| Summary: | Abstract Introduction As microbes play important roles in many hosts and niches, linking microbiota across niches is becoming an important area of research. Studying microbiota across multiple niches would allow understanding of ecosystem‐level interactions and potential points of better manipulation for agriculture gains. However, a suitable methodology to characterize microbiota from vast different niches is currently lacking. We used the plant shoot and soil as two important niches for this case study. Materials and Methods Considering the important linkage plant play in connecting above‐ and below‐ground ecosystems and challenges of working with plant microbiota, we first compared three commonly used 16S ribosomal RNA gene primer pairs targeting V3–V4 or V5–V7 regions coupled with Illumina sequencing for bacterial communities associated with ryegrass shoot. Then the selected primer was used to amplify bacterial communities in soil and rhizosphere samples for comparison with the commonly used 338F/806R. Finally, core operational taxonomic units (OTUs) across soil and plant niches were identified. Results Primer pair 799F/1193R amplified the lowest number of plant organelle sequences (<0.2% of total sequences) and consistently showed the highest α‐diversity compared with 338F/806R and 335F/769R. For soil and rhizosphere samples, 799F/1193R also demonstrated significantly higher α‐diversity indices compared with 338F/806R. The bacterial phyla commonly detected by both primer pairs comprised >97% of the total relative abundance in soil and rhizosphere samples. In addition, the differences in bacterial communities of the soil and rhizosphere samples were more evident when using 799F/1193R than 338F/806R. Using the 799F/1193R data set, 50 core bacterial OTUs were identified across soil, rhizosphere and shoot niches, whereas only 38 core OTUs were identified when using 338F/806R. Most of these core OTUs were dominant in either shoot or soil niche. Conclusion Primer pair 799F/1193R is suitable for bacterial community studies targeting ryegrass plant and soil microbiota, in particular for cross‐niches studies.
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