Isolation and sequence analysis of wheat NBS-LRR type disease resistance gene analogs using degenerate PCR primers

Abstract

Isolation of disease resistance gene analogs (RGAs) using the conserved motifs of the resistance genes has attracted considerable attention since it was first reported more than a decade ago. In this study, RGAs are isolated using homology-based PCR to target the nucleotide binding site (NBS) conserved regions from hexaploid wheat varieties and a few accessions of wild types. Based on sequence similarity analysis, 83 of the sequenced clones were clustered as groups. Of these RGAs, 40 were in the NBS-LLR class, containing kinase-1a (GGVGKTT or GGVGKTA), kinase-2 (KRFLIVLDDXW), kinase-3a (GSXIVVITTR or GCXVLATTR), and the GLPL motif of the NBS-spanning region. Among these, 15 contained possible intron regions, similar to Avena sativa O2 NBS-LLR type disease resistance gene (AF078874), and one to Rpm1 of rice and Yr10 and Lr10 of wheat. To our knowledge, this is the first observation of an intronic site within the P-loop domain of wheat RGAs. We detected an unspecified motif (VMVCVS) between the kinase-1a and kinase-2 domains within our clones. Additionally, one of the clones showed replacement with the kinase-3a motif with an undefined sequence.