Rhizospheric bacterial community composition
DNA was extracted from 0.5 g of soil using Nucleospin Soil Kit (Macherey Nagel) with buffer SL1 and SX according to manufacturers’ instructions. Amplicon sequencing of the V4 hypervariable region of the 16S rRNA gene was performed on a MiSeq Illumina instrument (MiSeq Reagent Kit v3 (600 Cycle); Illumina, San Diego, CA, USA) using the universal eubacterial primers 515F (Parada, Needham & Fuhrman 2016) and 806R […], extended with sequencing adapters to match Illumina indexing primers. To identify potential contamination during DNA extraction and amplification, both extraction and PCR no template control samples were performed. PCR was done using NEBNext high fidelity polymerase (New England Biolabs, Ipswich, USA) in a total volume of 25 µl (5 ng DNA template, 12.5 µl polymerase, 5 pmol of each primer, 2.5 µl 3% BSA). PCR conditions were 5 min at 98 ˚C; 25 cycles of 10 s at 98 ˚C, 30 s at 55 ˚C, 30 s at 72 ˚C; 5 min 72 ˚C. PCR products were purified using MagSi NGSprep Plus beads (Steinbrenner, Wiesenbach, Germany) and quantified via PicoGreen assay. Subsequently, indexing PCR was performed using the Nextera XT Index Kit v2 (Illumina, Inc. San Diego, CA, US) in a total volume of 25 µl (10 ng DNA template, 12.5 µl NEBNext high fidelity polymerase, 2.5 µl of each indexing primer) and the following PCR conditions: 30s at 98 ˚C; 8 cycles of 10 s at 98 ˚C, 30s at 55 ˚C, 30s at 72 ˚C; 5min 72 ˚C. Indexing PCR products were purified using MagSi NGSprep Plus beads, qualified and quantified via a Fragment Analyzer™ instrument (Advanced Analytical Technologies, Inc., Ankeny, USA), and pooled in an equimolar ratio of 4nM.
Sequences were analyzed on the Galaxy web platform ( Afgan et al, 2016). FASTQ files were trimmed with a minimum read length of 50 using Cutadapt (Martin 2011). Quality control was performed via FastQC (Andrews 2010). For subsequent data analysis DADA2 pipeline (Galaxy Version 1.20) (Callahan et al., 2016) was used with the following trimming and filtering parameters: 20 bp were removed n-terminally, and reads were truncated at position 240 (forward) and 180 (reverse), respectively, with an expected error of 3 (forward) and 5 (reverse). The resulting unique amplicon sequence variants (ASV) were assigned to the SILVA v138.1 (release 99%) reference database. To exclude potential contamination, ASV occurring in no template controls, as well as unassigned, mitochondrial and chloroplast, reads, and singletons (ASV represented by only one read) were removed from the dataset, resulting in an average read loss of 5.2%. To compare samples without statistical bias, 38724 reads were subsampled in all samples, reflecting the lowest observed read number. Rarefaction analysis indicated that this sampling depth was sufficient for further analysis of samples (Supplementary Figure. 1). The sequence data obtained in this study are deposited in the short-read archive of NCBI under accession number PRNJA989406 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA989406?reviewer=ficg6d69j4e0tit266actkpjd9).