Intergeneric somatic hybridization in Gramineae: somatic hybrid plants between tall fescue (Festuca arundinacea Schreb.) and Italian ryegrass (Lolium multiflorum Lam.)

Summary Tall fescue (Festuca arundinacea Schreb.) protoplasts, inactivated by iodoacetamide, and non-morphogenic Italian ryegrass (Lolium multiflorum Lam.) protoplasts, both derived from suspension cultures, were electrofused and putative somatic hybrid plants were recovered. Two different genotypic fusion combinations were carried out and several green plants were regenerated in one of them. With respect to plant habitus, leaf and inflorescence morphology, the regenerants had phenotypes intermediate between those of the parents. Southern hybridization analysis using a rice ribosomal DNA probe revealed that the regenerants contained both tall fescue- and Italian ryegrass-specific-DNA fragments. A cloned Italian ryegrass-specific interspersed DNA probe hybridized to total genomic DNA from Italian ryegrass and from the green regenerated somatic hybrid plants but not to tall fescue. Chromosome counts and zymograms of leaf esterases suggested nuclear genome instability of the somatic hybrid plants analyzed. Four mitochondrial probes and one chloroplast DNA probe were used in Southern hybridization experiments to analyze the organellar composition of the somatic hybrids obtained. The somatic hybrid plants analyzed showed tall fescue, additive or novel mtDNA patterns when hybridized with different mitochondrial gene-specific probes, while corresponding analysis using a chloroplast gene-specific probe revealed in all cases the tall fescue hybridization profile. Independently regenerated F. arundinacea (+) L. multiflorum somatic hybrid plants were successfully transferred to soil and grown to maturity, representing the first flowering intergeneric somatic hybrids recovered in Gramineae.     

Use of genome-specific repetitive DNA sequences to monitor chromatin introgression from Festuca mairei into Lolium perenne

Repetitive DNA sequences contribute considerably to an understanding of the genomes of higher plants. Repetitive DNA sequences tend to be genome-specific due to the rate of amplification and extent of divergence. Two genome-specific probes from the genomic DNA library of Festuca arundinacea var. genuina Schreb.were selected and characterized. TF521 was found to be P genome-specific since it was able to hybridize with Festuca pratensis Huds. (PP) and Festuca arundinacea var. genuina (PPG₁G₁G₂G₂), but not, or only weakly, with tetraploid Festuca species. TF521 hybridized only with the diploid Festuca and not with the Lolium species (LL). TF436 was specific to tetraploid species of Festuca, such as F. arundinacea var. glauces-cens Boiss. (G₁G₁G₂G₂) and Festuca mairei St. Yves (M₁M₁M₂M₂). By means of Southern hybridization, TF436 was used to detect chromatin introgression of F. mairei in the progenies of the hybrid F. mairei×Lolium perenne L. Potential addition and translocation lines were identified in the BC₁F₁ derivatives of F. mairei×L. perenne. In situ hybridization was used to confirm the genetic identity of these lines. Sequence analyses indicated that TF436 and TF521 were two novel DNA sequences as no homologous sequences were found in Genebank. Received: 22 June 2000 / Accepted: 3 November 2000     

Variability of ribosomal DNA sites inFestuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH

This study focuses on the variability of chromosomal location and number of ribosomal DNA (rDNA) sites in some diploid and autotetraploidFestuca pratensis andLolium perenne cultivars, as well as on identification of rDNA-bearing chromosomes in their triploid and tetraploidF. pratensis ×L. perenne hybrids. The rDNA loci were mapped using fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes, and the origin of parental genomes was verified by genomic in situ hybridization (GISH) withL. perenne genomicDNAas a probe, andF. pratensis genomic DNA as a block. FISH detected variation in the number and chromosomal location of both 5S and 45S rDNA sites. InF. pratensis mostly additional signals of 5S rDNA loci occurred, as compared with standardF. pratensis karyotypes. Losses of 45S rDNA loci were more frequent inL. perenne cultivars and intergeneric hybrids. Comparison of theF. pratensis andL. perenne genomes approved a higher number of rDNA sites as well as variation in chromosomal rDNA location inL. perenne. A greater instability ofF. pratensis-genome-like andL. perenne-genome-like chromosomes in tetraploid hybrids was revealed, indicating gains and losses of rDNA loci, respectively. Our data indicate that the rDNA loci physically mapped on chromosomes 2 and 3 inF. pratensis and on chromosome 3 inL. perenne are useful markers for these chromosomes in intergenericFestuca ×Lolium hybrids.     

