High-throughput sequencing-based analysis of the composition and diversity of endophytic bacterial community in seeds of elite rice resources from Heilongjiang area

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This study aimed to explore core microbiota composition and the influence of host genetic background on microbiota assembly. Methods Using Illumina NovaSeq 6000 sequencing, we analyzed endophytic bacteria diversity in 57 samples from 19 elite rice cultivars, identifying 792 OTUs. Results At the phylum level, Proteobacteria dominated (80.3%-99.73%). Core microbiota at the genus level included Pantoea , Xanthomonas , Methylobacterium - Methylorubrum , Pseudomonas , and Microbacterium . Parental genetic background significantly influenced bacterial community structure similarity, with closer genetic relationships showing more complex bacterial interaction networks. Conclusion This study showed the compositional characteristics of the core microbiota in elite rice seeds from Heilongjiang and its relationship with parental genetic background, providing a theoretical basis for microbiome-assisted breeding in rice. High-throughput sequencing Endophytic bacteria Diversity Heilongjiang rice Parental genetic relatedness Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Japonica rice ( Oryza sativa L. subsp. japonica ) serves as a strategic staple crop, with its stable supply being paramount to sustaining national food security. Heilongjiang Province is China's largest major producing region for japonica rice (He et al., 2021 ). Since 2010, its contribution rate to the national rice yield increase has consistently remained between 30% and 40%. Heilongjiang Province's rice production demonstrates unique ecological characteristics and exceptional quality properties. The province boasts significant natural advantages: fertile soils with a chernozem layer thickness of 30–100 cm and an average organic matter content of 4–6%; long sunshine duration, with daytime sunshine lasting 15–16 hours and the average diurnal temperature difference during the rice growing season in Heilongjiang exceeds 12°C, which is conducive to dry matter accumulation and quality enhancement; relatively high temperatures during the flowering and fertilization stage, suitable temperatures during the grain filling stage, and abundant water resources with high water quality. These features collectively contribute to the unique quality of Heilongjiang rice—aromatic, glutinous texture, and sweet flavor—making it a nationally recognized geographical indication product. The plant endophytic microbial community is a symbiotic system composed of bacteria, fungi, and other microorganisms, capable of colonizing healthy plant tissues without eliciting pathological responses in the host (Wang et al., 2021a ; Wang et al., 2022 ). Studies has shown that endophyte diversity and community structure is closely related to factors such as plant variety, genotype, growth environment, and geographical location (Santoyo et al., 2016 ; Afzal et al., 2019 ; Liu et al., 2019 ). Endophytic microbiota demonstrate the ability to enhance plant growth and development, regulate the allocation of photosynthates in the host, and suppress phytopathogenic infections (Wang et al., 2014 ; Thampi et al.,2024; Feng et al., 2006 ; Matsumoto et al., 2021 ). Furthermore, plant seeds harbor rich endophytic bacterial communities, whose species composition undergoes dynamic changes (Liu et al., 2013 ; Verma and White, 2019 ; Barret et al., 2015 ), however, due to the inherent enclosure of seeds, the diversity and abundance of their endophytic bacteria are generally lower compared to other plant organs (Truyens et al., 2015 ). As pivotal biological vectors for propagation, plant seeds play a crucial role in the vertical transmission of beneficial microorganisms to offspring (Sahu and Mishra, 2021 ). Studies indicate that seeds serve not only as the foundation for plant reproduction but also as important vectors for transmitting maternal microorganisms to progeny. Through seed transmission, beneficial microorganisms are stably propagated across generations, thereby directly or indirectly regulating plant growth and development, health status, quality formation, yield enhancement, and the accumulation of functional bioactive compounds (Liu et al., 2019 ; Berg and Raaijmakers, 2018 ; Li et al., 2019 ). Currently, compared to extensive investigations into microbial communities in the rhizosphere and phyllosphere, research on seed endophytes remains at a relatively nascent stage (Pal et al., 2021 ). Current research efforts on endophytes in elite rice cultivars from the Heilongjiang Province are scarce both domestically and internationally, particularly those utilizing high-throughput sequencing technology. Existing research predominantly focuses on the isolation and purification of beneficial endophytes from rice and the validation of their individual functions, or on investigating their growth-promoting effects through artificial inoculation (Hassan, 2017 ; Niu et al., 2022 ), however, the relationship between host phenotypic traits and endophytic microbiota interaction networks remains unelucidated. A study reveals that hybrid rice and its parental lines share conserved core endophytic bacterial communities (Liu et al., 2019 ). Therefore, this study employed high-throughput sequencing to comprehensively analyze the diversity and community structure of endophytic bacteria in high-quality rice seeds from Heilongjiang, aiming to identify their core dominant microbiota and elucidate its correlation with parental genetic background. Materials and methods The source of rice seeds The 19 sets of rice seed samples used in this study were provided by Beijing Lantu Technology Co., Ltd. The detailed information for these rice seed samples is presented in Table 1 . The seeds were transferred to sterile bags, sealed, and stored at 4°C until use. Table 1 Information statistics of rice seed samples. Sample ID Variety names Planting area Maternal parent Male parent A Longjing3025 Youyi county Longjing31 Longjing29 B Longjing3023 Youyi county Longjing31 Longjing29 C Longjing4569 Jiamusi Longjing29 Tongxi112 D Longjing1838 Jiamusi Sui098038 Longjing1525 E Longken2021 Jiamusi Longjing40/Kongyu131 Wuyoudao4 F Longjing1938 Jiamusi Sui098038 Longjing1525 G X56 Jiamusi Wuyoudao4 H Longjing31 Jiamusi Longhua96-1513 Kendao8 I Longjing1719 Jiamusi Longhua00-835 Longjing31 J Suijing18 Jiamusi Suijing4 Suijing3 K Wuyoudao4 Haerbin L Songjing5 Jixi Liaojing87-675 Tong5307 M SN123 Jixi Wuyoudao4 Songjing6 N Longdao20 Jixi Dongnong423 Longdao3 O Zhongkefa5 Jiamusi Kongyu131/Southern long grain japonica Jijing88 P Longqingjing39 Jiamusi Longjing61 Longqingdao3 Q 18 Jiamusi Suijing4 Suijing3 R 31 Jiamusi Longhua96-1513 Kendao8 S 2021 Jiamusi Longjing40/Kongyu131 Wuyoudao4 Note: Maternal parent details of Sample G are undisclosed. Sample K was derived via mutant line selection of the Wuyoudao1 cultivar. Sample surface sterilization and treatment Three replicates of each sample were selected in this study, resulting in 57 samples in total. Firstly, surface disinfection by soaking all seed samples in 75% ethanol for 30 minutes, subsequent operations were performed aseptically within a laminar flow hood in the listed order: husked seeds were washed three times with sterile water; 5 g of seeds were transferred into a 50-mL sterile centrifuge tube containing 25 mL of phosphate buffer (per liter: 7.15 g of NaH₂PO₄·2H₂O, 22.04 g of Na₂HPO₄·12H₂O, 200 µL of Silwet L-77) (Wang et al., 2021; Liu et al., 2019 ), and the seeds were sonicated twice by an Ultrasonic Processor Scientz-IID sonicator (NingBo Scientz Biotechnology Co., Ltd., China) at low power (237.5 W; 950 W × 25%) in an ice bath for 5 min (alternating thirty 2-s bursts and thirty 2-s rests) (Wang et al., 2021; Liu et al., 2019 ). To validate the effectiveness of surface sterilization, surface- sterilized seeds were pressed into LB medium using sterile tweezers and the samples were incubated at 30°C for 72 h. DNA extraction Five grams of surface-sterilized rice seeds from each sample was frozen with liquid nitrogen and was quickly ground into a fine powder with a pre-cooled sterile mortar, and then the DNA was extracted using the FastDNA® SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA) following the manufacturer’s instructions of the Kit. Amplicon library preparation and sequencing For rice seeds, 799F (5’-AACAGGATTAGATACCCTG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) was used for the first-round amplification. The thermocycling steps were as follows: 5 min at 95°C, 25 cycles of 30 s at 94°C, 35 s at 53°C, 30 s at 72°C, and 8 min at 72°C. Then the 750 bp fragment amplified from endophytic bacteria was used as the template for the second-round amplification. Primers for