Virus Detection and Quantification

RNA from cassava was extracted using an RNA extraction kit following the manufacturer’s protocol (Epoch, United States). The integrity of the RNA was analyzed by gel electrophoresis, and RNA was quantified in a Qubit^®fluorometer (Thermo Fisher Scientific, United States) using the Qubit RNA BR Assay Kit (Thermo Fisher Scientific, United States). Virus analysis by qRT-PCR was carried out by either an ep realplex4 (Eppendorf, Germany) Mastercycler equipped with the realplex4 software or a qTOWER³ (Analytik Jena, Germany) equipped with the qPCRsoft evaluation software.

For virus quantification in the varieties Albert and Namikonga, a SYBR Green qRT-PCR assay was performed using published primer sets (Adams et al., 2013) and cassava 4.1_010236 (acyl co-binding A) as an internal control for relative virus quantification (Hu et al., 2016; Supplementary Table 1).

One microliter of total RNA (20 ng/μl) was converted into cDNA in a reaction mixture containing 5 μl of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) buffer (2×), 0.25 μl of dNTPs, 0.3 μM oligo(dt), 0.3 μM random hexamer primers, 0.2 μl of M-MLV RT, and nuclease-free sterile water to reach a reaction volume of 25 μl. cDNA synthesis was done for 30 min at 43°C and diluted fivefold prior to SYBR Green Kapa (PEQLAB, Germany) qPCR. Reaction mixtures contained 10 μl of SYBR Master Mix, 0.15 μM CBSV primers (0.1 μM for UCBSV and for cassava 4.1_010236 primers), 5 μl of diluted cDNA template, and nuclease-free sterile water to a total reaction volume of 20 μl. Control reactions, non-template water, RNA from healthy cassava, and RNA from UCBSV-infected cassava were included in every reaction series to conduct qRT-PCR with two technical replicates. After an initial denaturation step for 3 min at 95°C, each two-step cycle consisted of denaturation for 10 s at 95°C and synthesis for 30 s at 60°C. PCR was done for 36 cycles followed by melting curve analysis to assess the specificity of the amplification.

To confirm presence/absence of U/CBSV in cassava, a one-step TaqMan assay was conducted (TaqMan Kit Maxima Probe/ROX qPCR Master Mix, Thermo Fisher Scientific, Germany) with virus-specific primers and probes and COX (cytochrome oxidase) as an internal control (Kaweesi et al., 2014; Supplementary Table 1). Reaction mixes for qRT-PCR contained 12.5 μl of Maxima Probe qPCR Master Mix (2×), 0.3 μM CBSV primers, and probe CBSV primers (0.4 μM for COX primers and probe, 0.4 μl UCBSV primers and probes), 5 μl of template, 0.15 μl of M-MLV RT, and nuclease-free sterile water to a total reaction volume of 25 μl. Each RNA was analyzed in two qRT-PCRs, and controls were included in every series. One-step qRT-PCRs were incubated for 30 min at 43°C for cDNA synthesis followed by an initial denaturation step for 2 min at 95°C and 40 cycles of denaturation (15 s at 95°C), annealing (30 s at 60°C), and synthesis (30 s at 72°C).

Cycle threshold (CT) values were used to calculate U/CBSV expression using the 2^−ΔΔCt method (Livak and Schmittgen, 2001). Virus expression values were estimated relative to CBSV-Mo83 concentrations determined in the uppermost youngest leaf. For variety, Albert recordings at 10 dag were taken for reference, while for the variety Namikonga, qRT-PCR reference measurements were taken 20 days after infection (dag) because of the delayed infection. To quantify virus in the South American cassava germplasm, expression values were calculated relative to CBSV-Mo83 concentrations recorded in the sixth uppermost leaf of the variety TMS-96/0304.