Molecular Characterization of Australian Isolates of Puccinia graminis f. sp. tritici Supports Long-Term Clonality but also Reveals Cryptic Genetic Variation.

Long-term surveys of pathogenicity in Puccinia graminis f. sp. tritici in Australia have implicated mutation as a major source of virulence, at times leading to the demise of stem-rust-resistant wheat cultivars and substantial yield losses. Since 1925, these surveys have identified at least four occasions on which exotic isolates of P. graminis f. sp. tritici appeared in Australia, with each acting as a founding isolate that gave rise sequentially to derivative pathotypes via presumed single-step mutation. The current study examined the relationship between virulence and molecular patterns using simple-sequence repeat (SSR) markers on selected isolates of P. graminis f. sp. tritici collected in Australia during a 52-year period in order to propose an evolutionary pathway involving these isolates. Studies of SSR variability among this collection of isolates within a putative clonal lineage based on pathotype 21-0, first detected in 1954 (the "21/34 lineage"), provided compelling evidence of clonality over the 52-year period, coupled with single-step acquisition of virulence for resistance genes. It also supported the postulation that two triticale-attacking pathotypes (34-2,12 and 34-2,12,13) detected in the early 1980s were derived from pathotype 21-0 via stepwise sequential acquisition of virulence for Sr5, Sr11, Sr27, and then SrSatu. Some of the isolates examined that were regarded as members of the race 21/34 lineage based on pathogenicity differed significantly in their SSR genotypes, indicating that they may have originated from processes more complex than simple mutation. This included two isolates of pathotype 21-0, which were collected in 1994 and 2006. Given that sexual recombination in P. graminis is rare or absent in Australia, the cryptic complexity observed could indicate that one or more of these isolates arose as a consequence of asexual recombination.

Leaf rust of cultivated barley: pathology and control.

Leaf rust of barley is caused by the macrocyclic, heteroecious rust pathogen Puccinia hordei, with aecia reported from selected species of the genera Ornithogalum, Leopoldia, and Dipcadi, and uredinia and telia occurring on Hordeum vulgare, H. vulgare ssp. spontaneum, Hordeum bulbosum, and Hordeum murinum, on which distinct parasitic specialization occurs. Although Puccinia hordei is sporadic in its occurrence, it is probably the most common and widely distributed rust disease of barley. Leaf rust has increased in importance in recent decades in temperate barley-growing regions, presumably because of more intensive agricultural practices. Although total crop loss does not occur, under epidemic conditions yield reductions of up to 62% have been reported in susceptible varieties. Leaf rust is primarily controlled by the use of resistant cultivars, and, to date, 21 seedling resistance genes and two adult plant resistance (APR) genes have been identified. Virulence has been detected for most seedling resistance genes but is unknown for the APR genes Rph20 and Rph23. Other potentially new sources of APR have been reported, and additivity has been described for some of these resistances. Approaches to achieving durable resistance to leaf rust in barley are discussed.

The wheat Sr50 gene reveals rich diversity at a cereal disease resistance locus.

We identify the wheat stem rust resistance gene Sr50 (using physical mapping, mutation and complementation) as homologous to barley Mla, encoding a coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) protein. We show that Sr50 confers a unique resistance specificity different from Sr31 and other genes on rye chromosome 1RS, and is effective against the broadly virulent Ug99 race lineage. Extensive haplotype diversity at the rye Sr50 locus holds promise for mining effective resistance genes.

Comparative genomics of Australian isolates of the wheat stem rust pathogen Puccinia graminis f. sp. tritici reveals extensive polymorphism in candidate effector genes.

The wheat stem rust fungus Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. In this study, a draft genome was built for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly were performed resulting in a 92 Mbp reference genome for Pgt isolate 21-0. Approximately 13 Mbp of de novo assembled sequence in this genome is not present in the p7a reference assembly. This novel sequence is not specific to 21-0 as it is also present in three other Pgt rust isolates of independent origin. The new reference genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ∼10% of the genes each in germinated spores and haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 520 were classified as haustorial secreted proteins (HSPs). Comparison of 21-0 with two presumed clonal field derivatives of this lineage (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 25 HSP effector candidates. Some of these mutations could explain their novel virulence phenotypes.

Molecular mapping of leaf rust resistance gene Rph14 in Hordeum vulgare.

An incompletely dominant gene conferring resistance to Puccinia hordei, Rph14, identified previously in an accession of Hordeum vulgare, confers resistance to all known pathotypes of P. hordei in Australia. Knowledge of the chromosomal location of Rph14 and the identification of DNA markers closely linked to it will facilitate combining it with other important leaf rust resistance genes to achieve long lasting resistance. The inheritance of Rph14 was confirmed using 146 and 106 F(3) lines derived from the crosses 'Baudin'/'PI 584760' (Rph14) and 'Ricardo'/'PI 584760' (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks using DArT markers located Rph14 to the short arm of chromosome 2H. DArT marker bPb-1664 was identified as having the closest genetic association with Rph14. PCR based marker analysis identified a single SSR marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cm in the 'Baudin'/'PI 584760'and 'Ricardo'/'PI 584760' populations, respectively.

Development of polymorphic microsatellite markers for Cryptococcus neoformans.

Cryptococcus neoformans is a haploid basidiomycetous yeast that causes life-threatening infections in patients with and without impaired immune function. Present typing systems for C. neoformans are limited by either poor standardization or high cost. We present eleven microsatellite loci that were developed from the published genomes of C. neoformans var. neoformans, and are applicable to the varieties and hybrids within C. neoformans.

Survey of simple sequence repeats in completed fungal genomes.

The use of simple sequence repeats or microsatellites as genetic markers has become very popular because of their abundance and length variation between different individuals. SSRs are tandem repeat units of 1 to 6 base pairs that are found abundantly in many prokaryotic and eukaryotic genomes. This is the first study examining and comparing SSRs in completely sequenced fungal genomes. We analyzed and compared the occurrences, relative abundance, relative density, most common, and longest SSRs in nine taxonomically different fungal species: Aspergillus nidulans, Cryptococcus neoformans, Encephalitozoon cuniculi, Fusarium graminearum, Magnaporthe grisea, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Ustilago maydis. Our analysis revealed that, in all of the genomes studied, the occurrence, abundance, and relative density of SSRs varied and was not influenced by the genome sizes. No correlation between relative abundance and the genome sizes was observed, but it was shown that N. crassa, the largest genome analyzed had the highest relative abundance of SSRs. In most genomes, mononucleotide, dinucleotide, and trinucleotide repeats were more abundant than the longer repeated SSRs. Generally, in each organism, the occurrence, relative abundance, and relative density of SSRs decreased as the repeat unit increased. Furthermore, each organism had its own common and longest SSRs. Our analysis showed that the relative abundance of SSRs in fungi is low compared with the human genome and that longer SSRs in fungi are rare. In addition to providing new information concerning the abundance of SSRs for each of these fungi, the results provide a general source of molecular markers that could be useful for a variety of applications such as population genetics and strain identification of fungal organisms.