the second round were 968F (5’-********AACGCGAAGAACCTTAC-3’) and 1378R (5’-CGGTGTGTACAAGGCCCGGGAACG-3’), where the eight asterisks (*) represent an 8-bp barcode incorporated at the 5′ end for sample multiplexing. PCR was performed using Phusion High-Fidelity DNA Polymerase (High-Fidelity PCR Master Mix with GC Buffer). The thermocycling steps were as follows: 5 min at 95°C, 26 cycles of 30 s at 94°C, 35 s at 55 °, 30 s at 72°C, and 8 min at 72°C. PCR amplicons were electrophoresed on a 1.5% agarose gel, and the target bands were excised and purified using a Gel Extraction Kit (Life Technology, USA). Purified DNA concentration was accurately quantified using Qubit 3.0 (Life Technology, USA). After using the NEBNext Ultra (NEB#e7370S/L) DNA Library Prep Kit for Illumina to perform the library, all samples were sequenced by 250-bp-paired-end sequencing with an Illumina NovaSeq 6000 platform. Sequence data processing The assembly of paired FASTQ files was performed by Mothur (version v.1.35.0). After removing raw reads with low quality (such as read length < 50 bp, average Qscore < 15), paired-end reads were assembled using FLASH. The high-quality DNA sequences were aligned to SILVA reference database (db131.1), and using chimera. uchime module to remove chimera sequences. Then the reads were classified and grouped into OTUs (operational taxonomic units) under the threshold of 97% identity. OTU clustering and taxonomic annotation were performed using Usearch. Data statistics Community richness, evenness, and diversity analysis (Shannon, Simpson, ACE, and Chao) were performed using Mothur. Principal Coordinates Analysis (PCoA) was analyzed based on the tayc matrix by Mothur. The t -test (with 95% confidence intervals) was employed to determine whether differences in evaluation indices were statistically significant, and p-value < 0.05 was considered as a significant standard. Taxonomy was assigned using the online software RDP Classifier at the 97% similarity threshold based on the SILVA reference database (db131.1). Differences in the relative abundance of taxa at the phylum and genus levels between samples were analyzed by Metastat. Spearman correlation coefficients between pairs of variables were calculated using the R command “cor. test”. Results Grouping of rice seed samples from Heilongjiang area The rice seed samples were categorized into 19 groups based on variety, with three biological replicates per group, resulting in a total of 57 samples. OTUs were clustered based on 16S rRNA gene sequences at the 97% similarity threshold, a total of 792 OTUs were obtained across the 57 samples. Bacterial endophyte community compositions and structures of rice seeds from Heilongjiang Analysis of the endophytic community composition across the 19 rice seed sample groups revealed Proteobacteria and Actinobacteria as the dominant phyla (Fig. 1 ). Proteobacteria constituted 80.3–99.73% of the relative abundance across all 19 sample groups and Actinobacteria accounted for 0.18%-19.49%, indicating high conservation of the endophytic microbiota at the phylum level in rice seeds. At the genus level (Fig. 2 ), the top five most abundant genera were Pantoea (28.87–95.81%), Xanthomonas (0.64–30.28%), Methylobacterium-Methylorubrum (0.20–26.54%), Pseudomonas (0.30–27.77%), and Microbacterium (0.12–15.55%). Notably, these five genera were consistently the most abundant across all 19 rice seed sample groups. Therefore, Pantoea , Xanthomonas , Methylobacterium-Methylorubrum , Pseudomonas , and Microbacterium were identified as the core microbiota of the high-quality rice seed samples from Heilongjiang area. Alpha diversity analysis of endophytes in rice seeds from Heilongjiang Alpha diversity indexes of 19 rice seed sample groups include ACE, Chao, Shannon and Simpson values, in which ACE and Chao values are used for sample abundance assessment, specifically reflecting the abundance of rare species; higher values indicate greater numbers of rare species, and Shannon and Simpson values are used for sample diversity assessment. Both the Shannon and Simpson indices exhibited considerable variation across the 19 sample groups (Fig. 3 ). However, samples sharing the same parental lines (samples A and B, E and S) displayed similar endophytic diversity, as did half-siblings (samples E and G). In contrast, samples D and F, despite also sharing the same parental lines, exhibited substantial differences in endophytic diversity. Similarly, significant variation was observed in the Chao1 and ACE indices, yet samples sharing the same parental lines (samples A and B) demonstrated comparable richness. Beta diversity analysis and heatmap of endophytes in rice seeds from Heilongjiang Beta diversity analysis of the 19 seed sample groups was performed, with the results visualized in the PCoA (Fig. 4 ) and the Non-Metric Multidimensional Scaling (NMDS, Fig. 5 ). Analysis of similarities (ANOSIM) yielded an R-value of 0.2754 and a P-value of 0.001, indicating statistically significant differences among the 19 sample groups. Furthermore, the PCoA and NMDS analyses demonstrated a dispersed distribution pattern of the 19 sample groups. Collectively, these results demonstrate significant differences in the endophytic bacterial diversity among these 19 seed samples, which is consistent with the findings from the alpha-diversity analysis described above. The heatmap (Fig. 6 ) depict the endophytic bacterial composition at the genus level for the 19 rice seed sample groups. This visualization illustrates the relative abundance of different endophytic bacteria within the seeds, with color intensity positively correlated with relative abundance. The results demonstrate that Pantoea was the most dominant genus in all seed samples. Although the relative abundances of endophytes varied among the different groups, the predominant genera identified were consistent with the taxonomic composition at the genus level described in the previous analysis. This finding further confirms that the core microbiota of these 19 rice seed samples comprises the five genera mentioned above. The correlation network analysis of endophytic in rice seeds from Heilongjiang The 19 rice seed sample groups were categorized into two cohorts based on parental genetic background: those sharing both parental lines (Double group) and those with differing parental lines (No group). The correlation network analysis was performed for each group (Fig. 7 ). Panel A of Fig. 7 depicts the intra-group microbial interaction network for the Double group, exhibiting a greater number of edges and increased complexity of interactions between nodes. In contrast, panel B shows the intra-group network for the No group, which displayed fewer edges and simpler inter-node connections compared to the Double group, indicating more frequent and complex microbial interactions within the Double group. This conclusion is further supported by the network parameters presented in panel C, where the Double group demonstrated a higher number of edges, greater average weight, and increased network density. Differential analysis of the intra-group correlation network patterns between the two groups (Fig. 7 D) revealed a statistically significant difference. Collectively, these results demonstrate that the parental genetic background influences the interactions within the endophytic bacterial community. Endophytic communities in rice seed samples sharing the same genetic background exhibited more frequent and complex interactions. Discussion Rice, as a staple food crop globally, harbors microbial resources that constitute a strategic reservoir for agricultural microbiotechnology development. Rice endophytic bacteria establish specific niche distributions across different plant tissues (e.g., roots, stems, leaves, and seeds) (Wang et al., 2015 ; Ali et al., 2021 ). Seeds represent the starting point of the plant life cycle and are associated with diverse microbial communities. Seed endophytes constitute a vital component of the plant microbiome, playing crucial roles in maintaining seed viability, promoting germination, and enhancing plant growth and yield (Chee-Sanford et al., 2006 ). Multiple studies have demonstrated that seed endophytic bacteria enhance plant adaptability (Langill et al., 2023 ; Ren et al., 2023 ). Rice seed endophytic bacteria not only directly promote rice growth by modulating phytohormone synthesis and siderophore release, but also indirectly enhance development by improving stress resistance and antagonism against phytopathogens. Notably, the vertical transmission capability of rice seed endophytes positions them as key mediators in rice environmental adaptation. RANA et al. (2024) revealed that the core seed endophyte Xanthomonas sontii can be vertically transmitted via seeds and assists the host in enriching beneficial