Cloning, genetic and physical mapping of resistance gene analogs in barley (Hordeum vulgare L.)

Abstract

The majority of verified plant disease resistance genes isolated to date belong to the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. Using degenerate primers, designed from the conserved motifs of the NBS region in tobacco N and Arabidopsis RPS2 genes, we isolated 190 resistance gene analogs (RGA) clones from barley genomic DNA. A total of 13 single- and low-copy RGAs were genetically mapped onto chromosomes 1H-7H (except 5H) using three barley double haploid (DH) mapping populations: Steptoe × Morex, Harrington × TR306 and LUGC × Bowman. Sequence analysis of the RGAs showed that they are members of a diverse group. As a result of BLAST searches, one RGA proved unique as it did not detect any significant hit. Another RGA is putatively functional, because it detected several barley expressed sequence tag (EST) matches. To physically map the RGAs, 13 sequences were used to screen a 6.3 × cv. 'Morex' bacterial artificial chromosome (BAC) library. After fingerprint analysis, eight contigs were constructed incorporating 62 BAC clones. These BAC contigs are of great value for positional cloning of disease resistance genes, because they span the regions where various barley R genes have been genetically mapped. © 2006 Blackwell Verlag.