Comparative RFLP mapping of meadow and tall fescue

 Molecular markers based on restriction fragment length polymorphism (RFLP) were used to construct a genetic linkage map in diploid meadow fescue, Festuca pratensis Huds. (2n=2x=14, genomic designation PP), and to compare its genomic relationship with a related species, hexaploid tall fescue (Festuca arundinacea Schreb.; 2n=6x=42, PPG₁G₁G₂G₂). Using a collection of 66 tall-fescue (heterologous) markers, an RFLP linkage map was constructed in F. pratensis. This map, which has a total length of 280.1 cM, includes seven linkage groups. A comparison of 33 markers that were mapped in both F. pratensis and F. arundinacea detected highly conserved linkage groups between these two species. Our data are consistent with the proposal that one of the genomes of F. arundinacea was derived from F. pratensis. However, since significant changes in marker sequences, map distances, and homoeologous linkage groups were also detected between the two species, it appears that the P genome diverged substantially during evolution from the diploid to the hexaploid Festuca. Received: 23 May 1997 / Accepted: 15 January 1998     

Phylogeny of chromosome races ofFestuca arundinacea andF. mairei (Poaceae) as indicated by seed protein electrophoresis

Seed protein electrophoresis of four chromosomes races ofFestuca arundinacea, F. mairei and their progenitors showed variation in banding patterns. High protein similarities betweenF. arundinacea, F. mairei, F. scariosa, andF. pratensis indicate close phylogenetic relationships of these species. The ancestry ofF. arundinacea cytotypes could be narrowed to three diploid species:F. scariosa, F. pratensis, andF. rubra or to their close relatives.     

Phylogeographical history of the widespread meadow fescue (Festuca pratensis Huds.) inferred from chloroplast DNA sequences

Aim Here we explore the variation in chloroplast DNA (cpDNA) in a widespread Eurasian diploid forage grass, meadow fescue (Festuca pratensis Huds.), to address its phylogeographical history. In particular, we aim to answer whether the post‐glacial migration routes of meadow fescue are associated with the spread of agriculture or concurrent with well‐documented natural migration pathways from glacial refugia. Location A total of 56 Eurasian accessions of F. pratensis were analysed, representing the entire native distribution area as well as non‐native areas in northernmost Europe. Methods Based on initial sequencing of 10 non‐coding cpDNA regions, three regions were sequenced for all F. pratensis accessions. For reference, three closely related species [the diploid Lolium perenne L. and the polyploids Festuca arundinacea Schreb. and Festuca gigantea (L.) Vill.] were also sequenced, as well as the more distantly related Festuca ovina L. Divergence times were estimated assuming a simple molecular clock, calibrated using a previously published estimate of 9 Myr for the divergence between fine‐leaved (F. ovina) and broad‐leaved fescues (F. pratensis, F. arundinacea and F. gigantea). Results Limited, but geographically structured, cpDNA variation was observed in F. pratensis. Three haplotypes, estimated to have diverged 0.16 Ma, were identified: one western European (A), one with a wide eastern distribution from central‐eastern Europe into Asia (B) and one Caucasian (C). The haplotypes of the polyploids and L. perenne were estimated to have diverged from haplotype A in F. pratensis 0.8–1.3 Ma. Main conclusions We found no definite evidence for migration of the diploid F. pratensis associated with the spread of agriculture from the Fertile Crescent after the last glaciation. The distinct geographical structuring of the present‐day variation in cpDNA can rather be explained by northwards expansion of the western haplotype from an Iberian refugium, expansion of the eastern haplotype from an unlocated (south‐)eastern refugium and glacial survival without subsequent expansion from a Caucasian refugium. The high level of cpDNA divergence observed between this diploid and the polyploids which have probably been derived from it may suggest that the very low level of cpDNA variation in the diploid is caused by a recent bottleneck. Today, F. pratensis is widespread in the open agricultural landscape but appears otherwise confined to naturally open habitats such as river banks, and its populations may have been decimated when dense forests dominated in the previous interglacial.     