microbiota to counter biotic stress. It has also been reported in a related study (Radhakrishnanr et al., 2023) that the seed endophyte Paenibacillus polymyxa S3, isolated from rice seeds, exhibits antifungal activity against pathogens and produces bioactive compounds to promote plant growth. Further investigations have shown that pretreatment of rice seeds with endophytic bacteria significantly enhances seed germination rates, plant growth, and improves salt tolerance (Kavitha et al., 2020 ; Chowdhury et al., 2024 ). Overall, these studies provide compelling theoretical support for exploiting rice seed endophytes as microbial fertilizers or biocontrol agents. Heilongjiang Province, situated as the northernmost rice-growing region in China, possesses a unique cold-region rice cropping system. Rice produced in this area is renowned for its soft and sweet texture, with representative varieties such as Wuchang, Daohuaxiang, and Xiangshui rice. Therefore, this study collected 19 groups of high-quality rice seed samples from diverse locations at Heilongjiang Province as experimental materials. The primary objectives were to systematically characterize the structural features of their endophytic bacterial communities and to provide a theoretical foundation and germplasm innovation basis for improving the quality and productivity of cold-region rice. This study employed high-throughput sequencing to systematically analyze the community structure and diversity of seed endophytic bacteria in 19 rice seed groups from Heilongjiang Province. A total of 792 OTUs were obtained from 57 samples. Analysis of the endophytic species composition across the 19 rice seed sample groups revealed that Proteobacteria and Actinobacteria were the predominant phyla. Proteobacteria , representing the most dominant bacterial phylum in all seed samples, accounted for 80.3–99.73% of the relative abundance. This finding is consistent with previous reports by Wang et al. ( 2021a ; 2021b ) on endophytic bacteria in saline-alkali tolerant and upland rice seeds. At the genus level, Pantoea , Xanthomonas , Methylobacterium-Methylorubrum , Pseudomonas , and Microbacterium constituted the core microbiota, exhibiting relatively high conservation across the different rice varieties. Furthermore, existing research has summarized the composition and associated functions of the rice seed microbiome (Kumar et al., 2024 ), which provides valuable references for our future exploration of functional bacteria. Analysis of diversity revealed an association between endophytic bacterial diversity and genetic background, although genetic background was not the sole determinant. For instance, varieties sharing both parental lines (A and B, E and S) exhibited relatively similar levels of richness and diversity. In contrast, significant differences in endophytic richness were observed between other varieties sharing both parental lines (e.g., H and R) and between varieties with differing genetic backgrounds. This observation aligns with existing research indicating that the composition of seed-associated microbial communities is influenced by multiple factors, including plant species, seed developmental stage, geographical location, and the presence or absence of phytopathogens (Nelson et al., 2018; Rezki et al., 2016 ; Rodríguez et al., 2018 ). Furthermore, beta-diversity analysis confirmed significant differences in endophytic community structure among the 19 sample groups. However, varieties with similar genetic backgrounds displayed a clustering tendency in their community structures. Consequently, correlation network analysis demonstrated that seeds sharing both parental lines (Double group) harbored endophytic bacterial communities with more frequent and complex interactions compared to those with differing parental lines (No group). Collectively, these results indicate that the community structure and diversity of endophytic bacteria within high-quality rice seeds from Heilongjiang area are not driven by a single factor but are likely influenced by rice variety, genotype, environment, among others. Nevertheless, endophytic bacteria in seeds sharing a genetic background exhibited more intricate interactions. This study unveils the community composition and potential functions of endophytic bacteria in rice seeds, providing novel evidence for a deeper understanding of their significance within the plant microbiome. Furthermore, while conventional breeding predominantly focuses on intrinsic plant traits, it often overlooks the co-evolutionary potential of their symbiotic microbiota. Insights from this study suggest that optimizing the seed microbiota composition through targeted selection or microbiome transplantation techniques holds promise for developing novel rice varieties with enhanced stress tolerance and nutrient use efficiency. Consequently, this study also provides a robust theoretical foundation for "holobiont breeding" (integrating both plant and associated microbes). Such a "plant-microbe co-evolutionary" breeding strategy not only expands the dimensions of crop improvement but also breathes new life into seed endophyte research, thereby steering agriculture towards a more ecologically sustainable trajectory. Conclusion Leveraging high-throughput sequencing, this study showed the characterization of the diversity and community structure of endophytic bacteria within seeds of high-quality rice varieties from Heilongjiang Province, and it revealed the "core microbiota" and potential associations between endophytic bacterial community composition and parental genetic background. The observed conservation of the core microbiota suggests its likely role in key functional aspects, such as supporting rice health or stress resistance. Future research involving isolation, cultivation, and functional validation holds promise for unlocking its application potential. Furthermore, the observed influence of parental genetic background on the endophytic bacterial correlation network indicates that harnessing the principles governing vertical transmission of the seed microbiome during breeding offers a promising avenue for developing novel rice varieties harboring stable beneficial microbiota. This approach broadens crop genetic improvement strategies from the perspective of "plant-microbe interactions," thereby contributing significant theoretical and practical value to advancing sustainable agriculture and food production. Declarations Authors’ Contributions Peng H and You YT designed and participated in all experimental procedures, performed data analysis, and drafted the manuscript. Cao SW participated in the samples collection and preparation. Liu Y supervised the study and critically revised the manuscript. All authors read and approved the final manuscript. 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). Availability of Data and Materials All data generated or analyzed during this study are included in this published article. Ethics Approval and Consent to Participate Not applicable. Consent for Publication Not applicable. 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Supplementary Files Highlights.docx Cite Share Download PDF Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Plant and Soil → Version 1 posted Editorial decision: Major revisions 10 Nov, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviewers invited by journal 15 Sep, 2025 Editor invited by journal 04 Sep, 2025 Editor assigned by journal 04 Sep, 2025 First submitted to journal 03 Sep, 2025 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7481527","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515150107,"identity":"67874649-b887-4af2-b339-89d1a14a4d3f","order_by":0,"name":"He Peng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Peng","suffix":""},{"id":515150108,"identity":"9ab75455-3d3b-4e2f-af2f-b537f71e8c10","order_by":1,"name":"Yongtao 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1","display":"","copyAsset":false,"role":"figure","size":21076,"visible":true,"origin":"","legend":"\u003cp\u003eCommunity structure of endophytic bacteria in rice seeds from Heilongjiang. It showed the relative abundance of endophytic bacteria in rice seeds from Heilongjiang at phylum level respectively.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7481527/v1/c2e79d09d72fb7cd63dec5c7.png"},{"id":92001120,"identity":"f0560988-247d-459d-9989-c1f2c390ed29","added_by":"auto","created_at":"2025-09-23 14:26:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16730,"visible":true,"origin":"","legend":"\u003cp\u003eCommunity structure of endophytic bacteria in rice seeds from Heilongjiang. It showed the relative abundance of endophytic bacteria in rice seeds from Heilongjiang at genus level respectively.