Novel perennial ryegrass-Neotyphodium endophyte associations: relationships between seed weight,seedling vigour and endophyte presence

Two forage cultivars of perennial ryegrass (Lolium perenne) colonised by the mutualistic fungal endophyte Neotyphodium lolii, strain AR37, were used to investigate relationships between seed weight, seed vigour and endophyte presence. Seed was separated into six fractions according to weight, with each fraction divided into two further groups with the first being subject to accelerated ageing. Seed germination rates and proportions of viable and total endophyte frequencies were assessed for each fraction. Heavier fractions of seed produced a higher number of endophyte infected seedlings sooner than the lighter fractions for both cultivars. The highest proportion of viable endophyte was found in the fastest germinating perennial ryegrass seed for all weight fractions, from both cultivars, indicating a strong relationship between endophyte presence and seed germination rate. For one of the cultivars, after accelerated ageing, as seed weight increased the proportion of viable endophyte increased and the discrepancy between the proportion of endophyte in fresh and accelerated aged seed was reduced. This implies that for this cultivar heavier ryegrass seed provides a more favourable habitat to the dormant endophyte than lighter seed during storage and/or allowed for a greater biomass of endophyte hyphae to proliferate in the seed tissues prior to seed dormancy, thus allowing the fungus to develop more propagules and greater nutritional reserves.     

Grass species and their fungal symbionts affect subsequent forage growth

Persistence of forage grasses is enhanced through the deliberate and selective use of symbiotic fungal endophytes that confer benefits, particularly pest resistance. However, they have also been implicated in reduced plant community diversity as a result of directly or indirectly enhancing competitive ability. A relatively underexplored mechanism by which endophytes might influence pasture plant composition is by altering the biotic or abiotic soil conditions. To examine the soil conditioning effects of forage grass species and their fungal symbionts we tested the responses of three pasture plants, perennial ryegrass, prairie grass, and white clover in nine different soils that had been conditioned by monocultures of endophyte-containing (E+), or endophyte-free (E?), perennial ryegrass, tall fescue, or meadow fescue. Conditioning grass species had little effect on the responses of perennial ryegrass and prairie grass regardless of E+ or E? treatments. In contrast, conditioning species had a strong effect on the response of white clover, resulting in reduced biomass when grown in perennial ryegrass conditioned soils. The presence of endophyte also had significant growth consequences for white clover, but was either positive or negative depending on the conditioning grass species. In comparison to their respective E? treatments, E+ tall and meadow fescue conditioned soils resulted in reduced biomass of white clover, whereas E+ perennial ryegrass conditioned soils resulted in increased biomass of white clover. Among the conditioning strains (AR1, AR37, NEA2, WE) of E+ perennial ryegrass, white clover showed significantly different responses, but all responses were positive in comparison to the E? treatment. By examining the effects of several grass species and endophyte strains, we were able to determine the relative importance of grass species vs. fungal symbiont on soil conditioning. Overall, the conditioning effect of grass species was stronger than the effects associated with endophyte, particularly with regard to the response of white clover. We conclude that both grass species and their fungal endophytes can influence pasture plant community composition through plant–soil feedback.     

High nitrogen supply and carbohydrate content reduce fungal endophyte and alkaloid concentration in Lolium perenne

The relationship between cool-season grasses and fungal endophytes is widely regarded as mutualistic, but there is growing uncertainty about whether changes in resource supply and environment benefit both organisms to a similar extent. Here, we infected two perennial ryegrass (Lolium perenne) cultivars (AberDove, Fennema) that differ in carbohydrate content with three strains of Neotyphodium lolii (AR1, AR37, common strain) that differ intrinsically in alkaloid profile. We grew endophyte-free and infected plants under high and low nitrogen (N) supply and used quantitative PCR (qPCR) to estimate endophyte concentrations in harvested leaf tissues. Endophyte concentration was reduced by 40% under high N supply, and by 50% in the higher sugar cultivar. These two effects were additive (together resulting in 75% reduction). Alkaloid production was also reduced under both increased N supply and high sugar cultivar, and for three of the four alkaloids quantified, concentrations were linearly related to endophyte concentration. The results stress the need for wider quantification of fungal endophytes in the grassland-foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes or increasing N inputs affect the role of endophytes in grassland ecosystems.     

