EcoTILLING for the identification of allelic variation in the powdery mildew resistance genes mlo and Mla of barley

In this investigation the successful implementation of a CEL I-based mutation detection technique for the discovery and detection of DNA polymorphism in the genes mlo and Mla of barley is described. The technique is called EcoTILLING, which is a high-throughput method to detect and discover new point mutations and small insertions/deletions in DNA. That the method not only reveals polymorphism between different alleles but can also be used as a powerful genetic marker is demonstrated. The genes mlo and Mla are involved in the defence of barley against the fungal pathogen powdery mildew. The powdery mildew resistance gene mlo is a single copy gene, whereas multiple alleles exist at the Mla locus. With EcoTILLING it was possible to identify point mutations and deletions in each of the 11 mlo mutants tested. For Mla 25 natural barley variants were tested, and although the identification was complex due to the presence of highly similar paralogues of Mla, most of the recently identified alleles from Hordeum vulgare ssp. spontaneum were identified. This method offers the possibility to combine different mlo alleles with different Mla alleles from wild barley to obtain cultivars with more durable resistance.