Rapid isolation of Yr9 via MutIsoSeq and QTL analysis of durable stripe rust resistance in wheat cultivar Xingzi 9104.

The fungus Puccinia striiformis f. sp. tritici (Pst) is the causal agent of wheat stripe rust which constitutes a major limitation to wheat production. Cloning and applying disease-resistant genes are considered as an effective solution. Chinese wheat cultivar Xingzi 9104 (XZ9104) has exhibited durable resistance across multiple environments since its release. Through quantitative trait loci (QTL) analysis, eight QTL were found on chromosome arms 1BS, 1BL, 2AL, 2BL, 3BS, 4BL, 5BL and 7BL. YrXZ identified as 1RS.1BL translocation conferred race-specific all-stage resistance to Pst race CYR23. QYrxz.nwafu-1BL.6 and QYrxz.nwafu-3BS.7 were considered as the adult plant resistance genes Yr29 and Yr30, respectively. Notably, QYrxz.nwafu-2BL.5 accounted for 15.75-47.63% of the phenotypic variation across diverse environments and its pyramiding with Yr29 and Yr30 can confer high level of resistance. Other QTL were environment-dependent with minor effects. To clone the above resistance genes, we created a population of over 2,000 M₅ mutants in XZ9104 using ethylmethane sulfonate (EMS) mutagenesis and screened various types of susceptible mutants. Using the MutIsoseq approach with five mutant lines susceptible to race CYR23, we rapid isolated a candidate gene for YrXZ encoding coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) protein. Integrating cytological analysis, gene-based association analysis, transcriptomic profiling and virus-induced gene silencing (VIGS), we confirmed that the causal gene for YrXZ was indeed Yr9. This study demonstrated that multiple QTL with different effects contributed to the durable resistance in XZ9104. Understanding the molecular mechanisms and pathways involved in plant defense can inform future strategies for deploying resistance gene and engineering of genetic resistance against evolving diseases.