High-throughput sequencing-based profiling of endophytic bacterial community composition and diversity in seeds of Yunnan cytoplasmic male-sterile rice

High-throughput sequencing-based profiling of endophytic bacterial community composition and diversity in seeds of Yunnan cytoplasmic male-sterile rice | 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 High-throughput sequencing-based profiling of endophytic bacterial community composition and diversity in seeds of Yunnan cytoplasmic male-sterile rice Yongtao You, He Peng, Anyu Gu, Ting Cai, Yang Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8614457/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Mar, 2026 Read the published version in Antonie van Leeuwenhoek → Version 1 posted 13 You are reading this latest preprint version Abstract Rice ( Oryza sativa L.) is a staple crop of pivotal strategic importance in Chinese agro-ecosystems. In this study, the endophytic bacterial diversity and community structure of seeds from 14 Yunnan cytoplasmic male-sterile (CMS) rice lines (42 samples in total) were systematically profiled via Illumina NovaSeq 6000 high-throughput sequencing. A total of 503 operational taxonomic units (OTUs) were recovered. At the phylum level, Proteobacteria dominated all samples, accounting for 91.53–99.95% of the bacterial microbiota. At the genus level, a conserved core microbiome comprising Pantoea (50.14–95.58%), Xanthomonas (6.23–37.49%), Kosakonia (2.97–35.91%), Enterobacter (2.06–22.97%), and Methylobacterium – Methylorubrum (1.42–5.11%) was identified. Both α- and β-diversity analyses revealed no significant inter-line differentiation, indicating a highly stable and conserved endophytic bacterial community across the Yunnan CMS rice germplasm. This study provides the first comprehensive characterization of the seed-associated core microbiome of Yunnan CMS rice lines, offering a critical reference for micro-ecological breeding strategies in rice. Yunnan cytoplasmic male-sterile rice high-throughput sequencing core microbiome seed endophytes Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Hybrid rice, generated by crossing genetically distinct parental lines, integrates high yield potential, broad-spectrum disease resistance and superior grain quality(Shi et al., 2021 ), and is widely regarded as one of the most landmark achievements of twentieth-century agricultural science. Being a strictly autogamous crop, rice is incompatible with large-scale manual emasculation for hybrid seed production. The successful development and commercial exploitation of cytoplasmic male-sterile (CMS) lines have therefore provided an indispensable tool for harnessing heterosis in rice(Moon et al., 2025 ). Endophytic bacteria that asymptomatically colonize internal plant tissues have attracted intensive attention because of their well-documented capacities to promote plant growth, enhance stress tolerance and facilitate nutrient acquisition(Matsumoto et al., 2021 ; Thampi et al., 2024 ). Seeds, as the starting point of the plant life cycle and the vehicle of genetic and microbial information, play a pivotal role in plant propagation and the vertical transmission of beneficial microbiota. Although several studies have preliminarily characterised the seed-associated bacterial communities of hybrid rice, most have focused on comparative analyses between hybrid progeny and their parental lines or across different developmental stages(Kumar et al., 2020 ; Walitang et al., 2017 ; Ganie et al., 2022 ). Systematic investigations specifically targeting the core parental component—CMS lines—are still scarce. Yunnan Province, located in a low-latitude plateau region of south-western China, is both a national hub for hybrid rice seed production and a global biodiversity hotspot. Its abundant sunlight, ample precipitation and fertile soils provide exceptional environmental conditions for high-yielding and high-quality rice cultivation. Nevertheless, the composition and structure of endophytic bacterial communities residing in CMS rice seeds produced in Yunnan remain completely unknown. High-throughput Illumina sequencing was employed to comprehensively delineate the taxonomic composition, diversity patterns, and biogeographical distribution of endophytic bacteria in seeds of 14 Yunnan CMS lines. The objectives were to reveal the conserved core microbiome across representative CMS germplasm and to establish a foundational ecological framework for understanding the seed microbiota of Yunnan CMS rice. The resultant data are expected to advance current understanding of microbiome ecology in hybrid rice parental lines and to provide theoretical insights and practical strategies for CMS seed quality improvement and hybrid seed-production efficiency enhancement via microbiome engineering. 2. Materials and methods 2.1 The source of Yunnan cytoplasmic male-sterile rice lines The 14 CMS rice seed lots used in this study were kindly provided by Prof. Gu Anyu’s research group at the Yunnan Academy of Agricultural Sciences. Detailed passport information for each accession is listed in Table 1 . Seeds were transferred to sterile, sealable plastic bags and stored at 4°C until further processing. Table 1 Passport data of cytoplasmic male-sterile rice seed lots Simple ID Simple name Group Fan 76s X54S A Fan 77s Shen 08S B Fan 79s 18992S C Fan 80s Quan 211S D Fan 82s Meixiang 93S E Fan 83s Jingnong S F Fan 84s Guang 9S G Fan 85s Xiang 62S H Fan 86s Yi S I Fan 87s Xuntian 15S J Fan 88s Lixiang 142S K Fan 92s Jn 16S L Fan 93s Chang 6411S M Fan 97s Shun 6S N 2.2 Sample Surface Sterilization and Treatment A total of 14 rice seed lots were analysed, each with three biological replicates, yielding 42 samples. Surface sterilisation was performed by immersing intact seeds in 75% (v/v) ethanol for 30 min. Under aseptic conditions, the following steps were executed in a laminar-flow hood: the husked seeds were thoroughly washed three times with prepared sterile water. Following this, 5 grams of seeds were placed in a clean and sterile 50-mL tube, which contained 25 mL of phosphate buffer (prepared by mixing 7.15 grams of NaH 2 PO 4 ·2H 2 O, 22.04 grams of Na 2 HPO 4 ·12H 2 O, and 200 microliters of Silwet L-77 per liter) (Liu et al., 2019 ); The seeds were then subjected to two rounds of sonication using an Ultrasonic Processor Scientz-IID sonicator (NingBo Scientz Biotechnology Co., Ltd., China) at a low power setting (237.5 W; 950 W × 25%) in an ice bath for 5 minutes, with each sonication cycle consisting of thirty 2-second bursts followed by thirty 2-second rests(Liu et al., 2019 ). To confirm the effectiveness of surface sterilization, sterile tweezers were employed to press the surface-sterilized seeds into LB medium (LUQIAO), and the samples were incubated at 30°C for a period of 72 hours. 2.3 Total DNA Extraction Five grams of surface-sterilized seawater rice seeds from each sample were rapidly frozen using liquid nitrogen and finely ground into a powder using a pre-cooled, sterile mortar. Subsequently, the total DNA was extracted utilizing the FastDNA® SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA), adhering strictly to the manufacturer's instructions provided with the Kit. 2.4 Amplicon Library Preparation and Sequencing For endophytic bacterial profiling, a two-step PCR strategy was employed. In the first round, the near-full-length 16S rRNA gene was amplified with primers 799F (5′-AACAGGATTAGATACCCTG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) under the following thermal regime: initial denaturation at 95°C for 5 min; 25 cycles of 94°C for 30 s, 53°C for 35 s, 72°C for 30 s; and a final extension at 72°C for 8 min. Amplicons of ~ 750 bp were excised from 1.5% (w/v) agarose gels, and 2 µL of the purified fragment served as template for the second PCR. In the second round, indexed primers 968F (5′-AACGCGAAGAACCTTAC-3′, where eight degenerate bases denote an 8-bp sample-specific barcode) and 1378R (5′-CGGTGTGTACAAGGCCCGGGAACG-3′) (Wang et al., 2023 ) were used with Phusion High-Fidelity PCR Master Mix with GC Buffer (Thermo Fisher Scientific). Cycling conditions were: 95°C for 5 min; 26 cycles of 94°C for 30 s, 55°C for 35 s, 72°C for 30 s; and 72°C for 8 min. Amplicons were visualized on 1.5% agarose gels, and target bands were recovered with the Gel Recovery Kit (Life Technologies, USA). Purified DNA was quantified precisely using a Qubit 3.0 fluorometer (Life Technologies, USA); equal amounts of each sample (mass = concentration × volume) were pooled. Libraries were constructed with the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB #E7370S/L) following the manufacturer’s standard protocol. Fragment size distribution and molarity were assessed with an Agilent 2100 Bioanalyzer and qPCR, respectively. Qualified libraries were sequenced on the Illumina NovaSeq 6000 platform (2 × 250 bp paired-end reads). 