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To test the hypothesis that resistance in Phytophthora cinnamomi to control by the fungicide phosphite (phosphonate) would arise in sites with prolonged use of phosphite, 30 P. cinnamomi isolates were collected from a range of sites with different phosphite-use histories, including phosphite-treated and untreated avocado orchards, and phosphite-treated and untreated native vegetation sites. The colonizing ability of these isolates was tested by different inoculation methods against a range of host tissues, treated and untreated with phosphite, including mycelial stem inoculation on clonally propagated Leucadendron sp., mycelial root inoculation of lupin seedlings and zoospore inoculation of Eucalyptus sieberi cotyledons. Isolates from avocado orchards with a long history of phosphite use were, on average, more extensive colonizers of the phosphite-treated Leucadendron sp., lupin seedling roots and Eucalyptus sieberi cotyledons. These isolates did not colonize untreated plant tissue (Leucadendron sp.) more extensively than isolates from sites with no history of phosphite use and no isolates were resistant to control by phosphite. Analysis of all isolates with microsatellite markers revealed the majority were from a single clonal lineage. Selection for decreased sensitivity to phosphite in planta has taken place within asexual clonal lineages of P. cinnamomi in sites with prolonged use of phosphite.

Development of accurate high-throughput molecular marker systems such as SNPs permits evaluation and selection of favourable gene variants to accelerate elite varietal production. SNP discovery in perennial ryegrass has been based on PCR amplification and sequencing of multiple amplicons designed to scan all components of the transcriptional unit. Full-length genes (with complete intron-exon structure and promoter information) corresponding to well-defined biochemical functions such as lignin biosynthesis and oligosaccharide metabolism are ideal for complete SNP haplotype determination. Multiple SNPs at regular intervals across the transcriptional unit were detected within and between the heterozygous parents and validated in the progeny of the F (1)(NA(6) x AU(6)) genetic mapping family. Haplotype structures in the parental genotypes were defined and haplotypic abundance, structure and variation were assessed in diverse germplasm sources. Decay of LD to r (2) values of c. 0.2 typically occurs over 500-3,000 bp, comparable with gene length and with little apparent variation between diverse, ecotypic and varietal population sub-groups. Similar patterns were revealed as limited blocks of intragenic LD. The results are compatible with the reproductive biology of perennial ryegrass and the effects of large ancestral population size. This analysis provides crucial information to validate strategies for correlation of haplotypic diversity and phenotypic variation through association mapping.

Microsatellites were evaluated as genetic markers for the mitochondrial genome (mtDNA) of Phytophthora cinnamomi for population studies. Two (A)n microsatellite loci were cloned from the mtDNA of P. cinnamomi. Amplification products from these loci showed little polymorphism among Phytophthora isolates due to their location in coding regions of mtDNA. A further three (A)n microsatellite loci obtained from the complete mtDNA sequence of P. infestans were also not highly polymorphic, although located in non-coding mtDNA. The presence of the (A)n microsatellites was not conserved in the genus Phytophthora. Unlike those of the chloroplast genome of plants, (A)n microsatellite loci of mtDNA do not have potential as highly polymorphic markers in Phytophthora.

The genetic structure of populations of Phytophthora cinnamomi, a pathogen of an enormous variety of woody plants, was investigated using microsatellites. Three intensively sampled disease sites in southwest Australia were analyzed along with a large culture collection of Austra-lian isolates and some isolates from elsewhere in the world. The mutation in the four microsatellite loci analyzed revealed spatial patterns at the disease sites that correlated with the age of the infestation. Only three clonal lineages were identified in Australian populations and these same clonal lineages were present in worldwide populations, where it is suggested that a limited number of clonal lineages have spread in most regions. No evidence for sexual reproduction between these clonal lineages in Australia has been found even though the pathogen has the opportunity. Instead, mitotic recombination is frequent within the clonal lineages. The implications of this are discussed.

Susceptibility to foliar pathogens commonly causes significant reductions in productivity of the important temperate forage perennial ryegrass. Breeding for durable disease resistance involves not only the deployment of major genes but also the additive effects of minor genes. An approach based on in vitro single nucleotide polymorphism (SNP) discovery in candidate defence response (DR) genes has been used to develop potential diagnostic genetic markers. SNPs were predicted, validated and mapped for representatives of the pathogenesis-related (PR) protein-encoding and reactive oxygen species (ROS)-generating gene classes. The F(1)(NA(6) x AU(6)) two-way pseudo-test cross population was used for SNP genetic mapping and detection of quantitative trait loci (QTLs) in response to a crown rust field infection. Novel resistance QTLs were coincident with mapped DR gene SNPs. QTLs on LG3 and LG7 also coincided with both herbage quality QTLs and candidate genes for lignin biosynthesis. Multiple DR gene SNP loci additionally co-located with QTLs for grey leaf spot, bacterial wilt and crown rust resistance from other published studies. Further functional validation of DR gene SNP loci using methods such as fine-mapping and association genetics will improve the efficiency of parental selection based on superior allele content.