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7481527/v1/fe57ada2144afcfe682bdccf.png"},{"id":92001122,"identity":"41bc3807-7ab4-412a-a7b5-8323b2e25a6c","added_by":"auto","created_at":"2025-09-23 14:26:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17140,"visible":true,"origin":"","legend":"\u003cp\u003eThe statistical results of endophytic bacterial α-diversity in each seed sample. The diversity indices were calculated using three replicates for each sample. \u003cstrong\u003eA/B/C/D\u003c/strong\u003e respectively corresponded to Shannon/Simpson/Chao/ACE diversity indices.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7481527/v1/008d83f81ba67375f96f31d8.png"},{"id":92002804,"identity":"e4febb60-d92d-4779-99ee-be191a8d425d","added_by":"auto","created_at":"2025-09-23 14:42:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103190,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Co-ordinates Analysis (PCoA) and anosim analysis. The abscissa and ordinate represent the contribution rate of the principal components 1 and 2 to the distribution of the samples. Each point in the figure represents a sample, and the points of the same color come from the same group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7481527/v1/c59e51f591296b6e63ae78dd.png"},{"id":92001450,"identity":"6248c45d-18ad-442a-aa19-68f515f331e6","added_by":"auto","created_at":"2025-09-23 14:34:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":24272,"visible":true,"origin":"","legend":"\u003cp\u003eNonmetric multidimensional scaling (NMDS). Each point in the figure represents a sample, and the points of the same color come from the same group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7481527/v1/1314f3ab7a49e4ffaca76f36.png"},{"id":92001451,"identity":"c8e48102-3adc-4c1e-953a-bc47c8602372","added_by":"auto","created_at":"2025-09-23 14:34:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":32363,"visible":true,"origin":"","legend":"\u003cp\u003eThe sample classifcation at a 97% sequence similarity level (genus, top 10). The horizontal represents the name of each sample, while the vertical denotes the name of the top 10 bacteria in the sample. The color gradients corresponding to the bacterial abundance from 0 to 1 are marked in yellow, orange, and red. The red color becomes more pronounced as the yellow becomes lighter, indicating a higher bacterial abundance.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7481527/v1/a824a17198c405d30e5f08b8.png"},{"id":92001452,"identity":"7922998c-5974-4440-b51e-36d28dbf452e","added_by":"auto","created_at":"2025-09-23 14:34:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":495645,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlation network analysis of the endophytic bacteria in the rice seeds. Double and No represent the hybrid rice variety of the same female parent and male parents, the hybrid rice variety of the different female parent and male parents. \u003cstrong\u003eA. B.\u003c/strong\u003e The cooccurrence network of the endophytic bacteria in the rice seeds with different genetic relationships based on Spearman’s correlation analysis. The nodes represent OTUs and are colored by their corresponding species. The size of each node is proportional to the relative OTU abundance. The connection between the nodes indicates a strong positive correlation (Spearman’s ρ≥0.6) and significance (an FDR-corrected p-value of\u0026lt;0.01). The thickness of the connection between two nodes (edge) is proportional to the value of Spearman’s correlation coefficients.\u003cstrong\u003e C.\u003c/strong\u003e Details of the networks in different groups.\u003cstrong\u003e D.\u003c/strong\u003eThe unique node-level topological features and degree of endophytic bacterial taxa in the rice seeds with different genetic relationships. The degree differed significantly between the two groups based on Wilcoxon rank sum tests (***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7481527/v1/93a57b352b4ce1245609e78f.png"},{"id":100069128,"identity":"76d9d7ed-f42a-4021-b187-b29fe186995f","added_by":"auto","created_at":"2026-01-12 16:10:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1244450,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7481527/v1/5e89d75a-7177-4b8c-86f1-ad770479934b.pdf"},{"id":92001133,"identity":"48c0229d-9618-4243-bed2-eb4514a595af","added_by":"auto","created_at":"2025-09-23 14:26:52","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":16277,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-7481527/v1/a68f994c2e8b3838a1a20e40.docx"}],"financialInterests":"","formattedTitle":"High-throughput sequencing-based analysis of the composition and diversity of endophytic bacterial community in seeds of elite rice resources from Heilongjiang area","fulltext":[{"header":"Introduction","content":"\u003cp\u003eJaponica rice (\u003cem\u003eOryza sativa\u003c/em\u003e L. subsp. \u003cem\u003ejaponica\u003c/em\u003e) serves as a strategic staple crop, with its stable supply being paramount to sustaining national food security. Heilongjiang Province is China's largest major producing region for japonica rice (He et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Since 2010, its contribution rate to the national rice yield increase has consistently remained between 30% and 40%. Heilongjiang Province's rice production demonstrates unique ecological characteristics and exceptional quality properties. The province boasts significant natural advantages: fertile soils with a chernozem layer thickness of 30\u0026ndash;100 cm and an average organic matter content of 4\u0026ndash;6%; long sunshine duration, with daytime sunshine lasting 15\u0026ndash;16 hours and the average diurnal temperature difference during the rice growing season in Heilongjiang exceeds 12\u0026deg;C, which is conducive to dry matter accumulation and quality enhancement; relatively high temperatures during the flowering and fertilization stage, suitable temperatures during the grain filling stage, and abundant water resources with high water quality. These features collectively contribute to the unique quality of Heilongjiang rice\u0026mdash;aromatic, glutinous texture, and sweet flavor\u0026mdash;making it a nationally recognized geographical indication product.\u003c/p\u003e\u003cp\u003eThe plant endophytic microbial community is a symbiotic system composed of bacteria, fungi, and other microorganisms, capable of colonizing healthy plant tissues without eliciting pathological responses in the host (Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies has shown that endophyte diversity and community structure is closely related to factors such as plant variety, genotype, growth environment, and geographical location (Santoyo et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Afzal et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Endophytic microbiota demonstrate the ability to enhance plant growth and development, regulate the allocation of photosynthates in the host, and suppress phytopathogenic infections (Wang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Thampi et al.,2024; Feng et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Matsumoto et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, plant seeds harbor rich endophytic bacterial communities, whose species composition undergoes dynamic changes (Liu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Verma and White, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Barret et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), however, due to the inherent enclosure of seeds, the diversity and abundance of their endophytic bacteria are generally lower compared to other plant organs (Truyens et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). As pivotal biological vectors for propagation, plant seeds play a crucial role in the vertical transmission of beneficial microorganisms to offspring (Sahu and Mishra, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Studies indicate that seeds serve not only as the foundation for plant reproduction but also as important vectors for transmitting maternal microorganisms to progeny. Through seed transmission, beneficial microorganisms are stably propagated across generations, thereby directly or indirectly regulating plant growth and development, health status, quality formation, yield enhancement, and the accumulation of functional bioactive compounds (Liu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Berg and Raaijmakers, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Currently, compared to extensive investigations into microbial communities in the rhizosphere and phyllosphere, research on seed endophytes remains at a relatively nascent stage (Pal et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCurrent research efforts on endophytes in elite rice cultivars from the Heilongjiang Province are scarce both domestically and internationally, particularly those utilizing high-throughput sequencing technology. Existing research predominantly focuses on the isolation and purification of beneficial endophytes from rice and the validation of their individual functions, or on investigating their growth-promoting effects through artificial inoculation (Hassan, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Niu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), however, the relationship between host phenotypic traits and endophytic microbiota interaction networks remains unelucidated. A study reveals that hybrid rice and its parental lines share conserved core endophytic bacterial communities (Liu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, this study employed high-throughput sequencing to comprehensively analyze the diversity and community structure of endophytic bacteria in high-quality rice seeds from Heilongjiang, aiming to identify their core dominant microbiota and elucidate its correlation with parental genetic background.