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Expressed sequence tag (EST) data for the grass fungal endophyte species Neotyphodium lolii and N. coenophialum has been used to generate a comprehensive set of simple sequence repeat (SSR) markers for the genetic analysis of the grass-endophyte symbiosis. Initial analysis of reference isolates from the perennial ryegrass endophyte N. lolii revealed low levels of genetic variation. Twenty-two EST-SSR markers were assembled into multiplex panels for in planta detection and genetic polymorphism analysis of the endophyte. The markers were shown to be endophyte-specific and effective at low mass-ratios of endophyte to plant genomic DNA. Genetic variation has been assessed in plants obtained from a globally distributed perennial ryegrass germplasm collection, as well as from Australian farms with variable incidence of endophyte-dependent livestock toxicosis and a perennial ryegrass variety containing an endophyte associated with reduced toxic effects. The reference isolates span the majority of the observed genetic variation in this sample, suggesting that relatively little genetic variation is present within the species, and that quantitative variation for expression of endophyte characters may be more associated with host grass genotypic differences. The geographical pattern of diversity of European-derived endophytes shows some similarity with the distribution of chloroplast DNA haplotype in perennial ryegrass, as may be expected for symbionts with matrilinear inheritance. This data supports the model of a monophyletic origin for N. lolii and dispersion from the Near East associated with ancient human migrations.

A number of plant species are used individually and in combination in pastures for the grazing of livestock. Temperate forages include grasses such as perennial ryegrass, tall fescue and phalaris and legumes such as white clover, red clover and alfalfa. Tropical forages include grasses such as buffelgrass and brachiaria and legumes such as cassia, siratro and stylo. Temperate and warm-season grass species are also important for turf and amenity purposes. In outbreeding forage species such as perennial ryegrass and white clover, natural and synthetic populations are genetically heterogeneous and show a high degree of developmental plasticity. Strategies for molecular marker development, genetic mapping, QTL detection and marker assisted selection must be designed in the context of these constraints. The current status of molecular marker technology with regard to genetic mapping, marker assisted selection, DNA profiling and molecular taxonomy is reviewed. The development of framework genetic maps based on highly informative co-dominant genetic markers (anchor RFLPs and SSRs) is described. Innovative strategies for implementation will be applied to these efficient marker systems in order to enhance forage breeding.