Root microbiota analysis of Oryza rufipogon and Oryza sativa reveals an orientation selection during the domestication process

ABSTRACT The root-associated microbiota has a close relation to the life activities of plants, and its composition is affected by the rhizospheric environment and plant genotypes. Rice (Oryza sativa) was domesticated from the ancestor species Oryza rufipogon. Many important agricultural traits and adversity resistance of rice have changed during a long time of natural domestication and artificial selection. However, the influence of rice genotypes on root microbiota in important agricultural traits remains to be explained. In this study, we performed 16S rRNA and internal transcribed spacer (ITS) gene amplicon sequencing to generate bacterial and fungal community profiles of O. rufipogon and O. sativa, both of which were planted in a farm in Guangzhou and had reached the reproductive stage. We compared their root microbiota in detail by alpha diversity, beta diversity, different species, core microbiota, and correlation analyses. We found that the relative abundance of bacteria was significantly higher in the cultivated rice than in the common wild rice, while the relative abundance of fungi was the opposite. Significant differences in agricultural traits between O. rufipogon and O. sativa showed a high correlation with core microorganisms in the two Oryza species, which only existed in either or had obviously different abundance in both two species, indicating that rice genotype/phenotype had a strong influence on recruiting specific microorganisms. Our study provides a theoretical basis for the in-depth understanding of rice root microbiota and the improvement of rice breeding from the perspective of the interaction between root microorganisms and plants. IMPORTANCE Plant root microorganisms play a vital role not only in plant growth and development but also in responding the biotic and abiotic stresses. Oryza sativa is domesticated from Oryza rufipogon which has many excellent agricultural traits especially containing resistance to biotic and abiotic stresses. To improve the yield and resistance of cultivated rice, it is particularly important to deeply research on differences between O. sativa and O. rufipogon and find beneficial microorganisms to remodel the root microbiome of O. sativa.

which are not only essential substances for root bacterial survival and activity but also recruit beneficial microorganisms and inhibit some plant pathogens (12,13).However, few successful cases occurred in directly utilizing the activity of root microbiota to improve the plant production systems except for the symbiotic relationship between arbuscular mycorrhizal fungi and leguminous plants (14).Therefore, it is of great value to reveal the relationship between root microbiota structure and function, in order to promote highly efficient utilization in green agriculture (15).
Rice is one of the most important cereal crops all over the globe, which feeds more than half the population of the world (16) and China contributes nearly 30% of global rice production (17).During a long time of natural evolution and artificial selection, the cultivated rice (Oryza sativa) evolves from wild rice (Oryza rufipogon), with a significant change in phenotypes of the important agronomic traits and resistance to the biotic and abiotic stresses (18,19).From the view of rice domestication, root microbiota resources that co-evolved and interacted with the host over a long time may benefit to efficiently improve crop production (20,21).For example, Rhizobium spp., Pseudomonas spp., and Bacillus subtilis FB17 produced signals to induce plant defense against pathogens and promoted plant growth and development by regulating the metabolism of carbon and nitrogen (21,22).
Compared with cultivated rice, the common wild rice shows creep growth, divergent fringe, longer awn, stronger dormancy, easier shattering, and has a stronger ability to survive in hostile environments may be because of some special root microorgan isms that provide essential function of fighting against pathogens, absorbing nutrients, and responding to abiotic stress in order to improve the resistivity to adversity and promote host healthy development (44)(45)(46).However, some root microorganisms were lost during the domestication process, so we need to explore the common and the different microbiota between O. sativa and O. rufipogon in order to find and utilize the beneficial microorganisms to improve rice production and quality.In this study, we used the amplicon sequencing technique [16S rRNA and internal transcribed spacer (ITS)] to analyze the population structure, diversity, and assembly process of symbiotic microorganisms between O. rufipogon and O. sativa.We independently and contrastively analyzed the correlation of bacterial and fungal microbiota in each and both of the two Oryza species and established the core microorganisms.We also evaluated the correlation between the different agronomic traits and different abundance of micro biota in the two Oryza species.This study could not only provide different or new microorganisms as supplementary resources to improve rice production or resistance to adversity but also put forward a new insight into rice domestication based on the change in microbiota from O. rufipogon to O. sativa.

