Development of polymerase chain reaction-based marker for identification of a nonsense mutation in a Pseudomonas-responsive receptor like protein gene of tomato

Abstract

ABSTRACT The present study was undertaken to develop a simple, robust, and reliable polymerase chain reaction-based molecular marker that can identify a critical nonsense mutation in a receptor like protein (RLP) gene of tomato. The Solyc04g014400 locus was found to code for a RLP with leucine-rich repeats, a trans-membrane domain, and characteristic three-dimensional structure. In silico analysis revealed the gene to be Pseudomonas-responsive. The gene was previously reported to contain a single nucleotide polymorphism (SNP) in the second exon, which converts the Trp 76 codon (TGG) to a premature stop (TAG) codon. Hence, a tetra-primer amplification refractory mutation system (T-ARMS) assay was developed to detect this functional SNP in 20 tomato genotypes in a co-dominant manner. Authenticity of the allele-specific amplicons was also tested, where the primers were observed to be discriminatory for the allelic variants. Suitability of the developed assay for determining heterozygosity in developed F1 hybrids was also confirmed on the basis of the results. Thus, the developed T-ARMS marker will be useful for functional characterisation of this gene in the light of its Pseudomonas-responsiveness.