogenomes of wild rice from Guangdong province appeared relatively conserved, a pattern that may reflect either geographic sampling concentration or limited underlying genetic differentiation. In contrast, the two Jiangxi accessions exhibited markedly distinct profiles in both the number and size of SVs (Fig. S5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhole-genome alignment using Mauve further characterized large-scale structural rearrangements, revealing 17 locally collinear blocks (LCBs) with variable ordering across accessions (Fig. S6). Three major rearrangement events were detected in Jiangxi Dongxiang wild rice, whereas only five LCBs were identified in GX4 and GX34. These results suggest that the mitochondrial genomes of Guangxi D1-CMS wild rice have lost numerous conserved genomic modules during evolution, consistent with previous reports of extensive structural variation in accessions from this region.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePan-mitochondrial genome of 11 selected D1 CMS wild rice and Nipponbare\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate mitochondrial genomic diversity across different origins, we selected 11 D1-CMS wild rice accessions representing distinct groups, along with the previously published Nipponbare mitochondrial genome, for pan-genome construction and comparative analysis. The gene accumulation curve (Fig. 5C) indicated an open pan-genome, suggesting that new genes are likely to be identified as more mitochondrial genomes from rice are sequenced.\u003c/p\u003e\n\u003cp\u003eComparative analysis of the eleven D1-CMS wild rice mitogenomes revealed no private genes among these accessions. Mitochondrial genomes from Guangdong accessions were generally the most conserved. However, one accession, GD4, exhibited a reduced number of core genes and a higher proportion of variable genes within the pan-genome.\u003c/p\u003e\n\u003cp\u003eIn contrast, accessions from Guangxi showed greater variability in pan-genome composition. Among these, GX34 contained fewer variable genes, implying a more stable evolutionary history and possible adaptation to a narrower ecological niche. Further comparison between Jiangxi accessions indicated that JX5 possessed more variable genes than JX2. Notably, private genes were exclusively identified in the Nipponbare reference genome, highlighting substantial divergence in mitochondrial gene content between the reference and the D1-CMS wild rice lineages examined in this study (Fig. 5A).\u003c/p\u003e\n\u003cp\u003eWe also quantified the proportional distribution of different genomic components. Core gene families accounted for the largest share (44.23%), followed by private (30.77%) and variable (25%) gene families. In terms of copy number, core genes constituted 66.55% of all genes, while private genes represented only 2.89%.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified the chimeric mitochondrial gene \u003cem\u003eorf182\u003c/em\u003e as a unique genetic element present exclusively in a subset of Chinese wild rice accessions. This finding offers significant insights into the evolutionary dynamics of plant mitochondrial genomes, the origin of cytoplasmic male sterility (CMS)-associated genes, and the potential utility of \u003cem\u003eorf182\u003c/em\u003e in hybrid rice breeding. The restricted distribution of \u003cem\u003eorf182\u003c/em\u003e suggests that it likely originated from a localized mitochondrial recombination event. This observation supports the hypothesis that CMS-related genes often emerge independently in different populations through rapid and extensive mitochondrial restructuring. Similar to \u003cem\u003eorf79\u003c/em\u003e and \u003cem\u003eorfH79\u003c/em\u003e, \u003cem\u003eorf182\u003c/em\u003e exemplifies the high structural plasticity of plant mitochondrial genomes, wherein recurrent gene fusion events give rise to chimeric open reading frames. The occurrence of \u003cem\u003eorf182\u003c/em\u003e solely in Chinese wild rice further indicates that CMS genes are not universally conserved but rather represent region-specific evolutionary innovations. Its discovery highlights the distinctive genetic value of Chinese wild rice germplasm within the global rice gene pool. As a naturally occurring CMS-inducing gene, \u003cem\u003eorf182\u003c/em\u003e provides direct evidence of functional diversity preserved in wild populations. Among the collected Chinese \u003cem\u003eOryza rufipogon\u003c/em\u003e accessions, 23.75% were found to carry \u003cem\u003eorf182\u003c/em\u003e. Based on these findings, we propose that the mitochondrial genome harboring \u003cem\u003eorf182\u003c/em\u003e has not evolved through conservative mechanisms but rather via dynamic structural rearrangements.