Qiaxcel advanced instrument

Sequence-based typing (SBT) was conducted in accordance with the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Legionella Infections (ESGLI) sequence-based typing (SBT) scheme [14 (link)]. Briefly, PCR amplification with Taq DNA polymerase (Qiagen, Hilden, Germany) was performed for extracted DNA, then PCR products were analyzed by capillary gel electrophoresis on a QIAxcel Advanced instrument (Qiagen, Hilden, Germany). PCR products were purified and prepared for the sequencing reaction. Sanger sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit protocol and read with Genetic Analyzer 3500 (Applied Biosystems, Waltham, MA, USA). New allelic profiles were submitted to the ESGLI SBT database [15 ].

The QIAGEN One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify the genes of NoV in a 50-μL reaction volume. RNase Inhibitor (Promega, Madison, WI, USA) was added at a final concentration of 5–10 units/reaction. The revised primers 289/290 (p290, nt4568–4590, GATTACTCCAGGTGGGAYTCMAC; p289, nt4865–4886, TGACGATTTCATCATCMCCRTA; positions are indicated relative to the M87661 reference sequence) were used to amplify the partial RdRp gene of NoV (319 bp) [11 (link)]. RT-PCR was performed at 42 °C for 60 min and 95 °C for 15 min followed by 40 cycles of 94 °C for 30 s, 58 °C for 80 s, and 72 °C for 60 s; a final extension was run at 72 °C for 7 min. Primers G1SKF/G1SKR and G2SKF/G2SKR were used to amplify the partial VP1 genes of GI and GII NoVs, generating 330 bp and 344 bp PCR products [12 (link)]. RT-PCR was performed at 50 °C for 30 min and 95 °C for 15 min, followed by 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s; a final extension was run at 72 °C for 7 min. The ORF1/ORF2 junction region (1095 bp) was amplified using primers 290 and G2SKR, and RT-PCR was performed under the same reaction conditions for the VP1 gene of NoV. The PCR products were analyzed using a QIAxcel Advanced Instrument with a QIAxcel DNA Screening Kit (QIAGEN, Hilden, Germany).

Prior to DNA extraction, the bacterial cultures were grown on blood agar (Biolife Italiana, Monza, Italy). For the DNA extraction, the commercially available kit “NucleoSpin Tissue” (Macherey-Nagel, Düren, Germany) was used. Since enterococci are Gram-positive bacteria, a protocol for hard-to-lyse bacteria was used. The protocol included an additional lysozyme step to hydrolyse the peptidoglycan glycosidic bonds in order to obtain better-quality DNA; after that, the standard protocol nr.5 was followed. The purity of the samples was assessed using a NanoDrop spectrophotometer and the quantity was measured using a Qubit fluorometer (both ThermoFisher Scientific, Landsmeer, The Netherlands).
To prepare the libraries for the whole-genome sequencing, an Illumina DNA prep (Illumina, San Diego, CA, USA) library preparation kit was used by following the provided instructions. For a sample quality assessment before pooling, the gel capillary electrophoresis (QIAxcel Advanced Instrument, QIAGEN, Venlo, Limburg, The Netherlands) was used with a high-resolution cartridge and a 50–1500 bp size marker.
The library was sequenced using the Illumina MiSeq next-generation sequencing system (Illumina, San Diego, CA, USA).

The genetic diversity analysis was conducted using 11 SSRs already identified by Urbanavičiūtė et al. (2023) (link). The markers were already demonstrated as highly associated with chromosomal regions related to root traits (Supplementary Table S2). The genomic DNA of each accession was extracted from fresh leaves using the PureLink Plant Total DNA Purification kit (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). The PCRs were carried out following the manufacturer’s instruction of GoTaq G2 DNA Polymerase (Promega, Madison, WI, USA) for mixture protocols and cycling conditions. The amplifications were run in SwiftMaxi Thermal Cyclers (Esco Technologies, St. Louis, MO, USA) using different annealing temperatures for each primer (Supplementary Table S3). The amplicons were further analyzed through capillary electrophoresis with the device QIAxcel Advanced Instrument (Qiagen, Hilden, Germania) using the QIAxcel DNA High-Resolution Kit, and the amplifications were validated using QIAxcel ScreenGel Software (Qiagen, Hilden, Germany). Each amplicon was named according to its molecular weight; the raw data are reported in Supplementary Table S4 and the Zenodo repository at the 10.5281/zenodo.10439459.

Genomic DNA was extracted following the genomic Wizard^® Genomic DNA KIT protocol (cat. No. A1120, Promega, Madison, WI, USA). PCRs of 16S rDNA sequences were completed using a Sensoquest Sensodirect thermocycler from Germany. PCRs were performed using a forward primer: 5′-AGA GTT TGA TCM TGG CTC AG-3′ [19 (link)]. Additionally, a reverse primer: 5′-CCG TAC TCC CCA GGC GGG G-3′ [20 (link)], which amplified 810 bp, was used. The PCR reactions were completed using a 2G Fast ReadyMix Kit (cat. No. KK5102, Merck, Taufkirchen, Germany). The PCR reactions were carried out at a total volume of 25 µL, containing 5 µL of DNA template (50 ng/L), 12.5 µL of 2G Fast ReadyMix, 6.5 µL of RNAse-free water, 0.5 µL of forward primer, and 0.5 µL of the reverse primer. Amplification was carried out in 35 cycles as follows: denaturation for 60 s at 92 °C, primer annealing for 60 s at 54 °C, and polymerization for 90 s at 72 °C. Bends were detected with the Qiaxcel Advanced instrument (Qiagen, Hilden, Germany) according to the protocol, then sequenced using the Sanger method. The results of the sequencing were analyzed phylogenetically using Mega version 11 software.