Development and characterization of EST-SSR markers for the crown rust pathogen of ryegrass (Puccinia coronataf.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.

Transferability of SSR markers from related Uredinales species to the coffee rust Hemileia vastatrix

“…Assays of cross-amplification in other taxa with some of the SSR markers used in this study indicated a moderate level of transferability with genetically distant organisms (Dracatos et al, 2006a). The low rate of transferability observed in this study could be evidence of mutations in the flanking regions of the SSR, the possibility of interruptions within the repeat motif or the presence of introns in the amplified region, as evidenced by sequencing of amplification products of these EST-SSRs by Dracatos et al (2006a).…”

“…The low rate of transferability observed in this study could be evidence of mutations in the flanking regions of the SSR, the possibility of interruptions within the repeat motif or the presence of introns in the amplified region, as evidenced by sequencing of amplification products of these EST-SSRs by Dracatos et al (2006a). Additionally, it would be ideal to perform a further optimisation of PCR conditions for the markers designed in H. vastatrix isolates, which was performed in this study.…”

Genetic Diversity in Australasian Populations of the Crown Rust Pathogen of Ryegrasses (Puccinia coronata f.sp. lolii)

“…Initial genetic diversity analysis using 16 single-pustule isolates from geographically diverse temperate regions (New Zealand, UK, Japan and Australia) identified 15 genetically distinct genotypes. A preliminary analysis using UPGMA generated a dendrogram showing a lack of correlation between clustering and geographic origin of isolates, which may indicate early evolution of population complexity and diversity (Dracatos et al 2006). …”

“…lolii provides the means to assess intraspecific genetic diversity within and between geographic locations and to define aspects of population structure (Dracatos et al 2006). SSR markers have previously been used to assess genetic diversity in the wheat stripe rust pathogen (P. striiformis f.sp.…”

Diversity of oat crown rust (Puccinia coronata f.sp. avenae) isolates detected by virulence and AFLP analyses

“…which probably resulted from exotic introduction of genetically distinct pathogen isolates. Dracatos et al (2006) developed EST-SSR markers for the P. coronata f.sp. lolii P. Syd.…”