Nucleospin gel and pcr clean up

A portion of λDNA (NIPPON GENE CO., LTD, Japan) was duplicated by PCR amplification using a biotin-modified forward primer and a biotin-unmodified reverse primer to obtain biotin-modified 400-bp dsDNA. PCR amplification was performed using MiniAmp Plus Thermal Cyclers (Thermo Fisher Scientific, Waltham, MA). Both ssDNA and primer were purchased from Sigma Aldrich, Japan. The sequences of the forward and reverse primers were 5′-TGCAACGAACAGGTCACTATCA-3′ and 5′-GAGCAAAGCAAAACAGGCGTA-3′, respectively. PCR amplification was performed in 50-μL reaction volumes consisting of 0.3 μM forward primer, 0.3 μM reverse primer, 50 ng λDNA, 1 U KOD-Plus- Neo, 1X PCR buffer for KOD -Plus- Neo, 0.2 mM dNTPs, and 1.5 mM MgSO₄ (TOYOBO CO., LTD., Japan). The cycling parameters of PCR were 94 °C for 2 min → [98 °C for 10 sec → 58 °C for 30 sec → 68 °C for 30 sec] × 35 times. The product obtained after PCR was purified using NucleoSpin® Gel and PCR Clean-up (Takara Bio Inc., Japan). The purified product was then quantitated with a Qubit fluorometric system (Life Technologies). A biotin-streptavidin binding reaction was performed in 18.3-μL reaction volumes consisting of 1.5 μM biotin-modified dsDNA and 10 μM streptavidin. The time and temperature of the binding reaction were 37 °C and 30 min, respectively.

cDNA of the full-length BVF genome was synthesized from total RNA by using Superscript IV reverse transcriptase (Invitrogen) with the primer BVF_NotIEcoRI Rev (Table 1). The entire viral genome (6.8 kb) was amplified with the CloneAmp HIFI PCR mix (Takara) using the primers BVF_XbaI Fw and BVF_NotIEcoRI Rev (Table 1). The forward primer BVF_XbaIT7 Fw contained an engineered XbaI site and the T7 promoter sequence followed by part of the BVF 5′ end sequence of the viral genome. The reverse primer BVF_NotIEcoRI Rev introduced a poly (A)₁₈ tail and NotI and EcoRI sites at the 3′ end of the viral genome for linearization and cloning purposes. The resultant PCR product was gel purified with NucleoSpin Gel and PCR Clean-up (Takara, San Jose, CA, USA) and ligated into pUC19 vector between XbaI and EcoRI sites by using the T4 DNA ligase (ThermoScientific, Waltham, MA, USA) to generate the clone pUC19-BVF. Recombinant plasmid DNA were extracted and purified using the Zyppy plasmid miniprep kit (Zymo Research, Irvine, CA, USA) and checked for the presence of the BVF genome by restriction digestion and sanger sequencing. The strategy followed to construct the full-length cDNA clone of BVF is shown in Figure 3A.

Allelic variation in the Ecp20-2 gene was determined for 120 strains of C. fulvum (Supplementary Table 2), including a previously reported collection (Iida et al., 2015 (link)). Genomic DNA isolation and PCR amplification were described as above. Of note, 618 bp containing the Ecp20-2 gene was PCR-amplified using primers as described in Supplementary Table 1. PCR amplicons were excised from 0.8% agarose gels and purified from agarose gels using the NucleoSpin Gel and PCR Clean-up (TakaraBio, Shiga, Japan). Sequences were determined using an Applied Biosystems 3730xl DNA analyzer (Thermo Fisher Scientific, Waltham, MA, United States). To evaluate DNA sequences surrounding the Ecp20-2 gene for the presence of transposable elements (TE), a size of 10 kb sequence upstream and downstream of Ecp20-2 (scf7180000130934) was obtained from the C. fulvum genome at JGI (de Wit et al., 2012 (link)). To screen the query sequence against a reference collection of repeats, it was uploaded to RepBas (Kohany et al., 2006 (link)) with default settings. Output data revealed the type and class of transposable elements.