Development of functional markers specific for seven Pm3 resistance alleles and their validation in the bread wheat gene pool

In the ideal case, molecular markers used for marker-assisted selection are allele-specific even if the alleles differ only by a few nucleotide polymorphisms within the coding sequence of target genes. Such ‘perfect' markers are completely correlated with the trait of interest. In hexaploid wheat (Triticum aestivum L.) the Pm3 locus encodes seven alleles (Pm3a-Pm3g) conferring resistance to different races of Blumeria graminis f.sp. tritici, the agent of powdery mildew, a major disease of bread wheat. All Pm3 alleles are known at the molecular level. Here, we generated specific markers for the Pm3 alleles based on nucleotide polymorphisms of coding and adjacent non-coding regions. The specificity of these markers was validated in a collection of 93 modern or historically important cultivars and breeding lines of wheat and spelt (Triticum spelta L.). These markers confirmed the presence of the predicted Pm3 alleles in 31 varieties and lines known to carry Pm3 resistance alleles. In a few varieties, Pm3 alleles different from alleles previously described based on pathogenicity tests or tightly linked markers were observed. In all these cases, the identity of the marker-detected Pm3 alleles was confirmed by DNA sequence analysis. Pm3 markers confirmed the absence of known Pm3 resistance alleles in 54 European wheat and spelt varieties in which Pm3 alleles had not been previously identified. These results indicate that the developed markers are highly diagnostic for specific Pm3 resistance alleles in a wide range of varieties and breeding lines, and will be useful (1) for identifying Pm3 alleles in the wheat gene pool, (2) for efficient marker-assisted selection of these genes, and (3) for combining multiple Pm3 alleles within a single cultivar through transgenic approache

Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen

Accelerated adaptive evolution is a hallmark of plant-pathogen interactions. Plant intracellular immune receptors (NLRs) often occur as allelic series with differential pathogen specificities. The determinants of this specificity remain largely unknown. Here, we unravelled the biophysical and structural basis of expanded specificity in the allelic rice NLR Pik, which responds to the effector AVR-Pik from the rice blast pathogen Magnaporthe oryzae. Rice plants expressing the Pikm allele resist infection by blast strains expressing any of three AVR-Pik effector variants, whereas those expressing Pikp only respond to one. Unlike Pikp, the integrated heavy metal-associated (HMA) domain of Pikm binds with high affinity to each of the three recognized effector variants, and variation at binding interfaces between effectors and Pikp-HMA or Pikm-HMA domains encodes specificity. By understanding how co-evolution has shaped the response profile of an allelic NLR, we highlight how natural selection drove the emergence of new receptor specificities. This work has implications for the engineering of NLRs with improved utility in agriculture

No Adverse Effect of Genetically Modified Antifungal Wheat on Decomposition Dynamics and the Soil Fauna Community – A Field Study

The cultivation of genetically modified (GM) plants has raised several environmental concerns. One of these concerns regards non-target soil fauna organisms, which play an important role in the decomposition of organic matter and hence are largely exposed to GM plant residues. Soil fauna may be directly affected by transgene products or indirectly by pleiotropic effects such as a modified plant metabolism. Thus, ecosystem services and functioning might be affected negatively. In a litterbag experiment in the field we analysed the decomposition process and the soil fauna community involved. Therefore, we used four experimental GM wheat varieties, two with a race-specific antifungal resistance against powdery mildew (Pm3b) and two with an unspecific antifungal resistance based on the expression of chitinase and glucanase. We compared them with two non-GM isolines and six conventional cereal varieties. To elucidate the mechanisms that cause differences in plant decomposition, structural plant components (i.e. C∶N ratio, lignin, cellulose, hemicellulose) were examined and soil properties, temperature and precipitation were monitored. The most frequent taxa extracted from decaying plant material were mites (Cryptostigmata, Gamasina and Uropodina), springtails (Isotomidae), annelids (Enchytraeidae) and Diptera (Cecidomyiidae larvae). Despite a single significant transgenic/month interaction for Cecidomyiidae larvae, which is probably random, we detected no impact of the GM wheat on the soil fauna community. However, soil fauna differences among conventional cereal varieties were more pronounced than between GM and non-GM wheat. While leaf residue decomposition in GM and non-GM wheat was similar, differences among conventional cereals were evident. Furthermore, sampling date and location were found to greatly influence soil fauna community and decomposition processes. The results give no indication of ecologically relevant adverse effects of antifungal GM wheat on the composition and the activity of the soil fauna community

Infestation of Transgenic Powdery Mildew-Resistant Wheat by Naturally Occurring Insect Herbivores under Different Environmental Conditions

