Plant & Animal Genome VI Conference

P175

CLONING AND CHARACTERIZATION OF RESISTANCE GENE ANALOGS FROM WHEAT

USDA-ARS, Department of Botany and Plant Pathology, 1155 Lilly Hall, Purdue University, West Lafayette, Indiana 47907-1155

Degenerate primers based on the conserved nucleotide binding sequence (NBS) of the cloned plant disease resistance genes N, L6 and RPS2 were used to amplify genomic DNA of wheat lines that were near-isogenic for a powdery mildew resistance gene. Amplification products in the anticipated 500 base-pair size range were extracted from agarose gels and cloned using a TA cloning kit. Following color selection, clones containing inserts of approximately 500 base pairs were identified by digesting plasmid DNA with the restriction enzyme Eco RI and separating the fragments by gel electrophoresis. Restriction analysis of approximately 300 randomly chosen clones using four enzymes with 4- or 6-base recognition sequences separated the putative NBS sequences into at least 10 different classes. Preliminary sequencing of some clones and analyses of the probable translation products revealed that all encoded amino acid sequences are highly conserved and shared the same conserved motifs found in the cloned plant disease resistance genes N, L6 and RPS2. Representative clones from each of the 10 resistance gene analog classes are currently being sequenced, and tested for linkage to known powdery mildew resistance genes by Southern analysis of near-isogenic wheat lines.