title: lettuce seed endoshere
design: Seeds of lettuce (Lactuca sativa var. crispa L.) of the Ozornik cultivar, produced by LLC "Agrofirma AELITA," were selected for the experiment. To assess the endophytic seed microbiome, surface sterilization of the seeds was performed. Specifically, the lettuce seeds were treated with 2.5% sodium hypochlorite for 2 minutes, rinsed with sterilized tap water, and then immersed in 70% ethanol for 3 minutes. The sterile seeds were thoroughly washed five times with sterile tap water. The final rinse water was plated onto LB agar Petri dishes. In the absence of bacterial or fungal colony growth, the sterilization process was considered successful, and such seeds were used for subsequent DNA extraction from the endosphere microbiome.  DNA extraction was carried out using the MoBio PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) following the manufacturers instructions. DNA concentration and quality were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).  Sequencing of the bacterial community was performed using the Illumina platform (Illumina, San Diego, CA, USA). Genomic library preparation was conducted according to the Illumina MiSeq 16S metagenomic sequencing library preparation protocol (Illumina MiSeq, San Francisco, CA, USA). Amplification of the V3V4 region of the 16S rRNA gene was performed using a DNA Engine Tetrad 2 thermal cycler (Bio-Rad, Hercules, CA, USA) with the following specific primers: primer A (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGGNGGCWGCAG-3) and primer B (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3). The PCR conditions were as follows: initial denaturation at 95 C for 3 minutes; 27 cycles of denaturation at 95 C for 30 seconds, annealing at 55 C for 30 seconds, and extension at 72 C for 30 seconds; followed by a final extension at 72 C for 3 minutes. Amplicons were purified using the Agencourt AMPure XP kit (Beckman Coulter, Brea, CA, USA). A second round of amplification was performed under the same conditions. Amplicon concentration was measured using a Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA, USA) with the Quant-iT High Sensitivity DNA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Quality control of the amplicons was performed on a LabChip GX Touch 24 instrument (PerkinElmer, Waltham, MA, USA). Sequencing was then conducted on the MiSeq platform (Illumina, San Diego, CA, USA) according to the manufacturers instructions.  The 16S rRNA sequencing data were analyzed using the DADA2 algorithm . Taxonomy was assigned to the resulting amplicon sequence variant (ASV) table based on the SILVA reference database version 132 using the assignTaxonomy function. Prior to downstream analyses, all ASVs identified as chloroplast or mitochondria were removed.