Influence of temperature on alkaloid levels and fall armyworm performance in endophytic tall fescue and perennial ryegrass

The symbiotic relationships between Neotyphodium endophytes (Clavicipitacea) and certain cool‐season (C3) grasses result in the synthesis of several alkaloids that defend the plant against herbivory. Over a 3 month period we evaluated the effects of temperature on the expression of these alkaloids in tall fescue, Festuca arundinacea Schreb, and perennial ryegrass, Lolium perenne L. (Poaceae). Response surface regression analysis indicated that month, temperature, and their interaction had an impact on the alkaloid levels in both grasses. We aimed to identify the alkaloids most closely associated with enhanced resistance to the fall armyworm, Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae), and clarify the role of temperature in governing the expression of these alkaloids. The dry weights and survival of fall armyworms feeding on endophyte‐infected tall fescue or perennial ryegrass were significantly lower than for those feeding on uninfected grass, whereas endophyte infection had no significant influence on survival. For tall fescue, a four‐alkaloid model consisting of a plant alkaloid, perloline, and the fungal alkaloids ergonovine chanoclavine, and ergocryptine, explained 47% of the variation in fall armyworm dry weight, whereas a three‐alkaloid model consisting of the plant alkaloid perloline methyl ether and the fungal alkaloids ergonovine and ergocryptine explained 70% of the variation in fall armyworm dry weight on perennial ryegrass. Although temperature had a significant influence on overall alkaloid expression in both grasses, the influence of temperature on individual alkaloids varied over time. The levels of those alkaloids most closely linked to armyworm performance increased linearly or curvilinearly with increasing temperature during the last 2 months of the study. We conclude that the growth temperature of grasses can influence the performance of fall armyworm, and that this effect may be mediated through a set of plant‐ and endophyte‐related alkaloids.     

Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

Three grass host species--tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.--each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.     

Recovery from drought stress in Lolium perenne (Poaceae): are fungal endophytes detrimental?

Perennial ryegrass (Lolium perenne) is a cool-season, perennial species widely used for forage and turf. It is often infected by a clandestine, endophytic fungus (Neotyphodium lolii) that has the potential to affect host growth responses to abiotically stressful conditions. In some species, the grass-endophyte symbiosis is mutualistic, but the relationship is reported to be contingent on environmental conditions and host genotype in L. perenne. The objective of this research was to determine the potential effects of endophyte infection on recovery from severe drought stress in variable genotypes of a perennial ryegrass cultivar. Sixteen infected (+E) and 16 uninfected (-E) ramets were planted in the greenhouse for each of 10 ryegrass genotypes. Eight +E and eight -E plants per genotype were exposed to three sequential droughts where water was withheld for 11-14 d, resulting in <5% soil moisture; the others (control) were watered as needed. Response variables were tiller numbers 1 wk and 4 wk after drought, and leaf area and dry mass of shoots and roots 7 wk after drought. In both control and drought, -E plants had more tillers, and greater leaf area and total mass, than +E plants, suggesting a detrimental effect of endophytic fungi. Fungal hyphae survived the drought and were abundant in post-drought, +E plants. The effects of endophytes were specific for particular host genotypes, as exemplified by significant genotype × endophyte interactions. Root : shoot ratio and percent of mass allocated to tiller bases (a rough measure of resource storage) showed genotype × endophyte × drought interactions. There was plasticity for root : shoot ratio and genetic variation in the ability to restore root growth during recovery from drought. For 7 of 10 genotypes, -E plants showed an equal or greater allocation to tiller bases than +E plants following drought recovery, illustrating a cost to endophyte infection for some genotypes. The symbiotic relationship between L. perenne and its endophyte primarily benefits the fungus, not the host, under many environmental conditions.     

Competition between Lolium perenne and Digitaria sanguinalis: Ecological consequences for harbouring an endosymbiotic fungus

Abstract. This study examined the influence of the fungal endophyte Neotyphodium lolii on the competitive interactions between its perennial, cool season host, Lolium perenne (perennial ryegrass), and a warm season, annual grass, Digitaria sanguinalis (large crabgrass), in densely planted stands (>1000 plant.m^?2) in the glasshouse. Endophyte infection had little or no effect on L. perenne tiller production, above‐ or below‐ground biomass or root: shoot ratio in monoculture. However, endophyte infection significantly reduced L. perenne tiller production and above‐ground biomass in mixtures with D. sanguinalis. Conversely, D. sanguinalis had significantly higher above‐ground biomass and yielded more seed (g) when competing with endophyte infected L. perenne. An apparent trade‐off between allocation of resources to reproductive vs root tissues was observed in D. sanguinalis– root: shoot ratio was significantly lower when competing with endophyte infected L. perenne. Results indicate negative ecological consequences for harbouring the fungal endophyte when competing with the fast growing annual grass in newly established stands. These findings underscore the existence of a physiological cost of harbouring the fungal endophyte which is often overlooked.     