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe source of rice seeds\u003c/p\u003e\u003cp\u003eThe 19 sets of rice seed samples used in this study were provided by Beijing Lantu Technology Co., Ltd. The detailed information for these rice seed samples is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The seeds were transferred to sterile bags, sealed, and stored at 4\u0026deg;C until use.\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\u003eInformation statistics of rice seed samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVariety names\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePlanting area\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMaternal parent\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMale parent\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongjing3025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYouyi county\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLongjing31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLongjing29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongjing3023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYouyi county\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLongjing31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLongjing29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongjing4569\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLongjing29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTongxi112\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongjing1838\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSui098038\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLongjing1525\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eE\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongken2021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLongjing40/Kongyu131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWuyoudao4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongjing1938\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSui098038\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLongjing1525\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eG\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWuyoudao4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eH\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongjing31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLonghua96-1513\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eKendao8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongjing1719\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLonghua00-835\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLongjing31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eJ\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSuijing18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSuijing4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSuijing3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eK\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWuyoudao4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHaerbin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eL\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSongjing5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJixi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLiaojing87-675\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTong5307\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSN123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJixi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWuyoudao4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSongjing6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eN\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongdao20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJixi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDongnong423\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLongdao3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eO\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZhongkefa5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eKongyu131/Southern long grain japonica\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eJijing88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLongqingjing39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLongjing61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLongqingdao3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eQ\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSuijing4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSuijing3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLonghua96-1513\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eKendao8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJiamusi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLongjing40/Kongyu131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWuyoudao4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: Maternal parent details of Sample G are undisclosed. Sample K was derived via mutant line selection of the Wuyoudao1 cultivar.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSample surface sterilization and treatment\u003c/p\u003e\u003cp\u003eThree replicates of each sample were selected in this study, resulting in 57 samples in total. Firstly, surface disinfection by soaking all seed samples in 75% ethanol for 30 minutes, subsequent operations were performed aseptically within a laminar flow hood in the listed order: husked seeds were washed three times with sterile water; 5 g of seeds were transferred into a 50-mL sterile centrifuge tube containing 25 mL of phosphate buffer (per liter: 7.15 g of NaH₂PO₄\u0026middot;2H₂O, 22.04 g of Na₂HPO₄\u0026middot;12H₂O, 200 \u0026micro;L of Silwet L-77) (Wang et al., 2021; Liu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and the seeds were sonicated twice by an Ultrasonic Processor Scientz-IID sonicator (NingBo Scientz Biotechnology Co., Ltd., China) at low power (237.5 W; 950 W \u0026times; 25%) in an ice bath for 5 min (alternating thirty 2-s bursts and thirty 2-s rests) (Wang et al., 2021; Liu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To validate the effectiveness of surface sterilization, surface- sterilized seeds were pressed into LB medium using sterile tweezers and the samples were incubated at 30\u0026deg;C for 72 h.\u003c/p\u003e\u003cp\u003eDNA extraction\u003c/p\u003e\u003cp\u003eFive grams of surface-sterilized rice seeds from each sample was frozen with liquid nitrogen and was quickly ground into a fine powder with a pre-cooled sterile mortar, and then the DNA was extracted using the FastDNA\u0026reg; SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA) following the manufacturer\u0026rsquo;s instructions of the Kit.\u003c/p\u003e\u003cp\u003eAmplicon library preparation and sequencing\u003c/p\u003e\u003cp\u003eFor rice seeds, 799F (5\u0026rsquo;-AACAGGATTAGATACCCTG-3\u0026rsquo;) and 1492R (5\u0026rsquo;-GGTTACCTTGTTACGACTT-3\u0026rsquo;) was used for the first-round amplification. The thermocycling steps were as follows: 5 min at 95\u0026deg;C, 25 cycles of 30 s at 94\u0026deg;C, 35 s at 53\u0026deg;C, 30 s at 72\u0026deg;C, and 8 min at 72\u0026deg;C. Then the 750 bp fragment amplified from endophytic bacteria was used as the template for the second-round amplification. Primers for the second round were 968F (5\u0026rsquo;-********AACGCGAAGAACCTTAC-3\u0026rsquo;) and 1378R (5\u0026rsquo;-CGGTGTGTACAAGGCCCGGGAACG-3\u0026rsquo;), where the eight asterisks (*) represent an 8-bp barcode incorporated at the 5\u0026prime; end for sample multiplexing. PCR was performed using Phusion High-Fidelity DNA Polymerase (High-Fidelity PCR Master Mix with GC Buffer). The thermocycling steps were as follows: 5 min at 95\u0026deg;C, 26 cycles of 30 s at 94\u0026deg;C, 35 s at 55 \u0026deg;, 30 s at 72\u0026deg;C, and 8 min at 72\u0026deg;C. PCR amplicons were electrophoresed on a 1.5% agarose gel, and the target bands were excised and purified using a Gel Extraction Kit (Life Technology, USA). Purified DNA concentration was accurately quantified using Qubit 3.0 (Life Technology, USA). After using the NEBNext Ultra (NEB#e7370S/L) DNA Library Prep Kit for Illumina to perform the library, all samples were sequenced by 250-bp-paired-end sequencing with an Illumina NovaSeq 6000 platform.