Fine mapping of a stable bacterial wilt resistance QTL and development of a linked KASP marker in tobacco germplasm QZ-1

Bacterial wilt caused by Ralstonia solanacearum is a major constraint to tobacco production, whereas effective resistance donors and molecular markers remain limited. Here, we established a standardized seedling-stage inoculation assay and evaluated 1437 tobacco germplasm accessions for bacterial wilt resistance. The optimized assay showed a strong association with adult-plant field resistance across 40 accessions, enabling rapid and reproducible phenotyping. Among 1437 tobacco germplasm accessions screened, QZ-1, a sun-cured tobacco accession, was identified as highly resistant and showed stable resistance in both seedling-stage and field evaluations. Genetic analysis of 227 F 2:3 families derived from QZ-1 × Honghua dajinyuan identified qzBW5.1 on chromosome 5 as the strongest resistance-associated QTL, with a LOD score of 12.64 and explaining 11.16% of the phenotypic variance. Sequential recombinant-based fine mapping using 1587 BC 3 F 2 and 4000 BC₄F₂ individuals narrowed qzBW5.1 to a 2.42-Mb interval. Comparison with previously reported loci suggested that this interval represents a previously unreported resistance-associated region. Transcriptome analysis prioritized six genes within the interval for further functional validation. Among three KASP assays developed across the qzBW5.1-associated region, QM10.2 showed the strongest association with resistance and complete genotype–phenotype co-segregation in a QZ-1-derived BC 2 F 4 family. Marker-assisted selection subsequently enabled the development of a homozygous resistant BC₂F₅ breeding line. These results provide a new resistance resource, a finely mapped resistance-associated QTL region, and a closely linked KASP marker for tobacco bacterial wilt resistance breeding.