\u003c/p\u003e\n\u003ch3\u003eMitochondrial genome structural plasticity and chimeric gene formation\u003c/h3\u003e\n\u003cp\u003eOur results demonstrate that large-scale structural variations (LSVs) are prevalent in the mitochondrial genomes of wild rice, including among accessions from the same geographic region. Accessions such as GD4 and GX4 exhibit pronounced structural divergence compared to others. Notably, the physical scale of SVs did not correlate with their quantity; for instance, GX34 displayed the largest SV size but ranked among the lowest in terms of SV number. Substantial variation in mitochondrial genome size was observed across the wild rice panel (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The assembly and annotation of 11 mitochondrial genomes provide new insights into this variability. None of the wild rice accessions shared a common presence\u0026ndash;absence variation (PAV) region associated with \u003cem\u003eorf182\u003c/em\u003e, indicating that this gene did not originate from a recent insertion event. Instead, analysis of repetitive sequences revealed that some \u003cem\u003eorf182\u003c/em\u003e chimeric genes likely arose from duplication events, while others were generated through recombination mediated by a 71-bp repetitive element, which appears to have undergone multiple recombination cycles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCMS gene diversity and implications for domestication and hybrid breeding\u003c/h2\u003e\u003cp\u003eDynamic structural changes, including gene duplications, have facilitated the emergence of new gene copies in the mitochondrial genome. Nucleotide alignment of \u003cem\u003eorf182\u003c/em\u003e sequences identified several SNP sites, which were categorized into three haplotypes (Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). All detected SNPs were synonymous, suggesting neutral evolution at the sequence level. CMS genes such as \u003cem\u003eorf182\u003c/em\u003e may have historically functioned as genetic barriers that contributed to reproductive isolation, thereby influencing domestication pathways and exhibiting geographic specificity. The D1-CMS system, characterized by \u003cem\u003eorf182\u003c/em\u003e, represents a promising resource for the development of novel hybrid rice breeding systems.\u003c/p\u003e\u003c/div\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOryza rufipogon\u003c/em\u003e collected from three province, Guangdong(GD), Guangxi(GX) and Jiangxi (JX), were used in the present research. The Guangdong common wild rice materials were generously contributed and identified by professor Yonggen Lu (South China Agricultural University), Guangxi wild rice were obtained from Researcher Yuntao Liang (Guangxi Academy of Agricultural Sciences), while Jiangxi wild rice were kindly provided by the Dongxiang Wild Rice Resource Nursery and identified by Hongwei Xie. \u003cem\u003eOryza spontanea\u003c/em\u003e (N104625) was provided by international rice research institute, Philippines. The voucher specimens (refer to Table S1) are stored in the herbarium of Jiangxi Academy of Agricultural Sciences, relevant data are stored in the Germplasm Resource Database of the Jiangxi Academy of Agricultural Sciences (currently an internal website, and are available from the corresponding author upon reasonable request). All the wild rice and \u003cem\u003eO. spontanea\u003c/em\u003e were grown in the Wild Rice Germplasm Resource Nursery of the Jiangxi Academy of Agricultural Sciences (28\u0026deg;56\u0026prime;20\u0026Prime;N, 115\u0026deg;39\u0026prime;59\u0026Prime;E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of maximum likelihood tree based on the Mitotype-specific sequences (MSSs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo selected the representative D1-CMS relatives among the 76 common wild rice collected from worldwide. The previous developed 20 pairs of evenly distribution D1-CMS mitochondrial specific sequences were used (Table S2). The 0/1 matrix was generated by MTTs gel results (Table S3). The phylogenetic tree was constructed using the maximum likelihood method in IQ-TREE software v1.6.12 [24], and the resulting maximum likelihood tree was visualized using ITOL software [25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSamples, DNA extraction and Mitochondrial genome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 11 representative wild rice were selected based on the phylogenetic tree constructed by MSSs in this study (Table 1). Fresh leaves from the wild rice species were collected from a local filed experiment field of Jiangxi Academy of Agricultural Science, Nanchang, China. High-molecular-weight genomic DNA was extracted from 1-month-old leaves following the standard CTAB method. The mitochondrial genome of 11 wild rice was sequenced using the Illimina, Pacbio and Nanopore methods. Paired-end libraries with insert sizes of \u0026sim;300\u0026thinsp;bp were prepared following Illumina\u0026rsquo;s standard genomic DNA library preparation procedure. The qualified Illumina pair-end library would be used for Illumina NovaSeq 2500 sequencing (150\u0026thinsp;bp \u0026times; 2, Shanghai BIOZERON Co., Ltd., Shanghai, China). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial genome assembly and annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw sequencing reads were trimmed using Btrim software to filter out low-quality bases and sequences. The high quality reads were assembled into bp using SOAP de novo v2.04 ( http://soap.genomics.org.cn/) and Celera Assembler 8.0. Gaps in the SSPACE scaffolds were filled using GapCloser v.1.12 [26] with default parameters. Finally, redundant sequences were manually removed to obtain the final mitochondrial genome. \u003c/p\u003e\n\u003cp\u003eThe denovo annotation strategy and homology-based comparison were conducted for Genome annotation. We utilized the protein sequences of the reference genome for homology alignment prediction. First, we rapidly aligned the protein sequences to each genome sequence, filtered out the poor alignment results, removed redundancies, and then using Genewise for precise alignment. The AUGUSTUS software (http://bioinf.uni-greifswald.de/augustus/) was performed to de novo gene prediction for plant mitochondrial genomes. Finally, we integrated the gene sets using the EVidenceModeler v1.1.1 software [27] to obtain the comprehensive annotation file. The output genebank format file was manually confirmed, and the mitochondrial circular map was drawn using Organellar Genome DRAW (MPI-MP CHLOROBOX - OGDRAW (mpg.de)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of repeated sequences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlastN was used to determine whether there were intragenomic duplications of large fragments or interspersed repeats in the 11 wild rice mitogenomes. Microsatellite identification tool was used to detect simple sequence repeats. The repeats of 1, 2, 3, 4, 5, and 6 bases with 8, 4, 4, 3, 3, and 3 repeats numbers, repeatively, were identified in this analysis. The tandem repeats with \u0026gt; 7p repeats units were detected using Tandem Repeats Finder v4.10 [28]. The direct and inverted repeats were identified using REPuter software (BiBiServ - Temporariliy not available (uni-bielefeld.de)) with the minimal repeat size set to 20 bp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollinear analysis and comparative analysis among the D1-CMS wild rice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mitogenome of Nipponbare was characterized by Notsu [29] were used for comparision. Collinear analysis between the two mitogenomes were conducted in Mummer [30]. Collinear regions were identified using the \u003cem\u003enucmer\u003c/em\u003e module with an identity threshold of 95% under the many-to-many alignment mode. The resulting collinearity maps were visualized with Plotsr implemented in the SYRI software[31].\u003c/p\u003e\n\u003cp\u003eThe genome sizes, GC contents, base compositions, start and stop codons, gene counts, and intron numbers of 11wild rice mitogenomes were compared to assess their variations and similarities. The copy number of protein coding genes among the mitogenome were identified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRearrangement event identification in D1-type wild rice mitogenome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMauve[32] genome alignments serve as a powerful tool for identify conserved genomic regions and structural rearrangements across different samples. In this study, we conducted a series of Mauve alignments under default parameters to compare mitochondrial genomes of GD, GX and JX, representing three distinct province region of China.\u003c/p\u003e\n\u003cp\u003eTo infer the rearrangement rate between eleven wild rice, multiple alignment of all pairwise combinations of the mitogenomes were conducted using Mauve (version 20150226) to identify locally collinear blocks (LCBs) in each mitogenome with default parameters. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural variation between the wild rice mitochondrial genome and cultivate rice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo construct the D1-type wild rice mitochondrial SV maps, we divided the SVs into four categories-insertions (INSs), deletions (DELs), inversions (INVs) and duplications (DUPs). A whole genome alignment by using mummer combined with SYRI software based on the NIP reference mitochondrial genome. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of mitochondrial genome variant maps \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SNP and INDEL calling of 11 D1-wild rice accessions was also conducted using NIP (BA000029.3) as a reference mitochondrial genome. Raw data of 11 wild rice were trimmed by Trimmomatic v0.39 [33]. Clean data was mapped to the reference genome using BWA[34]. Samtools [35] was used to convert the SAM into BAM format file. Picards (Picard Tools - By Broad Institute) was used to remove the duplicated reads. Variation identification and filtering were conducted by using GATK 4.1[36]. Finally, all SNP and INDELs with MAF large than 0.05 and max-missing rate less than 0.1 was kept for subsequent analysis. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalyses of the protein-coding-gene based \u003c/strong\u003e\u003cstrong\u003ePan-mitochondrial genome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe all-to-all comparison of the peptide sequence of protein-coding genes was carried out using BLASTP with an E-value of e-10, and followed by clustering of the blast results using OrthoFinder [37] with default parameters. The clusters were categorized into three groups: the core genome shared by all 12 genomes, the dispensable genome containing genes present in 2 to 11 genomes, and specific gene families unique to only one genome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mitochondrial genome sequences of long reads and short reads supporting in this study have been submitted to the National Center for Biotechnology Information (NCBI) with BioProject ID: PRJNA1338361 and PRJNA1338456, the material that support the findings in this study are available from the corresponding author, Hongwei Xie, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate professor Yonggen Lu, and Researcher Yuntao Liang for\u0026nbsp;kindly providing the wild rice materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Major Talent Development Program of the Jiangxi Academy of Agricultural Sciences (JXSNKYJCRC202501), Jiangxi Province Science Foundation for Youths (20224BAB215008), Major Discipline Academic and Technical Leaders Training Program of Jiangxi Province (20213BCJL22044), China Agriculture Research System (CARS-01-08).