Mapping of major Fusarium oxysporum resistance QTL fw8.1 and identification of candidate gene LsWAKL13 in bottle gourd

• This study is the first to systematically dissect the genetic basis of Fusarium wilt resistance in bottle gourd ( Lagenaria siceraria ). • A multi-method mapping strategy (GWAS + QTL-Seq + classical linkage mapping) was employed, successfully narrowing the major FW resistance QTL fw8.1 to a 264.4 kb interval on chromosome 8. • The wall-associated kinase gene LsWAKL13 was identified as the causal candidate for FW resistance, with a nonsynonymous Pro235Thr mutation altering its 3D structure and correlating with resistance phenotypes. • The findings provide valuable genetic resources to accelerate marker-assisted breeding of FW-resistant bottle gourd varieties. Bottle gourd fusarium wilt (FW), caused by Fusarium oxysporum f. sp. lagenariae (Fol), is a destructive soil-borne disease that severely impairs plant growth and reduces yields in bottle gourd production. Breeding FW-resistant varieties is the most sustainable management strategy, but the genetic underpinnings of resistance to FW in bottle gourd remain uncharacterized. To investigate the FW resistance genes, we first performed a genome-wide association study (GWAS) using a multiparent advanced generation intercross (MAGIC) population. Subsequently, we constructed a secondary population from FW-resistant line M238 and FW-susceptible line M363 within the MAGIC population, and further mapped FW resistance genes via quantitative trait loci sequencing (QTL-Seq) and linkage analysis. GWAS identified 83 significant single-nucleotide polymorphisms (SNPs) associated with FW resistance, primarily on chromosomes 3, 8, and 11. QTL-Seq identified two major QTLs ( fw5.1 and fw8.1 ), and regional linkage mapping refined fw8.1 to a 264.4 kb interval containing 6 wall-associated kinase (WAK) family genes. Among these, LsWAKL13 was significantly upregulated in M238 vs. M363 after FW inoculation. A nonsynonymous G-to-T mutation in LsWAKL13 altered its 3D structure and correlated with FW resistance (lower disease index in M238-type allele accessions). Our findings identify LsWAKL13 as a causal candidate gene for FW resistance, while offering valuable genetic resources to speed up the breeding of resistant varieties.