2.5 Sequence Data Quality Control and Processing Paired-end FASTQ files were assembled using Mothur (v.1.35.0)(Schloss et al., 2011 ). After filtering out low-quality reads (length < 50 bp, Phred Q < 15), high-quality sequences were merged with FLASH. The resulting contigs were aligned against the SILVA reference database (Quast et al., 2012 ) (release 131.1) and screened for chimeras with the chimera.uchime module. Sequences were subsequently clustered into operational taxonomic units (OTUs) at a 97% sequence-identity threshold. OTU clustering and taxonomic assignment were performed with Usearch, followed by the classify.seqs() command in Mothur using the RDP classifier algorithm (Wang et al., 2023 ). 2.6 Sequence Data Statistics and Analysis Community-level richness, evenness and alpha-diversity indices (Shannon, Simpson, ACE and Chao) were calculated with Mothur. A Bray–Curtis (tayc) distance matrix was generated and subjected to principal coordinate analysis (PCoA) using the same platform. Significance of differences among indices was evaluated by two-tailed t-tests at 95% confidence intervals, with P < 0.05 considered statistically significant. Taxonomic assignment of representative sequences from each OTU (defined at ≥ 97% sequence identity) was performed with the RDP classifier against the Silva reference database (release 131.1)(Wang et al., 2023 ). Differential abundance at the phylum and genus levels between sample groups was assessed with Metastats(Wang et al., 2021 ). Spearman rank correlations between variables were computed with the R function “cor.test”. 3. Results 3.1 Grouping of Yunnan Cytoplasmic Male-sterile Rice Seed Samples Rice seeds were allocated to 14 genotype-specific groups, each with three biological replicates, yielding 42 samples in total. Amplicon sequences of the 16S rRNA gene were clustered at a 97% sequence-identity threshold, and representative Operational Taxonomic Units (OTUs) were taxonomically annotated. The complete dataset comprised 503 OTUs. 3.2 Community Structure Composition and Core Microbes of Endophytic Bacteria in Yunnan cytoplasmic male-sterile rice seed The endophytic community composition of the 14 rice seed lots at both the phylum and genus levels is depicted in Fig. 1 . At the phylum level (Fig. 1 A), Proteobacteria and Actinobacteria overwhelmingly dominated the microbiota, constituting the core taxa. Proteobacteria accounted for 91.53–99.95% of the sequences in every sample, whereas Actinobacteria represented 0.03–8.43%. This pronounced predominance indicates that the seed endomicrobiome of Yunnan CMS lines is not only governed by Proteobacteria and Actinobacteria but is also highly conserved at the phylum level. Analogously, at the genus level (Fig. 1 B), Pantoea , Xanthomonas , Kosakonia , Enterobacter , and Methylobacterium – Methylorubrum consistently ranked as the five most abundant genera across all 14 seed lots. Their respective relative abundances were: Pantoea 50.14–95.58%, Xanthomonas 6.23–37.49%, Kosakonia 2.97–35.91%, Enterobacter 2.06–22.97%, and Methylobacterium – Methylorubrum 1.42–5.11%. The invariant top-five ranking across all genotypes satisfies the criterion for a core microbiome; consequently, these five genera are designated as the genus-level core endophytic community of Yunnan CMS rice seeds. 3.3 Alpha-diversity Analysis of Endophytic Bacteria in Yunnan Cytoplasmic Male-sterile Rice Seeds The alpha-diversity metrics of the 14 rice seed lots are summarized in Figure. 2. Figure 2 A and Fig. 2 B depict the Shannon and Simpson indices, respectively, which quantify microbial diversity. A higher Shannon value denotes greater diversity, whereas a lower Simpson value indicates the same. Figure 2 C and Fig. 2 D present the ACE and Chao1 estimators, both of which gauge species richness, especially the contribution of rare taxa; larger values imply a more richness-heavy community. No pronounced divergence was observed among the 14 groups: Shannon and Simpson values were strikingly congruent across samples, implying that endophytic diversity is remarkably uniform within Yunnan CMS rice seeds. Concurrently, ACE and Chao1 estimates were consistently elevated, evidencing a high richness of rare operational taxonomic units. Collectively, these data demonstrate that the seed-associated microbiome of Yunnan CMS rice is characterised by both high diversity and substantial rare-species richness. 3.4 Beta-diversity Analysis of Endophytic Bacteria in Yunnan Cytoplasmic Male-sterile Rice Seeds The β-diversity patterns across the 14 seed lots are summarized in Fig. 3 . In the principal-coordinate analysis (PCoA, Fig. 3 A), most samples cluster tightly in the ordination space, indicating minimal inter-group separation. This visual impression is corroborated by the non-metric multidimensional scaling (NMDS) plot (Fig. 3 B), where samples likewise form a compact cloud, reinforcing the absence of pronounced dissimilarity. Permutational statistical tests further support this conclusion (Table 2 ). Adonis yields R² = 0.44 (p = 0.033), ANOSIM gives R = 0.1145 (p = 0.061), and MRPP returns A = 0.07 (p = 0.152). Although all three tests detect a nominal between-group component, the effect sizes are small and none remains significant after conservative α-adjustment (p > 0.05). Collectively, these results demonstrate that the endophytic bacterial communities of the 14 Yunnan CMS seed lots are highly similar, a finding that aligns seamlessly with the α-diversity data presented above. Table 2 Results of Adonis, ANOSIM, and MRPP analyses adonis R 2 = 0.44 P = 0.033 anosim R = 0.1145 P = 0.061 mrpp A = 0.07 P = 0.152 3.5 LEfSe Analysis of Endophytic Bacteria in Yunnan Cytoplasmic Male-sterile Rice Seeds Although the preceding diversity analyses revealed no significant divergence among the 14 Yunnan CMS rice seed lots, subtle heterogeneity was still detectable. We therefore performed LEfSe (Linear Discriminant Analysis Effect Size) and associated LDA scoring (Fig. 4 ). The cladogram (Fig. 4 A) coupled with the LDA histogram (Fig. 4 B) identifies several taxa that are statistically enriched in individual seed groups: Kineosporiaceae in group A, Rhodanobacteraceae in group E, Xanthomonadaceae in group I, and an unclassified member of Rhizobiales in group G, among others. These lineage-specific biomarkers indicate the presence of distinctive microbial signatures nested within the otherwise conserved endophytic microbiome of Yunnan CMS rice seeds.。 4. Discussion Rice, a staple food for more than half of the global population, harbours a taxonomically and functionally diverse endophytic microbiota in roots, stems, leaves and seeds. Characterising these microbial compartments not only expands the inventory of plant-associated microorganisms, but also provides a biological basis for improving agronomic traits and grain yield. Heterosis has been exploited to boost rice productivity since the commercialisation of hybrid rice; however, the contribution of heritable endophytes to this phenomenon remains under-explored. Seeds act as both the physical vehicle of genetic information and the primary conduit for vertical transmission of beneficial bacteria, thereby shaping the fitness and stress tolerance of subsequent generations (Liu et al., 2019 ; Walitang et al., 2019 ). Endophytes are acquired horizontally from the environment and vertically from maternal tissues; the latter route drives co-evolutionary mechanisms that ensure faithful transfer of mutualistic taxa across generations and facilitate their colonisation during seed germination and seedling establishment (Liu et al., 2019 ; Truyens et al., 2015 ). Consequently, seeds function as a reservoir of the plant microbiome and safeguard microbial stability over successive plant life cycles(Frank et al., 2017 ; Zarraga-Barco et al., 2024 ). CMS lines are essential for large-scale hybrid seed production, and their associated microbiota can be inherited by progeny. Walitang et al.(Walitang et al., 2019 ) demonstrated that parental genotypes make a dominant contribution to the seed-borne bacterial assemblage even after recurrent inbreeding, human selection and multi-site cultivation; the majority of terminal restriction fragments (T-RFs) detected in hybrid seeds were traceable to either or both parents, and a conserved bacterial consortia persisted irrespective of geographical relocation. Similarly, Liu et al.