amplicons of 16S rRNA gene generated from seeds
Metagenomic DNA isolated from plant material was isolated using FastDNA SPIN Kit for soil, DNA was purified using Genomic DNA Clean and ConcentratorTM (ZYMO Research, USA) according to the manufacturers instructions. DNA concentration and purity were determined spectrophotometrically using NanoDrop ND-1000 (NanoDrop Technologies, USA). Amplicons 16S rRNA were prepared using three sequential PCRs. In the first PCR, amplicons of 16S rRNA gene were generated using 515 forward and 1068 reverse (5-CTGRCGRCRRCCATGCA-3) primers together with anti-mitochondrial and anti-plastid peptide-nucleic acids (PNAs) (PNABio, USA) to inhibit the amplification of plant organelle DNA. The first 15l reactions contained 0.3 M of each PNA probe, 0.3 M of each primer (Sigma-Aldrich, USA), 0.02 U/l of KAPA HiFi HotStart ReadyMix (Kapa Biosystems, USA), template DNA (~10 ng/l) and water for molecular biology (Sigma-Aldrich, USA). Amplification of each sample was performed in 6 copies and analysed on agarose gel (1.5%) electrophoresis. The bands at 553 bp was excised and purified using Zymoclean Gel DNA Recovery Kit (ZYMORESEARCH, USA). Extracted DNA was then used in the second PCR, where 515 forward (5-GTGYCAGCMGCNGCGG-3) and 926 reverse (5-CCGYCAATTYMTTTRAGTTT-3) primers were targeted on V4  V5 region of the gene. The reaction of 15 l contained: 0.02 U/l KAPA HiFi HotStart ReadyMix (Kapa Biosystems, USA), metagenomic DNA (~10 ng), 0.3 M of each primer (Sigma-Aldrich, USA), and water for molecular biology (Sigma-Aldrich, USA). In the third amplification run, the product of the second PCR was used as a template DNA, and the same primers modified with adaptor tags and internal barcodes for Illumina sequencing were used. The second 25 l reactions contained 0.02 U/l KAPA HiFi HotStart ReadyMix (Kapa Biosystems, USA), 1 M of each primer (Sigma-Aldrich, USA), 0.5 l of the previous PCR product, and water for molecular biology (Sigma-Aldrich, USA). Temperature program for both reactions: 95 C / 5 min, 98 C / 20 s, 56 C (for 16S rRNA gene) or 50 C (for ITS region) / 15 s, 72 C / 15 s, 72 C / 5 min. The first amplification were prepared in 28  30 cycles; the next amplification was performed in 8  10 cycles. The resultant amplicons of 16S rRNA and ITS region gene of plant and soil origin were purified with SPRIselect magnetic beads (Beckman Coulter, USA) and sent to the Core Facility for Nucleic Acid at the University of Alaska Fairbanks for Illumina Miseq platform sequencing.