Cloning and characterization of gene-resistant analogs (RGAs) involved in rust (Puccinia psidii) resistance in Eucalyptus grandis

Abstract

Disease-resistant genes play an important role in defending against a variety of pathogens and insect pests in plants. Most of the disease-resistant genes encode proteins with conserved leucine rich repeat and nucleotide binding site domains. In this study, we cloned and characterized gene-resistant analogs (RGAs) from Eucalyptus grandis using degenerate PCR, with primers specifically targeting these two domains. The amplified fragments were cloned into the pGEM-T vector and transformed into Escherichia coli. Among the 90 clones obtained, 13 were sequenced and compared with each other and with previously identified gene-resistant diseases. A BLASTX search in GenBank revealed high similarities among the conserved domains of these cloned genes with RGA genes. Some clones, however, showed no significant similarity with DNA sequences in GenBank. Southern blotting analysis identified several polymorphic RFLP loci between distinct genotypes. However, none of them co-segregated with the Puccinia psidii Winter resistance gene 1 (Ppr1) in a population study.