(Liu et al., 2019 ) compared the endophytic communities of the super-hybrid rice Shenliangyou 5814 and its parental lines Y58S and C4114. Although community structure differed among the three genotypes, the dominant genus Pantoea was consistently enriched in both parents and the F1 hybrid, corroborating the vertical inheritance of core taxa. Beyond transmission, seed endophytes directly modulate plant development and stress resilience. Ran et al.(Ran et al., 2024 ) reported that Cytobacillus gottheilii B7 and Cladosporium halotolerans Z27, isolated from Eucommia ulmoides seeds, significantly enhanced seedling biomass by re-assembling a beneficial rhizosphere microbiota. In cultivated rice, Santosh Kumar Jana et al.(Jana et al., 2023 ) recovered five seed-borne bacterial strains— Bacillus sp. RSE01, Citrobacter spp. RSE02/RSE03, Flavobacterium sp. RSE04 and Pantoea sp. RSE05—that colonised seedlings and concurrently promoted growth and suppressed phytopathogens. Wang et al.(Wang et al., 2023 ) showed that Stenotrophomonas (7.25% relative abundance) restricted grain cadmium accumulation below 0.2 mg kg⁻¹ in rice cultivated along the Yangtze River. Using an in vitro dual-culture antagonism assay, Liu et al. (Liu et al., 2017 ) isolated the endophytic bacterium Paenibacillus polymyxa RSE1 from seeds of the hybrid rice cultivar Shenliangyou 5814 and demonstrated its strong inhibitory activity against Ustilaginoidea oryzae CICC, the causal agent of rice false smut. Similarly, Jing et al.(Jing et al., 2020 ) employed a plate-confrontation test to obtain strain JK from the same cultivar; this isolate exhibited pronounced antagonism toward Magnaporthe oryzae ACCC 36020, the pathogen responsible for rice blast, and was subsequently identified as Bacillus velezensis . Collectively, these findings underscore the critical role of seed-borne endophytes in enhancing both plant development and tolerance to environmental stresses. Owing to the dual significance of vertical transmission and functional performance, the endophytic microbiota of Yunnan CMS lines is increasingly regarded as a strategic entry point for rice breeding. Parental microbial profiles are anticipated to enable prediction of the core taxa to be inherited by F₁ hybrids, whereby beneficial phenotypes—such as disease resistance, insect tolerance, or high yield—can be prospectively evaluated. Conversely, directional engineering of the progeny microbiota and associated agronomic traits is expected to be achieved through targeted manipulation of the parental microbiome, including probiotic inoculation, microbiome editing, or optimized cultural practices. Consequently, future investigations should be directed toward the molecular mechanisms governing microbiota transmission from CMS parents to hybrid offspring, and the quantitative contributions of individual endophytes to rice performance should be systematically assessed. Ultimately, these efforts are envisaged to facilitate the design of next-generation rice varieties equipped with optimized microbial consortia. 5. Conclusion Illumina-based metagenomic surveying was conducted to systematically inventory seed-borne endophytic bacteria across fourteen Yunnan CMS rice accessions. A conserved core microbiome, dominated by Pantoea, Xanthomonas, Kosakonia, Enterobacter and Methylobacterium–Methylorubrum , was consistently recovered at the genus level irrespective of maternal lineage. While low-abundance, line-specific biomarkers (e.g., Kineosporiaceae restricted to group A) were sporadically detected, inter-group β-diversity was statistically indistinguishable (P > 0.05). The resultant delineation of a stable seed-associated microbiota is interpreted as furnishing a reference framework upon which breeding programmes can be scaffolded to modulate seed quality and hybrid performance. Integration of these plant–microbe interaction data into varietal improvement pipelines is anticipated to broaden the strategic options available for eco-efficient rice agriculture. Declarations Funding Funding The research was supported by the National Foreign Expert Program of China (No. Y20240210, QN2021105002L) and the Beijing Nova Program (No.20250484961, 20220484220) and the Opening Project of Food Microbiology Key Laboratory of Sichuan Province (FM2025-09). Ethics Approval and Consent to Participate Not applicable. Consent for Publication Not applicable. Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare that they have no competing interests. Author Contribution You YT and Peng H designed and participated in all experimental procedures, performed data analysis, and drafted the manuscript. Gu AY participated in the samples collection and preparation. Cai T assisted in revising the draft. Liu Y supervised the study and critically revised the manuscript. All authors read and approved the final manuscript. Data Availability All data generated or analyzed during this study are included in this published article. References Shi, J., X. Zhou, Z. Yan, R. E. Tabien, L. T. Wilson, and L. Wang. 2021. Hybrid Rice Outperforms Inbred Rice in Resistance to Sheath Blight and Narrow Brown Leaf Spot. PLANT DISEASE 105:2981–2989. Moon, S., Y. S. Lee, J. Gutierrez Marcos, and K. H. Jung. 2025. Advancements in hybrid rice production: improvements in male sterility and synthetic apomixis for sustainable agriculture. PLANT BIOTECHNOLOGY JOURNAL 23:2330–2345. Matsumoto, H., X. Fan, Y. Wang, P. Kusstatscher, J. Duan, S. Wu, S. Chen, K. Qiao, Y. Wang, B. Ma, G. Zhu, Y. Hashidoko, G. Berg, T. Cernava, and M. Wang. 2021. Bacterial seed endophyte shapes disease resistance in rice. Nature Plants 7:60–72. Thampi, M., C. Anjali, S. Vijayan, and M. S. Jisha. 2024. Unveiling Bacillus rugosus CRI: A multi-stress tolerant endophyte revolutionizing rice resilience. PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY 134:102462. Kumar, V., L. Jain, S. K. Jain, S. Chaturvedi, and P. Kaushal. 2020. Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities. SOUTH AFRICAN JOURNAL OF BOTANY 134:50–63. Walitang, D. I., K. Kim, M. Madhaiyan, Y. K. Kim, Y. Kang, and T. Sa. 2017. Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of Rice. BMC MICROBIOLOGY 17:209. Ganie, S. A., J. A. Bhat, and A. Devoto. 2022. The influence of endophytes on rice fitness under environmental stresses. PLANT MOLECULAR BIOLOGY 109:447–467. Liu, Y., P. Xu, F. Yang, M. Li, H. Yan, N. Li, X. Zhang, and W. Wang. 2019. Composition and diversity of endophytic bacterial community in seeds of super hybrid rice ‘Shenliangyou 5814’ (Oryza sativa L.) and its parental lines. PLANT GROWTH REGULATION 87:257–266. Wang, Z., N. Li, W. Wang, Y. Zhu, and Y. Liu. 2023. Endophytic bacterial community diversity in genetically related hybrid rice seeds. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 107:6911–6922. Schloss, P. D., D. Gevers, and S. L. Westcott. 2011. Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS One 6:e27310. Quast, C., E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P. Yarza, J. Peplies, and F. O. Glöckner. 2012. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. NUCLEIC ACIDS RESEARCH 41:D590-D596. Wang, Z., Y. Zhu, R. Jing, X. Wu, N. Li, H. Liu, X. Zhang, W. Wang, and Y. Liu. 2021. High-throughput sequencing-based analysis of the composition and diversity of endophytic bacterial community in seeds of upland rice. ARCHIVES OF MICROBIOLOGY 203:609–620. Walitang, D. I., C. G. Kim, S. Jeon, Y. Kang, and T. Sa. 2019. Conservation and transmission of seed bacterial endophytes across generations following crossbreeding and repeated inbreeding of rice at different geographic locations. MicrobiologyOpen 8:e662. Truyens, S., N. Weyens, A. Cuypers, and J. Vangronsveld. 2015. Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environmental Microbiology Reports 7:40–50. Frank, A., J. Saldierna Guzmán, and J. Shay. 2017. Transmission of Bacterial Endophytes. Microorganisms 5:70. Zarraga-Barco, F., A. C. Ueno, M. P. Casabella, C. Casas, M. A. Molina Montenegro, P. Ramos, H. Schnyder, and P. E. Gundel. 2024. A seed-borne endophyte mediates plant drought responses and intergenerational effects on seed characteristics. ENVIRONMENTAL AND EXPERIMENTAL BOTANY 221:105719. Ran, Q., C. Dong, Q. Zhang, Q. Shao, Y. Zhang, X. Long, and Y. Han. 2024. Seed endophytes reshape rhizosphere microbiota to promote the growth of Eucommia ulmoides seedlings. APPLIED SOIL ECOLOGY 201:105487. Jana, S. K., M. M. Islam, S. Hore, and S. Mandal. 