Figure 1. Mapping of epigenetic quantitative trait loci (epiQTL) controlling transgenerational resistance against Hyaloperonosopra arabidopsidis (Hpa).

(a) Levels of Hpa resistance in 123 epiRIL lines, the ddm1-2 line (F4; red triangle) and six Wt lines (Col-0; green triangles). Top graph shows distribution of infection classes in each epiRIL; blue triangles pinpoint the eight most resistant epiRILs with statistically similar levels of Hpa colonisation as the ddm1-2 line (Pearson’s Chi-squared test, p>0.05). Bottom graph shows variation in Hpa resistance index (RI). Green bars: Wt lines; red bar: ddm1-2; blue bars eight most resistant epiRILs (n > 100). (b) Linkage analysis of RI (blue line) and green leaf area (GLA) of three-week-old seedlings (green). Green bars at the bottom represent chromosomes. Red line represents the threshold of significance. Peak DMR markers with the highest LOD scores are shown on top. (c) Correlation plots between peak marker haplotype (methylated Wt versus hypomethylated ddm1-2) and RI (blue) or GLA (green). (d) Percentages of resistance variance explained by the peak DMR markers, including covariance between markers (orange).

Figure 1—figure supplement 1. Representative examples of infection classes used for quantification of Hpa resistance.

Shown are trypan blue-stained Arabidopsis leaves at 6 days after spray-inoculation with Hpa. White (class I), absent or minimal colonisation; light blue (class II),≤50% leaf area colonised by the pathogen; dark blue (class III), ≤75% leaf area colonised by the pathogen, presence of conidiophores; black (class IV), >75% leaf area colonised by the pathogen, conidiophores and abundant sexual spores. Green arrows indicate colonisation by pathogen hyphae; blue arrows indicate hyphae surrounded by trailing necrosis, red arrows indicate conidiophores, black arrows indicate sexual oospores. Insets on the right show higher magnifications of colonisation markers. Scale bar = 50 μm.

Figure 1—figure supplement 2. Average green leaf area (GLA) of the 123 epiRILs (light green), the ddm1-2 line (F4; red) and six Wt lines (Col-0; dark green).

Shown are average GLA values of three-week-old plants (±SEM).

Figure 1—figure supplement 3. Resistance phenotypes of the eight most Hpa-resistant epiRILs against different (a) biotic stresses.

(a) Confirmation of resistance against biotrophic Hpa. Shown are levels of infection at 6 days post-inoculation (dpi) of 3-week-old plants. Trypan blue-stained leaves were analysed by microscopy and assigned to 4 Hpa infection classes (insets on the right; see Figure 1—figure supplement 1 for further details). Statistically significant differences in class distribution (asterisks) were analysed using Pearson’s Chi-squared tests (p<0.05) in pairwise comparisons with Wt line (#602); n > 80. (b) Quantification of resistance against necrotrophic Pletosphaerella cucumerina (Pc). Shown are average lesion diameters (±SEM) at 9 days after droplet inoculation with Pc spores onto similarly aged leaves of 5-week-old plants. Insets show representative examples of necrotic lesions by Pc. Statistically significant differences in necrotic lesions diameter (asterisks) were quantified by two-tailed Student’s t-test (p<0.05) in pairwise comparisons with Wt line (#602); n = 40–48. (c) Quantification of salt (NaCl) tolerance. Shown are percentages of seedlings developing full cotyledons after 6 days of growth on agar with increasing NaCl concentrations. Statistically significant differences in germination rates (asterisks) were quantified by Fisher’s exact test (p<0.05) in pairwise comparisons with Wt line (#602) at each salt concentration; n > 50.

Figure 1—figure supplement 4. Defence marker phenotypes of the eight most Hpa-resistant lines.

(a) Relative expression of SA-dependent PR1 at 48 and 72 hpi with Hpa (red) or water (blue). Shown are mean relative expression values (±SEM). Statistically significant differences in relative expression (asterisks) were quantified by two-tailed Student’s t-test (p<0.05) in pairwise comparisons with Hpa-treated Wt line (#602); n = 3 (b) Resistance efficiency of callose deposition in Hpa-inoculated plants. Shown are percentages of arrested (light) and non-arrested (dark) germ tubes at 48 hpi. Insets show representative examples of aniline-blue/calcofluor-stained leaves by epi-fluorescence microscopy (bars = 100 μm; yellow indicates callose; blue indicates Hpa). Statistically significant differences in resistance efficiency of callose (asterisks) were analysed using Pearson’s Chi-squared tests (p<0.05) in pairwise comparisons with Wt line (#602); n > 150.

Figure 1—figure supplement 5. Transgenerational stability of Hpa resistance in Hpa-resistant epiRILs.

Five individual F9 plants from the eight most resistant epiRILs and the Wt line (#602) were self-pollinated to generate 40 F10 families. Plants of each F10 family were analysed for Hpa colonisation at 6 dpi. (a) Shown are frequency distributions of leaves across 4 Hpa colonisation classes (insets on the right; see Figure 1—figure supplement 1 for further details). (b) Resistance index (RI) values of the F10 families. The red line indicates the average RI value of the Wt (#602). Asterisks at the top of each graph indicate statistically significant differences in class distribution between pooled F10 families of the epiRIL relative to pooled F10 families of the Wt line (Pearson’s Chi-squared test; p<0.05). Crosses (†) at the bottom of each graph indicate statistically significant differences between F10 families within each epiRIL (Pearson’s Chi-squared test; p<0.05).