A concern associated with the growing of genetically modified (GM) crops is that they could adversely affect non-target organisms. We assessed the impact of several transgenic powdery mildew-resistant spring wheat lines on insect herbivores. The GM lines carried either the Pm3b gene from hexaploid wheat, which confers race-specific resistance to powdery mildew, or the less specific anti-fungal barley seed chitinase and β-1,3-glucanase. In addition to the non-transformed control lines, several conventional spring wheat varieties and barley and triticale were included for comparison. During two consecutive growing seasons, powdery mildew infection and the abundance of and damage by naturally occurring herbivores were estimated under semi-field conditions in a convertible glasshouse and in the field. Mildew was reduced on the Pm3b-transgenic lines but not on the chitinase/glucanase-expressing lines. Abundance of aphids was negatively correlated with powdery mildew in the convertible glasshouse, with Pm3b wheat plants hosting significantly more aphids than their mildew-susceptible controls. In contrast, aphid densities did not differ between GM plants and their non-transformed controls in the field, probably because of low mildew and aphid pressure at this location. Likewise, the GM wheat lines did not affect the abundance of or damage by the herbivores Oulema melanopus (L.) and Chlorops pumilionis Bjerk. Although a previous study has revealed that some of the GM wheat lines show pleiotropic effects under field conditions, their effect on herbivorous insects appears to be low

Development of functional markers specific for seven Pm3 resistance alleles and their validation in the bread wheat gene pool

In the ideal case, molecular markers used for marker-assisted selection are allele-specific even if the alleles differ only by a few nucleotide polymorphisms within the coding sequence of target genes. Such ‘perfect' markers are completely correlated with the trait of interest. In hexaploid wheat (Triticum aestivum L.) the Pm3 locus encodes seven alleles (Pm3a-Pm3g) conferring resistance to different races of Blumeria graminis f.sp. tritici, the agent of powdery mildew, a major disease of bread wheat. All Pm3 alleles are known at the molecular level. Here, we generated specific markers for the Pm3 alleles based on nucleotide polymorphisms of coding and adjacent non-coding regions. The specificity of these markers was validated in a collection of 93 modern or historically important cultivars and breeding lines of wheat and spelt (Triticum spelta L.). These markers confirmed the presence of the predicted Pm3 alleles in 31 varieties and lines known to carry Pm3 resistance alleles. In a few varieties, Pm3 alleles different from alleles previously described based on pathogenicity tests or tightly linked markers were observed. In all these cases, the identity of the marker-detected Pm3 alleles was confirmed by DNA sequence analysis. Pm3 markers confirmed the absence of known Pm3 resistance alleles in 54 European wheat and spelt varieties in which Pm3 alleles had not been previously identified. These results indicate that the developed markers are highly diagnostic for specific Pm3 resistance alleles in a wide range of varieties and breeding lines, and will be useful (1) for identifying Pm3 alleles in the wheat gene pool, (2) for efficient marker-assisted selection of these genes, and (3) for combining multiple Pm3 alleles within a single cultivar through transgenic approache

Molecular approaches for characterization and use of natural disease resistance in wheat

Wheat production is threatened by a constantly changing population of pathogen species and races. Given the rapid ability of many pathogens to overcome genetic resistance, the identification and practical implementation of new sources of resistance is essential. Landraces and wild relatives of wheat have played an important role as genetic resources for the improvement of disease resistance. The use of molecular approaches, particularly molecular markers, has allowed better characterization of the genetic diversity in wheat germplasm. In addition, the molecular cloning of major resistance (R) genes has recently been achieved in the large, polyploid wheat genome. For the first time this allows the study and analysis of the genetic variability of wheat R loci at the molecular level and therefore, to screen for allelic variation at such loci in the gene pool. Thus, strategies such as allele mining and ecotilling are now possible for characterization of wheat disease resistance. Here, we discuss the approaches, resources and potential tools to characterize and utilize the naturally occurring resistance diversity in wheat. We also report a first step in allele mining, where we characterize the occurrence of known resistance alleles at the wheat Pm3 powdery mildew resistance locus in a set of 1,320 landraces assembled on the basis of eco-geographical criteria. From known Pm3 R alleles, only Pm3b was frequently identified (3% of the tested accessions). In the same set of landraces, we found a high frequency of a Pm3 haplotype carrying a susceptible allele of Pm3. This analysis allowed the identification of a set of resistant lines where new potentially functional alleles would be present. Newly identified resistance alleles will enrich the genetic basis of resistance in breeding programmes and contribute to wheat improvement