Breeding for Bio-ethanol Production in Lolium perenne L.: Association of Allelic Variation with High Water-Soluble Carbohydrate Content

Increasing the extractable sugar yield from perennial crops is one strategy to generate renewable fuels such as bio-ethanol. Lolium perenne L. (perennial ryegrass) can contain significant (>30% dry matter) water-soluble sugars in the form of polymeric fructan which is readily extracted, broken down and fermented to bio-ethanol. A population of L. perenne generated from four parents which differed in water-soluble carbohydrate (WSC) content was subjected to multiple rounds of selection and recombination on the basis of early spring WSC content to produce a high WSC, and a low WSC population. A control population was generated by selecting the same number of plants at random. The alleles present at six candidate gene loci were analysed before and after selection and correlated to WSC content. Significant differences in the allele frequency of L. perenne soluble-acid invertase1:4 were observed between the three populations with one haplotype significantly associated with the high WSC C2^S+ population (after three rounds of selection and two rounds of recombination). Moreover, WSC content was also associated with biomass accumulation. Thus, in addition to a 2.84-fold increase in WSC yield, the C2^S+ population also had 1.48-fold more biomass per plant, resulting in 3.9-fold higher WSC yield per plant than the control population.     

Distribution of the fungal endophyte Neotyphodium lolii is not a major determinant of the distribution of fungal alkaloids in Lolium perenne plants

The relationships of the distributions of the insect and mammalian mycotoxins, lolitrem B and ergovaline, and the insect-feeding deterrent, peramine, with the distribution of fungal mycelium were investigated in three genotypes of perennial ryegrass (Lolium perenne L.) infected with the endophyte Neotyphodium lolii. In planta levels and distribution of the endophyte and of the three alkaloids were assessed in parallel, and different spatial or temporal concentration gradients were observed for each. Variation in the tissue distribution of the endophyte accounted only for 20%, 6%, and 31% of the variation in ergovaline, lolitrem B, and peramine, respectively. Alkaloid-endophyte ratios, determined in individual grass tissues, showed distinct in planta distribution patterns for each alkaloid and differed in magnitude among genotypes. The ergovaline-endophyte ratio was higher in the very basal plant tissues than in the apical tissues, while the lolitrem B and peramine ratios tended to be higher in apical tissues. The lolitrem B-endophyte ratio increased with leaf age, while no consistent temporal trends were detected for the other alkaloids. The results indicate that endophyte colonisation is a minor determinant of alkaloid levels, and that accumulation of the alkaloids relative to the endophyte mycelium is affected by plant genotype and tissue in a manner specific to each alkaloid. Possible factors in the regulation of alkaloid levels in the grass plant are discussed.     

Overseeding endophytic perennial ryegrass into stands of Kentucky bluegrass to manage bluegrass billbug (Coleoptera: Curculionidae)

Studies were conducted to determine the utility of overseeding endophyte enhanced varieties of perennial ryegrass, Lolium perenne L., into existing stands of Kentucky bluegrass, Poa pratensis L., to manage bluegrass billbug, Sphenophorus parvulus Gyllenhal, larval populations and their damage. Two commercial varieties of endophyte enhanced perennial ryegrass (Repell II and Triple Play) were overseeded at two different rates in the field. S. parvulus larval populations, their damage, and turfgrass composition were monitored over the course of 3 yr. Significantly fewer larvae were found in plots overseeded with Repell II, whereas Triple Play had no significant effect on larval population density. Endophyte infection was confirmed in approximately 58% of all Repell II shoots, whereas only approximately 31% of Triple Play shoots were infected. Regression analysis, using a segmented model, demonstrated that larval populations declined as the proportion of endophyte-infected perennial ryegrass increased. However, no additional reductions in larval population density were achieved in stands containing >40% endophyte-infected perennial ryegrass. Visual damage varied with cultivar, seeding rate, and year, but generally decreased as the proportion of endophyte-infected perennial ryegrass increased. Although seeding rate had an initial effect on turfgrass composition, these differences were not detected after the first year of the study. Results indicate overseeding existing stands of Kentucky bluegrass with endophytic varieties of perennial ryegrass can reduce S. parvulus larval populations and their damage. However, plant genetic characteristics (i.e., different cultivars) may be overriding determinants of many aspects of plant performance including establishment, tolerance to insect feeding, and expression of endophyte-mediated resistance.     

