Haydar Karaoglu

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 142 microsatellite marker loci to the Molecular Ecology Resources database. Loci were developed for the following species: Agriophyllum squarrosum, Amazilia cyanocephala, Batillaria attramentaria, Fungal strain CTeY1 (Ascomycota), Gadopsis marmoratus, Juniperus phoenicea subsp. turbinata, Liriomyza sativae, Lupinus polyphyllus, Metschnikowia reukaufii, Puccinia striiformis and Xylocopa grisescens. These loci were cross‐tested on the following species: Amazilia beryllina, Amazilia candida, Amazilia rutila, Amazilia tzacatl, Amazilia violiceps, Amazilia yucatanensis, Campylopterus curvipennis, Cynanthus sordidus, Hylocharis leucotis, Juniperus brevifolia, Juniperus cedrus, Juniperus osteosperma, Juniperus oxycedrus, Juniperus thurifera, Liriomyza bryoniae, Liriomyza chinensis, Liriomyza huidobrensis and Liriomyza trifolii.

Characterization of two new Puccinia graminis f. sp. tritici races within the Ug99 lineage in South Africa

Two new races of the wheat (Triticum aestivum L.) stem rust pathogen, representing the fifth and sixth variants described within the Ug99 lineage, were detected in South Africa. Races TTKSP and PTKST (North American notation) were detected in 2007 and 2009, respectively. Except for Sr24 virulence, race TTKSP is phenotypically identical to TTKSF, a commonly detected race of Puccinia graminis f. sp. tritici (Pgt) in South Africa. PTKST is similar to TTKSP except that it produces a lower infection type on the Sr21 differential and has virulence for Sr31. Simple sequence repeat (SSR) analysis confirmed the genetic relationship amongst TTKSF, TTKSP, PTKST and TTKSK (Ug99). TTKSK, PTKST and TTKSF grouped together with 99% similarity, while sharing 88% genetic resemblance with TTKSP. These four races in turn shared only 31% similarity with other South African races. It is proposed that both TTKSP and PTKST represent exotic introductions of Pgt to South Africa.

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.

Recrudescent cryptococcosis, caused by Cryptococcus gattii (molecular type VGII), over a 13-year period in a Birman cat

A 17-year-old desexed male Birman cat presented with a fleshy mass protruding from the left ear canal. A culture from the mass revealed a heavy growth of Cryptococcus gattii (molecular type VGII, serotype B). The lesion resolved with antifungal therapy over 8 weeks. Itraconazole was continued indefinitely due to persistent high serum cryptococcal antigen titres. The cat was euthanased 12 months later due to the acute development of hindlimb ataxia and collapse which may or may not have been attributable to cryptococcosis. This cat had first presented when 4 years of age with a 3-week history of inappetance, sneezing and serous nasal discharge. Culture of swabs from both nostrils were positive for C. gattii (VGII). Fluconazole therapy produced steady improvement over a 6 month period, at which time therapy was discontinued. The cat presented 9 months later for sneezing, again with a positive culture of C. gattii from the nasal cavity. Antifungal therapy was continued for 8 months, after which time cultures were negative and symptoms resolved. Three episodes of cryptococcosis in a cat over a 13-year period were thus documented. Importantly, the two C. gattii isolates, obtained 13 years apart, were identical using DNA fingerprinting and random amplification of polymorphic DNA (RAPD) analysis.

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.

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.

Isolation and characterization of microsatellite markers for the causal agent of barley leaf rust, Puccinia hordei

In this study, we isolated and characterized seventy-six polymorphic microsatellite loci in the barley leaf rust pathogen, Puccinia hordei. The loci were screened with 19 isolates of P. hordei to assess polymorphism. The markers exhibited high variability, with the number of alleles ranging from 3 to 9 with an average of 4.92 alleles per locus. The polymorphic information content (PIC) varied from 0.401 to 0.843 with an average value of 0.624 per locus. These polymorphic microsatellite loci are the first of its type developed for P. hordei and will be extremely useful in studies of population genetics and in monitoring the global movement of this important cereal rust pathogen.

Development, characterization and application of genomic SSR markers for the oat stem rust pathogen Puccinia graminis f. sp. avenae

Oat stem rust, caused by Puccinia graminis f. sp. avenae (Pga), is one of the most severe diseases of oats worldwide. Population studies are scarce for this pathogen, mainly due to the lack of polymorphic molecular markers suitable for genetic analysis. We sequenced an Australian Pga isolate, determined the abundance of simple sequence repeats (SSRs), and developed PCR-based polymorphic markers suitable for genetic diversity analysis. The amplification of 194 primer pairs was initially assessed across a validation panel including 12 isolates of different cereal rust species and their formae speciales. A high cross-species amplification frequency was observed for most markers, however 19% SSRs were diagnostic for only P. graminis. A subset of 19 genomic-derived SSRs were deemed useful for genetic diversity analysis of Pga and were assessed on 66 Pga isolates from Australia, Brazil and Sweden. Brazilian and Australian isolates were characterized by one and two predominant clonal lineages, respectively. In contrast, the Swedish isolates previously shown to undergo sexual recombination were highly diverse (nine distinct genotypes out of 10 isolates) and divided into two sub-populations. The genomic-derived SSR markers developed in this study were well suited to the population studies undertaken, and have diagnostic capabilities that should aid in the identification of unknown rust pathogen species. This article is protected by copyright. All rights reserved.

Genetic Variability among Presumed Clonal Pathotypes of Puccinia graminis f. sp. tritici in Australia

Evolution of aggressiveness Puccinia graminis f. sp. tritici pathotypes with high virulence against important resistance genes constitutes a major threat to the global wheat production, and consequently food security. Changes in pathogenicity and environmental adaptation of these fungi have resulted in serious epidemics and total crop loss. In order to develop appropriate rust control measures, there is a need to understand the current pathogen-environment interactions; mechanisms of pathotype evolution, and pathogenic dynamics against the existing resistance genes. This research examined the genetic variability among 157 Australian Puccinia graminis f. sp. tritici isolates collected over 39 years. The isolates represented two putative clonal lineages derived from founding pathotypes 326-1,2,3,5,6 (Lineage 3) and 194-1,2,3,5,6 (Lineage 4). Pathogenicity assessments found general consistency with previous determinations, indicating that pathotypes in Lineage 3 and Lineage 4 generally displayed similar avirulence and virulence patterns. Cluster analysis of 111 isolates based on phenotypic data collected into one group comprising multiple subgroups. The level of variation in pathogenicity observed among the subgroups of the 99 derivatives of 326-1,2,3,5,6 and 194- 1,2,3,5,6 resulted from the pathotypes with uncommon or additional virulence. Six SSR markers showed 100% genetic similarity among Lineages 2, 3 and 4, and their distinctiveness from Lineage 1, consistent with independent origins. Seven highly polymorphic SSRs or microsatellites revealed 34 genotypes among 142 isolates with low genetic variation F-statistics (FST = 0.042) indicating high genetic similarity confirming their clonality arising via mutation. However, the existing pathotype genetic variations originate from the different forms and rates of mutation which vary per locus.