Multiplex PCR assay for the simultaneous identification of race specific and non-specific leaf resistance genes in wheat (Triticum aestivum L.)

Race-nonspecific resistance is a key to sustainable management of pathogens in bread wheat (Triticum aestivum L.) breeding. It is more durable compared to race-specific immunity, conferred by the major genes (R), which are often overcome by pathogens. The accumulation of the genes, which provide the resistance to a specific race of a pathogen, together with the introduction of race-non-specific resistance genes is the most effective strategy aimed at preventing the breakdown of genetically conditioned immunity. PCR markers improved the productivity and accuracy of classical plant breeding by means of marker-assisted selection (MAS). Multiplexing assays provide increased throughput, reduced reaction cost, and conservation of limited sample material, which are beneficial for breeding purposes. Here, we described the process of customizing multiplex PCR assay for the simultaneous identification of the major leaf rust resistance genes Lr19, Lr24, Lr26, and Lr38, as well as the slow rusting, race-nonspecific resistance genes: Lr34 and Lr68, in thirteen combinations. The adaptation of PCR markers for multiplex assays relied on: (1) selection of primers with an appropriate length; (2) selection of common annealing/extension temperature for given primers; and (3) PCR mixture modifications consisting of increased concentration of primers for the scanty band signals or decreased concentration of primers for the strong bands. These multiplex PCR protocols can be integrated into a marker-assisted selection of the leaf rust-resistant wheat genotypes.