Effects of fungal endophytes on the seed and seedling biology of Lolium perenne and Festuca arundinacea

Summary Many grasses are infected by endophytic fungi that grow intercellularly in leaves, stems, and flowers and are transmitted maternally by hyphal growth into ovules and seeds. The seed biology and seedling growth of endophyte-infected and uninfected perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) were investigated under controlled environmental conditions. The percentage of filled seeds produced by infected tall fescue was over twice of uninfected tall fescue; infected and uninfected perennial reegrass had similar percentages. Weights of seeds from infected and uninfected plants were similar in both species. Seeds from infected plants of both species exhibited a higher rate of germination than seeds from uninfected plants. Shoot growth in the greenhouse was compared by making three sequential harvests of above-ground plant parts from infected and uninfected plants of both species. Infected perennial ryegrass plants produced significantly more biomass and tillers than uninfected plants after 6 and 10 weeks of growth and significantly more biomass after 14 weeks of growth. Infected tall fescue plants produced significantly more biomass and tillers than uninfected plants after 10 and 14 weeks of growth. The physiological mechanism of enhancement of growth is not known. The results of this study suggest that infected plants may have a selective advantage in populations with uninfected members.     

Accumulation of selenium by different plant species grown under increasing sodium and calcium chloride salinity

High levels of naturally occurring selenium (Se) are often found in conjunction with different forms of salinity in central California. Plants considered for use in phytoremediation of high Se levels must therefore be salt tolerant. Selenium accumulation was evaluated for the following species under increasing salt (NaCl and CaCl) conditions:Brassica napus L. (canola),Hibiscus cannibinus L. (kenaf),Festuca arundinacea L. (tall fescue), andLotus tenuis L. (birdsfoot trefoil). The experimental design was a complete randomized block with four salt treatments of <1, 5, 10, and 20 dS m^-1, four plant species, three blocks, and six replicates per treatment. Ninety days after growing in the respective salt treated soil with a Se concentration of 2 mg Se kg^-1 soil, added as Na₂SeO₄, all plant species were completely harvested. Among the species tested, shoot and root dry matter yield of kenaf was most significantly (p<0.001) affected by the highest salt treatment and tall fescue and canola were the least affected species. Generally there was a decrease in tissue accumulation of Se with increasing salt levels, except that low levels of salinity stimulated Se accumulation in canola. Canola leaf and root tissue accumulated the highest concentrations of Se (315 and 80 mg Se kg^-1 DM) and tall fescue the least (35 and 7 mg Se kg^-1 DM). Total soil Se concentrations all harvest were significantly (p<0.05) lower for all species at all salt treatments. Removal of Se from soil was greatest by canola followed by birdsfoot trefoil, kenaf and tall fescue. Among the four species, canola was the best candidate for removing Se under the tested salinity conditions. Kenaf may be effective because of its large biomass production, while tall fescue and birdsfoot trefoil may be effective because they can be repeatedly clipped as perennial crops.     

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.     

Relationships between three species ofFestuca sect.Bovinae (Poaceae)

Seed protein electrophoresis confirms the existence of polymorphism among hexaploid populations ofFestuca arundinacea. Both protein and morphological results suggest thatF. pratensis andF. arundinacea should retain independent specific status. High protein homology of these two species withF. gigantea points towards phylogenetic links between these taxa.     

Temporal and spatial patterns of soil water following wildfire-induced changes in plant communities in the Great Basin in Nevada,USA

