The presence of the seven-transmembrane (7-TM) domain Mlo (mildew resistance locus o) protein is a prerequisite for successful colonization of barley (Hordeum vulgare L.) by the biotrophic powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh). The mlo-mediated resistance response is dependent on at least two genes, Ror1 and Ror2 (required for mlo resistance). Double mutant mlo ror1 partially restores susceptibility to the fungus and exhibits reduced spontaneous leaf cell death. The Ror1 gene represents an interesting target for characterization, since its isolation could reveal an unknown pathway or additional molecular components necessary for effective mlo resistance. Nevertheless, despite extensive prior efforts to clone the Ror1 gene, its nature remains unknown. In this project, we pursued an alternative approach to isolate the Ror1 gene performing chromosome walking using a barley YAC library combined with Next Generation Sequencing (NGS) techniques. Previously, the Ror1 gene was located on barley chromosome 1H to an interval of 0.15cM between two predicted flanking genes, Cons and Pol. These two genes were used to design primers to screen a barley YAC library by PCR. The isolated YAC clones and additional overlapping ones, discovered by chromosome walking, formed the basis of our YAC contig at the Ror1 region. The YAC clones were paired-end sequenced by Illumina. Thorough analysis of the sequences revealed that we obtained two non-overlapping YAC contigs around the Ror1 locus. Eight annotated genes present in the contigs were selected as candidate genes. However, by “pseudo-mapping” in a Ror1 recombinant population and positioning in the YAC contigs, seven of the eight genes were excluded to encode Ror1. Furthermore, comparative genomics of barley with three other model grasses, Oryza sativa, Brachypodium distachyon and Sorghum bicolor revealed re-arrangements in the Ror1 region. Additionally, for the first time, we could physically locate the Ror1 region on barley chromosome 1H using Fluorescence In Situ Hybridization. Further chromosome walking steps are required to bridge the gap between Pol and Cons and complete the Ror1 YAC contig. The analysis of newly isolated YAC clones/pools that can be used to extend the YAC contigs is currently in progress. Our approach combining classical genetics and second-generation sequencing technologies has opened a new door that can potentially lead to the isolation of the Ror1 gene
