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Assignment or exclusion of an individual to specific populations or cultivars based on molecular genetic markers provides an attractive approach for varietal identification at the individual level in cross-pollinated plant species. The objectives of this study were (i) to explore the molecular diversity and relationships between Australasian perennial ryegrass (Lolium perenne L.) populations; (ii) to investigate accuracy of assignment of individuals to different types of populations including ecotypic, nonrestricted- and restricted-based cultivars; and (iii) to determine the effect of variable number of SSR loci and different statistical analysis methods on assignment accuracy. Eight forage perennial ryegrass populations comprising 48 individual plants per population were genotyped with 29 simple sequence repeat (SSR) marker loci. The number of alleles per locus ranged from 3.72 to 6.76. The mean observed heterozygosity varied from 0.419 to 0.538. Various genetic distance estimates and clustering methods obtained results consistent with breeding history. Genetic variation among (8.7%) and within populations (91.3%) was significant. Accuracy of individual assignment differed by population type, and it was higher (>90%) for restricted-based cultivars than for ecotypic and nonrestricted-based populations. These results indicate that SSR marker profiles can be effectively used to assign individuals for outbreeding populations such as perennial ryegrass. The approach used in this study may be useful for ryegrass germplasm management issues such as cultivar identification at the individual level.

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.

The symbiotic association between perennial ryegrass (Lolium perenne L.) and the fungal endophyte Neotyphodium lolii is associated with host-specific adaptations, particularly in response to abiotic and biotic stresses. Knowledge of the origin of the symbiosis and the contribution of endophyte genotype to host phenotypic variation is currently limited. Simple sequence repeat (SSR) markers were used to assess endophyte genetic diversity in a globally distributed collection of perennial ryegrass accessions. Consistent in planta detection was achieved with 18 of 22 SSR markers (primer pairs). Endophytes representing as many as four different taxa were detected in 42 accessions from 20 different countries, N. lolii being predominant. A total of 33 unique N. lolii genotypes were discriminated, of which 29 clustered into three major groups with limited within-group variation. The three major N. lolii groups were associated with distinct perennial ryegrass chloroplast haplotypes. The alkaloid profiles of accessions were apparently associated with the presence of specific N. lolii genotypes. Genotypic analysis provides a powerful method for genetic dissection of the grass-endophyte interaction and prediction of phenotypic variation based on genotypic variation.

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.

Crown rust fungus, Puccinia coronata f.sp. lolii is an obligate biotrophic pathogen of ryegrasses which causes significant reductions of herbage yield, palatability and digestibility. Genetic diversity in virulence has been reported in all the major temperate regions of the world and is therefore a major problem for pasture and turf breeders developing varieties with durable resistance for crown rust. Knowledge of the genetic variation present both within and between Australasian crown rust populations is essential for the efficient production of resistant varieties. A total of 11 efficient simple sequence repeat (SSR) markers developed from a urediniospore-derived expressed sequence tag (EST) resource have been used for Australasian intraspecific genetic diversity analysis. Seventy-two single pustule samples comprising three main populations from both North and South Islands of New Zealand and from south-eastern Australia were genotyped. The analysis identified 59 distinct genotypes, high levels of genetic diversity being detected both within and between populations. All methods of analysis detected no significant difference between isolates from the North and South Islands of New Zealand (p > 0.05), and high intrapopulation diversity between Victorian isolates. However high population differentiation (p < 0.001) was detected between Victorian isolates and those from the South Island (PhiPT [estimate of genetic variability = 0.101) and especially the North Islands (PhiPT = 0.162) of New Zealand. Genetic dissection of crown rust population structure within Australasia will inform the magnitude of gene pyramiding required for the development of varieties with durable resistance.

The key components for implementation of functionally associated genetic markers in perennial ryegrass and white clover breeding have been established. They include: generation of substantial genomic sequence resources; methods for functional analysis and verification of candidate gene status, including transcriptomics and transgenesis; efficient methods for in silico and in vitro discovery and validation of gene-associated SNP loci; evaluation of intra- and inter-population diversity; estimation of SNP haplotype complexity and LD; development of robust protocols for phenotypic analysis of key agronomic traits; design and implementation of experiments for haplotype-phenotype correlation; and development of efficient strategies for gene-based marker deployment in existing germplasm improvement programmes. These processes are mature for perennial ryegrass, and despite the added complexities of genetic architecture in white clover, optimized methods for SNP discovery should be equally effective for the latter species. The strategies are also suitable for other agronomically important temperate forage species, such as the grasses Italian ryegrass, tall fescue (Festuca arundinacea), meadow fescue (Festuca pratensis), cocksfoot (Dactylis glomerata) and timothy (Phleum pratense), as well as the legumes red clover (Trifolium pratense) and lucerne (Medicago sativa).

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.

