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Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression., Summary: Mir-9a overexpression can suppress the overgrowth phenotype caused by Lgl knock-down in epithelia. Gene profiling identifies pathways dysregulated in lgl mutants and shared features between flies and human cancer cells.

Asexual Epichloë sp. endophytes in association with pasture grasses produce agronomically important alkaloids (e.g., lolitrem B, epoxy-janthitrems, ergovaline, peramine, and lolines) that exhibit toxicity to grazing mammals and/or insect pests. Novel strains are primarily characterised for the presence of these compounds to ensure they are beneficial in an agronomical setting. Previous work identified endophyte strains that exhibit enhanced antifungal activity, which have the potential to improve pasture and turf quality as well as animal welfare through phytopathogen disease control. The contribution of endophyte-derived alkaloids to improving pasture and turf grass disease resistance has not been closely examined. To assess antifungal bioactivity, nine Epichloë related compounds, namely peramine hemisulfate, n-formylloline-d3, n-acetylloline hydrochloride, lolitrem B, janthitrem A, paxilline, terpendole E, terpendole C, and ergovaline, and four Claviceps purpurea ergot alkaloids, namely ergotamine, ergocornine, ergocryptine, and ergotaminine, were tested at concentrations higher than observed in planta in glasshouse and field settings using in vitro agar well diffusion assays against three common pasture and turf phytopathogens, namely Ceratobasidium sp., Drechslera sp., and Fusarium sp. Visual characterisation of bioactivity using pathogen growth area, mycelial density, and direction of growth indicated no inhibition of pathogen growth. This was confirmed by statistical analysis. The compounds responsible for antifungal bioactivity of Epichloë endophytes hence remain unknown and require further investigation.

The neuroactive mycotoxin lolitrem B causes a neurological syndrome in grazing livestock resulting in hyperexcitability, muscle tremors, ataxia and, in severe cases, clonic seizures and death. To define the effects of the major toxin lolitrem B in the brain, a functional metabolomic study was undertaken in which motor coordination and tremor were quantified and metabolomic profiling undertaken to determine relative abundance of both toxin and key neurotransmitters in various brain regions in male mice. Marked differences were observed in the duration of tremor and coordination between lolitrem B pathway members, with some showing protracted effects and others none at all. Lolitrem B was identified in liver, kidney, cerebral cortex and thalamus but not in brainstem or cerebellum which were hypothesised previously to be the primary site of action. Metabolomic profiling showed significant variation in specific neurotransmitter and amino acid profiles over time. This study demonstrates accumulation of lolitrem B in the brain, with non-detectable levels of toxin in the brainstem and cerebellum, inducing alterations in metabolites such as tyrosine, suggesting a dynamic catecholaminergic response over time. Temporal characterisation of key pathways in the pathophysiological response of lolitrem B in the brain were also identified.

Asexual Epichloë fungi are strictly seed-transmitted endophytic symbionts of cool-season grasses and spend their entire life cycle within the host plant. Endophyte infection can confer protective benefits to its host through the production of bioprotective compounds. Inversely, plants provide nourishment and shelter to the resident endophyte in return. Current understanding of the changes in global gene expression of asexual Epichloë endophytes during the early stages of host-endophyte symbiotum is limited. A time-course study using a deep RNA-sequencing approach was performed at six stages of germination, using seeds infected with one of three endophyte strains belonging to different representative taxa. Analysis of the most abundantly expressed endophyte genes identified that most were predicted to have a role in stress and defence responses. The number of differentially expressed genes observed at early time points was greater than those detected at later time points, suggesting an active transcriptional reprogramming of endophytes at the onset of seed germination. Gene ontology enrichment analysis revealed dynamic changes in global gene expression consistent with the developmental processes of symbiotic relationships. Expression of pathway genes for biosynthesis of key secondary metabolites was studied comprehensively and fuzzy clustering identified some unique expression patterns. Furthermore, comparisons of the transcriptomes from three endophyte strains in planta identified genes unique to each strain, including genes predicted to be associated with secondary metabolism. Findings from this study highlight the importance of better understanding the unique properties of individual endophyte strains and will serve as an excellent resource for future studies of host-endophyte interactions.