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJiangxi Super-Rice Research and Development Center, Jiangxi Provincial Key Laboratory of Rice Germplasm Innovation and Breeding, National Engineering Research Center for Rice, Jiangxi Academy of Agricultural Sciences, Nanchang, 330200, China\u003c/p\u003e\n\u003cp\u003eWeixiong Long, Jie Wang, Lihua Luo, Lujian Zhou, Wei Chen, Laiyang Luo, Weibiao Xu, Yonghui Li, Longan Yan, Yaohui Cai \u0026amp; Hongwei Xie\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eWeixiong Long, Longan Yan, Yaohui Cai, and Hongwei Xie conceived and designed the project. Jie wang, Lihua Luo, Lujian Zhou and Wei Chen were responsible for the management of the wild rice materials. Laiyang Luo, Weibiao Xu, and Yonghui Li helped in the preparation of the manuscript. Weixiong Long and Hongwei Xie wrote and supervised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eCorresponding authors\u003c/p\u003e\n\u003cp\u003eCorrespondence to Hongwei Xie.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOryza rufipogon\u003c/em\u003e seedings collected from Guangdong province in China and were identified by professor Yonggen Lu, while the Guangxi wild rice (\u003cem\u003eOryza rufipogon\u003c/em\u003e) seedlings were collected from Guangxi Academy of Agricultural Sciences and were identified by researcher Yuntao Liang, the Jiangxi common wild rice were collected from Dongxiang, Jiangxi, China and were identified by researcher Hongwei Xie. The methods involved in the study were carried out in compliance with local and national regulations.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMilani L, Ghiselli F: \u003cstrong\u003eMitochondrial activity in gametes and transmission of viable mtDNA\u003c/strong\u003e. \u003cem\u003eBiol Direct \u003c/em\u003e2015, \u003cstrong\u003e10\u003c/strong\u003e:22.\u003c/li\u003e\n\u003cli\u003eEyre-Walker A, Gaut RL, Hilton H, Feldman DL, Gaut BS: \u003cstrong\u003eInvestigation of the bottleneck leading to the domestication of maize\u003c/strong\u003e. \u003cem\u003eProc Natl Acad Sci U S A \u003c/em\u003e1998, \u003cstrong\u003e95\u003c/strong\u003e(8):4441-4446.\u003c/li\u003e\n\u003cli\u003eWang N, Li C, Kuang L, Wu X, Xie 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\u003cstrong\u003e20\u003c/strong\u003e(1):238.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Oryza rufipogon, orf182, wild rice, CMS, structural variation","lastPublishedDoi":"10.21203/rs.3.rs-7649942/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7649942/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe utilization of hybrid rice has successfully addressed food security challenges in China. However, the predominant use of wild-abortive (WA) cytoplasmic male sterility (CMS) systems in hybrid rice cultivation raises significant safety concerns. In this study, we have elucidated the distribution patterns of \u003cem\u003eorf182\u003c/em\u003e, a novel CMS gene identified in our previous work, in Chinese common wild rice. Mitochondrial sequencing and assembly of 11 representative wild rice species harboring \u003cem\u003eorf182\u003c/em\u003e revealed 16 novel genes. A total of 469 mitochondrial genes were classified into 36 gene families, with nine families containing single-copy homologous genes, indicating significant gene duplication in mitochondria. We observed a strong positive correlation between mitogenome size and the quantity and size of repetitive sequences. Collinearity analysis revealed extensive mitochondrial variation and large-scale inversions in Guangdong wild rice. Comparative genome analysis uncovered inversions, translocations, and several variations surrounding \u003cem\u003eorf182\u003c/em\u003e, with a 71 bp repeat sequence mediating the formation of the \u003cem\u003eorf182\u003c/em\u003e-\u003cem\u003enad6\u003c/em\u003e chimeric gene. Gene copy number analysis (GCNV) revealed variable \u003cem\u003eorf182\u003c/em\u003e gene copy counts (1, 2, and 3) in wild rice species. Additionally, successful transformation of \u003cem\u003eorf182\u003c/em\u003e from various sources into sterile lines was achieved. These findings provide valuable resources for advancing hybrid rice development in China, thus contributing to enhanced food security.\u003c/p\u003e","manuscriptTitle":"Comparative Analysis of orf182 Widespread in Chinese Wild Rice Species for Hybrid Rice Breeding","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 16:35:12","doi":"10.21203/rs.3.rs-7649942/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94f0a097-8e85-43ce-abcc-ff1dbd12fb5a","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-14T12:40:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 16:35:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7649942","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7649942","identity":"rs-7649942","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}