White clover (Trifolium repens L.) is an important temperate pasture legume that plays a key role as a companion to grass species, such as perennial ryegrass (Lolium perenne L.). Due to the outbreeding nature of white clover, cultivars are highly heterogeneous. Genetic diversity was assessed using 16 elite cultivars from Europe, North and South America, Australia, and New Zealand. Fifteen simple sequence repeat markers that detect single, codominant polymorphic genetic loci were selected for the study. The genetic relationships among individuals were compared using phenetic clustering, and those among cultivars were compared using nonmetric multidimensional scaling. Intrapopula tion variability exceeded interpopulation variability, with substantial overlap among populations and weak interpopula tion differentiation. No obvious or significant differentiation was observed on the basis of morphology or geographic origin of the cultivars. The number of parental genotypes used to derive each cultivar was not a major determinant of genome-wide genetic diversity. The outcomes of this assessment of genetic variation in elite white clover germplasm pools have important implications for the feasibility of molecular marker-based cultivar discrimination, and will be used to assist the design of linkage disequilibrium mapping strategies for marker-trait association.Key words: white clover, allotetraploid, genetic diversity, polymorphism, simple sequence repeat, cluster analysis, nonmetric multidimensional scaling.

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 1100 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 evalu- ate 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. Resume : L'agent causal de la rouille couronnee chez les ray-grass (P. coronata f. sp. lolii) est un biotrophe obliga- toire qui occasionne des pertes economiques importantes dans les zones temperees de production fourragere pour les fins de production de lait, de viande et de laine. Cette etude rapporte le developpement, la transportabilite et l'utilite de marqueurs microsatellites (SSR) associes a des genes chez la rouille couronnee. L'analyse de 1 100 etiquettes de sequences exprimees (EST) issus d'une banque d'ADNc provenant d'uredospores a decele 55 SSR. La plupart de ces EST-SSR contenaient des repetitions trinucleotidiques parfaites dont le nombre de repetitions etait constamment faible et chez lesquelles les motifs (ACC)n et (CAT)n etaient les plus communs. L'extraction d'ADN de pustules individuels, en conjonction avec une amplification a deplacement multiple (MDA), a rendu possible un crible PCR pour evaluer la performance des marqueurs genetiques. Un exemple de detection de diversite genetique intraspecifique a ete obtenu en analysant 16 isolats du P. coronata provenant du Royaume-Uni, de l'Australie, de la Nouvelle-Zelande et du Japon. Un sous-ensemble de 12 marqueurs EST-SSR robustes s'est avere utilisable chez des taxons fongiques proches (Puccinia spp.) ainsi que chez des champignons filamenteux appartenant aux ascomycetes. Mots cles : Puccinia coronata, microsatellite, etiquette de sequence exprimee, uredospore, diversite genetique, pathogene.

We report the development of four microsatellite loci into genetic markers for the diploid oomycete plant pathogen Phytophthora cinnamomi and that (AC)(n) and (AG)(n) microsatellites are significantly less frequent than in plant and mammal genomes. A minisatellite motif 14 bp long was also discovered. The four microsatellite loci were used to analyze sexual progeny from four separate crosses of P. cinnamomi. A large proportion of non-Mendelian inheritance was observed across all loci in all four crosses, including inheritance of more than two alleles at a locus and noninheritance of alleles from either parent at a locus. The aberrant inheritance is best explained by nondisjunction at meiosis in both the A1 parent and the A2 trisomic parents, resulting in aneuploid progeny. Two loci on the putative trisomic chromosome showed linkage and no loci were linked to mating type. One aneuploid offspring was shown to have lost alleles at two loci following subculture over 4 years, indicating that aneuploid progeny may not be mitotically stable.

Due to the complex genetic architecture of perennial ryegrass, based on an obligate outbreeding reproductive habit, association-mapping approaches to genetic dissection offer the potential for effective identification of genetic marker–trait linkages. Associations with genes for agronomic characters, such as components of herbage nutritive quality, may then be utilised for accelerated cultivar improvement using advanced molecular breeding practices. The objective of the present study was to evaluate the presence of such associations for a broad range of candidate genes involved in pathways of cell wall biosynthesis and carbohydrate metabolism. An association-mapping panel composed from a broad range of non-domesticated and varietal sources was assembled and assessed for genome-wide sequence polymorphism. Removal of significant population structure obtained a diverse meta-population (220 genotypes) suitable for association studies. The meta-population was established with replication as a spaced-plant field trial. All plants were genotyped with a cohort of candidate gene-derived single nucleotide polymorphism (SNP) markers. Herbage samples were harvested at both vegetative and reproductive stages and were measured for a range of herbage quality traits using near infrared reflectance spectroscopy. Significant associations were identified for ~50% of the genes, accounting for small but significant components of phenotypic variance. The identities of genes with associated SNPs were largely consistent with detailed knowledge of ryegrass biology, and they are interpreted in terms of known biochemical and physiological processes. Magnitudes of effect of observed marker–trait gene association were small, indicating that future activities should focus on genome-wide association studies in order to identify the majority of causal mutations for complex traits such as forage quality.

The products of RAPD-PCR amplification of Phytophthora cinnamomi DNA were separated by electrophoresis in agarose. Parallel Southern blots of the gels were hybridized with nick translated DNA from different species of Phytophthora. Fragments that hybridized specifically to P. cinnamomi DNA were identified. These fragments were purified and cloned into pUC18. Their specificity for P. cinnamomi was confirmed.