Multiplex PCR assay for the detection of Lr24 and Lr68 in salt tolerant wheat genotype

Abstract

In this study, we developed and optimized a multiplex polymerase chain reaction (PCR) for simultaneous detection of two important leaf rust resistance genes Lr24 and Lr68. Seedling resistance gene Lr24 and slow rusting gene Lr68 encoding resistance to leaf rust were identified in forty-one advanced salt-tolerant wheat genotypes using molecular markers viz. Sr24#50 and csGS, respectively. For re-validation of PCR reaction results, a seedling resistance test (SRT) was performed in controlled glasshouse conditions. Genotypes were evaluated under epiphytotic conditions for SRT by artificial inoculation with a spectrum of Puccinia triticina pathotypes. Results of SRT were supplemented by PCR results, indicated that the gene Lr24/Sr24 conferred leaf rust resistance in two wheat genotypes namely HD 2851 and KRL2029. In advanced line KRL2029, along with Lr24/Sr24 other rust resistance genes (Lr26/Sr31/ Yr9) were also present. Molecular marker csGS for a known slow rusting gene Lr68 was amplified in fifteen salt-tolerant wheat genotypes indicated the presence of Lr68 in these lines. These lines could also be utilized as donors for slow rusting characteristics. Combination of both the genes could provide resistance to leaf rust and stem rust. Presences of respective genes in salt-tolerant lines were confirmed by SRT as well as molecular markers separately and multiplex PCR assay was designed. Multiplex PCR method allowed to reduce the cost and time of analyses as well as reducing the workload.