Asexual fungal endophytes of the genus Epichloë form mutually beneficial associations with cool-season pasture grasses such as ryegrasses (Lolium spp.). Alkaloid production by the fungus confers both beneficial (deterrence of invertebrate herbivory) and detrimental (toxicity to mammalian livestock) attributes. A few novel strains with desirable metabolite profiles have been advanced into commercial production by inoculation of perennial ryegrass cultivars. In the present study, an integrated process for discovery of novel endophytes based on exploitation of genotypic information has been designed and implemented. A survey of genetic diversity was performed on a large-scale, customised germplasm collection (containing 244 accessions) in order to identify previously uncharacterised endophyte genotypes. Preliminary qualitative metabolic profiling in the endogenous genetic background permitted elimination of undesirable combinations, and definition of a subset of priority candidates. A novel method was developed for inoculation of endophytes into meristem-culture-derived callus tissue of single genotypes from multiple perennial ryegrass cultivars, in order to allow isogenic comparisons with respect to both host and endophyte genotype. Beneficial toxin profiles were confirmed for associations formed with the grass genotypic panel, and semi-quantitative metabolite analysis provided evidence for genotype-specific effects of both host and genotype on levels of alkaloid production. Vegetative stability was also assessed over both shorter and longer terms. A final set of three prioritised candidates was obtained, two of which (belonging to the known taxa E. festucae var. lolii and LpTG-2) produce the alkaloids ergovaline and peramine. The third candidate endophyte, belonging to a putative novel taxon, solely produces representatives of an additional alkaloid class, the epoxy-janthitrems.

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.

Genetic analysis of host resistance to the crown rust pathogen of ryegrasses has traditionally involved the use of composite urediniospore collections, which may contain multiple genotypes and virulence races. Consequently, discrimination between major gene responses to alternative races and quantitative resistance has proved challenging for disease trait-dissection studies.Amethod of propagation on detached leaves has been used to derive genetically homogenised crown rust pathogen lines from bulked spore collections obtained from five distinct Australian locations. Spores from individual pustules were repeatedly reinoculated onto fresh leaves. Amplicon complexity was assessed at the conclusion of each cycle using crown rust pathogen expressed sequence tag (EST)-derived simple sequence repeat (SSR) primer pairs. The mean number of amplification products per isolate declined over successive rounds of reinfection. The number of rounds of sequential infection required for decay to stable levels was dependent on the diversity present within the bulked spore collection. Large-scale propagation of the putative homogenised populations developed in the present study may provide inoculum for infection studies to allow quantitative trait loci mapping of specific gene-for-gene responses. The methodology has significant applications for the pyramiding of resistance genes specific to diverse crown rust pathogen genotypes during cultivar development and for pathotyping of existing elite germplasm. This approach is also applicable to other Puccinia species.

Australian canola (Brassica napus L.) has been relatively isolated from the global gene pool and limited knowledge is available for genetic variability based on DNA profiling. In the present study, genetic diversity of recent Australian canola cultivars was determined by simple sequence repeat (SSR) marker analysis. In total, 405 individuals from 48 varieties were genotyped with 18 primer pairs, resulting in 112 polymorphic features. The number of polymorphic features amplified by each SSR primer pair varied from 3 to 16. Analysis of molecular variance (AMOVA) detected 53.7% and 46.3% within- and between-cultivar variation, respectively. Intra-cultivar genetic variability differed according to cultivar. The number of polymorphic features per cultivar varied from 35 (Ag-Spectrum) to 72 (Ag-Insignia), while mean sum of squares (MSS) varied from 6.29 (Tornado TT) to 24.76 (Ag-Emblem). Genetic differentiation of cultivars generally reflected pedigree structure and origin by breeding organisation. Clustering and principal coordinate analysis (PCoA) indicated that the individuals were separated into 4 major groups. The genetic diversity information from this study will be useful for future Australian canola breeding programs.

Alkaloids produced by the perennial ryegrass pasture and turf grass endophyte Neotyphodium lolii confer both beneficial (insect feeding deterrence) and deleterious (mammalian herbivore toxicoses) effects. Novel endophyte strains with altered and desirable in planta metabolic profiles have been introduced into cultivars by inoculation. The potentially severe effects of contamination with standard toxic endophytes provide an important incentive for quality control during varietal development. Genotyping with expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers in order to monitor endophyte content was evaluated in this study. The NEA2 endophyte, described as producing low levels of lolitrem B and moderate quantities of ergovaline and peramine, has been deployed in the cultivar (cv.) Tolosa and transferred to other proprietary varieties by crossing or controlled inoculation. In planta genotypic analysis of cv. Tolosa-derived plants revealed the presence of 2 distinct NEA2 genotypes (A and B). Variable proportions of NEA2 A and B were observed at different stages of cultivar multiplication, and NEA2 B was present in inoculated varieties. Several accessions also revealed the presence of a third genotype (C), identical to standard toxic types. Alkaloid measurement in plants containing individual A and B endophytes identified distinct profiles which collectively account for the average NEA2 profile. The C variant is apparently a contaminant arising from seed or adventitious seedlings during varietal multiplication or incomplete removal of endophyte from seed before inoculation. SSR-based genotyping provides an efficient means to continuously assess endophyte prevalence and identity in pasture grass breeding programs.