Small sample amounts from rhizosphere of barley maintain microbial community structure and diversity revealed by total RNA sequencing

Total RNA sequencing is a crucial technique in microbial ecology for profiling active microbial communities in various environments, including the rhizosphere. Since total RNA sequencing yields both 16 S and 18 S ribosomal RNA (rRNA), it is effective for taxonomic profiling of the full microbial community in a sample. However, the effectiveness of this approach with limited initial sample amounts remains unclear. In this study, we grew barley in a growth system designed for highly controlled plant experiments using an inert growth medium inoculated with a soil microbiome. Our objectives were two-fold: firstly, to test the feasibility of extracting total RNA from the rhizosphere of barley grown in an inert growth medium consisting of sand and perlite. Secondly, we aimed to address the challenge of extracting comprehensive taxonomic information from minimal amounts of rhizosphere samples from barley plants, using three different amounts of freeze-dried rhizosphere material: 10, 40, and 200 mg. We showed that although smaller sample amounts yielded lower concentrations of extracted RNA, this did not significantly influence the diversity or composition of the rhizosphere microbiome as indicated by SSU rRNA. Our results demonstrate that total RNA sequencing, focusing on SSU rRNA, robustly captures the taxonomic diversity of active rhizosphere microbial communities, even in small initial sample amounts. Effective use of smaller samples opens new possibilities for detailed studies in environments where sample quantity is limited. We also conclude that the growth system applied in this experiment is suitable for highly controlled plant experiments focusing on total RNA extraction from the rhizosphere.