Infection of tall fescue (Festuca arundinacea Schreb.) with its endemicNeotyphodium coenophialum-endophyte (Morgan-Jones and Gams) Glenn, Bacon and Hanlin appears to reduce copper (Cu) concentrations in forage and serum of grazing animals, contributing to a range of immune-related disorders. A greenhouse experiment was conducted to identify effects of novel endophyte strains on Cu acquisition by tall fescue (Festuca arundinacea Schreb.) varieties Grasslands Flecha and Jesup infected with a novel, non ergot producing endophyte strain AR542, and two perennial ryegrass (Lolium perenne L.) varieties Aries and Quartet infected with a novel, non lolitrem B producing strain AR1, and their noninfected (E−) forms. Individual endophyte/grass associations were cultivated in nutrient solutions at 1.0 (P+) and 0.0 mM (P−) phosphorus concentrations. The Cu²⁺-binding activity of extracellular root exudates, and concentrations of Cu and other heavy metals in roots and shoots were measured. Extracellular root exudates of AR542-infected vs. E− tall fescue had higher Cu²⁺-binding activity only in P− nutrient solution as shown by lower concentration of free Cu²⁺ (0.096 vs. 0.188 mmol Cu²⁺ g⁻¹ root DM, respectively). The Cu²⁺-binding activity by root exudates of perennial ryegrass was not affected by endophyte infection, but was higher (i.e., lower concentration of free Cu²⁺) in P− vs. P+ nutrient solution (0.068 vs. 0.114 mmol Cu²⁺ g⁻¹ root DM). In this hydroponic experiment, Cu concentrations in shoots of both grasses were not a function of Cu²⁺-binding activity and endophyte effects on heavy metal concentrations in shoots and roots were specific for each variety. The Cu²⁺-binding activity of extracellular root exudates may affect Cu accumulation by field-grown, endophyte-infected tall fescue under P-limiting growth conditions and warrants verification by more specific methods.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

Relationship between Contents of Chlorophyll (a+b) (SPAD values) and Nitrogen of Some Temperate Grasses

In a field experiment the chlorophyll (a+b) (SPAD readings) and nitrogen contents of three grass species (Festuca arundinacea Schreb., Lolium perenne L., and Lolium multiflorum Lam.) and three intergeneric hybrids of Festuca pratensis Huds. × Lolium multiflorum Lam. and Festuca arundinacea Schreb. × Lolium multiflorum Lam. were measured. Close relationships were found between SPAD readings and nitrogen leaf content (r ² = 0.873^** 0.491^** and 0.938^**) for the 1^st, 2^nd, and 3^rd cut, respectively. SPAD readings and N contents were closely correlated (r = 0.836^**) confirming that SPAD measurements could be used in grass selection and/or breeding for high N concentration in herbage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside     

Soil boron and selenium removal by three plant species

High concentrations of boron (B) and selenium (Se) naturally found in the environment are detrimental to sustainable agriculture in the western USA. Greenhouse pot experiments were conducted to study B and Se uptake in three different plant species; Brassica juncea (L.) Czern (wild brown mustard), Festuca arundinacea Schreb. L. (tall fescue), and Brassica napus (canola) were grown in soil containing naturally occurring concentrations of 3.00 mg extractable B kg^–1 and 1.17 mg total Se kg^–1 soil. During the growing season, four intermediate harvests were performed on wild mustard and tall fescue. Final harvest I consisted of harvesting wild mustard, canola, and clipping tall fescue. Final harvest II consisted of harvesting wild mustard, which had been planted in soil in which wild mustard was previously grown, and harvesting previously clipped tall fescue. The greatest total amount of above ground biomass and below surface biomass was produced by tall fescue. Plants were separated into shoots and roots, weighted, and plant tissues were analyzed for total B and Se. The highest concentrations of tissue B were recovered in shoots of wild mustard and canola at final harvest I, while roots from tall fescue contained the highest concentrations of B irrespective of the harvest. Tissue Se concentrations were similar in all plants species. Soils were analyzed for residual B and Se. Extractable soil B concentrations at harvest times were lowered no less than 32% and total Se no less than 24% for all three species. The planting of wild mustard, canola, or tall fescue can reduce water-extractable B and total Se in the soil.     

Genomic studies on killer yeasts belonging to the genusPichia

Twenty-four species belonging to the genusPichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA.Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii andCandida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of theEcoRI andXhoI RFLP patterns withP. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis andC. albicans). However, one hybridized band, located at 3.9–4.1 Kb, seems to be peculiar to thePichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.     

