Abstract
<title>Abstract</title> <p> Barley leaf spot, caused by <italic>Bipolaris sorokiniana</italic> , severely threatens global barley production by compromising grain yield and quality. Identifying resistance genes is therefore vital for breeding resilient cultivars. In this study, we developed F <sub>2</sub> and F <sub>6</sub> recombinant inbred line (RIL) populations from a cross between the resistant Mengpimai 3 and susceptible Mengpimai 1. Genetic analysis of the F <sub>2</sub> population revealed a normal distribution of disease severity, indicating quantitative inheritance of resistance. Combining genotypic data with spray inoculation assays, we performed QTL mapping using SNP arrays. Two QTLs, <italic>qSRH7-59</italic> and <italic>qSRH7-60</italic> , were identified in the RIL population. Validation using newly developed KASP markers confirmed these results, with three markers showing over 70% selection efficiency for the resistant genotype, highlighting their utility in marker-assisted breeding. To refine the mapping interval, high-density SSR markers were developed for the <italic>qSRH7-60</italic> region. Subsequent linkage mapping identified two resistance loci, <italic>qSRH7-7</italic> and <italic>qSRH7-8</italic> , and pinpointed the candidate gene <italic>HORVU7Hr1G017950</italic> , a cytochrome P450 family member. RT-qPCR analysis showed significantly higher transcript levels of <italic>HORVU7Hr1G017950</italic> in the resistant parent post-inoculation. Furthermore, BSMV-VIGS-mediated silencing of this gene significantly reduced resistance to <italic>B. sorokiniana</italic> , confirming its functional role in the defense response. These findings establish <italic>HORVU7Hr1G017950</italic> as a key candidate gene, providing valuable molecular resources for enhancing barley leaf spot resistance. </p>