Resistance gene analogue isolation and RGA-based marker development for identifying downy mildew resistance in radish (Raphanus sativus L.)

Abstract

Resistance Gene Analogs (RGAs)-based molecular markers can significantly enhance the breeding efficiency for disease resistance in various crops. However, RGAs isolation and RGA-based marker development have not been conducted in radish. In this study, two prevalent approaches, PCR amplification with degenerate primers and data-mining of radish EST databases, were used to isolate radish resistance gene candidate sequences. A total of 26 RsRGAs with high homology to known R-genes or RGAs were obtained by PCR amplification. Meanwhile, 257 R-gene-like unigenes/ESTs were identified using data-mining method. In total, 115 out of 124 designed RGA-specific primer pairs can successfully amplified the target bands. To explore the RGA genetic variability, 32 radish accessions were genotyped and 269 RGA loci were successfully identified. It was found that 44 RGA primers only generated monomorphic band and the remaining 71 RGA primers generated 225 RGA polymorphic bands among the 32 radish accessions. Based on RGA marker analysis, 32 radish accessions were clustered into four main groups at the similarity index of 0.68, which was in a high accordance with disease index from a downy mildew evaluation. This study might be the first report on candidate R-genes identification and RGA-based markers development in radish. These findings will facilitate the disease resistance identification and speed up the genetic improvement of DM resistances in radish breeding programs.