Isolation, sequence analysis, and linkage mapping of resistance-gene analogs in cowpea (Vigna unguiculata L. Walp.)

Degenerate oligonucleotides designed to recognize conserved coding regions within the nucleotide binding site (NBS) and hydrophobic region of known resistance (R)genes from various plant species were used to target PCR to amplify resistance gene analogs (RGAs) from a cowpea (Vigna unguiculata L. Walp.) cultivar resistant to Striga gesnerioides. PCR products consisted of a group of fragments approximately 500 bp in length that migrated as a single band during agarose gel electrophoresis. The nucleotide sequence of fifty different cloned fragments was determined and their predicted amino acid sequences compared to each other and to the amino acid sequence encoded by known resistance genes, and RGAs from other plant species. Cluster analysis identified five different classes of RGAs in cowpea. Gel blot analysis revealed that each class recognized a different subset of loci in the cowpea genome. Several of the RGAs were associated with restriction fragment length polymorphisms, which allowed them to be placed on the cowpea genomic map. The potential for using these sequences to isolate R genes, and subsequent direct manipulation of disease and pest resistance using genetic engineering is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.