striiformis f. sp. tritici, causing stripe rust, have a sexual cycle on North American Berberis spp. and have a greater race variability where the alternate host is present. The Pt aeciospore stage is on Thalictrum spp. and Isopyrum, found mainly in the Mediterranean region, however, other Thalictrum species present in North America can support a reduced level of infection but are generally resistant to Pt. Populations are essentially asexual, supported by the lack of recombin ation found in numerous North American races. A parasexual cycle may exist allowing recombination since germtube Inhibitors,Modulators,Libraries fusion, nuclear migration, and bridging structures between nuclei have been observed in Pt. The obligate biotrophic nature of cereal rusts makes experimental manipulation difficult, however, genomics provides a means of studying evolution and gene function.
We set out to understand the genome variation of two rust fungi at three regions. Inhibitors,Modulators,Libraries A Pt bacterial artificial chromosome library was made and clones were identified using three probes that would isolate regions of predicted secreted proteins and Drug_discovery avirulence. Sequenced DNA regions of Pt were compared to syntenic regions in two rust species with complete genome sequences, Pgt, and Mlp, and evaluated for genomic conservation, expansion and mutations. Results BAC library construction Urediniospores harbor two haploid nuclei with an esti mated total genome complexity for Pt of approximately 135 Mb, based on comparative DNA fluorescence and the current total size of the genome assembly. The generated P.
triticina BAC library contained 15,360 clones arrayed Inhibitors,Modulators,Libraries in 384 well plates with an average insert size of 105 kb representing an estimated 10 to 12 genome equivalents. A single copy probe identified nine positive clones on high density filters, and assuming fragments were randomly cloned during library construction, this is in agreement with the estimated genome coverage. BAC clone selection, sequencing and characterization Three genomic regions were targeted for comparison. Previous work had mapped a Pgt RAD18 homolog in a genomic region harboring an avirulence gene. Using PgtRAD18 as Inhibitors,Modulators,Libraries a reference, PT0313. J16. C21 was identified from a Pt EST database using TBLASTN and used as a probe. Nine positive BAC clones were found and clone 1F16 was selected for sequencing because of its longer length and the centralized location of PtRAD18 within the BAC clone.
Sequences from Pt1F16 were assembled into two contiguous sequences of 39,219 and 63,874 bp, totaling 103,093 bp. The GC content of these sequences was 47%. Subclones In a functional screen of Uromyces viciae fabae, secreted peptide effector protein UF5 was related to the flax rust Melampsora lini haustorial expressed secreted protein HESP 379. A Pgt genome search revealed several predicted secreted protein homologs in close proximity, suggesting the presence of small clusters of predicted secreted proteins.