\u003c/p\u003e\u003cp\u003eSequence data processing\u003c/p\u003e\u003cp\u003eThe assembly of paired FASTQ files was performed by Mothur (version v.1.35.0). After removing raw reads with low quality (such as read length\u0026thinsp;\u0026lt;\u0026thinsp;50 bp, average Qscore\u0026thinsp;\u0026lt;\u0026thinsp;15), paired-end reads were assembled using FLASH. The high-quality DNA sequences were aligned to SILVA reference database (db131.1), and using chimera. uchime module to remove chimera sequences. Then the reads were classified and grouped into OTUs (operational taxonomic units) under the threshold of 97% identity. OTU clustering and taxonomic annotation were performed using Usearch.\u003c/p\u003e\u003cp\u003eData statistics\u003c/p\u003e\u003cp\u003eCommunity richness, evenness, and diversity analysis (Shannon, Simpson, ACE, and Chao) were performed using Mothur. Principal Coordinates Analysis (PCoA) was analyzed based on the tayc matrix by Mothur. The \u003cem\u003et\u003c/em\u003e-test (with 95% confidence intervals) was employed to determine whether differences in evaluation indices were statistically significant, and p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as a significant standard. Taxonomy was assigned using the online software RDP Classifier at the 97% similarity threshold based on the SILVA reference database (db131.1). Differences in the relative abundance of taxa at the phylum and genus levels between samples were analyzed by Metastat. Spearman correlation coefficients between pairs of variables were calculated using the R command \u0026ldquo;cor. test\u0026rdquo;.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eGrouping of rice seed samples from Heilongjiang area\u003c/p\u003e\u003cp\u003eThe rice seed samples were categorized into 19 groups based on variety, with three biological replicates per group, resulting in a total of 57 samples. OTUs were clustered based on 16S rRNA gene sequences at the 97% similarity threshold, a total of 792 OTUs were obtained across the 57 samples.\u003c/p\u003e\u003cp\u003eBacterial endophyte community compositions and structures of rice seeds from Heilongjiang\u003c/p\u003e\u003cp\u003eAnalysis of the endophytic community composition across the 19 rice seed sample groups revealed \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eActinobacteria\u003c/em\u003e as the dominant phyla (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eProteobacteria\u003c/em\u003e constituted 80.3\u0026ndash;99.73% of the relative abundance across all 19 sample groups and \u003cem\u003eActinobacteria\u003c/em\u003e accounted for 0.18%-19.49%, indicating high conservation of the endophytic microbiota at the phylum level in rice seeds. At the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the top five most abundant genera were \u003cem\u003ePantoea\u003c/em\u003e (28.87\u0026ndash;95.81%), \u003cem\u003eXanthomonas\u003c/em\u003e (0.64\u0026ndash;30.28%), \u003cem\u003eMethylobacterium-Methylorubrum\u003c/em\u003e (0.20\u0026ndash;26.54%), \u003cem\u003ePseudomonas\u003c/em\u003e (0.30\u0026ndash;27.77%), and \u003cem\u003eMicrobacterium\u003c/em\u003e (0.12\u0026ndash;15.55%). Notably, these five genera were consistently the most abundant across all 19 rice seed sample groups. Therefore, \u003cem\u003ePantoea\u003c/em\u003e, \u003cem\u003eXanthomonas\u003c/em\u003e, \u003cem\u003eMethylobacterium-Methylorubrum\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eMicrobacterium\u003c/em\u003e were identified as the core microbiota of the high-quality rice seed samples from Heilongjiang area.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlpha diversity analysis of endophytes in rice seeds from Heilongjiang\u003c/p\u003e\u003cp\u003eAlpha diversity indexes of 19 rice seed sample groups include ACE, Chao, Shannon and Simpson values, in which ACE and Chao values are used for sample abundance assessment, specifically reflecting the abundance of rare species; higher values indicate greater numbers of rare species, and Shannon and Simpson values are used for sample diversity assessment. Both the Shannon and Simpson indices exhibited considerable variation across the 19 sample groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, samples sharing the same parental lines (samples A and B, E and S) displayed similar endophytic diversity, as did half-siblings (samples E and G). In contrast, samples D and F, despite also sharing the same parental lines, exhibited substantial differences in endophytic diversity. Similarly, significant variation was observed in the Chao1 and ACE indices, yet samples sharing the same parental lines (samples A and B) demonstrated comparable richness.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBeta diversity analysis and heatmap of endophytes in rice seeds from Heilongjiang\u003c/p\u003e\u003cp\u003eBeta diversity analysis of the 19 seed sample groups was performed, with the results visualized in the PCoA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and the Non-Metric Multidimensional Scaling (NMDS, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Analysis of similarities (ANOSIM) yielded an R-value of 0.2754 and a P-value of 0.001, indicating statistically significant differences among the 19 sample groups. Furthermore, the PCoA and NMDS analyses demonstrated a dispersed distribution pattern of the 19 sample groups. Collectively, these results demonstrate significant differences in the endophytic bacterial diversity among these 19 seed samples, which is consistent with the findings from the alpha-diversity analysis described above.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) depict the endophytic bacterial composition at the genus level for the 19 rice seed sample groups. This visualization illustrates the relative abundance of different endophytic bacteria within the seeds, with color intensity positively correlated with relative abundance. The results demonstrate that \u003cem\u003ePantoea\u003c/em\u003e was the most dominant genus in all seed samples. Although the relative abundances of endophytes varied among the different groups, the predominant genera identified were consistent with the taxonomic composition at the genus level described in the previous analysis. This finding further confirms that the core microbiota of these 19 rice seed samples comprises the five genera mentioned above.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe correlation network analysis of endophytic in rice seeds from Heilongjiang\u003c/p\u003e\u003cp\u003eThe 19 rice seed sample groups were categorized into two cohorts based on parental genetic background: those sharing both parental lines (Double group) and those with differing parental lines (No group). The correlation network analysis was performed for each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePanel A of Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e depicts the intra-group microbial interaction network for the Double group, exhibiting a greater number of edges and increased complexity of interactions between nodes. In contrast, panel B shows the intra-group network for the No group, which displayed fewer edges and simpler inter-node connections compared to the Double group, indicating more frequent and complex microbial interactions within the Double group. This conclusion is further supported by the network parameters presented in panel C, where the Double group demonstrated a higher number of edges, greater average weight, and increased network density. Differential analysis of the intra-group correlation network patterns between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) revealed a statistically significant difference. Collectively, these results demonstrate that the parental genetic background influences the interactions within the endophytic bacterial community. Endophytic communities in rice seed samples sharing the same genetic background exhibited more frequent and complex interactions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRice, as a staple food crop globally, harbors microbial resources that constitute a strategic reservoir for agricultural microbiotechnology development. Rice endophytic bacteria establish specific niche distributions across different plant tissues (e.g., roots, stems, leaves, and seeds) (Wang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ali