The ergot alkaloid ergotamine is produced by Claviceps purpurea, a parasitic fungus that commonly infects crops and pastures of high agricultural and economic importance. In humans and livestock, symptoms of ergotism include necrosis and gangrene, high blood pressure, heart rate, thermoregulatory dysfunction and hallucinations. However, ergotamine is also used in pharmaceutical applications to treat migraines and stop post-partum hemorrhage. To define its effects, metabolomic profiling of the brain was undertaken to determine pathways perturbed by ergotamine treatment. Metabolomic profiling identified the brainstem and cerebral cortex as regions with greatest variation. In the brainstem, dysregulation of the neurotransmitter epinephrine, and the psychoactive compound 2-arachidonylglycerol was identified. In the cerebral cortex, energy related metabolites isobutyryl-L-carnitine and S-3-oxodecanoyl cysteamine were affected and concentrations of adenylosuccinate, a metabolite associated with mental retardation, were higher. This study demonstrates, for the first time, key metabolomic pathways involved in the behavioural and physiological dysfunction of ergot alkaloid intoxicated animals.

Development of grass–endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (Lolium perenne L.)-associated Epichlo&euml, endophyte strains using A. tumefaciens-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail.

The rapid identification and quantitation of alkaloids produced by Epichlo&euml, endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichlo&euml, infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography&ndash, mass spectrometry (LC&ndash, MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments, a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC&ndash, MS (for peramine). This work provides the first highly sensitive quantitative LC&ndash, MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Indole-diterpenes are an important class of chemical compounds which can be unique to different fungal species. The highly complex lolitrem compounds are confined to Epichloë species, whilst penitrem production is confined to Penicillium spp. and Aspergillus spp. These fungal species are often present in association with pasture grasses, and the indole-diterpenes produced may cause toxicity in grazing animals. In this review, we highlight the unique structural variations of indole-diterpenes that are characterised into subgroups, including paspaline, paxilline, shearinines, paspalitrems, terpendoles, penitrems, lolitrems, janthitrems, and sulpinines. A detailed description of the unique biological activities has been documented where even structurally related compounds have displayed unique biological activities. Indole-diterpene production has been reported in two classes of ascomycete fungi, namely Eurotiomycetes (e.g., Aspergillus and Penicillium) and Sordariomycetes (e.g., Claviceps and Epichloë). These compounds all have a common structural core comprised of a cyclic diterpene skeleton derived from geranylgeranyl diphosphate (GGPP) and an indole moiety derived from tryptophan. Structure diversity is generated from the enzymatic conversion of different sites on the basic indole-diterpene structure. This review highlights the wide-ranging biological versatility presented by the indole-diterpene group of compounds and their role in an agricultural and pharmaceutical setting.

Temperate pasture grasses form symbiotic associations with asexual fungal endophytes of the genus Epichloe. Viabilities of both seed and endophyte were assessed in cultivars of perennial ryegrass and tall fescue following different accelerated ageing (AA) treatments based on variations of relative humidity (RH) (80-100%) and duration at elevated temperature (42 degrees C), to identify particularly stable and compatible host-endophyte associations. The cultivars were inoculated with three endophytes (NEA10, NEA11 and NEA12) isolated from perennial ryegrass and belonging to distinct taxa (E. festucae var. lolii, LpTG-2 and LpTG-3, respectively), and two endophytes isolated from tall fescue (belonging to the taxon E. coenophiala). Consistent with previous studies, endophyte viability following AA treatment was inversely correlated with both increasing RH and duration of treatment in both species. Significant interaction effects between cultivar variety, endophyte genotype, humidity level and treatment duration were observed for both seed germination and endophyte viability. NEA12 consistently displayed higher viability following AA treatment than NEA10 and NEA1 1 in each of the perennial ryegrass cultivars. Stable host-endophyte combinations were observed for 'Alto'-NEA10, 'Bealey'-NEAll and 'Trojan'-NEA12, 'Bronsyn' being the least compatible host population. The applicability of AA for selecting stable host-endophyte associations, or to identify and eliminate unstable associations, is discussed.

Asexual Epichlo&euml, spp. fungal endophytes have been extensively studied for their functional secondary metabolite production. Historically, research mostly focused on understanding toxicity of endophyte-derived compounds on grazing livestock. However, endophyte-derived compounds also provide protection against invertebrate pests, disease, and other environmental stresses, which is important for ensuring yield and persistence of pastures. A preliminary screen of 30 strains using an in vitro dual culture bioassay identified 18 endophyte strains with antifungal activity. The novel strains NEA12, NEA21, and NEA23 were selected for further investigation as they are also known to produce alkaloids associated with protection against insect pests. Antifungal activity of selected endophyte strains was confirmed against three grass pathogens, Ceratobasidium sp., Dreschlera sp., and Fusarium sp., using independent isolates in an in vitro bioassay. NEA21 and NEA23 showed potent activity against Ceratobasidium sp. and NEA12 showed moderate inhibition against all three pathogens. Crude extracts from liquid cultures of NEA12 and NEA23 also inhibited growth of the phytopathogens Ceratobasidium sp. and Fusarium sp. and provided evidence that the compounds of interest are stable, constitutively expressed, and secreted. Comparative analysis of the in vitro and in planta metabolome of NEA12 and NEA23 using LCMS profile data revealed individual metabolites unique to each strain that are present in vitro and in planta. These compounds are the best candidates for the differential bioactivity observed for each strain. Novel endophyte strains show promise for endophyte-mediated control of phytopathogens impacting Lolium spp. pasture production and animal welfare.