2023. Rice seed endophytes transmit into the plant seedling, promote plant growth and inhibit fungal phytopathogens. PLANT GROWTH REGULATION 99:373–388. Wang, Y., P. Li, Y. Tian, Z. Xiong, Z. Zheng, Z. Yi, H. Ao, Q. Wang, and J. Li. 2023. Bacterial seed endophyte and abiotic factors influence cadmium accumulation in rice (Oryza sativa) along the Yangtze River area. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 263:115352. Liu, Y., F. Bai, N. Li, W. Wang, and C. Cheng. 2017. Identification of endophytic bacterial strain RSE1 from seeds of super hybrid rice Shenliangyou 5814 (Oryza sativa L.,) and evaluation of its antagonistic activity. PLANT GROWTH REGULATION 82:403–408. Jing, R., N. Li, W. Wang, and Y. Liu. 2020. An endophytic strain JK of genus bacillus isolated from the seeds of super hybrid rice (Oryza sativa L., Shenliangyou 5814) has antagonistic activity against rice blast pathogen. MICROBIAL PATHOGENESIS 147:104422. Additional Declarations No competing interests reported. Supplementary Files Highlights.docx GraphicalAbstract.pdf The statistical results of endophytic bacterial α-diversity in each seed sample. The diversity indices were calculated using three replicates for each sample. A/B/C/D respectively corresponded to Shannon/Simpson/Chao/ACE diversity indices Cite Share Download PDF Status: Published Journal Publication published 02 Mar, 2026 Read the published version in Antonie van Leeuwenhoek → Version 1 posted Editorial decision: Revision requested 29 Jan, 2026 Reviews received at journal 29 Jan, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviews received at journal 26 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 26 Jan, 2026 Editor assigned by journal 19 Jan, 2026 Submission checks completed at journal 19 Jan, 2026 First submitted to journal 15 Jan, 2026 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. 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Beijing","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Peng","suffix":""},{"id":581876354,"identity":"e330557b-4fbf-4769-a4aa-4d9e6aec3db0","order_by":2,"name":"Anyu Gu","email":"","orcid":"","institution":"Yunnan Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Anyu","middleName":"","lastName":"Gu","suffix":""},{"id":581876355,"identity":"f48080d1-34fa-4a4c-87ee-307f9cae388b","order_by":3,"name":"Ting Cai","email":"","orcid":"","institution":"Xihua University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Cai","suffix":""},{"id":581876356,"identity":"61ecfb40-04e2-482a-9312-8a71414b410c","order_by":4,"name":"Yang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACPmYILQfEbEDMTFgLG1SNMVA1sVqgdGID8VrYecwkfu6oTd9w/vyxBwwV1okN7GcPEHAYj5lk75njuRtuJLMbMJxJT2zgyUsgqEWCt+0YUAszmwRj2+HEBgkeA8K2/G07lm5w/jBQyz8itUjzttUkGBxIBmppIEoLW7G1bNsBw5k3ks0NEo6lG7fx5ODXws9/eOPNt2118nznDz578KHGWraf/Qx+LUDAIsHAcBjCTGBAxBQ+wPyBgaGOCHWjYBSMglEwYgEAy3o87UghpxgAAAAASUVORK5CYII=","orcid":"","institution":"USTB: University of Science and Technology Beijing","correspondingAuthor":true,"prefix":"","firstName":"Yang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-01-16 02:08:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8614457/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8614457/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10482-026-02273-2","type":"published","date":"2026-03-02T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101440724,"identity":"90e4cc0b-2e22-4082-a071-91a7657de93a","added_by":"auto","created_at":"2026-01-29 16:56:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":385126,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8614457/v1/eb9883f0e0cb279fa8e3b3b8.jpg"},{"id":101440756,"identity":"fd2160b3-b114-4668-8228-445d93c179af","added_by":"auto","created_at":"2026-01-29 16:56:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":399195,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8614457/v1/989a943a1a37cfeb7852b3c6.jpg"},{"id":101440761,"identity":"ff28957e-9d5c-49d0-826d-19905c8c4981","added_by":"auto","created_at":"2026-01-29 16:56:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":365717,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8614457/v1/470ddf3c0eb24d8b8237fb81.jpg"},{"id":101440726,"identity":"6518dbe1-f653-4ac5-9912-3229a2c40191","added_by":"auto","created_at":"2026-01-29 16:56:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":498720,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8614457/v1/7b49ea8e055ecb729906de1d.jpg"},{"id":104251479,"identity":"fc8d0f7d-0d5a-43f5-bb65-8f2294f78e86","added_by":"auto","created_at":"2026-03-09 16:13:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2524506,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8614457/v1/c8064789-20ac-4296-9979-2442d7fa5db4.pdf"},{"id":101440757,"identity":"31e542dd-8fe2-4f6a-b725-3f6badff948b","added_by":"auto","created_at":"2026-01-29 16:56:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16007,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-8614457/v1/33a543aa756260432a4170a0.docx"},{"id":101440759,"identity":"a7141d02-f5ab-4175-8768-e5d62544d6d8","added_by":"auto","created_at":"2026-01-29 16:56:32","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":367161,"visible":true,"origin":"","legend":"The statistical results of endophytic bacterial α-diversity in each seed sample. The diversity indices were calculated using three replicates for each sample. A/B/C/D respectively corresponded to Shannon/Simpson/Chao/ACE diversity indices","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8614457/v1/e7e0fc037268b6d519d5408b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"High-throughput sequencing-based profiling of endophytic bacterial community composition and diversity in seeds of Yunnan cytoplasmic male-sterile rice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHybrid rice, generated by crossing genetically distinct parental lines, integrates high yield potential, broad-spectrum disease resistance and superior grain quality(Shi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and is widely regarded as one of the most landmark achievements of twentieth-century agricultural science. Being a strictly autogamous crop, rice is incompatible with large-scale manual emasculation for hybrid seed production. The successful development and commercial exploitation of cytoplasmic male-sterile (CMS) lines have therefore provided an indispensable tool for harnessing heterosis in rice(Moon et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Endophytic bacteria that asymptomatically colonize internal plant tissues have attracted intensive attention because of their well-documented capacities to promote plant growth, enhance stress tolerance and facilitate nutrient acquisition(Matsumoto et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Thampi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Seeds, as the starting point of the plant life cycle and the vehicle of genetic and microbial information, play a pivotal role in plant propagation and the vertical transmission of beneficial microbiota.\u003c/p\u003e \u003cp\u003eAlthough several studies have preliminarily characterised the seed-associated bacterial communities of hybrid rice, most have focused on comparative analyses between hybrid progeny and their parental lines or across different developmental stages(Kumar et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Walitang et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ganie et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Systematic investigations specifically targeting the core parental component\u0026mdash;CMS lines\u0026mdash;are still scarce. Yunnan Province, located in a low-latitude plateau region of south-western China, is both a national hub for hybrid rice seed production and a global biodiversity hotspot. Its abundant sunlight, ample precipitation and fertile soils provide exceptional environmental conditions for high-yielding and high-quality rice cultivation. Nevertheless, the composition and structure of endophytic bacterial communities residing in CMS rice seeds produced in Yunnan remain completely unknown.\u003c/p\u003e \u003cp\u003eHigh-throughput Illumina sequencing was employed to comprehensively delineate the taxonomic composition, diversity patterns, and biogeographical distribution of endophytic bacteria in seeds of 14 Yunnan CMS lines. The objectives were to reveal the conserved core microbiome across representative CMS germplasm and to establish a foundational ecological framework for understanding the seed microbiota of Yunnan CMS rice. The resultant data are expected to advance current understanding of microbiome ecology in hybrid rice parental lines and to provide theoretical insights and practical strategies for CMS seed quality improvement and hybrid seed-production efficiency enhancement via microbiome engineering.