Discovery of Resistance Genes in Rye by Targeted Long-Read Sequencing and Association Genetics

The majority of released rye cultivars are susceptible to leaf rust because of a low level of resistance in the predominant hybrid rye-breeding gene pools Petkus and Carsten. To discover new sources of leaf rust resistance, we phenotyped a diverse panel of inbred lines from the less prevalent Gülzow germplasm using six distinct isolates of <i>Puccinia recondita</i> f. sp. <i>secalis</i> and found that 55 out of 92 lines were resistant to all isolates. By performing a genome-wide association study using 261,406 informative SNP markers, we identified five resistance-associated QTLs on chromosome arms 1RS, 1RL, 2RL, 5RL and 7RS. To identify candidate <i>Puccinia recondita</i> (<i>Pr</i>) resistance genes in these QTLs, we sequenced the rye nucleotide-binding leucine-rich repeat (NLR) intracellular immune receptor complement using a Triticeae NLR bait-library and PacBio^® long-read single-molecule high-fidelity (HiFi) sequencing. Trait-genotype correlations across 10 resistant and 10 susceptible lines identified four candidate NLR-encoding <i>Pr</i> genes. One of these physically co-localized with molecular markers delimiting <i>Pr3</i> on chromosome arm 1RS and the top-most resistance-associated QTL in the panel.