Vast collections of barley germplasm have been established and conserved in various global gene banks. These collections hold tremendous genetic diversity for resistance genes to Puccinia hordei, a causal agent of barley leaf rust. This study was undertaken to discover, characterize and postulate the known Rph genes (resistance to Puccinia hordei) and identify novel sources of ASR (all-stage resistance) and APR (adult plant resistance) to P. hordei. A core set of 315 barley lines were rust-tested as seedlings for their response to eight Australian pathotypes of P. hordei and genotyped with molecular markers linked to the known characterised ASR and APR genes. These tests led to the postulation of ASR leaf rust resistance genes Rph1, Rph2, Rph3, Rph9.am, Rph12, Rph15, Rph19 and Rph25 singly or in combination. Field tests revealed that the vast majority of lines (84%) carried APR. Genotyping of the APR-carrying lines with markers bPb-0837, Ebmac0603 and sun43-44 identified lines that likely carry the known APR genes Rph20, Rph23 and Rph24 singly or in combination. Thirty-nine per cent of the lines were negative for all the three markers and were thus postulated to carry uncharacterized APR. The sources of resistance identified in this study provide a valuable resource to breeders for further utilization and diversifying the genetic basis of leaf rust resistance in barley.

Barley leaf rust (BLR), caused by Puccinia hordei, is best controlled through genetic resistance. An efficient resistance breeding program prioritizes the need to identify, characterize, and map new sources of resistance as well as understanding the effectiveness, structure, and function of resistance genes. In this study, three mapping populations were developed by crossing Israelian barley lines “AGG-396,” “AGG-397,” and “AGG-403” (carrying unknown leaf rust resistance) with a susceptible variety “Gus” to characterize and map resistance. Genetic analysis of phenotypic data from rust testing F3s with a P. hordei pathotype 5457 P+ revealed monogenic inheritance in all three populations. Targeted genotyping-by-sequencing of the three populations detected marker trait associations in the same genomic region on the short arm of chromosome 2H between 39 and 57 Mb (AGG-396/Gus), 44 and 64 Mb (AGG-397/Gus), and 31 and 58 Mb (AGG-403/Gus), suggesting that the resistance in all three lines is likely conferred by the same locus (tentatively designated RphAGG396). Two Kompetitive allele-specific PCR (KASP) markers, HvGBSv2-902 and HvGBSv2-932, defined a genetic distance of 3.8 cM proximal and 7.1 cM distal to RphAGG396, respectively. To increase the marker density at the RphAGG396 locus, 75 CAPS markers were designed between two flanking markers. Integration of marker data resulted in the identification of two critical recombinants and mapping RphAGG396 between markers- Mloc-28 (40.75 Mb) and Mloc-41 (41.92 Mb) narrowing the physical window to 1.17 Mb based on the Morex v2.0 reference genome assembly. To enhance map resolution, 600 F2s were genotyped with markers- Mloc-28 and Mloc-41 and nine recombinants were identified, placing the gene at a genetic distance of 0.5 and 0.2 cM between the two markers, respectively. Two annotated NLR (nucleotide-binding domain leucine-rich repeat) genes (r2.2HG0093020 and r2.2HG0093030) were identified as the best candidates for RphAGG396. A closely linked marker was developed for RphAGG396 that can be used for marker-assisted selection.