et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Seeds represent the starting point of the plant life cycle and are associated with diverse microbial communities. Seed endophytes constitute a vital component of the plant microbiome, playing crucial roles in maintaining seed viability, promoting germination, and enhancing plant growth and yield (Chee-Sanford et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Multiple studies have demonstrated that seed endophytic bacteria enhance plant adaptability (Langill et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ren et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Rice seed endophytic bacteria not only directly promote rice growth by modulating phytohormone synthesis and siderophore release, but also indirectly enhance development by improving stress resistance and antagonism against phytopathogens. Notably, the vertical transmission capability of rice seed endophytes positions them as key mediators in rice environmental adaptation. RANA et al. (2024) revealed that the core seed endophyte \u003cem\u003eXanthomonas sontii\u003c/em\u003e can be vertically transmitted via seeds and assists the host in enriching beneficial microbiota to counter biotic stress. It has also been reported in a related study (Radhakrishnanr et al., 2023) that the seed endophyte \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e S3, isolated from rice seeds, exhibits antifungal activity against pathogens and produces bioactive compounds to promote plant growth. Further investigations have shown that pretreatment of rice seeds with endophytic bacteria significantly enhances seed germination rates, plant growth, and improves salt tolerance (Kavitha et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chowdhury et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Overall, these studies provide compelling theoretical support for exploiting rice seed endophytes as microbial fertilizers or biocontrol agents.\u003c/p\u003e\u003cp\u003eHeilongjiang Province, situated as the northernmost rice-growing region in China, possesses a unique cold-region rice cropping system. Rice produced in this area is renowned for its soft and sweet texture, with representative varieties such as Wuchang, Daohuaxiang, and Xiangshui rice. Therefore, this study collected 19 groups of high-quality rice seed samples from diverse locations at Heilongjiang Province as experimental materials. The primary objectives were to systematically characterize the structural features of their endophytic bacterial communities and to provide a theoretical foundation and germplasm innovation basis for improving the quality and productivity of cold-region rice.\u003c/p\u003e\u003cp\u003eThis study employed high-throughput sequencing to systematically analyze the community structure and diversity of seed endophytic bacteria in 19 rice seed groups from Heilongjiang Province. A total of 792 OTUs were obtained from 57 samples. Analysis of the endophytic species composition across the 19 rice seed sample groups revealed that \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eActinobacteria\u003c/em\u003e were the predominant phyla. \u003cem\u003eProteobacteria\u003c/em\u003e, representing the most dominant bacterial phylum in all seed samples, accounted for 80.3\u0026ndash;99.73% of the relative abundance. This finding is consistent with previous reports by Wang et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e; \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) on endophytic bacteria in saline-alkali tolerant and upland rice seeds. At the genus level, \u003cem\u003ePantoea\u003c/em\u003e, \u003cem\u003eXanthomonas\u003c/em\u003e, \u003cem\u003eMethylobacterium-Methylorubrum\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eMicrobacterium\u003c/em\u003e constituted the core microbiota, exhibiting relatively high conservation across the different rice varieties. Furthermore, existing research has summarized the composition and associated functions of the rice seed microbiome (Kumar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which provides valuable references for our future exploration of functional bacteria.\u003c/p\u003e\u003cp\u003eAnalysis of diversity revealed an association between endophytic bacterial diversity and genetic background, although genetic background was not the sole determinant. For instance, varieties sharing both parental lines (A and B, E and S) exhibited relatively similar levels of richness and diversity. In contrast, significant differences in endophytic richness were observed between other varieties sharing both parental lines (e.g., H and R) and between varieties with differing genetic backgrounds. This observation aligns with existing research indicating that the composition of seed-associated microbial communities is influenced by multiple factors, including plant species, seed developmental stage, geographical location, and the presence or absence of phytopathogens (Nelson et al., 2018; Rezki et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rodr\u0026iacute;guez et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, beta-diversity analysis confirmed significant differences in endophytic community structure among the 19 sample groups. However, varieties with similar genetic backgrounds displayed a clustering tendency in their community structures. Consequently, correlation network analysis demonstrated that seeds sharing both parental lines (Double group) harbored endophytic bacterial communities with more frequent and complex interactions compared to those with differing parental lines (No group). Collectively, these results indicate that the community structure and diversity of endophytic bacteria within high-quality rice seeds from Heilongjiang area are not driven by a single factor but are likely influenced by rice variety, genotype, environment, among others. Nevertheless, endophytic bacteria in seeds sharing a genetic background exhibited more intricate interactions.\u003c/p\u003e\u003cp\u003eThis study unveils the community composition and potential functions of endophytic bacteria in rice seeds, providing novel evidence for a deeper understanding of their significance within the plant microbiome. Furthermore, while conventional breeding predominantly focuses on intrinsic plant traits, it often overlooks the co-evolutionary potential of their symbiotic microbiota. Insights from this study suggest that optimizing the seed microbiota composition through targeted selection or microbiome transplantation techniques holds promise for developing novel rice varieties with enhanced stress tolerance and nutrient use efficiency. Consequently, this study also provides a robust theoretical foundation for \"holobiont breeding\" (integrating both plant and associated microbes). Such a \"plant-microbe co-evolutionary\" breeding strategy not only expands the dimensions of crop improvement but also breathes new life into seed endophyte research, thereby steering agriculture towards a more ecologically sustainable trajectory.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLeveraging high-throughput sequencing, this study showed the characterization of the diversity and community structure of endophytic bacteria within seeds of high-quality rice varieties from Heilongjiang Province, and it revealed the \"core microbiota\" and potential associations between endophytic bacterial community composition and parental genetic background. The observed conservation of the core microbiota suggests its likely role in key functional aspects, such as supporting rice health or stress resistance. Future research involving isolation, cultivation, and functional validation holds promise for unlocking its application potential. Furthermore, the observed influence of parental genetic background on the endophytic bacterial correlation network indicates that harnessing the principles governing vertical transmission of the seed microbiome during breeding offers a promising avenue for developing novel rice varieties harboring stable beneficial microbiota. This approach broadens crop genetic improvement strategies from the perspective of \"plant-microbe interactions,\" thereby contributing significant theoretical and practical value to advancing sustainable agriculture and food production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeng H and You YT designed and participated in all experimental procedures, performed data analysis, and drafted the manuscript. Cao SW participated in the samples collection and preparation. Liu Y supervised the study and critically revised the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\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).