RFLP analysis for species separation in the generaBipolaris andCurvularia

Restriction fragment length polymorphism (RFLP) of the total DNA ofBipolaris andCurvularia species was analysed using arbitrarily chosen genomic clones of DNA fromCurvularia lunata andBipolaris maydis as probes. Clear differences among species in both genera, resulting in different banding positions, were obtained with some probe-enzyme combinations. Intraspecific polymorphism in banding positions with these probe-enzyme combinations was slight. These analyses allow discrimination between the species. DNA fingerprinting with intrageneric probes is a potentially useful tool for species separation and identification inBipolaris andCurvularia when coupled with another characteristic such as conidial morphology.Curvularia aeria comb. nov. was proposed forCurvularia lunata var.aeria on the basis of differences in RFLP banding patterns and differences in conidial morphology.     

Moisture conditions and the presence of bryophytes determine fescue species abundance in a dry calcareous grassland

Festuca ovina is the abundant matrix-forming species and F. rubra a subordinate species in shallow-soil calcareous grasslands. F. pratensis is a transient species, occurring sparsely in this community. We hypothesised that the different abundances of these three species are primarily due to the differential effect of moisture conditions on their germination and early establishment, and that the effect of the pattern of rainfall intensity depends on the presence or absence of a bryophyte layer. We studied the dependence of the germination and establishment of the three fescue species on the moisture conditions both in the laboratory and in the patches of intact grassland community (microcosms). In a laboratory germination experiment, F. pratensis showed the highest, F. rubra , the intermediate and F. ovina, the lowest drought tolerance. In microcosms, the establishment of F. ovina was the highest. At the same time, the annual mortality of seedlings of F. ovina was the lowest. All three species responded positively to an increasing irrigation level. Differently from F. ovina, F. rubra showed a positive response only in plots from which the bryophyte layer had been removed, while F. pratensis responded positively to both irrigation and bryophyte removal. We conclude that moisture conditions have a differential effect on the three fescue species mainly in the seedling establishment, not in the germination phase. For the successful establishment of F. rubra and F. pratensis, the coincidence of high rainfall and local disturbance, removing bryophytes, is required. The presence or absence of bryophytes had no effect on establishment in dry years, while in rainy years the removal of bryophytes has a clear positive effect.     

The effect ofAcremonium coenophialum on the growth and nematode infestation of tall fescue

The presence of the endophytic fungusAcremonium coenophialum Morgan-Jones et Gams in tall fescue (Festuca arundinacea Schreb.) induces toxicity when this grass is grazed by cattle; however, there is evidence that removing the endophyte reduces the stand vigor and longevity of fescue. A field trial was conducted to determine the effects of water supply and the presence of the endophytic fungus on plant growth, drought tolerance, and soil nematode populations in Kentucky 31 tall fescue. The design included two factors, level of endophyte infection (0 and 75%) and irrigation regime (none, low, and high). Where water deficits occurred, herbage yield and leaf area were lower, and percentage dead tissue and canopy minus air temperature were greater in endophyte-free compared with endophyte-infected fescue. Soil populations ofPratylenchus scribneri andTylenchorhynchus acutus were substantially higher in the noninfected than in the endophyte-infected plots. The endophyte apparently confers drought tolerance to Kentucky 31 tall fescue, and this effect may be at least partially mediated through enhanced resistance to soil-borne nematodes.Published with the approval of the Director of the Ark. Agric. Exp. Stn.     

Isozyme variation and species relationships in the genus Lolium L. (ryegrasses,Graminaceae)

Thirty-two natural populations belonging to the eight species of the genus Lolium (ryegrass) or to Festuca pratensis (meadow fescue) were recorded for allelic frequencies at 13 isozyme loci. Cultivated ryegrass (L. perenne and L. multiflorum), meadow fescue, and the annual L. rigidum, are true outbreeders. The other species are true inbreeders, except for L. canariense, which shows a moderate level of cross fertilisation (20%). Hierarchical clustering from Nei's unbiased distance leads to four groups. The three self-pollinating, weed species, L. temulentum, L. remotum and L. persicum, belong to the first cluster, which is the most differentiated one. The second cluster comprises L. multiflorum, L. subulatum and most populations of L. rigidum. All L. perenne populations belong to the third cluster, as do two of L. rigidum. The average genetic distance within the L. perenne group is very low. Surprisingly, the fourth cluster groups together L. canariense and Festuca pratensis. The data suggest that L. rigidum is the species with the greatest diversity, and could be a common ancestor of the genus. Knowledge of historical processes of domestication could help to calibrate the molecular clock.