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Key message Comparative sequence analysis was used to design a SNP marker that aided in the identification of new sources of oat stem rust resistance. New races of  Puccinia graminis  f. sp.  avenae  ( Pga ) threaten global oat production. An  A. strigosa  accession known to carry the broadly effective oat stem rust resistance gene,  Pg6 , was crossed with two susceptible  A. strigosa  accessions to generate 198 F 2:3  families and 190 F 5:6  RILs. The RIL population was used to determine that  Pg6  was a single dominant gene located between 475 and 491 Mbp on diploid chromosome AA2 of the  A. atlantica  genome. This region was further refined by identifying SNPs associated with  Pg6  resistance in a panel of previously sequenced A-genome accessions. Twenty-four markers were developed from SNPs that showed perfect association between the  Pg6  phenotype and 11 sequenced  Avena  diploid accessions. These markers were validated in the RILs and F 2:3  families, and the markers most closely linked with resistance were tested in a diverse panel of 253 accessions consisting of oat stem rust differentials, all available diploid  Avena  spp. accessions, and 41  A. vaviloviana  accessions from the National Small Grains Collection. One SNP marker located at 483, 439, 497 bp on AA2, designated as AA2_483439497, was perfectly associated with the  Pg6  phenotype in  Avena strigosa  diploids and was within several Kb of a resistance gene analog, RPP13. The marker results and seedling testing against  Pga  races DBD, KBD, TJS, and TQL enabled the postulation of  Pg6  and potential new sources of resistance in the  Avena  panel. These results will be used to infer  Pg6  presence in other germplasm collections and breeding programs and can assist with introgression, gene pyramiding, and cloning of  Pg6 .

Comparative sequencing and SNP marker validation for oat stem rust resistance gene Pg6 in a diverse collection of Avena accessions