Experimental materials and sampling
In this study, 10 common wild rice accessions (Or01 to Or10) and 10 cultivated rice varieties (Os01 to Os10) were, respectively, collected in the paddy field in Lyutian town, Conghua district, Guangzhou city, Guangdong province, China.Root samples with adherent soil were collected from each rice plant (Table S1).For consistency, 10 cm of root was cut off by scissors from each rice plant and immediately washed in 40 mL PBS buffer (pH 7.0, per liter 16.5 g of Na 2 HPO 4 •7H 2 O, 6.33 g of NaH 2 PO 4 •H 2 O, 200 mL Silwet L-77) shaking three times at 180 r/min, followed by blotting up water attached on the root and frozen in liquid nitrogen for storing at −80°C (1).

DNA extraction and high-throughput sequencing
Total genome DNA from samples were extracted using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) and, respectively, checked by 1% agarose gel electrophoresis for purity detection and ultraviolet spectrophotometry for concentration detection.DNA was diluted to 1 ng/uL using sterile water for amplification.16S rRNA and ITS genes were, respectively, amplified using the specific primers with the barcode.The specific primers 515F (forward primer, 5′-GTGCCAGCMGCCGCGG-3′) and 806R (reverse primer, 5′-GGACTACHVGGGTWTCTAAT-3′) with barcode were used for bacterial 16S rRNA gene tags (V4 region) amplification, while the specific primers 1723F (forward primer, 5′-CTTG GTCATTTAGAGGAAGTAA-3′) and 2043R (reverse primer, 5′-GCTGCGTTCTTCATCGATGC-3′) were used for fungal ITS gene (ITS1 region) amplification.PCR amplification was carried out with Phusion High-Fidelity PCR Master Mix (New England Biolabs) consisting of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s, and then a final extension at 72°C for 5 min.PCR products were checked by 2% agarose gel electrophoresis for mixing the same volume of 1× loading buffer (contained SYB green).Samples with bright main strips between 400 and 450 bp were chosen to be mixed in equidensity ratios and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions and quantified using QuantiFluor-ST (Promega, USA).Sequencing libraries were produced by using the Illumina TruSeq DNA PCR-Free Library Preparation Kit (Illumina, San Diego, CA, USA) following the manufactur er's instructions, and index codes were added.The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system.The library was sequenced on an Illumina Novaseq platform and 250 bp paired-end reads were generated.

Bioinformatics analysis
After removing the adaptors, primers, and low-quality reads, the pair-end reads were overlapped to assemble the final sequences.The criterion of overlapping was the overlapping region lengths larger than 10 bp and a mismatch ratio lower than 0.2.Chimera tags were filtered out using the Gold database by UCHIME (version 4.2.40).Then operational taxonomic unit (OTU) analysis was performed using the Uparse package (version 7.0.1001)with a 97% sequence identity.Each OTU was taxonomically assigned to the silva database using the Ribosomal Database Project (RDP) classifier OTUs were processed by removing chloroplast sequences, chondriosome sequences, and unclassi fied sequences.The OTUs with relative abundance values >0.001% (above three tags in at least one sample) in at least one sample were retained.In order to compare the samples at the same sequencing depth, we selected 35,000 sequences obtained by random sampling.

Alpha and beta diversity analysis
In-house Perl scripts were used to analyze alpha (within samples) and beta (among samples) diversity.In order to compute alpha diversity, we analyzed the OTU table and calculated three indexes: Chao1, Observed species, and PD whole tree.Chao1 can reflect the community abundance; Observed species indicated the number of OTU; and PD whole tree reflects the total represent sequences of OTU.Beta diversity analysis was conducted to examine the similarity of the community structure among different samples.The principal coordinates analysis (PCoA) was performed on the community composition structure at the genus level to explore the similarities or dissimilarities between O. rufipogon and O. sativa, which was applied to reduce the dimension of the original variables using the QIIME software package.Cluster analysis mainly refers to the hierarchical clustering analysis method using any distance to evaluate the similarity between the two Oryza species.

Statistical test
A permutational multivariate analysis of variance was performed using QIIME software and 999 displacement tests to determine the differences between bacteria and fungi in the two Oryza species.Redundancy analysis was analyzed using R to figure out the relationships between soil microorganisms and agricultural traits.

