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An isolate of the basidiomycete Puccinia striiformis, which causes yellow (stripe) rust on wheat, was selfed on the newly discovered alternate host, Berberis vulgaris. This allowed a study of the segregation of molecular markers and virulence in the progeny isolates, and of the development of fungal sexual structures and spore forms. Pycnia and aecia were obtained after inoculation of B. vulgaris with basidiospores resulting from germinating teliospores from infected wheat leaves. Subsequent inoculation of wheat with aeciospores from bulked aecia resulted in 16 progeny isolates of the S1 generation. Genotyping with 42 simple sequence repeat (SSR) markers confirmed a parental origin of progeny isolates. Of the 42 analyzed loci, 15 were heterozygous in the parental isolate and 14 revealed segregation in the progenies. This resulted in 11 new multilocus genotypes (MLGs), which confirmed segregation following sexual reproduction. Additionally, parental and progeny isolates were phenotyped using a genetic stock of wheat genotypes representing 21 resistance genes. All S1 progeny isolates had virulence for 14 out of 15 loci where the parental isolate was virulent. This was consistent with the hypothesis that virulence in plant pathogens is often recessive to avirulence, i.e., only expressed in a homozygous state. Furthermore, no segregation was observed for five out of six loci, for which the parental isolate had an avirulent phenotype. The results for one of the two segregating virulence/avirulence loci suggested that the parental isolate was heterozygous with Avr alleles resulting in different but clearly avirulent phenotypes. The other locus indicated that additional genes modifying the phenotypic expression of avirulence were involved., (Copyright © 2014 Elsevier Inc. All rights reserved.)