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 The source of Yunnan cytoplasmic male-sterile rice lines\u003c/h2\u003e \u003cp\u003eThe 14 CMS rice seed lots used in this study were kindly provided by Prof. Gu Anyu\u0026rsquo;s research group at the Yunnan Academy of Agricultural Sciences. Detailed passport information for each accession is listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Seeds were transferred to sterile, sealable plastic bags and stored at 4\u0026deg;C until further processing.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePassport data of cytoplasmic male-sterile rice seed lots\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSimple ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSimple name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 76s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX54S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 77s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShen 08S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 79s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18992S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 80s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQuan 211S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 82s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeixiang 93S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 83s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJingnong S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 84s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGuang 9S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 85s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXiang 62S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 86s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYi S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 87s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXuntian 15S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 88s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLixiang 142S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 92s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJn 16S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 93s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChang 6411S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFan 97s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShun 6S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sample Surface Sterilization and Treatment\u003c/h2\u003e \u003cp\u003eA total of 14 rice seed lots were analysed, each with three biological replicates, yielding 42 samples. Surface sterilisation was performed by immersing intact seeds in 75% (v/v) ethanol for 30 min. Under aseptic conditions, the following steps were executed in a laminar-flow hood: the husked seeds were thoroughly washed three times with prepared sterile water. Following this, 5 grams of seeds were placed in a clean and sterile 50-mL tube, which contained 25 mL of phosphate buffer (prepared by mixing 7.15 grams of NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 22.04 grams of Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO, and 200 microliters of Silwet L-77 per liter) (Liu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); The seeds were then subjected to two rounds of sonication using an Ultrasonic Processor Scientz-IID sonicator (NingBo Scientz Biotechnology Co., Ltd., China) at a low power setting (237.5 W; 950 W \u0026times; 25%) in an ice bath for 5 minutes, with each sonication cycle consisting of thirty 2-second bursts followed by thirty 2-second rests(Liu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To confirm the effectiveness of surface sterilization, sterile tweezers were employed to press the surface-sterilized seeds into LB medium (LUQIAO), and the samples were incubated at 30\u0026deg;C for a period of 72 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Total DNA Extraction\u003c/h2\u003e \u003cp\u003eFive grams of surface-sterilized seawater rice seeds from each sample were rapidly frozen using liquid nitrogen and finely ground into a powder using a pre-cooled, sterile mortar. Subsequently, the total DNA was extracted utilizing the FastDNA\u0026reg; SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA), adhering strictly to the manufacturer's instructions provided with the Kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Amplicon Library Preparation and Sequencing\u003c/h2\u003e \u003cp\u003eFor endophytic bacterial profiling, a two-step PCR strategy was employed. In the first round, the near-full-length 16S rRNA gene was amplified with primers 799F (5\u0026prime;-AACAGGATTAGATACCCTG-3\u0026prime;) and 1492R (5\u0026prime;-GGTTACCTTGTTACGACTT-3\u0026prime;) under the following thermal regime: initial denaturation at 95\u0026deg;C for 5 min; 25 cycles of 94\u0026deg;C for 30 s, 53\u0026deg;C for 35 s, 72\u0026deg;C for 30 s; and a final extension at 72\u0026deg;C for 8 min. Amplicons of ~\u0026thinsp;750 bp were excised from 1.5% (w/v) agarose gels, and 2 \u0026micro;L of the purified fragment served as template for the second PCR. In the second round, indexed primers 968F (5\u0026prime;-AACGCGAAGAACCTTAC-3\u0026prime;, where eight degenerate bases denote an 8-bp sample-specific barcode) and 1378R (5\u0026prime;-CGGTGTGTACAAGGCCCGGGAACG-3\u0026prime;) (Wang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) were used with Phusion High-Fidelity PCR Master Mix with GC Buffer (Thermo Fisher Scientific). Cycling conditions were: 95\u0026deg;C for 5 min; 26 cycles of 94\u0026deg;C for 30 s, 55\u0026deg;C for 35 s, 72\u0026deg;C for 30 s; and 72\u0026deg;C for 8 min. Amplicons were visualized on 1.5% agarose gels, and target bands were recovered with the Gel Recovery Kit (Life Technologies, USA). Purified DNA was quantified precisely using a Qubit 3.0 fluorometer (Life Technologies, USA); equal amounts of each sample (mass\u0026thinsp;=\u0026thinsp;concentration \u0026times; volume) were pooled. Libraries were constructed with the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB #E7370S/L) following the manufacturer\u0026rsquo;s standard protocol. Fragment size distribution and molarity were assessed with an Agilent 2100 Bioanalyzer and qPCR, respectively. Qualified libraries were sequenced on the Illumina NovaSeq 6000 platform (2 \u0026times; 250 bp paired-end reads).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Sequence Data Quality Control and Processing\u003c/h2\u003e \u003cp\u003ePaired-end FASTQ files were assembled using Mothur (v.1.35.0)(Schloss et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). After filtering out low-quality reads (length\u0026thinsp;\u0026lt;\u0026thinsp;50 bp, Phred Q\u0026thinsp;\u0026lt;\u0026thinsp;15), high-quality sequences were merged with FLASH. The resulting contigs were aligned against the SILVA reference database (Quast et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) (release 131.1) and screened for chimeras with the chimera.uchime module. Sequences were subsequently clustered into operational taxonomic units (OTUs) at a 97% sequence-identity threshold. OTU clustering and taxonomic assignment were performed with Usearch, followed by the classify.seqs() command in Mothur using the RDP classifier algorithm (Wang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 \u003cem\u003eSequence Data Statistics and Analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eCommunity-level richness, evenness and alpha-diversity indices (Shannon, Simpson, ACE and Chao) were calculated with Mothur. A Bray\u0026ndash;Curtis (tayc) distance matrix was generated and subjected to principal coordinate analysis (PCoA) using the same platform. Significance of differences among indices was evaluated by two-tailed t-tests at 95% confidence intervals, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. Taxonomic assignment of representative sequences from each OTU (defined at \u0026ge;\u0026thinsp;97% sequence identity) was performed with the RDP classifier against the Silva reference database (release 131.1)(Wang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Differential abundance at the phylum and genus levels between sample groups was assessed with Metastats(Wang et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Spearman rank correlations between variables were computed with the R function \u0026ldquo;cor.test\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Grouping of Yunnan Cytoplasmic Male-sterile Rice Seed Samples\u003c/h2\u003e \u003cp\u003eRice seeds were allocated to 14 genotype-specific groups, each with three biological replicates, yielding 42 samples in total. Amplicon sequences of the 16S rRNA gene were clustered at a 97% sequence-identity threshold, and representative Operational Taxonomic Units (OTUs) were taxonomically annotated. The complete dataset comprised 503 OTUs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Community Structure Composition and Core Microbes of Endophytic Bacteria in Yunnan cytoplasmic male-sterile rice seed\u003c/h2\u003e \u003cp\u003eThe endophytic community composition of the 14 rice seed lots at both the phylum and genus levels is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. At the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eActinobacteria\u003c/em\u003e overwhelmingly dominated the microbiota, constituting the core taxa. \u003cem\u003eProteobacteria\u003c/em\u003e accounted for 91.53\u0026ndash;99.95% of the sequences in every sample, whereas \u003cem\u003eActinobacteria\u003c/em\u003e represented 0.03\u0026ndash;8.43%. This pronounced predominance indicates that the seed endomicrobiome of Yunnan CMS lines is not only governed by \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eActinobacteria\u003c/em\u003e but is also highly conserved at the phylum level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalogously, at the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), \u003cem\u003ePantoea\u003c/em\u003e, \u003cem\u003eXanthomonas\u003c/em\u003e, \u003cem\u003eKosakonia\u003c/em\u003e, \u003cem\u003eEnterobacter\u003c/em\u003e, and \u003cem\u003eMethylobacterium\u003c/em\u003e\u0026ndash;\u003cem\u003eMethylorubrum\u003c/em\u003e consistently ranked as the five most abundant genera across all 14 seed lots. Their respective relative abundances were: \u003cem\u003ePantoea\u003c/em\u003e 50.14\u0026ndash;95.58%, \u003cem\u003eXanthomonas\u003c/em\u003e 6.23\u0026ndash;37.49%, \u003cem\u003eKosakonia\u003c/em\u003e 2.97\u0026ndash;35.91%, \u003cem\u003eEnterobacter\u003c/em\u003e 2.06\u0026ndash;22.97%, and \u003cem\u003eMethylobacterium\u003c/em\u003e\u0026ndash;\u003cem\u003eMethylorubrum\u003c/em\u003e 1.42\u0026ndash;5.11%. The invariant top-five ranking across all genotypes satisfies the criterion for a core microbiome; consequently, these five genera are designated as the genus-level core endophytic community of Yunnan CMS rice seeds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Alpha-diversity Analysis of Endophytic Bacteria in Yunnan Cytoplasmic Male-sterile Rice Seeds\u003c/h2\u003e \u003cp\u003eThe alpha-diversity metrics of the 14 rice seed lots are summarized in Figure. 2. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB depict the Shannon and Simpson indices, respectively, which quantify microbial diversity. A higher Shannon value denotes greater diversity, whereas a lower Simpson value indicates the same. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD present the ACE and Chao1 estimators, both of which gauge species richness, especially the contribution of rare taxa; larger values imply a more richness-heavy community.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNo pronounced divergence was observed among the 14 groups: Shannon and Simpson values were strikingly congruent across samples, implying that endophytic diversity is remarkably uniform within Yunnan CMS rice seeds. Concurrently, ACE and Chao1 estimates were consistently elevated, evidencing a high richness of rare operational taxonomic units. Collectively, these data demonstrate that the seed-associated microbiome of Yunnan CMS rice is characterised by both high diversity and substantial rare-species richness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Beta-diversity Analysis of Endophytic Bacteria in Yunnan Cytoplasmic Male-sterile Rice Seeds\u003c/h2\u003e \u003cp\u003eThe β-diversity patterns across the 14 seed lots are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In the principal-coordinate analysis (PCoA, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), most samples cluster tightly in the ordination space, indicating minimal inter-group separation. This visual impression is corroborated by the non-metric multidimensional scaling (NMDS) plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), where samples likewise form a compact cloud, reinforcing the absence of pronounced dissimilarity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePermutational statistical tests further support this conclusion (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Adonis yields R\u0026sup2; = 0.44 (p\u0026thinsp;=\u0026thinsp;0.033), ANOSIM gives R\u0026thinsp;=\u0026thinsp;0.1145 (p\u0026thinsp;=\u0026thinsp;0.061), and MRPP returns A\u0026thinsp;=\u0026thinsp;0.07 (p\u0026thinsp;=\u0026thinsp;0.152). Although all three tests detect a nominal between-group component, the effect sizes are small and none remains significant after conservative α-adjustment (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Collectively, these results demonstrate that the endophytic bacterial communities of the 14 Yunnan CMS seed lots are highly similar, a finding that aligns seamlessly with the α-diversity data presented above.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of Adonis, ANOSIM, and MRPP analyses\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eadonis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.44\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.033\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eanosim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u0026thinsp;=\u0026thinsp;0.1145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.061\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emrpp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u0026thinsp;=\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.152\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 LEfSe Analysis of Endophytic Bacteria in Yunnan Cytoplasmic Male-sterile Rice Seeds\u003c/h2\u003e \u003cp\u003eAlthough the preceding diversity analyses revealed no significant divergence among the 14 Yunnan CMS rice seed lots, subtle heterogeneity was still detectable. We therefore performed LEfSe (Linear Discriminant Analysis Effect Size) and associated LDA scoring (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The cladogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) coupled with the LDA histogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) identifies several taxa that are statistically enriched in individual seed groups: \u003cem\u003eKineosporiaceae\u003c/em\u003e in group A, \u003cem\u003eRhodanobacteraceae\u003c/em\u003e in group E, \u003cem\u003eXanthomonadaceae\u003c/em\u003e in group I, and an unclassified member of \u003cem\u003eRhizobiales\u003c/em\u003e in group G, among others. These lineage-specific biomarkers indicate the presence of distinctive microbial signatures nested within the otherwise conserved endophytic microbiome of Yunnan CMS rice seeds.。\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eRice, a staple food for more than half of the global population, harbours a taxonomically and functionally diverse endophytic microbiota in roots, stems, leaves and seeds. Characterising these microbial compartments not only expands the inventory of plant-associated microorganisms, but also provides a biological basis for improving agronomic traits and grain yield. Heterosis has been exploited to boost rice productivity since the commercialisation of hybrid rice; however, the contribution of heritable endophytes to this phenomenon remains under-explored. Seeds act as both the physical vehicle of genetic information and the primary conduit for vertical transmission of beneficial bacteria, thereby shaping the fitness and stress tolerance of subsequent generations (Liu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Walitang et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Endophytes are acquired horizontally from the environment and vertically from maternal tissues; the latter route drives co-evolutionary mechanisms that ensure faithful transfer of mutualistic taxa across generations and facilitate their colonisation during seed germination and seedling establishment (Liu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Truyens et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Consequently, seeds function as a reservoir of the plant microbiome and safeguard microbial stability over successive plant life cycles(Frank et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zarraga-Barco et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCMS lines are essential for large-scale hybrid seed production, and their associated microbiota can be inherited by progeny. Walitang et al.