Testing water-soluble carbohydrate QTL effects in perennial ryegrass (Lolium perenne L.) by marker selection

Water-soluble carbohydrates (WSC) are an important factor determining the nutritional value of grass forage and development of genetic markers for selection of WSC traits in perennial ryegrass would benefit future breeding programmes. Quantitative trait loci (QTLs) for WSC have been published for an F₂ ryegrass mapping family. Markers showing significant associations with these QTLs were used to design narrow-based populations with homozygosity for target QTLs. Founders were selected from within the mapping family. The divergent populations produced were analysed for WSC content in the glasshouse and the field. There was evidence of complex interactions between WSC content and other factors and traits, including the scale of assessment, time/degree of sward establishment and other forage quality parameters. Differences between the divergent pairs of the various populations were small. However, differences observed between the founder selection groups were maintained and the roles of the QTL regions in regulating forage WSC content were confirmed. In general, the individual divergent populations exploited only a limited extent of the large phenotypic variation available within the mapping family. However, this study sets the scene for exploring the opportunities for marker-assisted breeding strategies for complex traits in obligate out-breeding species, and the challenges of doing this are discussed.     

Advanced data-mining strategies for the analysis of direct-infusion ion trap mass spectrometry data from the association of perennial ryegrass with its endophytic fungus, Neotyphodium lolii

Direct-infusion mass spectrometry (MS) was applied to study the metabolic effects of the symbiosis between the endophytic fungus Neotyphodium lolii and its host perennial ryegrass (Lolium perenne) in three different tissues (immature leaf, blade, and sheath). Unbiased direct-infusion MS using a linear ion trap mass spectrometer allowed metabolic effects to be determined free of any preconceptions and in a high-throughput fashion. Not only the full MS(1) mass spectra (range 150-1,000 mass-to-charge ratio) were obtained but also MS(2) and MS(3) product ion spectra were collected on the most intense MS(1) ions as described previously (Koulman et al., 2007b). We developed a novel computational methodology to take advantage of the MS(2) product ion spectra collected. Several heterogeneous MS(1) bins (different MS(2) spectra from the same nominal MS(1)) were identified with this method. Exploratory data analysis approaches were also developed to investigate how the metabolome differs in perennial ryegrass infected with N. lolii in comparison to uninfected perennial ryegrass. As well as some known fungal metabolites like peramine and mannitol, several novel metabolites involved in the symbiosis, including putative cyclic oligopeptides, were identified. Correlation network analysis revealed a group of structurally related oligosaccharides, which differed significantly in concentration in perennial ryegrass sheaths due to endophyte infection. This study demonstrates the potential of the combination of unbiased metabolite profiling using ion trap MS and advanced data-mining strategies for discovering unexpected perturbations of the metabolome, and generating new scientific questions for more detailed investigations in the future.     

内生真菌对羽茅生长及光合特性的影响

通过温室栽培实验,以感染两种内生真菌(Neotyphodium sibiricum和Neotyphodium gansuence)和未感染内生真菌的羽茅(Achnatherum sibiricum)为实验材料,分析感染不同种内生真菌对宿主植物的生长及光合特性的影响。结果表明,感染两种内生真菌羽茅的株高和CO₂补偿点显著低于未染菌的羽茅,而染菌羽茅的蒸腾速率和气孔导度显著高于未染菌羽茅,但对于感染不同种内生真菌的羽茅,无论是分蘖数与生物量的积累还是光合生理值之间均无显著差异。     

A fungal endophyte of an annual weed reduces host competitive ability and confers associational protection to wheat

