Recent advances in cloning of disease resistance (R) genes from plants identified several classes of sequences responsible for the initiation of the plant response to pathogen attack (review in Hammond-Kosack and Jones, 1997). In addition, R genes have been frequently observed to reside in multigene clusters and appear to be widely distributed throughout plant genomes. Many of these genes across different plant species share conserved motifs, which can be utilized as convenient "tags" for the isolation of resistance gene analogs (RGAs). Our objectives are to isolate the majority of RGA sequences from barley, to map them, and to identify and characterize disease resistance gene clusters. We utilized primers designed for several classes of RGAs to amplify these gene families from the barley genome and used them as probes to screen a barley cv. Morex bacterial artificial chromosome (BAC) library (6.3 X genome equivalents, average insert size 106 kb) developed by Dr. Rod Wing at Clemson University. Screening this library with NBS-LRR RGA probes (derived from b2, b3, b4, b5, b6, b7, b9 sequences published by Leister et al., 1998) identified 81 positive BAC clones. Moreover, over 200 b7-containing BACs were identified that have not been characterized. Probes derived from the receptor kinase sequences Xa21 (Song et al., 1995), Lr10 (Feuillet et al., 1997) and LM identified 31, 10 and 4 BAC clones, respectively. We utilized a modified AFLP-based approach to fingerprint individual BACs, to identify overlapping clones and to create sequence contigs. BACs were digested with two restriction enzymes, e.g., EcoRI and MseI, PstI and MspI. After digestion, adaptors were ligated to the restriction fragments and the resulting template was subjected to PCR amplification with fluorescently tagged primers corresponding to the adaptors. Products were visualized using an ABI377 automatic sequencer. We observed 40-50 amplified fragments per 100 kb BAC. Molecular weights of the amplified fragments were determined and stored in a database. Contigs were assembled from multiple restriction enzyme digestions. This approach permits us to categorize and order a large number of individual BAC clones. Structural analysis (sequencing and contig building) of these gene clusters will provide insight into the possible mechanisms of duplication and divergence of the R genes and provide clues about the evolution of pathogen specificities. Furthermore, we anticipate that our results will be useful for marker assisted selection in barley breeding programs and for the eventual isolation of R genes from barley and other Triticeae.

References:

Feuillet C., G. Schachermayr and B. Keller. 1997. Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat. Plant J. 11:45-52.

Hammond-Kosack K., J. Jones. 1997. Plant disease resistance genes. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:575-607

Leister D., J. Kurth, D. Laurie, M. Yano, T. Sasaki, K. Devos, A. Graner, P. Schulze-Lefert. 1998. Rapid reorganization of resistance gene homologues in cereal genomes. Proc.Natl. Acad. Sci. USA 95:370-375

Song W.-Y., G. Wang, L. Chen, H. Kim, L. Pi, T. Holsten, J. Gardner, B. Wang, W. Zhai, L. Zhu, C. Fauquet, P. Ronald. 1995. A receptor kinase protein encoded by the rice disease resistance gene Xa21. Science 270:1804-1806