Wheat stripe rust, caused by Puccinia striiformis f. sp. Tritici, is one of the major fungal diseases threatening wheat yield globally. Breeding resistant varieties is the most economical and effective way to manage this disease. Yr54 derived from a CIMMYT spring wheat variety Quaiu#3 was located on the long arm of chromosome 2D (chr2DL) and expressed stable adult-plant resistance to stripe rust under many countries’ rust environments, but its sequence is still unknown.

In the present study, the monogenic lines Aoc_Yr54 only containing one major locus Yr54 was crossed with the susceptible wheat line Apav#1 to construct an RIL mapping population. Combined the linkage analysis between phenotype and genotype and BSE-seq analysis, Yr54 was limited to a 990 kb region on the end of chr2DL, with the aid of γ-ray fragment deletion susceptible mutants. In order to get the sequence information of target region, we sequenced and assembled a ~14.48 Gb Quaiu#3 genome using long-read sequencing and Hi-C.

We analyzed all of genes in the target region and verified the candidate genes by agrobacterium mediated genetic transformation. Finally, the candidate gene G22 was identified as capable of enhancing stripe rust resistance in wheat, and its possible resistance mechanism may be involved in the accumulation of H2O2.

The related gene based molecular markers were developed and distributed to wheat breeders worldwide.