title: Development and characterization of EST-SSR markers for the crown rust pathogen of ryegrass (Puccinia coronata f.sp. lolii).
abstract: The causative organism of crown rust in ryegrasses (Puccinia coronata f.sp. lolii) is an obligate biotroph that causes significant economic losses within the temperate grazing industries of dairy, meat, and wool production. This study reports on the development, transferability, and utility of gene-associated simple sequence repeat (SSR) molecular markers for crown rust. Analysis of 1,100 expressed sequence tag (EST) sequences from a urediniospore-derived cDNA library detected 55 SSR loci. The majority of EST-SSR arrays contained perfect trinucleotide repeats with consistently low repeat numbers, and the motifs (ACC)n and (CAT)n were most commonly represented. DNA extraction from single pustules, in conjunction with multiple displacement amplification, provided the basis for PCR-based screening to evaluate genetic marker performance. An example of the identification of intraspecific genetic diversity was obtained from the analysis of 16 P. coronata isolates originating from the United Kingdom, Australia, New Zealand, and Japan. A subset of 12 robust EST-SSR markers was informative for determination of pathogen diversity within and between these localities. It was also demonstrated that crown rust EST-SSR markers were capable of cross-amplification in closely related fungal taxa (Puccinia spp.) and filamentous fungi within the Ascomycota.
authors.affiliation: Primary Industries Research Victoria, Victorian AgriBiosciences Centre, Australia 1 Park Drive, La Trobe Research and Development Park, Bundoora, Victoria 3083, Australia.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Dracatos
authors.forename: Jeremy L
authors.initials: JL
authors.lastname: Dumsday
authors.forename: Rhiannon S
authors.initials: RS
authors.lastname: Olle
authors.forename: Noel O I
authors.initials: NO
authors.lastname: Cogan
authors.forename: Mark P
authors.initials: MP
authors.lastname: Dobrowolski
authors.forename: Masahiro
authors.initials: M
authors.lastname: Fujimori
authors.forename: Hywel
authors.initials: H
authors.lastname: Roderick
authors.forename: Alan V
authors.initials: AV
authors.lastname: Stewart
authors.forename: Kevin F
authors.initials: KF
authors.lastname: Smith
authors.forename: John W
authors.initials: JW
authors.lastname: Forster
mesh_terms: D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis; D004271:DNA, Fungal / Q000145:classification; D020224:Expressed Sequence Tags; D005658:Fungi / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D005819:Genetic Markers; D014644:Genetic Variation; D016681:Genome, Fungal; D008129:Lolium / Q000382:microbiology*; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010802:Phylogeny; D010935:Plant Diseases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid
chemical_list: D018076:DNA, Complementary; D004271:DNA, Fungal; D005819:Genetic Markers