Isolation and characterization of resistance gene analogs (RGAs) from sorghum (Sorghum bicolor L. Moench)

Degenerate primers designed based on known resistant genes (R-genes) and resistance gene analogs (RGAs) were used in combinations to elucidate RGAs from Sorghum bicolor, cultivar M 35-1. Most of the previously tried primer combinations resulted in amplicons of expected 500-600 bp sizes in sorghum along with few novel combinations. Restriction analysis of PCR amplicons of expected size revealed a group of fragments present in a single band indicating the heterogeneous nature of the amplicon. Many of these were cloned and some were considered for analysis. The nucleotide sequence of different cloned fragments was done and their predicted amino acid sequences compared to each other and to the amino acid sequences of known R-genes revealed significant sequence similarity. A cluster analysis based on neighbor-joining (N-J) method was carried out using sorghum RGAs (SRGAs) together with several analogous known R-genes resulting in two major groups; cluster-I comprising only SRGAs and cluster-II comprised of known R-gene sequences along with three SRGAs. Further analysis clearly indicated similarity of SRGAs in overall sense with already known ones from other crop plants. These sequences can be used as guidelines to detect, map and eventually isolate numerous R-genes in sorghum.