Wheat is one of the most important food crops worldwide with ranking second based on the total production annually. Wheat leaf rust is one of the important factors affecting the global wheat yield, which can reduce wheat yield by about 50% during the epidemic period.

Breeding resistant wheat varieties is the most economical, environmentally friendly and effective way to control wheat leaf rust. In the present study, we identified a total of 27 stable leaf rust resistance loci at seedling stageby GWAS analysis of 569 wheat germplasms, of which six may be the known resistance genes Lr10, Lr16, Lr37, Lr73, Lr79 and Lr80. Interestingly, one locus on chromosome 2BS was mapped in all three leaf rust isolates and it was located nearby the known leaf rust resistance gene Lr16.

Thus, we used the Lr16 monogenic line Thather+Lr16 to cross with the Chinese susceptible wheat variety Yangmai 18 to develop an F2 population with 3459 plants. Five polymorphic SSR molecular markers were selected for genotyping of the population, and combined with the phenotypic against leaf rust isolate 18054, a molecular marker SSRN.N was co-segerated with the phenotype.

Combined GWAS and linkage analysis, the transcription factor TaWN was identified as a candidate gene for Lr2BS, and its function was verified by using VIGS and transgenic techniques. In addition, the interaction between TaWN and TaGAPDH-4B was confirmed by yeast two-hybrid(Y2H) method and firefly luciferase complementation experiment (LCA). We also developed the gene based molecular marker for Lr2BS in breeding leaf rust resistant wheat varieties.