Isolation of disease resistance gene analogs from Italian Ryegrass (Lolium multiflorum Lam.)

Abstract

AbstractLarge‐scale sequencing of disease resistance gene analogs (RGA) in Italian ryegrass was carried out using degenerate primers and a nested polymerase chain reaction (PCR) strategy. A total of 24 000 PCR clones were subjected to single pass sequencing to enable similarity searches of public databases. As a result, 9344 clones showed significant levels of homology to either known resistance genes or RGA derived from other plant species. These clones were grouped into 115 clusters based on nucleotide sequence similarities and representative clones were selected from each cluster. Of 115 representative clones, 62 had a continuous open reading frame and were defined as potentially functional RGA from Italian ryegrass. Specific sequenced tag site (STS) primer sets were designed for these 62 clones and 50 RGA sequences were subsequently amplified from genomic DNA of Italian ryegrass in one step PCR. A subset of these STS primers could also amplify the expected size PCR products from genomic DNA of tall fescue and meadow fescue, both of which are forage crops closely relate to Italian ryegrass.