Isolation and Evolution Mode Analysis of NBS-LRR Resistance Gene Analogs from Hexaploid Wheat

Based on conserved regions of a sequence of a previously isolated powdery mildew (Pm) resistance gene, Pm3b, from hexaploid wheat (Triticum aestivum L.), a pair of primers was designed, and 11 resistance gene analogs (RGAs) were obtained using a polymerase chain reaction-based cloning approach. Three RGAs were deemed as pseudogenes, while the remaining eight corresponded to protein-encoding genes. At the nucleotide level, all these RGAs shared a sequence identity of 99% and showed 86% sequence identity with Pm3b. Phylogenetic analysis revealed that the eight protein-encoding genes were paraphyletic with Pm3 alleles. Positively selected sites were identified using the Selecton 2.1 program, and these were located on secondary structures. Based on these findings, the following two inferences could be made on the mode of evolution of the nucleotide-binding site and leucine-rich repeat (LRR) class of resistance genes. First, the majority of evolution events must have occurred primarily in the LRR domain, and this might have contributed to an increase in the proportion of evolution events in other domains. Second, these evolutionary events in the LRR domain must have occurred initially in secondary structures and then in the beta-sheet. The crystalline structure models of RGAs were constructed.