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAfzal, I., Shinwari, Z.K., Sikandar, S., Shahzad, S., 2019. Plant beneficial endophytic bacteria: mechanisms, diversity, host range and genetic determinants. Microbiol. Res. 221, 36-49.\u003c/li\u003e\n \u003cli\u003eAli, M., Ali, Q., Sohail, M. A., Ashraf, M. F., Saleem, M. H., Hussain, S., Zhou, L., 2021. Diversity and taxonomic distribution of endophytic bacterial community in the rice plant and its prospective.\u0026nbsp;Int J Mol Sci. 22(18), 10165.\u003c/li\u003e\n \u003cli\u003eBarret, M., Briand, M., Bonneau, S.,\u0026nbsp;Pr\u0026eacute;veaux, A.,\u0026nbsp;Vali\u0026egrave;re, S., Bouchez, O., Hunault, G., Simoneau, P., Jacques, M., 2015. Emergence shapes the structure of the seed microbiota. Appl Environ Microb. 81(4), 1257-1266.\u003c/li\u003e\n \u003cli\u003eBerg, G., Raaijmakers, J.M., 2018. Saving seed microbiomes. ISME J. 12(5), 1167-1170.\u003c/li\u003e\n \u003cli\u003eChowdhury, M. Z. H., Mostofa, M. G., Mim, M. F., Haque, M. A., Karim, M. A., Sultana, R., Rohman, M. M., Bhuiyan, A., Rupok, M. R. B., Islam, S. M. N., 2024. 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Composition and diversity of endophytic bacterial community in seeds of super hybrid rice \u0026lsquo;Shenliangyou 5814\u0026rsquo; (\u003cem\u003eOryza sativa\u003c/em\u003e L.) and its parental lines. Plant Growth Regul. 87, 257\u0026ndash;266.\u003c/li\u003e\n \u003cli\u003eLiu, Y., Zuo, S., Zou, Y., Wang, J., Song, W., 2013. Investigation on diversity and population succession dynamics of endophytic bacteria from seeds of maize (Zea mays L., Nongda108) at different growth stages. Ann Microbiol. 63(1), 71-79.\u003c/li\u003e\n \u003cli\u003eLangill, T., Jorissen, L. P., Oleńska, E., W\u0026oacute;jcik, M., Vangronsveld, J., Thijs, S., 2023. Community profiling of seed endophytes from the Pb-Zn Hyperaccumulator Noccaea caerulescens and their plant growth promotion potential. 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Seed-inhabiting endophytes: Their role in plant development and disease protection. \u003cem\u003eIn New and Future Developments in Microbial Biotechnology and Bioengineering\u003c/em\u003e (pp. 35-44). Elsevier.\u003c/li\u003e\n \u003cli\u003eRen, Z., Chen, A. J., Zong, Q., Du, Z., Guo, Q., Liu, T., Chen, W., Gao, L., 2023. Microbiome signature of endophytes in wheat seed response to wheat dwarf Bunt caused by Tilletia controversa K\u0026uuml;hn. Microbiology Spectrum. 11(1), e00390-22.\u003c/li\u003e\n \u003cli\u003eRana, R., Patil, P. B., 2024. \u003cem\u003eXanthomonas sontii\u003c/em\u003e, and Not X. \u003cem\u003esacchari\u003c/em\u003e, Is the Predominant Vertically Transmitted Core Rice Seed Endophyte. Phytopathology. 114(9), 2017-2023.\u003c/li\u003e\n \u003cli\u003eRadhakrishnan, N. A., Ravi, A., Joseph, B. J., Jose, A., Jithesh, O., Krishnankutty, R. E., 2023. Phenazine 1-carboxylic acid producing seed harbored endophytic bacteria from cultivated rice variety of kerala and its broad range antagonism to diverse plant pathogens. Probiotics Antimicro. 15(3), 516-523.\u003c/li\u003e\n \u003cli\u003eRezki, S., Campion, C., Iacomi-Vasilescu, B., Preveaux, A., Toualbia, Y., Bonneau, S., Briand, M., Laurent, E., Hunault, G., Simoneau, P., Jacques, M.-A., Barret, M., 2016. Differences in stability of seed-associated microbial assemblages in response to invasion by phytopathogenic microorganisms. PeerJ. 4, e1923.\u003c/li\u003e\n \u003cli\u003eRodr\u0026iacute;guez, C. E., Mitter, B., Barret, M., Sessitsch, A., Compant, S., 2018. Commentary: seed bacterial inhabitants and their routes of colonization. Plant Soil. 422, 129-134.\u003c/li\u003e\n \u003cli\u003eSantoyo, G., Moreno-Hagelsieb, G., Orozco-MosquedaMdel, C., Glick, B.R., 2016. Plant growth-promoting bacterial endophytes. Microbiol. Res. 183, 92\u0026ndash;99.\u003c/li\u003e\n \u003cli\u003eSahu, P. K., Mishra, S., 2021. Effect of hybridization on endophytes: the endo-microbiome dynamics. Symbiosis. 84(3), 369-377.\u003c/li\u003e\n \u003cli\u003eThampi, M., Anjali, C., Vijayan, S., Jisha, M. S., 2024. Unveiling Bacillus rugosus CRI: A multi-stress tolerant endophyte revolutionizing rice resilience. Physiol Mol Plant P. 134, 102462.\u003c/li\u003e\n \u003cli\u003eTruyens, S., Weyens, N., Cuypers, A., Vangronsveld, J., 2015. Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Env Microbiol Rep. 7(1),40-50.\u003c/li\u003e\n \u003cli\u003eVerma, S. K., White, J. F., 2019. Seed endophytes. Springer, Cham, Switzerland. doi, 10, 978-3.\u003c/li\u003e\n \u003cli\u003eWang, Z., Zhu, Y., Li, N., Liu, H., Zheng, H., Wang, W., Liu, Y., 2021a. High throughput sequencing-based analysis of the composition and diversity of endophytic bacterial community in seeds of saline alkali tolerant rice. Microbiol Res. 250, 126794.\u003c/li\u003e\n \u003cli\u003eWang, Z., Zhu, Y., Jing, R., Wu, X., Li, N., Liu, H., Zhang, X., Wang, W., Liu, Y., 2021b. High-throughput sequencing-based analysis of the composition and diversity of endophytic bacterial community in seeds of upland rice. Arch Microbiol. 203(2), 609-620.\u003c/li\u003e\n \u003cli\u003eWang, Z., Wu, X., Li, N., Wang, W., Liu, Y., 2022. A high-throughput screening method for endophytic bacteria with antagonistic activity against Magnaporthe oryzae in rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) seeds. Plant Growth Regul. 96, 237-241.\u003c/li\u003e\n \u003cli\u003eWang, Y., Gao, B. L., Li, X. X., Zhang, Z. B., Yan, R. M., Yang, H. L., Zhu, D., 2015. Phylogenetic diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff), detection of polyketide synthase gene and their antagonistic activity analysis. 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Journal of Jilin Agricultural University. 36(1), 10-16.\u003c/li\u003e\n\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"High-throughput sequencing, Endophytic bacteria, Diversity, Heilongjiang rice, Parental genetic relatedness","lastPublishedDoi":"10.21203/rs.3.rs-7481527/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7481527/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBackground and aims\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u0026nbsp;\u003c/em\u003eRice is a major staple crop in China, and Heilongjiang Province is a core japonica rice production region. This study aimed to explore core microbiota composition and the influence of host genetic background on microbiota assembly.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eUsing Illumina NovaSeq 6000 sequencing, we analyzed endophytic bacteria diversity in 57 samples from 19 elite rice cultivars, identifying 792 OTUs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults \u003c/strong\u003e\u003c/em\u003eAt the phylum level, Proteobacteria dominated (80.3%-99.73%). Core microbiota at the genus level included \u003cem\u003ePantoea\u003c/em\u003e, \u003cem\u003eXanthomonas\u003c/em\u003e, \u003cem\u003eMethylobacterium\u003c/em\u003e-\u003cem\u003eMethylorubrum\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eMicrobacterium\u003c/em\u003e. Parental genetic background significantly influenced bacterial community structure similarity, with closer genetic relationships showing more complex bacterial interaction networks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusion \u003c/strong\u003e\u003c/em\u003eThis study showed the compositional characteristics of the core microbiota in elite rice seeds from Heilongjiang and its relationship with parental genetic background, providing a theoretical basis for microbiome-assisted breeding in rice.\u003c/p\u003e","manuscriptTitle":"High-throughput sequencing-based analysis of the composition and diversity of endophytic bacterial community in seeds of elite rice resources from Heilongjiang area","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 14:26:47","doi":"10.21203/rs.3.rs-7481527/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-11-10T05:09:35+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-18T00:07:35+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-15T08:33:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2025-09-04T22:05:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T11:54:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2025-09-03T09:27:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4f231989-dab3-43b1-9478-69dbca6f107e","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:01:34+00:00","versionOfRecord":{"articleIdentity":"rs-7481527","link":"https://doi.org/10.1007/s11104-025-08254-0","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2026-01-06 15:56:57","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-09-23 14:26:47","video":"","vorDoi":"10.1007/s11104-025-08254-0","vorDoiUrl":"https://doi.org/10.1007/s11104-025-08254-0","workflowStages":[]},"version":"v1","identity":"rs-7481527","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7481527","identity":"rs-7481527","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}