Development of an Efficient Real-time PCR Assay to Accurately Quantify Resistant Gene Analogue Expression in Taro (Colocasia esculenta)

Abstract

Taro (Colocasia esculenta (L.) Schott.), an important tropical tuber crop with high nutritive, and medicinal potential, is ranked fourteen among the most consumed vegetable worldwide. Leaf blight caused by Phytophthora colocasiae, is one of the most destructive diseases of taro which leads to severe yield loss up to 50%. The objective of this study was to standardize the quantification of RGA (resistant gene analogues) expression in resistant and susceptible taro varieties. For isolating the taro RGAs, PCR-based strategy with degenerate primers was used and the obtained sequences showed similarity with other RGA sequences in the NCBI database, which categorised them into the NBS-LRR class of gene family. The conserved domain search has proved the presence of Nucleotide Binding-ARC domain in all the sequences. RGA specific primer was designed based on sequence information, which is the first report in taro. The expression of RGAs in Muktakeshi and Sree Kiran genotypes was determined by the SYBR green PCR assay with actin as reference gene. The target gene was up-regulated during the course of infection in both the resistant and susceptible varieties, but the difference was that the hike in expression upon pathogenwas found earlier in resistant variety than the susceptible variety and the level of expression (fold change) was also more in the resistant variety. The found results should be used as a good start point for further studies such as candidate gene mapping for taro leaf blight.