Genome-wide characterisation of leaf rust resistance loci in an Eastern European barley collection

GWAS and inheritance testing uncovered known and potentially new genetic sources of leaf rust resistance in barley, mapping the dominant locus RphCaspian (RphCas) to chromosome 4HL in close physical proximity to Rph21. This study has unravelled the genetic architecture of leaf rust in barley and also provides closely linked SNP markers on the 40k Infinium wheat barley array suitable for marker assisted selection. Barley leaf rust (BLR) is a damaging foliar disease affecting grain yield and quality. Genetic resistance offers a sustainable solution, but often succumbs to breakdown in the absence of stewardship. Sourcing and incorporating novel diverse alleles in breeding programs is challenging yet enhanced by mining unexplored gene pools available in gene banks. In this study, we investigated 318 barley accessions of Eastern European origin for BLR resistance using detailed greenhouse-based host-pathogen interaction assays at the seedling stage. The panel was genotyped using two marker platforms, targeted genotyping-by-sequencing and Infinium™ Wheat Barley 40 K BeadChip Array. Following marker curation, 35,059 SNPs were used for genome-wide association studies (GWAS) using GAPIT/FarmCPU, with associated regions further supported using a BayesR approach. GWAS identified significant peaks corresponding to known resistance loci, including Rph2, Rph3 and Rph12, as well as potentially uncharacterised BLR resistance QTL on chromosomes 1HL, 2HL, 3HS and 6HS. Several biparental populations were also developed by crossing three diverse EEB donors carrying broadly effective BLR resistance including: Caspian, Anska and Athenais, with the leaf rust-susceptible barley cultivar Gus. Genetic analysis at the F₃ generation revealed that resistance Athenais was consistent with two-gene inheritance, whereas resistance in Caspian was controlled by a single dominant gene (RphCas) and the inheritance of resistance in Anska was inconclusive. Bulked segregant RNA sequencing and conventional bulked segregant analysis (BSA) mapped the Caspian resistance locus to chromosome 4HL. An allelism test using an F₃ population derived from Riccardo, carrying Rph21, and Caspian, tested with a Puccinia hordei pathotype (5457P+) avirulent to both resistance sources, susggested that the RphCas locus was distinct from Rph21 however their close physical proximity and haplotypic similarity requires further investigation for a formal gene designation for RphCas.