In silico molecular and morphological analysis of rice blast resistant gene Pi-ta in Sri Lankan rice germplasm

Abstract

Pi-ta is a major blast resistant gene, introgressed from indica rice varieties. In this study, diversity of the Pi-ta gene of 47 Sri Lankan rice accessions was studied by bioinformatics, and the results were validated with molecular and disease reaction assays. Sequences of rice accessions at the locus Os12g0281300 were retrieved from Rice SNP-Seek Database, and the coding sequence of reference Pi-ta gene of cultivar Tetep (accession no. GQ918486.1) was obtained from GenBank. Comparisons were made at nucleotide, amino acid, and protein structure level, and the 3D models predicted using Phyre2 software were superimposed using TM-align software. Results: In silico analysis revealed that 10 accessions possessed resistant allele of the Pi-ta gene. The remaining accessions recorded high polymorphism in the leucine-rich domain resulting in 9 allele types, leading to single– amino acid substitutions at 27 different positions including a functional mutation of alanine to serine at the 918th amino acid position. None of the genotypes led to truncations in the amino acid sequence. The in silico analysis results were validated on 23 accessions comprising resistant and susceptible genotypes and another 25 cultivars from Northern Sri Lanka, by molecular assay using YL183/YL87 and YL155/YL87 resistant and susceptible allele specific markers. Resistance of Pi-ta gene for the causal fungus, Magnaporthe oryzae, was further validated through pathogenicity assay. Conclusion: The Pi-ta gene, especially the LRD region, revealed significant variations within Sri Lankan rice cultivars leading to high levels of resistance against blast. This information would be highly useful in breeding programmes for resistance against rice blast