Stripe or yellow rust (YR) caused by Puccinia striiformis tritici (Pst) is an important foliar disease affecting wheat production globally. Resistant varieties are the most economically and environmentally effective way to manage this disease. The common winter wheat (Triticum aestivum L.) cultivar Luomai 163 exhibited resistance to the Pst races CYR32 and CYR33 at the seedling stage and showed a high level of adult plant resistance in the field. To understand the genetic basis of YR resistance in this cultivar, 142 F5 recombinant inbred lines (RILs) derived from cross Apav#1 × LM163 and both parents were genotyped with the 16K SNP array and bulked segregant analysis sequencing. The analysis detected a major gene, YrLM163, at the seedling stage associated with the 1BL.1RS translocation. Additionally, three genes for resistance at the adult plant stage were detected on chromosome arms 1BL (Lr46/Yr29/Pm39/Sr58), 6BS, and 6BL in Luomai 163, whereas Apav#1 contributed resistance at a quantitative trait locus (QTL) on 2BL. These QTL explained YR disease severity variations ranging from 6.9 to 54.8%. The kompetitive allele-specific PCR (KASP) markers KASP-2BL, KASP-6BS, and KASP-6BL for the three novel loci QYr.hzau-2BL, QYr.hzau-6BS, and QYr.hzau-6BL were developed and validated. QYr.hzau-1BL, QYr.hzau-2BL, and QYr.hzau-6BS showed varying degrees of resistance to YR when present individually or in combination based on genotype and phenotype analysis of a panel of 570 wheat accessions. Six RILs combining resistance alleles of all QTL, showing higher resistance to YR in the field than Luomai 163 with disease severities of 10.7 to 16.0%, are important germplasm resources for breeding programs to develop YR-resistant wheat varieties with good agronomic traits.
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