16S rRNA Gene Sequencing of SKFO-17-2 Rhizosphaera
Soil samples were collected from the root surface of 81 white poplar trees of both sexes growing under different geographical conditions. Soil was collected in the climatic spring (April-May) and autumn (September-October) during the period 2022-2023. The collected soil samples were stored at -75C until DNA extraction. DNA was extracted from 300 (90 isolates, 2 years: autumn and springer) soil samples (average mass was about 250-300 mg) according to the standard protocol of MagnoPrime kit (NextBio, Russia). DNA was quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific, USA); quality control was performed on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., USA). The A260/A280 ratio in the DNA samples was 1.82.0. Amplification of 16S rDNA regions was performed for 324 soil samples. Amplicon libraries were prepared according to the protocol with two-step PCR. Briefly, in the first step, target sequences were amplified using primers containing locus-specific sequences for 16S rDNA amplification [ 10.1093/nar/gks808] and overhang adapters (Table 2). For each sample, amplicons were pooled equimolarly and the second PCR was performed with Nextera XT Index primers consisting of dual-index barcodes and sequencing adapters. All PCR products were then pooled equimolarly and the library was assessed for quality using the 2100 Bioanalyzer (Agilent Technologies, USA) and for quantity using the Qubit fluorometer (Thermo Fisher Scientific). The library was sequenced on the MiSeq platform (Illumina, USA) with the Illumina MiSeq Reagent Kit v3 (600 cycles).