High-throughput amplicon sequencing and relative abundance analysis
After paired-end alignments and quality filtering, a total of 674,923 bacterial 16S and 741,398 fungal ITS taxon tags were recovered and assigned to 80,178 bacterial and 11,078 fungal OTUs, respectively (Table S2).Relative abundance analysis of bacteria based on taxonomic phylum between O. rufipogon and O. sativa showed that all of the five phyla, Proteobacteria, Thaumarchaeota, Bacteroidetes, Chloroflexi, and Acidobacteria, were top five of relative higher abundance both in the two Oryza species, although relative abundance of Proteobacteria and Bacteroidetes were higher in O. sativa while Thaumarchaeota was higher in O. rufipogon (Fig. 1A).The same analysis for fungi showed that Ascomycota, Basidiomycota, Chytridiomycota, Mortierellomycota, and Rozellomycota were the top five phyla with relative higher abundance, in which total of relative abundance of Ascomycota and Basidiomycota accounted for more than 70% both in the two Oryza species, although relative abundance of Basidiomycota was slightly higher and Ascomycota was lower in O. sativa (Fig. 1B).In addition, contrastive analysis of relative abundance of top 35 fungi and bacteria based on taxonomic genus between O. rufipogon and O. sativa were completed.As shown in Fig. S1A, eight bacterial genera had relatively higher abundance both in the two Oryza species, including Candidatus Solibacter, Pseudomonas, Geobacter, Acinetobacter, Candidatus Nephrothrix, Anaeromyxo bacter, Bryobacter, and Stenotrophomonas, while the relative abundance of Pseudomonas was higher in O. sativa than in O. rufipogon.The relative abundance of the top 35 genera accounted for only 10% indicating that bacterial species were very rich both in the two Oryza species.For fungi, eight genera had relatively higher abundance both in the two Oryza species, including Conocybe, Westerdykella, Echria, Mortierella, Hebeloma, Fusarium, Clitopilus, and Panaeolus.The relative abundance of Mortierella was significantly lower in O. sativa, which resulted as the dominant fungal genus in O. rufipogon, while Conocybe, Echria, and Fusarium had an obviously higher abundance in O. sativa, and the relative abundance of Conocybe was more than 20% to be the dominant fungal genus (Fig. S1B).

Diversity analysis of root microbial community between O. rufipogon and O. sativa
We used the Chao1 index and Observed species index to analyze the richness and the PD whole tree index to analyze the diversity of root microbial communities between O. rufipogon and O. sativa at the OTUs level.As shown in Fig. 2A, for the bacterium, significant enhancements in the Chao1 index (P = 0.0343) and Observed species index (P = 0.0343) indicated the richness in O. sativa was significantly higher than in O. rufipogon, and PD whole tree index (P = 0.0126) indicated a same tendency for the diversity.
Interestingly, an opposite variation of PD whole tree index (P = 0.0126) for fungi showed that the diversity in O. rufipogon was significantly higher than in O. sativa, although both the Chao1 index (P = 0.45) and Observed species index (P = 0.496) showed only slightly higher richness in O. rufipogon (Fig. 2B).
In order to observe the similarities and dissimilarities of fungi and bacteria between O. rufipogon and O. sativa, PCoA was performed based on Bray-Curtis.As shown in Fig. 3a, PCoA showed distinct differences in microbial communities existed between O. rufipogon and O. sativa, with the two principal components (PC1 and PC2) of PCoA explained 26% and 14% (bacteria, Fig. 3A), 33% and 15% (fungi, Fig. 3B), respectively.Analysis of Similarities (ANOSIM) and Multiple Response Permutation Procedure (MRPP) analysis (Table 1) showed that it is more similar to each other within species than that between the two Oryza species, for both bacteria (ANOSIM: R = 0.514, P = 0.001 and MRPP: A = 0.08, P = 0.001) and fungi (ANOSIM: R = 0.799, P = 0.001 and MRPP: A = 0.16, P = 0.001).

