Characterization of disease resistance gene homologues isolated from finger millet (Eleusine coracana L. Gaertn)

Resistance gene homologues were isolated from finger millet (Eleusine coracana L.) using degenerate oligonucleotide primers designed to the conserved regions of the nucleotide binding site (NBS) of previously cloned plant disease resistance genes (R-genes) using polymerase chain reaction (PCR). Of the eleven primer combinations tested, only five showed amplification of resistance gene homologues in finger millet. BLAST search of cloned finger millet DNA fragments showed strong homology to NBS-LRR-type R-genes of other crop species. Of the 107 clones sequenced, 41 showed homology to known R-genes, and are denoted as EcRGHs (Eleusine coracana resistance gene homologues), while 11 showed homology to pollen signalling proteins (PSiPs), and are denoted as EcPSiPs (Eleusine coracana pollen signalling proteins). The cloned EcRGH sequences were classified into four clusters, and EcPSiPs formed two separate clusters based on sequence homology at the amino acid level. The amino acid sequences of the cloned EcRGHs showed characteristic features of non-TIR-type R-genes, which have been identified in all the monocot species studied so far. Six EcRGHs-specific primers were designed based on the sequences obtained in finger millet; reverse transcription PCR was performed on the cDNA and revealed the expression of EcRGHs in finger millet. The ratio of non-synonymous to synonymous nucleotide substitution (dN/dS) in the NBS domains of finger millet RGHs varied from 0.3 to 0.7 for the different classes, which suggests a purifying selection, though the LRR region also needs to be considered to make predictions. This is the first report on RGHs in finger millet, which will serve as a valuable resource for finger millet improvement using molecular tools.