Epichloid fungal endophytes (Epichloë spp., Ascomycota: Clavicipitaceae) inhabit aerial tissues of several cool-season grasses, and enhance host growth and defence against herbivores. The presence of these symbionts can also affect interactions between the host and other non-epichloid plants. The role of an epichloid endophyte on interspecific competition has been tested using perennial grasses with contrasting results, but it has been scarcely tested using annual species in agroecosystems. We evaluated the impact of Epichloë-grass symbiosis on the competitive interaction between a non-host cereal crop (Triticum aestivum, wheat) and a host weed (Lolium multiflorum, ryegrass), growing in the presence of invertebrate herbivores (aphids) under no resource limitation. We conducted an outdoor mesocosm experiment with wheat plants growing in monoculture or in mixture with low or high proportions of ryegrass plants. Ryegrass plants presented either low (E-) or high (E+) incidence of Epichloë occultans (i.e. frequency of epichloid endophytic plants). We measured wheat vegetative and reproductive yield and its natural aphid infestation. Although epichloid endophyte incidence did not affect ryegrass biomass, wheat reproductive yield in mixtures (relative to wheat monocultures) was 45% higher when grown with E+ ryegrass plants than E- conspecific plants. Aphids preferred wheat plants grown with E- plants rather than wheat plants grown with E+ plants, but only in mixtures with high proportion of ryegrass. Our results demonstrate that epichloid endophyte incidence can decrease host competitive ability and confers associational protection to the non-endophytic neighbouring plants. Thus, ryegrass-endophyte symbiosis can increase crop yield by positive neighbourhood effects through different mechanisms probably related to the density of the weed. The benefits of this endosymbiont cannot be considered host-exclusive since they can be disseminated to non-endophytic plants. Furthermore, our results suggest that the epichloid endophyte incidence on annual weeds can contribute to agroecosystem sustainability by influencing pest management and increasing crop yield.     

The determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass

Many cool-season grasses harbor fungal endophytes in the genus Neotyphodium, which enhance host fitness, but some also produce metabolites--such as ergovaline--believed to cause livestock toxicoses. In Claviceps species the first step in ergot alkaloid biosynthesis is thought to be dimethylallyltryptophan (DMAT) synthase, encoded by dmaW, previously cloned from Claviceps fusiformis. Here we report the cloning and characterization of dmaW from Neotyphodium sp. isolate Lp1, an endophyte of perennial ryegrass (Lolium perenne). The gene was then disrupted, and the mutant failed to produce any detectable ergovaline or simpler ergot and clavine alkaloids. The disruption was complemented with the C. fusiformis gene, which restored ergovaline production. Thus, the biosynthetic role of DMAT synthase was confirmed, and a mutant was generated for future studies of the ecological and agricultural importance of ergot alkaloids in endophytes of grasses.     

Endophyte-mediated resistance to black cutworm as a function of plant cultivar and endophyte strain in tall fescue

To improve Neotyphodium endophyte-mediated resistance to black cutworm Agrotis ipsilon (Hufnagel) (BCW), a series of experiments was conducted by using several different cultivars of tall fescue, Schedonorus arundinaceus (Schreb.) Dumort. in combination with several different haplotypes of the endophyte Neotyphodium coenophialum (Morgan-Jones & Gams) (plant cultivar × endophyte haplotype = plant line), each producing unique alkaloid profiles. BCW settling response, survival at 5 and 10 d, and larval biomass varied significantly among plant lines. In general, greater variation BCW performance was observed within a single plant cultivar infected with different endophyte haplotypes than among different plant cultivars infected with the same endophyte haplotype, but comparisons among the former were far more numerous. Although five endophyte-mediated alkaloids representing three alkaloid classes were quantified in the plants, the pyrrolizidine alkaloid N-acetyl norloline was consistently the single best predictor of BCW performance. BCW settling response, 5-d survival, and 10-d survival decreased as levels of the alkaloid N-acetyl norloline increased. The same three response variables also decreased with increasing levels of peramine, but increased with increasing levels of ergovaline. Minor variation in endophyte infection levels occurring among infected plant lines had no significant influence on BCW performance. Results indicate a potentially important role for N-acetyl norloline and peramine in providing resistance to black cutworm whereas ergovaline appears to be much less important. Therefore, endophyte haplotypes expressing high levels of N-acetyl norloline and peramine may be of particular importance for developing 'friendly' endophyte-enhanced turf and pasture grasses that resist challenging lepidopteran pests, although remaining safe for wildlife and grazing mammals.     

