Identification, Characterization, and Phylogenetic analysis of Pigeonpea (Cajanus cajan L. Millsp.) Resistance Gene Analogs using PCR cloning and in silico methods

Abstract

Pigeonpea (Cajanus cajan), an important grain legume, is susceptible to Fusarium wilt (FW), sterility mosaic disease (SMD), and Phytophthora blight. Identification of resistance gene analogs (RGAs) is important for development of resistant varieties. In this study, degenerate primers targeting nucleotide binding sites (NBS) of known resistance (R) genes were used to amplify RGAs from two pigeonpea genotypes with differing disease resistance profiles. The translated cloned RGAs had high amino acid identity (68–71%) with putative disease resistance proteins in Glycine max. Five RGA open reading frames were found in the whole pigeonpea genome after BLASTN analysis with the cloned sequences. Translated RGA proteins contained several characteristic features such as the NB-ARC domain (characteristic of death-related disease resistance genes) and four NBS motifs. A tryptophan residue at the kinase-2 motif was indicative of the non-TIR-NBS class of proteins. Phylogenetic analysis revealed two major clusters. The seven pigeonpea RGAs were in a non-TIR group alongside wilt resistance proteins from tomato. Specific primers were designed against the RGAs identified by BLASTN, and these successfully amplified sequences from all eight pigeonpea genotypes. The 40 resultant sequences were combined according to genotype and subjected to phylogenetic analysis. Genotypes clustered according to breeding pedigree. Multiple alignment of the 40 sequences revealed a number of single nucleotide polymorphisms (SNPs) that are useful in identifying candidate resistance genes associated with FW and SMD.