High-Resolution Melting-Based Marker Development for Wheat Leaf Rust Resistance Gene .

Deploying adult plant resistance (APR) against rust diseases is an important breeding objective of most wheat-breeding programs. The gene is an effective and widely deployed broad-spectrum APR gene in wheat against leaf rust fungus . Various molecular markers have been developed for , but they either require post-PCR handling processes or are not economical. Herein, we developed a high-resolution melting (HRM)-based diagnostic assay for based on a 3-bp 'TTC' deletion in exon 11 of the resistant allele. The susceptible cultivar Thatcher (Tc) and the near-isogenic Thatcher line (RL6058) with yielded distinct melting profiles and were differentiated with high reproducibility. For further validation, all three copies of were cloned in plasmid vectors, and HRM analysis using individual and combination (equimolar mixture of three copies) homoeologs yielded distinct melting profiles. An additional layer of genotyping was provided by a LunaProbe assay. The allele-specific probes successfully distinguished the homoeologs but not Tc and RL6058. Furthermore, the practical deployment of the HRM assay was tested by running the marker on a set of breeding lines. When compared with a kompetitive allele-specific PCR (KASP) assay, the HRM assay had similar genotyping results and was able to accurately differentiate the resistant and susceptible breeding lines. However, our HRM assay was unable to detect the heterozygote. To our knowledge, this is the first report of an HRM assay for genotyping a wheat rust resistance gene.