Correlation analysis of root-associated microbiome of O. rufipogon and O. sativa
We analyzed the correlation between bacteria and bacteria, fungi and fungi, and bacteria and fungi based on the common genera of the top 35 genera in O. rufipogon and O. sativa.Twenty-four of the top 35 bacterial genera commonly existed in both O. rufipogon and O. sativa, while 28 common fungal genera existed in the two Oryza species (Fig. 4).Observed positive correlation among microorganisms indicated a mutual synergistic effect, while significant negative correlation among microorganisms showed an interactive antagonistic effect.
As shown in Fig. 4A and B, comparative analysis of correlation among bacterial genera in O. rufipogon and O. sativa showed the obvious positive correlation between Phenylo bacterium and Rhodomicrobium, and both Geothrix and Geobacter with either Novosphin gobium or Anaeromyxobacter in the two Oryza species, while only significant negative correlation between Occallatibacter and Desulfovirga.Correlation between Anaeromyxo bacter and either Geobacter or Spirochaeta, Ellin6067, and Granulicella were observed positive in O. rufipogon but significantly negative in O. sativa.
In both O. rufipogon and O. sativa, there was a noted positive correlation among certain fungal genera.Specifically, Sarocladium, Fusarium, and Microdochium exhibited positive correlations, as did Didymella, Nigrospora, Cladosporium, and Clonostachys, except for the correlation between Cladosporium and Nigrospora.Additionally, positive correlations were observed among Myxospora, Edenia, and Myrmecridium, as well as between Pseudeurotium and either Talaromyces or Coniochaeta, in both O. rufipogon and O. sativa.On the other hand, positive correlations among Myrmecridium, Acremo nium, Talaromyces, Boothiomyces, Panaeolus, and Pyrenochaetopsis were noted, except for the correlation between Boothiomyces and Talaromyces.Furthermore, positive correlations among Trichoderma, Neurospora, and Westerdykella were found exclusively in O. rufipogon.Compared with the microorganisms with observed positive correlation, the population of microorganisms with significant negative correlation was relatively less, for only Conioscypha and Psilocybe showing significant negative correlation in O. rufipogon and a few in O. sativa, such as Echria with either Cladosporium, Clonostachys, Edenia, or Pyrenochaetopsis (Fig. 4C and D).
A combination correlation analysis between bacteria and fungi showed that only observed positive correlation between Bryobacter and Clitopilus existed in both O. rufipogon and O. sativa, indicating that they are synergistic.Most correlations between bacteria and fungi in O. rufipogon showed no significant correlation in O. sativa, and vice versa.For example, in O. rufipogon, three bacterial genera (Ellin 6067, Anaerolinea, and  and Spirochaeta were significant correlation to Clitopilus, and Anaeromyxobacter was significant correlation to both Edenia and Pyrenochaetopsis), which had no significant correlation in O. rufipogon (Fig. S4A and B).

Analysis of core microorganisms in genus between O. rufipogon and O. sativa
We defined a bacterial or fungal genus as the core microorganism based on two standards, which accounted for more than 1% relative abundance in the whole bacterial or fungal microorganisms and existed in more than 90% of O. rufipogon or O. sativa samples.As shown in Fig. S3; Fig. 5, core bacteria of O. rufipogon and O. sativa were, respectively, consisted of 30 and 40 genera within 26 common ones which contained two significant relative higher abundance of genera (Bryobacter and Bradyrhizobium) in O. rufipogon and three significant relative higher ones (MND1, Nitrospira, and Ellin6067) in O. sativa, while 4 and 14 specific genera, respectively, existed in the two Oryza species that contained two (Denitratisoma and Opitutus) and 13 (Pseudomo nas, Acinetobacter, Stenotrophomonas, Duganella, Sphingobacterium, Dyella, Bdellovibrio, Terrimonas, Aquicella, Massilia, Pelobacter, Sphingomonas, and Thiobacillus) significant different genera (Fig. 5A).For fungus, core fungi of O. rufipogon and O. sativa were, respectively, consisted of 21 and 23 genera within 15 common ones which contained one significant relative higher abundance of genera (Clitopilus) in O. rufipogon and two significant relative higher ones (Clonostachys and Conocybe) in O. sativa, while six and eight specific genera, respectively, existed in the two Oryza species that contained two (Hebeloma and Neurospora) and seven (Echria, Sarocladium, Parasarocladium, Tricoderma, Zopfiella, Metapochonia, and Limnoperdon) significant different genera (Fig. 5B).