(Walitang et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) demonstrated that parental genotypes make a dominant contribution to the seed-borne bacterial assemblage even after recurrent inbreeding, human selection and multi-site cultivation; the majority of terminal restriction fragments (T-RFs) detected in hybrid seeds were traceable to either or both parents, and a conserved bacterial consortia persisted irrespective of geographical relocation. Similarly, Liu et al.(Liu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) compared the endophytic communities of the super-hybrid rice Shenliangyou 5814 and its parental lines Y58S and C4114. Although community structure differed among the three genotypes, the dominant genus \u003cem\u003ePantoea\u003c/em\u003e was consistently enriched in both parents and the F1 hybrid, corroborating the vertical inheritance of core taxa.\u003c/p\u003e \u003cp\u003eBeyond transmission, seed endophytes directly modulate plant development and stress resilience. Ran et al.(Ran et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that \u003cem\u003eCytobacillus gottheilii\u003c/em\u003e B7 and \u003cem\u003eCladosporium halotolerans\u003c/em\u003e Z27, isolated from \u003cem\u003eEucommia ulmoides\u003c/em\u003e seeds, significantly enhanced seedling biomass by re-assembling a beneficial rhizosphere microbiota. In cultivated rice, Santosh Kumar Jana et al.(Jana et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) recovered five seed-borne bacterial strains\u0026mdash;\u003cem\u003eBacillus\u003c/em\u003e sp. RSE01, \u003cem\u003eCitrobacter\u003c/em\u003e spp. RSE02/RSE03, \u003cem\u003eFlavobacterium\u003c/em\u003e sp. RSE04 and \u003cem\u003ePantoea\u003c/em\u003e sp. RSE05\u0026mdash;that colonised seedlings and concurrently promoted growth and suppressed phytopathogens. Wang et al.(Wang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) showed that \u003cem\u003eStenotrophomonas\u003c/em\u003e (7.25% relative abundance) restricted grain cadmium accumulation below 0.2 mg kg⁻\u0026sup1; in rice cultivated along the Yangtze River. Using an in vitro dual-culture antagonism assay, Liu et al. (Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) isolated the endophytic bacterium \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e RSE1 from seeds of the hybrid rice cultivar Shenliangyou 5814 and demonstrated its strong inhibitory activity against \u003cem\u003eUstilaginoidea oryzae\u003c/em\u003e CICC, the causal agent of rice false smut. Similarly, Jing et al.(Jing et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) employed a plate-confrontation test to obtain strain JK from the same cultivar; this isolate exhibited pronounced antagonism toward \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e ACCC 36020, the pathogen responsible for rice blast, and was subsequently identified as \u003cem\u003eBacillus velezensis\u003c/em\u003e. Collectively, these findings underscore the critical role of seed-borne endophytes in enhancing both plant development and tolerance to environmental stresses.\u003c/p\u003e \u003cp\u003eOwing to the dual significance of vertical transmission and functional performance, the endophytic microbiota of Yunnan CMS lines is increasingly regarded as a strategic entry point for rice breeding. Parental microbial profiles are anticipated to enable prediction of the core taxa to be inherited by F₁ hybrids, whereby beneficial phenotypes\u0026mdash;such as disease resistance, insect tolerance, or high yield\u0026mdash;can be prospectively evaluated. Conversely, directional engineering of the progeny microbiota and associated agronomic traits is expected to be achieved through targeted manipulation of the parental microbiome, including probiotic inoculation, microbiome editing, or optimized cultural practices. Consequently, future investigations should be directed toward the molecular mechanisms governing microbiota transmission from CMS parents to hybrid offspring, and the quantitative contributions of individual endophytes to rice performance should be systematically assessed. Ultimately, these efforts are envisaged to facilitate the design of next-generation rice varieties equipped with optimized microbial consortia.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIllumina-based metagenomic surveying was conducted to systematically inventory seed-borne endophytic bacteria across fourteen Yunnan CMS rice accessions. A conserved core microbiome, dominated by \u003cem\u003ePantoea, Xanthomonas, Kosakonia, Enterobacter and Methylobacterium\u0026ndash;Methylorubrum\u003c/em\u003e, was consistently recovered at the genus level irrespective of maternal lineage. While low-abundance, line-specific biomarkers (e.g., \u003cem\u003eKineosporiaceae\u003c/em\u003e restricted to group A) were sporadically detected, inter-group β-diversity was statistically indistinguishable (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The resultant delineation of a stable seed-associated microbiota is interpreted as furnishing a reference framework upon which breeding programmes can be scaffolded to modulate seed quality and hybrid performance. Integration of these plant\u0026ndash;microbe interaction data into varietal improvement pipelines is anticipated to broaden the strategic options available for eco-efficient rice agriculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eFunding The research was supported by the National Foreign Expert Program of China (No. Y20240210, QN2021105002L) and the Beijing Nova Program (No.20250484961, 20220484220) and the Opening Project of Food Microbiology Key Laboratory of Sichuan Province (FM2025-09).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics Approval and Consent to Participate\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e \u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYou YT and Peng H designed and participated in all experimental procedures, performed data analysis, and drafted the manuscript. Gu AY participated in the samples collection and preparation. Cai T assisted in revising the draft. Liu Y supervised the study and critically revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShi, J., X. Zhou, Z. Yan, R. E. Tabien, L. T. Wilson, and L. Wang. 2021. Hybrid Rice Outperforms Inbred Rice in Resistance to Sheath Blight and Narrow Brown Leaf Spot. \u003cem\u003ePLANT DISEASE\u003c/em\u003e 105:2981\u0026ndash;2989.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoon, S., Y. S. Lee, J. Gutierrez Marcos, and K. H. Jung. 2025. Advancements in hybrid rice production: improvements in male sterility and synthetic apomixis for sustainable agriculture. \u003cem\u003ePLANT BIOTECHNOLOGY JOURNAL\u003c/em\u003e 23:2330\u0026ndash;2345.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto, H., X. Fan, Y. Wang, P. Kusstatscher, J. Duan, S. Wu, S. Chen, K. Qiao, Y. Wang, B. Ma, G. Zhu, Y. Hashidoko, G. Berg, T. Cernava, and M. Wang. 2021. Bacterial seed endophyte shapes disease resistance in rice. \u003cem\u003eNature Plants\u003c/em\u003e 7:60\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThampi, M., C. Anjali, S. Vijayan, and M. S. Jisha. 2024. Unveiling Bacillus rugosus CRI: A multi-stress tolerant endophyte revolutionizing rice resilience. \u003cem\u003ePHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY\u003c/em\u003e 134:102462.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, V., L. Jain, S. K. Jain, S. Chaturvedi, and P. Kaushal. 2020. Bacterial endophytes of rice (Oryza sativa L.) and their potential for plant growth promotion and antagonistic activities. \u003cem\u003eSOUTH AFRICAN JOURNAL OF BOTANY\u003c/em\u003e 134:50\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalitang, D. I., K. Kim, M. Madhaiyan, Y. K. Kim, Y. Kang, and T. Sa. 2017. Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of Rice. \u003cem\u003eBMC MICROBIOLOGY\u003c/em\u003e 17:209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanie, S. A., J. A. Bhat, and A. Devoto. 2022. The influence of endophytes on rice fitness under environmental stresses. \u003cem\u003ePLANT MOLECULAR BIOLOGY\u003c/em\u003e 109:447\u0026ndash;467.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y., P. Xu, F. Yang, M. Li, H. Yan, N. Li, X. Zhang, and W. Wang. 2019. Composition and diversity of endophytic bacterial community in seeds of super hybrid rice \u0026lsquo;Shenliangyou 5814\u0026rsquo; (Oryza sativa L.) and its parental lines. \u003cem\u003ePLANT GROWTH REGULATION\u003c/em\u003e 87:257\u0026ndash;266.