Leaf rust is a highly destructive disease of barley caused by the fungal pathogen Puccinia hordei. Genetic resistance is considered to be the most effective, economical and eco-friendly approach to minimize losses caused by this disease. A study was undertaken to characterize and fine map a seedling resistance gene identified in a Hordeum vulgare ssp. spontaneum-derived barley line, HEB-04-101, that is broadly effective against a diverse set of Australian P. hordei pathotypes. Genetic analysis of an F3 population derived from a cross between HEB-04-101 and the H. vulgare cultivar Flagship (seedling susceptible) confirmed the presence of a single dominant gene for resistance in HEB-04-101. Selective genotyping was performed on representative plants from non-segregating homozygous resistant and homozygous susceptible F3 families using the targeted genotyping-by-sequencing (tGBS) assay. Putatively linked SNP markers with complete fixation were identified on the long arm of chromosome 5H spanning a physical interval between 622 and 669 Mb based on the 2017 Morex barley reference genome assembly. Several CAPS (cleaved amplified polymorphic sequences) markers were designed from the pseudomolecule sequence of the Morex assembly (v1.0 and v2.0), and 16 polymorphic markers were able to delineate the
RphHEB locus to a 0.05 cM genetic interval spanning 98.6 kb. Based on its effectiveness and wild origin, RphHEB is distinct from all other designated Rph genes located on chromosome 5H and therefore the new locus symbol Rph28 is recommended for RphHEB in accordance with the rules and cataloguing system of barley gene nomenclature.