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.

Allotetraploid (2n = 4x = 32) white clover (Trifolium repens L.) is the most commonly cultivated legume component of temperate pastures, sown in swards with a companion grass species. Genetic control of growth performance of white clover on saline land is highly important for dairy industries, due to increasing soil salinity problems. The objective of this study was to identify quantitative trait loci (QTLs) for salinity tolerance in terms of vegetative growth under stress. Two parental genetic maps consisting of 213 and 159 marker loci and spanning 1,973.0 and 1,837.6 cM, respectively, were constructed using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers from a two-way pseudo-test cross F(1) population derived from pair-crossing of the Haifa(2) and LCL(2) genotypes. A total of 8 unique genomic regions on 8 linkage groups (LGs) of the Haifa(2) parental map and 6 unique regions on 5 LGs in the LCL(2) parental map were associated with plant growth under salt stress and relative growth under stress, as compared to control conditions. The results of this study indicate that salt tolerance in white clover is controlled by multiple QTLs, some at common locations, but each of limited magnitude. Location of these QTLs provides the genetic basis and potential for pyramiding of salt tolerance genes in breeding improvement.

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.

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.