Correlation analysis of significantly different agronomic traits and microor ganisms between O. sativa and O. rufipogon
The common wild rice (O.rufipogon) is the ancestor of cultivated rice (O.sativa), with significant differences in many important agronomic traits such as leaf sheath color (most wild rice are purple while most cultivated rice are green), growth habit (most wild rice creep while most cultivated rice upright), awn (most wild rice have long awn while most cultivated rice have short awn or awnless), ligule color (most wild rice are purple while most cultivated rice are green), and height (most wild rice are tall while most cultivated rice are dwarf ).
As shown in Fig. 6A, the relative abundance of Bradyrhizobium, Opitutus, Bryobacter, and Denitratisoma in O. rufipogon was significantly higher than in O. sativa, indicating that the four bacterial genera had an obvious positive correlation with a difference of phenotypes of O. rufipogon compared with O. sativa, including purple ligule and leaf sheath, long awn, procumbent growth, and tall plants, except for the non-significant correlation between Denitratisoma and purple leaf sheath.Oppositely, four bacterial genera had a significant negative correlation with five agronomic traits of O. rufipogon above, such as Terrimonas, Thiobacillus, MND1, and Nitrospira.
As shown in Fig. 6B, the relative abundance of Clitopilus and Neurospora in O. rufipogon were significantly higher than in O. sativa, indicating that the two fungal genera had an obvious positive correlation with a difference of phenotypes above of O. rufipogon compared with O. sativa.Oppositely, four fungal genera had a significant negative correlation with the five agronomic traits of O. rufipogon above, such as Conocybe, Zopfiella, Trichoderma, and Limnoperdon.

DISCUSSION
Plant root microbiota is closely associated with plants, which have important roles in regulating many important life processes of plants (3,40,47), such as growth, develop ment, immunity, and so on.Previous research in rice (27)(28)(29)  plant species and genotype but also soil microbiota, soil physicochemical properties, and plant secondary metabolites could be influential in the composition of rhizosphere microbe.As is well known, cultivated rice is domesticated from its ancestor, the common wild rice, for why O. sativa and O. rufipogon have many differences in various agricultural traits (growth habit, awn, leaf sheath color, and so on) and biotic (pathogen and pest) and abiotic resistances (cold, hot, salty, and so on).In this study, rice root microbial community diversity and structure between the two Oryza species were examined via high-throughput sequencing of ITS and 16S rRNA genes.
Based on the analysis of the abundance of microorganisms, we found that for bacteria, Proteobacteria and Bacteroidetes had relatively higher abundance in O. sativa, the abundance of Thaumarchaeota was relatively higher in O. rufipogon, while for fungi, Chytridiomycota and Basidiomycota had relatively higher abundance in O. sativa, the abundance of Ascomycota was relatively higher in O. rufipogon.Although the abundance of each fungal and bacterial phylum is different between O. rufipogon and O. sativa, the dominant phyla in the two Oryza species are always similar, such as Proteobacteria, Thaumarchaeota, Bacteroidetes, Chloroflexi, and Acidobacteria were the five dominant bacterial phyla, while Ascomycota, Basidiomycota, Chytridiomycota, Mortierellomycota, and Rozellomycota were the five dominant fungal phyla.This result was similar to other plants, such as barley (48), cotton (49), Arabidopsis (50), and rice (51,52).
In our research, all three indexes (Chao1, Observed species, and PD whole tree) <0.05 showed a significantly higher bacterial diversity in O. sativa than in O. rufipogon, while only PD whole tree index <0.05indicated an obviously lower fungal diversity in O. sativa compared to O. rufipogon based on alpha diversity analysis.However, relatively higher fungal alpha diversity occurred in the cultivated varieties than in the wild accessions both in soybean (53) and maize (54), even in rice (51), which demonstrated that both the fungal and the bacterial diversity in O. sativa were relatively higher than in O. rufipogon based parent-child relationship between the two Oryza species.The wild rice accessions and the cultivated varieties in our research didn't have a direct parent-child relationship may be the cause of relatively higher fungal abundance in O. rufipogon and lower in O. sativa.The PCoA analysis of fungi and bacteria based on Bray-Curtis showed distinct two groups of O. rufipogon and O. sativa in our study, which is similar to other reports (51,55,56).
Based on the correlation analysis of bacterial and fungal microorganisms within different abundance between O. sativa and O. rufipogon, we found that most microor ganisms that had an obvious negative or positive correlation with other microorganisms existed either in O. sativa or in O. rufipogon, and only a few showed the same positive or negative correlation both in the two Oryza species.This result indicated that not only the composition of root microbiota but also the interaction among them was significantly different between O. rufipogon and O. sativa.A lot of research has demonstrated that rice root microbiota was significantly affected by a long time of domestication from O. rufipogon to O. sativa (31,51,55,56).Anaerolinea, Anaeromyxobacter, and Bradyrhizobium were identified to be nitrogen-fixing bacteria (57), which were the common core bacteria in the two Oryza species and showed the relative abundance of Bradyrhizobium was significantly higher in O. rufipogon than in O. sativa, although the abundance of the other two bacterial genera had no obvious difference in the two Oryza species, indicating that the wild rice had a more reasonable strategy for utilizing nitrogen by root nitrogen relative microorganisms to response to the environment stress.
Based on the core microbiota analysis and the correlation analysis of significantly different agriculture traits and microorganisms in O. rufipogon and O. sativa, we found that although significant changes in root microbiota had occurred during a long time of domestication (58), most core microorganisms were retained with relative stable abundance while only a few were lost or newly recruited.Interestingly, most of these changed microorganisms had a significant high positive or negative correlation with at least one domestication trait, indicating that rice root microbiota selectively discarded or recruited special microorganisms to adapt to the changing environment, including carbohydrate metabolism, cofactors and vitamin metabolism, transcription, replication, and repair (Fig. S5).
As is well known, during the process of cultivating rice varieties domesticated from wild rice accessions, many beneficial characteristics were lost, especially the resistance to adversity including pathogens, pests, cold, hot, drought, and so on, meanwhile, the root microbiota also had been obviously affected.Many root microorganisms have demon strated that they can effectually promote host plants absorbing nutrition and responding to unfriendly environments for survival and development.It is of great significance to improve the rice quality by digging and utilizing root microbial resources from the wild rice accessions.The results in our study systematically reflected the significance of root microbiota between O. rufipogon and O. sativa, providing a theoretical basis for improving the rice-cultivated varieties through an angle of interaction between root microbiota and host plants.

