Isolation, characterization and evolutionary analysis of resistance gene analogs in annual ryegrass, perennial ryegrass and their hybrid

Abstract

Recently, a number of disease‐resistance genes related to a diverse range of pathogens were isolated from a wide variety of plant species. The majority of plant disease‐resistance genes encoded a nucleotide‐binding site (NBS) domain. According to the comparisons of the NBS domain of cloned R‐genes, it has shown highly conserved amino acid motifs in this structure, which made it possible to isolate resistance gene analogs (RGAs) by PCR using degenerate primers. We have designed three pairs of degenerate primers based on two conserved motifs in the NBS domain of resistance proteins encoded by R‐genes to amplify genomic sequences from ryegrass (Lolium sp.). Sixteen NBS‐like RGAs were isolated from turf and forage type grasses. The sequence analysis of these RGAs revealed that there existed a high similarity (up to 85%) between RGA sequences among ryegrass species and other plants. The alignment of the predicted amino acid sequences of RGAs showed that ryegrass RGAs contained four conserved motifs (P‐Loop, kinase‐2, kinase‐3a, GLPL) present in other known plant NBS‐leucine rich repeat resistance genes. These ryegrass RGAs all belonged to non‐toll and interleukin‐1 receptor subclass. Phylogenetic analysis of ryegrass RGAs and other cloned R‐genes indicated that gene mutation was the predominant source of gene variations, and the sequence polymorphism was due to purifying selection rather than diversifying selection. We further analyzed the source of gene variation in other monocots, rice, barley, wheat, and maize based on the data published before. Our analysis indicated that the source of RGA diversity in these monocots was the same as in ryegrass. Thus, monocots were probably the same as dicots in the source of RGA diversity. Ryegrass RGAs in the present paper represented a large group of resistance gene homologs in monocots. We discussed the origin and the evolution of R‐genes in grass species.