title: lettuce plant endospheric microbiome
design: For DNA extraction from seed, leaves, roots, rhizosphere soil and hydroponic solution samples, the MoBio Power Soil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) was used according to the manufacturers instructions. The DNA concentration and quality were estimated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Detection of the bacterial 16S rRNA gene copy number was carried out via real-time PCR using primers 947f/1349r with the following sequences: F: AACGCGAAGAACCTTAC, R: CGGTGTGTACAAGGCCCGGGAACG. The master mix reaction mixture (25 L) contained the following components: DNA template1 L, forward and reverse primers (10 M)0.5 L each, dNTPs (10 M)2.5 L, 10x buffer with SYBR green2.5 L, MgCl2 (25 M)2.5 L, SynTaq polymerapse (5 U L 1 )0.2 L, and ddH2O15.3 L. Amplification was performed on a BioRad CFX96 cycler (BioRad, Hercules, CA, USA) using the following temperature program: primary denaturation at 95 C for 5 min and then 39 three-step cycles at 6062 C for 45 s at 95 C for 15 s and at 72 C for 30 s. DNA standard curves were constructed using the bacterium Pseudomonas putida. Sequencing of the bacterial community was performed on the Illumina base (Illumina, San Diego, CA, USA). The preparation of the genomic library was carried out according to the 16S Metagenomic Sequencing Library Preparation Protocol (Illumina MiSeq, San Diego, CA, USA). Amplification of the V3V4 region of the 16S rRNA was performed using a DNA Engine Tetrad 2 cycler thermal cycler (BioRad, Hercules, CA, USA) using specific primers: A (TCGTCGGCAGCGTCAGATGTGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and B (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGTATCTAATCC). Amplification was carried out according to the following regime: 95 C for 3 min; 27 cycles of 95 C (30 s), 55 C (30 s), and 72 C (30 s); and a final elongation at 72 C (3 min). Amplicons were purified using an Agencourt AMPure XP purification kit (Beckman Coulter, Brea, CA, USA). The second round was carried out using the same amplification parameters. Amplicon concentration was measured on a Qubit 3.0 Fluorometer (Invitrogen, Carslbad, CA, USA) using a Quant-iT dsDNA High-Sensitivity Assay Kit (Thermo Fisher, Waltham, MA, USA). The quality control of the obtained amplicons was carried out on a Labchip GX Touch 24 instrument (PerkinElmer, Waltham, MA, USA). Next, sequencing was carried out on a MiSeq instrument (Illumina, San Diego, CA, USA), according to the manufacturers instructions. The number of raw reads ranged between 12,947 and 50,326 for 16S rRNA amplicons. 16S rRNA sequencing data were analyzed using Quantitative Insights Into Microbial Ecology (QIIME) platform software (Version 1.9.1) and a table of sequence variants with taxonomy based on the Silva reference database version 132 was created. Operational taxonomic units (OTUs) were grouped at a similarity threshold of 97%. Before subsequent analyses, all OTUs identified as "chloroplasts" or "mitochondria", as well as singletones, were removed.