Nucleospin plant 2 mini kit

The SSU region, as well as the ITS1-5.8S-ITS2 region (hereafter, ITS-1,2 regions), and in particular groups, the ITS-2 region for green algae and the SSU-LSU rRNA (large subunit rRNA) intergenic spacer for cyanobacteria were used as molecular markers. The cells of green algae and cyanobacteria were disrupted by shaking with 1.25–1.55-mm glass beads in combination with a threefold freezing and thawing cycle (liquid N, heating block 65 °C). The DNA was extracted with the NucleoSpin Plant II mini kit (Macherey Nagel, Düren, Germany) following the instructions. PCR was performed as described by Mikhailyuk et al. [86 (link)] using the primers EAF3 and ITS055R for green algae [87 (link),88 (link)] and SSU-4-forw and ptLSU C-D-rev for cyanobacteria [89 (link)]. Sanger sequencing was conducted by GATC Sequencing Services (Eurofins Genomics Germany, Ebersberg, Germany) using the primers EAF3 and 1400R [87 (link)], N920R [88 (link)], 536R [90 ], 920F and 1400F [91 (link)], and GF and GR [92 (link)] for green algae and SSU-4-forw, Wil 6, Wil 12, Wil 14, Wil 5, Wil 9, Wil 16, and ptLSU C-D-rev [89 (link),93 (link)] for cyanobacteria.

Cyanobacteria and eukaryotic microalgae were isolated from the enrichment cultures used in our previous study [12 (link)]. Single colonies were transferred to the Petri dishes with Bold’s Basal Medium, and unialgal cultures were established. The cultures were then kept at 15 °C under 30 μmol photons m⁻² s⁻¹ (Osram Lumilux Cool White lamps L36W/840) with a light/dark regime of 16/8 h.
The DNA of cyanobacterial and microalgal strains was extracted using the NucleoSpin Plant II mini kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s instructions. The 16S (for cyanobacteria) and 18S rRNA (for eukaryotic microalgae) genes were amplified using primers BS-1F/CPL-10R and EAF3/ITS055R, respectively (Supplementary Materials Table S1). PCRs for both sets of primers were performed as follows: an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of DNA denaturation at 95 °C for 15 s, primer annealing at 55 °C for 15 s, strand extension at 72 °C for 1 min 30 s, and a final extension step at 72 °C for 2 min. Sanger sequencing was performed at GATC Sequencing Services (Eurofins Genomics Germany, Ebersberg, Germany) with the primers listed in the Supplementary Materials Table S1.

High-quality DNA was extracted from young leaves from bamboo culm of each genotype using NucleoSpin Plant II Mini kit (Macherey–Nagel) according to the manufacturer’s instructions. After dilution to 100 ng/μl, the 25 genomic DNA samples were used to generate 25 Tru-Seq Nano libraries with a mean insertion size of 450 bp and the libraries were sequenced, Paired-End 150 bp, using an Illumina NovaSeq S4 system (Illumina, San Diego, CA, United States).

The BCC was determined by metabarcoding of the V3/V4 region of the 16S rRNA gene. Filters dedicated to bacterial diversity were immediately flash-frozen in liquid nitrogen and stored at −80°C until processing. Blank dry filters were sampled simultaneously and used as a contamination control. The two experiments were processed simultaneously. Filters were cut into pieces and DNA was extracted using the NucleoSpin Plant II Mini Kit (Macherey Nagel Ref. 740770.50) according to the manufacturer’s instructions, with an additional lysis step performed for 2 h at 56°C with 25 μL of proteinase K (20 mg mL^–1, Macherey Nagel Ref. 740506) and 100 μL of lysozyme (20 mg mL^–1, Sigma ref 4403-5g). Libraries were prepared and sequenced by Génome Québec using the 341F/785R primers (Klindworth et al., 2013 (link)). Samples were sequenced on an Illumina MySeq using 2 × 300 pb and V3 chemistry. Data were processed using the SAMBA pipeline (v3.0.1)² developed by the IFREMER bioinformatics team (SeBiMER). This resulted in 2,120 amplicon sequence variants (ASVs), which were then clustered using dbOTU3 (Olesen et al., 2017 (link)), resulting in 1,502 ASVs (29% clustering) that were assigned against the Silva v138 database (Quast et al., 2013 (link)).

DNA was purified from the seeds present in the capsules (both from 2017 and 2018 samples), after pulverization using a mortar, pestle, and liquid nitrogen. In detail, DNA was extracted from the plant powder (20 mg) by a NucleoSpin Plant II Mini Kit (Macherey-Nagel Düren, Germany), following the manufacture’s guidelines. The nucleic acid was visualized under UV light (GelDoc 2000, BIO-RAD, Hercules, CA, USA), after electrophoretic separation on 1% agarose gel containing 10 mg/mL ethidium bromide (the molecular weights were MW1-MassRuler-MW1 and MW2-GeneRuler by Thermo Fisher Scientific (Waltham, MA USA).