The most potent of the indole diterpenes, lolitrem B, is found in perennial ryegrass (Lolium perenne L.) infected with the endophyte Epichlo&euml, festucae var. lolii (also termed LpTG-1). Ingestion causes a neurological syndrome in grazing livestock called ryegrass staggers disease. To enable the rapid development of new forage varieties, the toxicity of lolitrem B and its biosynthetic intermediates needs to be established. However, most of these indole diterpenes are not commercially available, thus, isolation of these compounds is paramount. A concentrated endophyte-infected perennial ryegrass seed extract was subjected to silica flash chromatography followed by preparative HPLC and purification by crystallization resulting in lolitrem B and the intermediate compounds lolitrem E, paspaline and terpendole B. The four-step isolation and purification method resulted in a 25% yield of lolitrem B. After isolation, lolitrem B readily degraded to its biosynthetic intermediate, lolitriol. We also found that lolitrem B can readily degrade depending on the solvent and storage conditions. The facile method which takes into consideration the associated instability of lolitrem B, led to the purification of indole diterpenes in quantities sufficient for use as analytical standards for identification in pastures, and/or for toxicity testing in pasture development programs.

Symbiotic associations between tall fescue grasses and asexual Epichloë fungal endophytes exhibit biosynthesis of alkaloid compounds causing both beneficial and detrimental effects. Candidate novel endophytes with favourable chemotypic profiles have been identified in germplasm collections by screening for genetic diversity, followed by metabolite profile analysis in endogenous genetic backgrounds. A subset of candidates was subjected to genome survey sequencing to detect the presence or absence and structural status of known genes for biosynthesis of the major alkaloid classes. The capacity to produce specific metabolites was directly predictable from metabolic data. In addition, study of duplicated gene structure in heteroploid genomic constitutions provided further evidence for the origin of such endophytes. Selected strains were inoculated into meristem-derived callus cultures from specific tall fescue genotypes to perform isogenic comparisons of alkaloid profile in different host backgrounds, revealing evidence for host-specific quantitative control of metabolite production, consistent with previous studies. Certain strains were capable of both inoculation and formation of longer-term associations with a nonhost species, perennial ryegrass (Lolium perenne L.). Discovery and primary characterisation of novel endophytes by DNA analysis, followed by confirmatory metabolic studies, offers improvements of speed and efficiency and hence accelerated deployment in pasture grass improvement programs.

Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual Epichlo&euml, endophytes have been reported to produce epoxy-janthitrems, LpTG-3 (Lolium perenne Taxonomic Group 3, e.g., NEA12) and LpTG-4 (e.g., E1). Epichlo&euml, epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of Epichlo&euml, A gene, jtmD, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes jtmD, jtmO, jtm01, and jtm02 that are unique to Epichlo&euml, spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I&ndash, IV in Epichlo&euml, endophytes is proposed.

The major insect pest of Australian cool temperate pastures is the root-feeding insect Heteronychus arator (African black beetle, ABB). Significant pasture damage can occur even at low ABB densities (11 individuals per square meter), and often re-sowing of the whole paddock is required. Mitigation of the effects of pasture pests, and in particular subterranean species such as the larval form of ABB, can be challenging. Early detection is limited by the ability to visualize above-ground symptoms, and chemical control of insects in soil is often ineffective. This review takes a look at the historical events that molded the pastoral landscape in Australia. The importation route, changes in land management and pasture composition by European settlers may have aided the establishment of ABB in Australia. Perennial ryegrass Lolium perenne is discussed as it is one of the most important perennial agricultural grasses and is widely-sown in moderate-to-high-rainfall temperate zones of the world. Endophytic fungi from the genus Epichloë form symbiotic relationships with cool season grasses such as Lolium perenne (perennial ryegrass). They have been studied extensively and are well documented for enhancing persistence in pasture via a suite of bioactive secondary metabolites produced by the fungal symbionts. Several well-characterized secondary metabolites are discussed. Some can have negative effects on cattle (e.g., ergovaline and lolitrems) while others have been shown to benefit the host plant through deterrence of insect pests from feeding and by insecticidal activity (e.g., peramine, lolines, ergopeptines). Various control methods for ABB are also discussed, with a focus on the potential role of asexual Epichloë endophytes.