ADDITIONAL FILES
The following material is available online.

FIG 1
FIG 1 Relation abundance of bacterial and fungal composition at phyla level of O. rufipogon and O. sativa.(A) Bacteria.(B) Fungi.

FIG 2
FIG 2 Root bacterial and fungal diversity indices of O. rufipogon and O. sativa (Chao1, PD whole tree, and Observed species) calculated by 16S rRNA and ITS gene sequence data at OTU level.(A) Bacteria.(B) Fungi.

FIG 3
FIG 3 PCoA based on Bray-Curtis dissimilarity metrics, showing the distance in the bacterial and fungal communities between O. rufipogon and O. sativa.(A) Bacteria.(B) Fungi.

Granulicella) had observedFIG 4
FIG 4 Spearman correlation of bacteria-bacteria, fungi-fungi at genera level in O. rufipogon and O. sativa.(A) Correlation among bacteria in O. rufipogon.(B) Correlation among bacteria in O. sativa.(C) Correlation among fungi in O. rufipogon.(D) Correlation among fungi in O. sativa.Red represents a positive correlation, blue represents a negative correlation.The bigger and darker diamond represents a higher correlation index.* represents P-value < 0.05, ** represents P-value < 0.01, *** represents P-value < 0.001.

FIG 6
FIG 6 Correlation analysis of significantly different agronomic traits and microorganisms between O. sativa and O. rufipogon.(A) Bacteria.(B) Fungi.

TABLE 1
ANOSIM and MRPP analysis of bacteria and fungi between O. rufipogon and O. sativa O. rufipogon vs O. sativaDifferent bacterial OTUs between O. rufipogon and O. sativa were classified into 20 genera (Table

TABLE 2
Difference analysis based on bacterial genus between O. rufipogon and O. sativa

TABLE 3
Difference analysis based on fungal genus between O. rufipogon and O. sativa

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