Production of phenolics and the emission of volatile organic compounds by perennial ryegrass (Lolium perenne L.)/Neotyphodium lolii association as a response to infection by Fusarium poae

Grasses very often form symbiotic associations with Neotyphodium/Epichloë endophytic fungi. These endophytes often allow the host grass to be protected from different pathogens. However, there is little known about the mechanisms of such endophyte influence on the host. Thus, the purpose of this research was to examine the effect of the N. lolii endophyte on the total production of phenolic compounds, VOCs emission and the resistance of three perennial ryegrass genotypes infected by pathogenic Fusarium poae. Analyses of total phenolics content were performed in control (not inoculated) and inoculated plants after 1, 2, 3, 4, 5, and 6 days (DAI) and for VOCs after 0, 3, 6 and 12 DAI. The presence of endophytes significantly reduced the disease index in two of the three genotypes relative to that in E−. Plants infected by N. lolii exhibited higher production of phenolics relative to the E− plants. The highest amounts of phenolics were observed on the second and sixth DAI. Genotype Nl22 showed the strongest effect of the endophyte on the production of phenolics, which increased by over 61%. Both the endophyte infected and non-infected plants emitted most abundantly two GLVs ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate), three terpenes (linalool, (Z)-ocimene, β-caryophyllene) and three shikimic acid pathway derivatives (benzyl acetate, indole, and methyl salicylate). The endophyte presence and the intervals of VOCs detection were a highly significant source of variation for all emitted volatiles (P < 0.001). The genotype of the perennial ryegrass significantly affected only the emission of methyl salicylate (P < 0.05) and β-caryophyllene (P < 0.05). Most of the VOCs ((Z)-3-hexen-1-yl acetate, (Z)-3-hexenal, linalool and methyl salicylate) reached their highest levels of emission on the sixth DAI, when averaged over genotypes and endophyte status. The results highlight the role of Neotyphodium spp. in the mediation of quadro-trophic interactions among plants, symbiotic endophytes, invertebrate herbivores and plant pathogenic fungi. Our results also confirm the fact that symbiotic plants can activate a defense reaction faster than non-symbiotic plants after a pathogen attack. Thus, N. lolii can be involved in the defense of perennial ryegrass against pathogens and potentially could be central to the host plants’ protection.     

Development and implementation of a multiplexed single nucleotide polymorphism genotyping tool for differentiation of ryegrass species and cultivars

Perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.) are important temperate forage grasses which are closely related, generating fertile interspecific hybrids. All groups are represented by multiple cultivars in the commercial pasture seeds market. Due to the close taxonomic relationship between the two species, differentiation based on morphophysiological criteria is not always readily achievable. In addition, an obligate outbreeding reproductive habit produces high levels of individual heterozygosity and intrapopulation diversity, which presents problems for discrimination between cultivars. Molecular genetic marker polymorphism provides an effective means of addressing these challenges. An iterative process of resequencing from loci distributed across the perennial ryegrass genome was used to identify single nucleotide polymorphism (SNP) markers, which were then validated and formatted in a highly multiplexed (384-plex) assay system. SNP genotyping was then performed across samples of 48–192 individuals from a total of 27 ryegrass cultivars (19 of perennial ryegrass, seven of Italian ryegrass and one hybrid cultivar). SNP markers from perennial ryegrass exhibited a high level of transfer to Italian ryegrass. Data analysis permitted quantification of intra- and inter-species diversity, as well as discrimination between cultivars within each species, including diploid and autotetraploid cultivars of perennial ryegrass. Lower levels of SNP-based diversity were detected in Italian ryegrass than in perennial ryegrass. A neighbour-joining tree based on genetic distance analysis located a hybrid cultivar to an intermediate position between the two species-specific cultivar groups. The resulting catalogue of ryegrass cultivars will provide support for the processes of cultivar accreditation and quality assurance.     

Molecular breeding of transgenic perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) with altered fructan biosynthesis through the expression of fructosyltransferases

Perennial ryegrass is the most important forage grass used in temperate agriculture. Transgenic perennial ryegrass events with altered fructan biosynthesis have the potential not only to increase animal production by improving digestibility of the grasses, but also to increase pasture intake by the animal due to lower neutral detergent fibre (NDF) concentrations. Transgenic perennial ryegrass plants were shown to have increased (P?<?0.05) in fructan concentrations of leaf blades in transgenic T₀ events and have thus an increase in water-soluble carbohydrate and in vivo dry matter digestibility concentrations as well as a decrease in the NDF concentration within the plant in spring and summer. These changes in nutritive value have led to an increase in metabolisable energy of up to 1.7 MJ ME·kg DM^?1 in selected T₀ events compared to FLp418-20 during spring and summer, with no differences in autumn or winter. The field evaluation of these events and the further development of these events using molecular breeding technologies are described in this paper.