Powdery mildew (causative pathogen: Blumeria graminis f. sp. tritici) is an important foliar disease of bread wheat in cooler environments. To date, a large number of both major genes (denoted Pm) and quantitative trait loci associated with powdery mildew resistance have been described in the literature (McIntosh et al., Reference McIntosh, Yamazaki, Dubcovsky, Rogers, Morris, Somers, Appels and Devos2008, Reference McIntosh, Dubcovsky, Rogers, Morris, Appels and Xia2009, Reference McIntosh, Dubcovsky, Rogers, Morris, Appels and Xia2010, Reference McIntosh, Dubcovsky, Rogers, Morris, Appels and Xia2011), and one (Pm3) of the former genes has been successfully isolated (Yahiaoui et al., Reference Yahiaoui, Strichumpa, Dudler and Keller2004). While the majority of these genes have been identified within the primary gene pool represented by Triticum spp., some have also originated from the closely related genera Secale and Aegilops. The potential of the more distantly related species Aegilops markgrafii (syn. Aegilops caudata L.; 2n = 2x = 14) as a donor for powdery mildew resistance prompted Schubert (Reference Schubert1991) to construct a series of bread wheat (cultivar ‘Alcedo’)/A. markgrafii (accession ‘S 740-69’) single chromosome addition lines. Spontaneous genetic rearrangements occurring during the necessary crossing programme later allowed for the selection of powdery mildew-resistant euploid derivatives having a largely cultivar ‘Alcedo’ background. Genetic analysis of some of these materials led to the conclusion that resistance factors had been transferred to both chromosomes 1A and 7A (Junghans, 1995). Here we deployed a standard genetic mapping approach to characterize the genomic location of powdery mildew resistance present in two of the best performing introgression lines.
Experimental
The two powdery mildew-resistant introgression lines ‘EgIII’ and ‘EgIV’ were each crossed with the powdery mildew-susceptible cultivar ‘Kanzler’, and the resulting F2 populations, along with the parental material and the cultivar ‘Alcedo’, were raised in a greenhouse. The first fully expanded seedling leaf was removed from each plant for use in a detached leaf in vitro resistance test. The distal centimetre of the leaf was discarded, and the remaining 3 cm laid on agar containing benzimidazol (35 ppm) and silver nitrate (1.5 ppm). The leaves were then inoculated with a mixed population of the pathogen (composed of spores produced by isolates carrying virulences against Pm1, Pm2, Pm3a, Pm3b, Pm3c, Pm3d, Pm4b, Pm5, Pm6, Pm8, Pm9 and Pm17) and held at 16°C under a 16 h/8 h light/dark photoperiod (light intensity >10,000 lx). At 10 d after the inoculation, a disease score was assigned to each segment, ranging from 0 (no visible symptoms) to 4 (clearly defined powdery mildew pustules), following McIntosh et al. (Reference McIntosh, Wellings and Park1995). For the characterization of the adult plant response, the same set of seedlings was first fully vernalized (8 weeks at 4°C), and then planted outside in plots of 20 plants each, with each plot surrounded by similarly sized, earlier sown plots comprising the fully susceptible cultivars ‘Pallas’ and ‘Kanzler’. Disease spread was achieved by including within the plots pot-grown plants of the cultivar ‘Kanzler’ which had been artificially inoculated in a greenhouse. The same scale of resistance/susceptibility was applied as for the seedling test. The F2 segregation of disease scores among both seedling and adult plants and plant numbers investigated are shown in Fig. 1.
Fig. 1 Segregation for powdery mildew resistance expressed at seedling (JPT) and adult plant (APT) stages in F2 populations derived from the crosses ‘EgIII’ x cultivar ‘Kanzler’ and ‘EgIV’ x cultivar ‘Kanzler’ (lower disease scores represent higher levels of resistance).
