Nanodrop nd 8000 v1.1.1 spectrophotometer

To perform metagenomic analysis, three biological repetitions, consisting in a pool of three raspberry fruits, were washed in 20 mL MgSO₄ 10 mM for 5 min under gentle agitation (70 rpm). Washing suspensions were frozen and stored at −80 °C. DNA was extracted according to the CTAB protocol [34 (link)], using the pellet obtained from centrifuging the washing solutions at 13,000× g for 10 min. DNA quality and quantity were measured by spectrophotometric quantification with a NanoDrop ND-8000 V1.1.1 spectrophotometer (Thermo Fisher Scientific, Dreieich, Germany). Bacterial V3-V4 regions were amplified with 16S Amplicon polymerase chain reaction (PCR) Forward = 5_TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and Reverse = 5_GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC primers according to Illumina protocols and subjected to automated sequencing by BioFab research (Rome, Italy).

At each sampling point, we randomly assigned three subplots (30 cm × 30 cm) on rocks where H. heterophylla was growing. H. heterophylla plants were collected from all corners and at the middle of all subplots with knife and combined as one composite sample per sampling point. H. heterophylla samples were cleaned, stripped of all debris, and shipped on ice to a molecular laboratory. H. heterophylla samples (including all parts of the plants) were intensively washed using Milli-Q water (three times, 2 min vortex), 70% ethanol (one time, 3 min), Milli-Q water (three times), and PCR water (one time, 1 h and vortex 2 min). Each H. heterophylla sample was homogenized and genomic DNA from each sample was extracted from 150 mg of the homogenized sample using the ZR Soil Microbe DNA MiniPrep kit (Zymo Research, Irvine, CA, United States) following the manufacturer’s instructions. DNA quality and quantity were measured by spectrophotometric quantification with a NanoDrop ND-8000 V1.1.1 spectrophotometer (Thermo Fisher Scientific, Dreieich, Germany). DNA extracts were then stored at −20°C for further analysis.