Asian soybean rust (ASR) is an economically significant disease caused by the fungus Phakopsora pachyrhizi. The soybean genes Rpp3 and Rpp?(Hyuuga) confer resistance to specific isolates of the pathogen. Both genes map to chromosome 6 (Gm06) (linkage group [LG] C2). We recently identified 12 additional soybean accessions that harbor ASR resistance mapping to Gm06, within 5 centimorgans of Rpp3 and Rpp?(Hyuuga). To further characterize genotypes with resistance on Gm06, we used a set of eight P. pachyrhizi isolates collected from geographically diverse areas to inoculate plants and evaluate them for differential phenotypic responses. Three isolates elicited different responses from soybean accessions PI 462312 (Ankur) (Rpp3) and PI 506764 (Hyuuga) (Rpp?[Hyuuga]). In all, 11 of the new accessions yielded responses identical to either PI 462312 or Hyuuga and 1 of the new accessions, PI 417089B (Kuro daizu), differed from all others. Additional screening of Hyuuga-derived recombinant inbred lines indicated that Hyuuga carries two resistance genes, one at the Rpp3 locus on Gm06 and a second, unlinked ASR resistance gene mapping to Gm03 (LG-N) near Rpp5. These findings reveal a natural case of gene pyramiding for ASR resistance in Hyuuga and underscore the importance of utilizing multiple isolates of P. pachyrhizi when screening for ASR resistance.

Soybean [Glycine max (L.) Merr.] rust is caused by the fungal pathogen Phakopsora pachyrhizi. Six rust resistance loci (Rpp1, 2, 3, 4, 5, and 6) have been reported. Crosses were made between 75 resistant plant introductions (PIs) and a susceptible elite line or cultivar. Bulked segregant analysis (BSA) was used to determine if the PI resistance genes mapped to a previously identified locus or to an unreported locus. Fifty‐two PIs had resistance genes that mapped to the Rpp3 region on chromosome 6 of the soybean genome. A set of P. pachyrhizi isolates was used to further characterize the resistance of these PIs. Forty‐two of the PIs exhibited the same reaction profiles as either PI 462312 (Rpp3) or ‘Hyuuga’ (Rpp3 and Rpp5) to the panel of isolates. The fine mapping of Rpp1, Rpp3, and Rpp4 and the availability of the SoySNP50K Infinium Chip data on the USDA Soybean Germplasm Collection made it possible to use BSA and isolate data on these PIs to determine in retrospect how effective haplotype analysis would be in narrowing down the PIs to those likely to have a unique source of resistance. Thirty‐seven of the 52 PIs (71%) mapping to the Rpp3 region had a haplotype identical to that of PI 462312. A combination of these analyses could prove useful in more rapidly narrowing down resistant PIs to those likely to carry a unique resistance gene.