The genotypic analysis initially involved 30 simple sequence repeat (SSR) loci, of which 15 are known to map to chromosome 1A and 15 to chromosome 7A. The polymerase chain reaction conditions applied were those detailed by Röder et al. (Reference Röder, Korzun, Wendehake, Plaschke, Tixier, Leroy and Ganal1998). Following the outcome for the cultivars ‘Kanzler’, ‘EgIII’ and ‘EgIV’, the SSR assays GWM1223, GWM0033b (chromosome 1A) and GWM0698, GWM1061 (chromosome 7A) were applied to DNA extracted from each of the F2 individuals. When the phenotypic (powdery mildew resistance score) and genotypic (Xgwm1223, Xgwm0033b, Xgwm0698 and Xgwm1061) scores were inserted into the QGENE program (Nelson, Reference Nelson1997), the resulting analysis indicated the presence of two loci, denoted QPm.ipk-1A and QPm.ipk-7A, mapping to the distal ends of chromosome arms 1AS and 7AL, respectively (Fig. 2). The LOD score associated with the former locus was 6.8 at the seedling stage and 5.2 at the adult stage, while the latter locus was only expressed significantly (LOD 13.4) at the seedling stage. The alleles Xgwm1223, Xgwm0033b, Xgwm0698 and Xgwm1061 present in ‘EgIII’ and ‘EgIV’ were inherited from ‘S 740-69’, so the assumption is that both resistances were derived from A. markgrafii.
Fig. 2 Map positions of the loci underlying powdery mildew resistance on the chromosome arms 1AS and 7AL. LOD scores associated with both resistances at seedling (JPT) and adult plant (APT) stages are shown.
Discussion
Various Aegilops species have been identified as potential sources of disease resistance in bread wheat (McIntosh et al., Reference McIntosh, Yamazaki, Dubcovsky, Rogers, Morris, Somers, Appels and Devos2008). The A. markgrafii parent of ‘EgIII’ and ‘EgIV’ was also resistant to leaf rust (Schubert, Reference Schubert2001; Weidner, Reference Weidner2004), and a leaf rust-resistant locus derived from the donor was successfully mapped to chromosome arm 2AS by Iqbal et al. (Reference Iqbal, Eticha, Khlestkina, Weidner, Röder and Börner2007). Here we have confirmed that powdery mildew resistance present in ‘EgIII’ and ‘EgIV’ relies on gene(s) located on chromosomes 1A and 7A, as suggested by Junghans (1995), and shown that these resistance factors both map to the distal ends of the two chromosome arms 1AS and 7AL. Both these genomic regions are known to carry genes responsible for powdery mildew resistance. The 1AS-located gene Pm3 maps 2.3 cM from Xgdm0033 (Bouget et al., Reference Bouget, Lemoine, Pavoine, Barloy and Doussinault2002), an SSR locus which is closely linked to Xgwm1223b (Ganal and Röder, Reference Ganal, Röder, Varshney and Tuberosa2007). The possibility is therefore that QPm.ipk-1A is a homoeoallele of the multi-allele locus Pm3. It should be noted that, to date, all known Pm3 resistance alleles have originated from within the primary bread wheat gene pool, so if QPm.ipk-1A does proves to be a Pm3 homoeoallele, this would represent a new departure, as may also be the case for Pm8 of rye (Keller, pers. commun.). The chromosome arm 7AL is the site of both Pm1 and Pm37, genes which can be traced to Triticum spp. donors. The two genes lie within 16 cM of one another, but while Pm37 is separated from Xgwm332 by just 0.5 cM (Perugini et al., Reference Perugini, Murphy, Marshall and Brown-Guedira2007), Pm1 co-segregates with Xpsr687 (Neu et al., Reference Neu, Stein and Keller2002). Based on the Paillard et al. (Reference Paillard, Schnurbusch, Winzeler, Messmer, Sourdille, Abderhalden, Keller and Schachermayr2003) linkage map (which includes both Xgwm332 and Xpsr687), QPm.ipk-7A is more likely to be related to Pm1 than to Pm37, because Xpsr687 maps to the distal region of chromosome 7AL, whereas Xgwm332 is located closer to the centromere.
Acknowledgements
The powdery mildew isolates used for inoculation were kindly provided by Dr K. Flath (Julius Kühn Institute, Kleinmachnow, Germany). We thank Mrs Marion Fischer for her assistance in performing the field experiments, and Mrs Ellen Weiß for her contribution to the genotypic analyses.
