Bacterial artificial chromosomes(BACs)or yeast artificial chromosomes(YACs)containing large inserts as probes for fluorescence in situ hybridization(FISH)have been used in the physical mapping of specific DNA sequences,especially for single-or low-copy sequences.Our earlier study identified Stpk-V,a powdery mildew resistance-related gene located on the 6VS chromosome arm of the wild grass Haynaldia villosa(tribe Triticeae),and obtained several Triticum aestivum–H.villosa alien chromosome lines carrying the Stpk-V gene.However,the precise physical location of the Stpk-V gene on chromosome 6VS is not known.In this study,we used TAC-FISH with TAC15 as the probe coupled with sequential genomic in situ hybridization(GISH)to determine the physical location of the Stpk-V gene in different T.aestivum–H.villosa 6V alien chromosome lines,including addition,substitution and translocation lines.The result indicated that the fraction length of the Stpk-V locus is 0.575±0.035 on the 6V chromosome short arm and this was confirmed by FISH using TAC15 as the probe for tracing the Stpk-V gene in other genetic stocks.The cytological mapping strategies used in this study will be of benefit for tracing the alien gene location in the course of introducing desirable traits from wild species. Bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs) containing large inserts as probes for fluorescence in situ hybridization (FISH) have been used in the physical mapping of specific DNA sequences, especially for single-or low-copy sequences. Earlier Study identified Stpk-V, a powdery mildew resistance-related gene located on the 6VS chromosome arm of the wild grass Haynaldia villosa (tribe Triticeae), and obtained several Triticum aestivum-H. villosa alien chromosome lines carrying the Stpk-V gene. , the precise physical location of the Stpk-V gene on chromosome 6VS is not known. this study, we used TAC-FISH with TAC15 as the probe coupled with sequential genomic in situ hybridization (GISH) to determine the physical location of the Stpk -V gene in different T. aestivum-H. villosa 6V alien chromosome lines, including addition, substitution and translocation lines. The result indicates that fraction length of the Stpk-V locus is 0.575 ± 0.035 on the 6V chromoso me short arm and this was was by FISH using TAC15 as the probe for tracing the Stpk-V gene in other genetic stocks.The cytological mapping strategies used in this study will be benefit for tracing the alien gene location in the course of traits from wild species.