Diversity and evolutionary relationship of nucleotide binding site-encoding disease-resistance gene analogues in...

Most plant disease-resistance genes (R-genes) isolated so far encode proteins with a nucleotide binding site (NBS) domain and belong to a superfamily. NBS domains related to R-genes show a highly conserved backbone of an amino acid motif, which makes it possible to isolate resistance gene analogues (RGAs) by degenerate primers. Degenerate primers based on the conserved motif (P-loop and GLPL) of the NBS domain from R -genes were used to isolate RGAs from the genomic DNA of sweet potato cultivar Qingnong no.2. Five distinct clusters of RGAs (22 sequences) with the characteristic NBS representing a highly diverse sample were identified in sweet potato genomic DNA. Sequence identity among the 22 RGA nucleotide sequences ranged from 41.2% to 99.4%, while the deduced amino acid sequence identity from the 22 RGAs ranged from 20.6%to 100%. The analysis of sweet potato RGA sequences suggested mutation as the primary source of diversity. The phylogenetic analyses for RGA nucleotide sequences and deduced amino acids showed that RGAs from sweet potato were classified into two distinct groups--toll and interleukin receptor-1 (TIR)-NBS-LRR and non-TIR-NBS-LRR. The high degree of similarity between sweet potato RGAs and NBS sequences derived from R-genes cloned from tomato, tobacco, flax and potato suggest an ancestral relationship. Further studies showed that the ratio of non-synonymous to synonymous substitution within families was low. These data obtained from sweet potato suggest that the evolution of NBS-encoding sequences in sweet potato occur by the gradual accumulation of mutations leading to purifying selection and slow rates of divergence within distinct R-gene families.