Isolation and characterization of resistance gene analogs (RGAs) in sorghum

Abstract

The largest group of plant disease resistance (R) genes that share similar structures contains a predicted nucleotide-binding site (NBS) domain. NBS domains of this class of R genes show highly conserved amino acid motifs, which makes it possible to isolate resistance gene analogs (RGAs) by PCR with degenerate primers and homology searches from public databases. Multiple combinations of degenerate primers were designed from three conserved motifs (one motif was used for a subgroup-specific primer design) in the NBS regions of R genes of various plants. All combinations of primer pairs were used to amplify genomic DNA from sorghum. TIR-specific primer combinations showed no PCR amplification in sorghum. Homology searches identified many NBS-encoding sequences among the expressed or genomic molecular database entries for sorghum. Motif analysis of the sorghum NBS sequences that were identified in this study revealed eight major conserved motifs plus two additional highly conserved motifs, but no TIR-specific motifs. Phylogenetic analysis of sorghum NBS sequences showed tree topology typical of NBS-LRR genes, including clustered nodes and longbranch lengths. Eleven distinct families of NBS sequences, representing a highly diverse sample, were isolated from Sorghum bicolor. With two exceptions, sorghum RGA families appeared to be closely related in sequence to at least one R-gene cloned from other species. In addition, deduced amino acid sequences of sorghum RGAs showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)- type R-genes. Mapping with sorghum RGA markers revealed one linkage group containing four out of ten randomly selected markers, suggesting non-random distribution of NBS sequences in the sorghum genome. Rice sequences homologous to sorghum NBS sequences were found from two-way BLAST searches. Some of them were shown to be orthologs, when determined by using phylogenetic approaches which combined five different evolution models and tree-building methods.