Core Ideas Quantitative disease resistance to F. graminearum was previously mapped in soybean. A major QDRL to F. graminearum characterized for putative candidate resistant genes. Hybrid genome assembly aided identification of genome rearrangement in the QDRL region. Positions of four expressed candidate genes were within the QDRL to F. graminearum. Fusarium graminearum Schwabe [teleomorph: Gibberella zeae (Schweintiz) Petch] has been identified as a pathogen of soybean [Glycine max (L.) Merr.] causing seed, seedling damping‐off and root rot in North America. A major quantitative disease resistance locus (QDRL) that contributed 38.5% of the phenotypic variance toward F. graminearum in soybean was previously identified through mapping of a recombinant inbred line (RIL) population derived from a cross between ‘Wyandot’ and PI 567301B. This major QDRL mapped to chromosome 8 to a predicted 305 kb region harboring 36 genes. This locus maps near the Rhg4 locus for soybean cyst nematode (SCN) and the I locus contributing to seed coat color. Long‐read sequencing of the region was completed and variations in gene sequence and gene order compared with the ‘Williams 82’ reference were identified. Molecular markers were developed for genes within this region and mapped in the original population, slightly narrowing the region of interest. Analyses of the hybrid genome reassembly using three previously published bacterial artificial chromosome (BAC) sequences (BAC56G2, BAC104J7, and BAC77G7‐a) combined with RNA‐sequencing narrowed the region making candidate gene identification possible. The markers within this region may be used for marker‐assisted selection (MAS). There were 10 differentially expressed genes between resistant and susceptible lines, with four of these candidates also located within the genomic interval defined by the flanking markers. These genes included an actin‐related protein 2/3 complex subunit, an unknown protein, a hypothetical protein, and a chalcone synthase 3.
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