Mining of resistance genes associated with anthracnose infection in greater yam (Dioscorea alata Linn.)

Abstract

Anthracnose caused by Colletotrichum gloeosporioides is the major fungal disease of greater yam (Dioscorea alata), which is one of the important tropical tuber crops with high production and nutritive potential. For identifying the Resistance Gene Analogues (RGAs) in greater yam, degenerate primers based on the conserved motifs were used to isolate nucleotide-binding site (NBS) type sequences. Cloning and sequencing of identified NBS-type sequences called resistance gene analogues (RGAs) showed similarity to other cloned RGA sequences available in the database and the presence of conserved domains, viz. P-loop, RNBS-B, RNBS-C, Kinase-2 and GLPL, categorising them with the NBS–leucine-rich repeat class gene family. Amino acid sequence alignment of the Dioscorea RGAs with RGAs of other plant species grouped them with the non-Toll interleukin receptor (TIR) subclasses of the NBS sequences. The expression profiles of RGAs determined using semi quantitative Reverse Transcriptase polymerase chain-reaction in Sree Keerthi (tolerant) and Orissa Elite (susceptible) genotypes in response to anthracnose infection demonstrated that, Dioscorea alata RGAs were up-regulated three days after disease inoculation in the tolerant genotype, whereas in the susceptible genotype it was observed on the 5th day. In contrast, RGAs were found to be expressed in both tolerant and susceptible control plants, but the level was found to be increased in the test plants following pathogen inoculation. The reverse transcription PCR product was normalized and the efficiency was evaluated using Actin primers, which serves as reference gene. The result suggests a role of Resistance Genes Analogues in the early pathogen recognition of Sree Keerthi against C. gloeosporioides, which may be one of the reasons for its tolerance to anthracnose disease. These genes could be a good start point for further studies such as candidate gene mapping or understand the bases for resistance in greater yam. The isolation and expression analysis of D. alata RGAs have been reported for the first time in this study.