High-throughput identification of stripe rust resistance resources and genetic mapping of a new gene from Triticum durum–Haynaldia villosa amphiploids

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is a devastating disease that threatens global wheat ( Triticum aestivum ) production. Given the rapid evolution of the Pst pathogen and the scarcity of effective resistance genes in cultivated wheat, resistance sources from wild relatives of wheat are being explored. Haynaldia villosa (2 n = 2 x = 14, VV), an annual wild relative of cultivated wheat, exhibits strong resistance to multiple wheat diseases. We previously synthesized a series of Triticum durum – H. villosa amphiploid lines to facilitate the transfer of beneficial traits into wheat. In this study, we evaluated the resistance of these amphiploid lines to stripe rust at the seedling stage and further identified a novel resistance gene. In total, eleven amphiploid lines derived from seven H. villosa accessions exhibited high resistance to Pst , among which STH55-1 showed high resistance to multiple Pst races. Genetic analysis using F 1 , F 2 , and F 2:3 populations indicated that the Pst resistance was controlled by a single dominant gene. We mapped the resistance gene, designated Yr2VS , to chromosome 2VS via bulked-segregant exome-capture sequencing, delimiting it to a 5.6-cM interval (spanning 1.84 Mb in the H. villosa RefSeq) flanked by markers InDel411 and InDel1393. Allelism tests revealed that the resistance gene in STH55-1 was likely allelic to that in STH63-1, but not to those in STH36-3 or STH59-1, and was closely linked to the resistance genes in STH50-4 and STH65-4. These findings demonstrate that the H. villosa -derived amphiploid lines harbor multiple, distinct stripe rust resistance genes, reflecting the high genetic diversity of the species. This combination of high-throughput synthesis, resistance screening, and allelism tests provides an efficient strategy for identifying superior resistance loci. The newly identified Yr2VS gene and the resistance germplasm developed herein represent valuable genetic resources for breeding stripe rust-resistant wheat.