The wheat Lr28 locus harbors three tandem NLR genes that independently confer leaf rust resistance

Leaf rust is a devastating wheat disease, and deploying resistant cultivars is the most effective control strategy. Here, we report a high-quality genome assembly for the Lr28 introgression line GSTR 428 and use this resource to clone Lr28 , a resistance locus introgressed from an Aegilops speltoides segment into wheat chromosome arm 4AL. The Lr28 locus comprises three tandem NLR genes ( CNL1 - CNL3 ), each of which is independently sufficient to confer resistance, as demonstrated by CRISPR/Cas9-induced mutations and transgenic complementation. CNL1 and CNL2 are identical and share 99.03% protein sequence identity with CNL3. The same three genes occur in four Ae. speltoides accessions. We further demonstrate that Lr28 underlies the major 4AL resistance QTL identified by genome-wide association study (GWAS) in European wheat cultivars. To reduce linkage drag, we generate shortened alien segments (< 1.22 Mb) harboring Lr28 using the ph1b mutant. The cloning of Lr28 will accelerate its deployment in wheat breeding programs.