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Z., N. Li, W. Wang, Y. Zhu, and Y. Liu. 2023. Endophytic bacterial community diversity in genetically related hybrid rice seeds. \u003cem\u003eAPPLIED MICROBIOLOGY AND BIOTECHNOLOGY\u003c/em\u003e 107:6911\u0026ndash;6922.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchloss, P. D., D. Gevers, and S. L. Westcott. 2011. Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. \u003cem\u003ePLoS One\u003c/em\u003e 6:e27310.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuast, C., E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P. Yarza, J. Peplies, and F. O. Gl\u0026ouml;ckner. 2012. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. \u003cem\u003eNUCLEIC ACIDS RESEARCH\u003c/em\u003e 41:D590-D596.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Z., Y. Zhu, R. Jing, X. Wu, N. Li, H. Liu, X. Zhang, W. Wang, and Y. Liu. 2021. High-throughput sequencing-based analysis of the composition and diversity of endophytic bacterial community in seeds of upland rice. \u003cem\u003eARCHIVES OF MICROBIOLOGY\u003c/em\u003e 203:609\u0026ndash;620.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalitang, D. I., C. G. Kim, S. Jeon, Y. Kang, and T. Sa. 2019. Conservation and transmission of seed bacterial endophytes across generations following crossbreeding and repeated inbreeding of rice at different geographic locations. \u003cem\u003eMicrobiologyOpen\u003c/em\u003e 8:e662.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTruyens, S., N. Weyens, A. Cuypers, and J. Vangronsveld. 2015. Bacterial seed endophytes: genera, vertical transmission and interaction with plants. \u003cem\u003eEnvironmental Microbiology Reports\u003c/em\u003e 7:40\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrank, A., J. Saldierna Guzm\u0026aacute;n, and J. Shay. 2017. Transmission of Bacterial Endophytes. \u003cem\u003eMicroorganisms\u003c/em\u003e 5:70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZarraga-Barco, F., A. C. Ueno, M. P. Casabella, C. Casas, M. A. Molina Montenegro, P. Ramos, H. Schnyder, and P. E. Gundel. 2024. A seed-borne endophyte mediates plant drought responses and intergenerational effects on seed characteristics. \u003cem\u003eENVIRONMENTAL AND EXPERIMENTAL BOTANY\u003c/em\u003e 221:105719.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRan, Q., C. Dong, Q. Zhang, Q. Shao, Y. Zhang, X. Long, and Y. Han. 2024. Seed endophytes reshape rhizosphere microbiota to promote the growth of Eucommia ulmoides seedlings. \u003cem\u003eAPPLIED SOIL ECOLOGY\u003c/em\u003e 201:105487.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJana, S. K., M. M. Islam, S. Hore, and S. Mandal. 2023. Rice seed endophytes transmit into the plant seedling, promote plant growth and inhibit fungal phytopathogens. \u003cem\u003ePLANT GROWTH REGULATION\u003c/em\u003e 99:373\u0026ndash;388.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y., P. Li, Y. Tian, Z. Xiong, Z. Zheng, Z. Yi, H. Ao, Q. Wang, and J. Li. 2023. Bacterial seed endophyte and abiotic factors influence cadmium accumulation in rice (Oryza sativa) along the Yangtze River area. \u003cem\u003eECOTOXICOLOGY AND ENVIRONMENTAL SAFETY\u003c/em\u003e 263:115352.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y., F. Bai, N. Li, W. Wang, and C. Cheng. 2017. Identification of endophytic bacterial strain RSE1 from seeds of super hybrid rice Shenliangyou 5814 (Oryza sativa L.,) and evaluation of its antagonistic activity. \u003cem\u003ePLANT GROWTH REGULATION\u003c/em\u003e 82:403\u0026ndash;408.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJing, R., N. Li, W. Wang, and Y. Liu. 2020. An endophytic strain JK of genus bacillus isolated from the seeds of super hybrid rice (Oryza sativa L., Shenliangyou 5814) has antagonistic activity against rice blast pathogen. \u003cem\u003eMICROBIAL PATHOGENESIS\u003c/em\u003e 147:104422.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"antonie-van-leeuwenhoek","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anto","sideBox":"Learn more about [Antonie van Leeuwenhoek](https://www.springer.com/journal/10482)","snPcode":"10482","submissionUrl":"https://submission.nature.com/new-submission/10482/3","title":"Antonie van Leeuwenhoek","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Yunnan cytoplasmic male-sterile rice, high-throughput sequencing, core microbiome, seed endophytes","lastPublishedDoi":"10.21203/rs.3.rs-8614457/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8614457/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) is a staple crop of pivotal strategic importance in Chinese agro-ecosystems. In this study, the endophytic bacterial diversity and community structure of seeds from 14 Yunnan cytoplasmic male-sterile (CMS) rice lines (42 samples in total) were systematically profiled via Illumina NovaSeq 6000 high-throughput sequencing. A total of 503 operational taxonomic units (OTUs) were recovered. At the phylum level, Proteobacteria dominated all samples, accounting for 91.53\u0026ndash;99.95% of the bacterial microbiota. At the genus level, a conserved core microbiome comprising \u003cem\u003ePantoea\u003c/em\u003e (50.14\u0026ndash;95.58%), \u003cem\u003eXanthomonas\u003c/em\u003e (6.23\u0026ndash;37.49%), \u003cem\u003eKosakonia\u003c/em\u003e (2.97\u0026ndash;35.91%), \u003cem\u003eEnterobacter\u003c/em\u003e (2.06\u0026ndash;22.97%), and \u003cem\u003eMethylobacterium\u003c/em\u003e\u0026ndash;\u003cem\u003eMethylorubrum\u003c/em\u003e (1.42\u0026ndash;5.11%) was identified. Both α- and β-diversity analyses revealed no significant inter-line differentiation, indicating a highly stable and conserved endophytic bacterial community across the Yunnan CMS rice germplasm. This study provides the first comprehensive characterization of the seed-associated core microbiome of Yunnan CMS rice lines, offering a critical reference for micro-ecological breeding strategies in rice.\u003c/p\u003e","manuscriptTitle":"High-throughput sequencing-based profiling of endophytic bacterial community composition and diversity in seeds of Yunnan cytoplasmic male-sterile rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 16:55:21","doi":"10.21203/rs.3.rs-8614457/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-29T09:56:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-29T09:27:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313657496258395225797119205763908584656","date":"2026-01-29T06:47:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179569932260734645623219876662718969541","date":"2026-01-29T06:19:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225778456016705893958807594864904684131","date":"2026-01-28T14:37:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-27T02:28:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237546213052568785312063087268782119496","date":"2026-01-27T00:42:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165317661865322870398201288646673267363","date":"2026-01-26T16:22:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112623663172149006623680110921109213587","date":"2026-01-26T15:54:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-26T15:52:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T09:42:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T09:36:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Antonie van Leeuwenhoek","date":"2026-01-16T01:53:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"antonie-van-leeuwenhoek","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anto","sideBox":"Learn more about [Antonie van Leeuwenhoek](https://www.springer.com/journal/10482)","snPcode":"10482","submissionUrl":"https://submission.nature.com/new-submission/10482/3","title":"Antonie van Leeuwenhoek","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"92f49fc5-4cdb-4464-b40a-6aa26b119449","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:08:19+00:00","versionOfRecord":{"articleIdentity":"rs-8614457","link":"https://doi.org/10.1007/s10482-026-02273-2","journal":{"identity":"antonie-van-leeuwenhoek","isVorOnly":false,"title":"Antonie van Leeuwenhoek"},"publishedOn":"2026-03-02 15:57:42","publishedOnDateReadable":"March 2nd, 2026"},"versionCreatedAt":"2026-01-29 16:55:21","video":"","vorDoi":"10.1007/s10482-026-02273-2","vorDoiUrl":"https://doi.org/10.1007/s10482-026-02273-2","workflowStages":[]},"version":"v1","identity":"rs-8614457","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8614457","identity":"rs-8614457","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}