| Summary: | The wheat stem rust fungus <i>Puccinia graminis</i> f. sp. <i>tritici</i> (<i>Pgt</i>), is one of the most destructive pathogens of wheat. In this study, a draft genome was built for a founder Australian <i>Pgt</i> isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced American <i>Pgt</i> isolate CDL 75-36-700-3 (p7a) and <i>de novo</i> assembly were performed resulting in a 92 Mbp reference genome for <i>Pgt</i> isolate 21-0. Approximately 13 Mbp of <i>de novo</i> assembled sequence in this genome is not present in the p7a reference assembly. This novel sequence is not specific to 21-0 as it is also present in three other <I>Pgt</I> rust isolates of independent origin.The new reference genome was subsequently used to build a pan-genome based on five Australian <i>Pgt</i> isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ~10% of the genes each in germinated spores and haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 520 were classified as haustorial secreted proteins (HSPs). Comparison of 21-0 with two presumed clonal field derivatives of this lineage (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (<i>Sr5</i>, <i>Sr11</i>, <i>Sr27</i>, <i>SrSatu</i>) identified mutations in 25 HSP effector candidates, some of which could explain their novel virulence phenotypes.
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