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Log10 (WYMV/Actin)
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-6
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ELISA absorbance
Supplementary Fig. S5. Validation of the TaqMan Real-time PCR and ELISA assays for segregating
plants. The log10W/A ratio and the ELISA extinction of leaf samples from segregating 200 F2 plants
of the cross TK3  Hokushin was shown. About 1000 F2 progeny bred from the cross TK3  Hokushin
were raised in a WYMV-contaminated field at Naka-Mareppu, Date, Hokkaido. We generated a set
of 200 F2 plants homozygous for the Madsen chromosome 2D WYMV resistance QTL as selected by
genotyping with markers gwm157 and wmc041 flanking the 2D QTL reported by Takeuchi et al.
(2010), but segregated for the Qym2 in chromosome 3B. For validation of the TaqMan assay, a
single leaf was sampled from each plant and was longitudinally split into two parts along the
midrib; one half of the leaf was used for the TaqMan-based assay, and the other for ELISA. The
plate-trapped antigen ELISA elaborated by Clark (1981) was used with a polyclonal anti-WYMV
antiserum prepared following Uyeda et al. (1998). The log10W/A ratios were bimodally distributed,
with one peak ranging from +2 to +4 (41 individuals) and the other (159 individuals) from –6 to zero
(Supplementary Fig. S5). A χ2 analysis of the F2 population was not done, because Suzuki et al.
(2015) recommended the necessity of an F3 progeny test, given the low penetrance of Qym2. In
contrast, the frequency distribution produced by the ELISA was not discontinuous, so it was not
possible to apply a threshold value to divide the population into a resistant and a susceptible class.
Unlike the ELISA, the TaqMan assay readily split the 200 TK3  Hokushin F2 progeny into two distinct
classes. Perovic et al. (2009) suggested that an ELISA value of 0.5 at A405nm could serve as a
threshold for distinguishing between SBCMV resistant and susceptible plants. Under this criterion,
the TK3  Hokushin F2 progeny AH-191 (ELISA of 0.37), AH-98 (0.38) and AH-136 (0.39) would all
have been classified as resistant, but their log10W/A ratios were, respectively, +3.5, +2.6 and +2.8,
well within the susceptible category according to the TaqMan test. The threshold ELISA value may
vary according to growing conditions, genetic background of the host or the sensitivity of the
polyclonal antiserum used. Although the ELISA test is more rapid than the TaqMan assay, the latter
is saturated, not quantitative, but rather qualitative. It provides a specific, sensitive and robust
technique to quantify WYMV, suitable as a means of genetically analyzing resistance to the virus.