A number of pasture and turf grass species form mutually beneficial symbiotic associations with endophytic fungal species. Within the fescue grasses, diploid meadow fescue (Huds.) interacts with while allohexaploid tall fescue (Schreb.) has been reported to associate with and two other morphologically distinct taxa (taxonomic groups 2 and 3 [TG-2 and TG-3]). The evolutionary history of hexaploid tall fescue is complex, as part of a species group with varying ploidy levels and exhibiting distinct ecogeographical morphotypes. To evaluate both naturally occurring variation and host grass taxon specificity, diversity was determined in collections representing multiple meadow fescue and tall fescue accessions. Initial screening with a minimal set of endophyte-specific simple sequence repeat (SSR) genetic markers detected endophyte incidence in 33% of 701 tested accessions. Subsequent analysis identified genotypes within Continental and rhizomatous hexaploid and octoploid tall fescue [ subsp. (St.-Yves) Auquier] accessions. taxonomic group 2 and TG-3 endophytes appeared to be restricted to Mediterranean hexaploid and decaploid tall fescue [ (St.-Yves) Gamisans] hosts. Endophytes of meadow fescue were confirmed as belonging to . This study has elucidated host specificity of fescue endophyte taxa and supported models for host–symbiont coevolution. A substantial number of candidate novel endophytes have been identified that are suitable for metabolic characterization and deployment by inoculation in fescue breeding programs.

This study aimed to estimate the heritabilities of three economically important traits (total weight, shell shape and meat yield) in Australian blue mussels. The estimates were derived using a pedigree reconstructed from a suite of both published and newly developed microsatellite markers. A total of 135 microsatellite loci were tested, of which 10 loci produced consistent PCR amplification and reliable results across all samples (74 full-sibling families including 74 pairs of parents and 2536 offspring). Lack of polymorphism at the non-repetitive region of the adhesive protein gene confirmed that the broodstock were derived from a single species. A total of 1538 progenies (62.5%) could be assigned to single parent pairs, and the remainder were assigned to two families or more, so were discarded from further analysis. Heritabilities for total weight, shell shape and meat yield were low (0.051±0.027, 0.085±0.038 and 0.049±0.028, respectively) but reflected large environmental variation rather than limited genetic variation, suggesting a family-based breeding programme could improve these traits. The genetic correlation between weight and meat yield, expressed as percentage of total mussel which was not shell, was negative, while the genetic correlation between meat yield and shell shape was weakly positive.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichloë isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichloë species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.Key words: Neotyphodium, simple sequence repeats, expressed sequence tags, amplified fragment length polymorphism, genetic diversity.

Summary A fructosyltransferase ( LpFT1 ) cDNA and gene from perennial ryegrass was isolated and sequenced. Nucleotide sequence analysis revealed an open reading frame of 1947 bp encoding for a protein of 648 amino acids. LpFT1 is 69 % identical to barley 6-SFT, and contains plant fructosyltransferase functional domains. LpFT1 is present as a single copy gene and maps to the distal region of LG7 in perennial ryegrass. The expression pattern analysis of LpFT1 revealed transcript accumulation in young roots and shoots and in mature sheaths.

Amplified fragment length polymorphism (AFLP) analysis has been used to measure genetic diversity in perennial ryegrass (Lolium perenne L.) and to relate intra- and interpopulation variation to breeding history. Cluster analysis of AFLP data from contrasting populations showed features consistent with the origins of these varieties. Significant differences in intrapopulation diversity were detected and partial separation of different cultivars was observed. Restricted base cultivars, derived from small numbers of foundation clones, were suitable for this type of study, allowing near complete discrimination of closely related cultivars. Analysis of bulked samples was based on the pooling of genomic DNA from 20 individuals from 6 selected populations. Cluster analysis of AFLP data from bulked samples produced a phenogram showing relationships consistent with the results of individual analysis. AFLP profiling provides an important tool for the detection and quantification of genetic variation in perennial ryegrass.

Background Tall fescue and meadow fescue are important as temperate pasture grasses, forming mutualistic associations with asexual Neotyphodium endophytes. The most frequently identified endophyte of Continental allohexaploid tall fescue is Neotyphodium coenophialum, while representatives of two other taxa (FaTG-2 and FaTG-3) have been described as colonising decaploid and Mediterranean hexaploid tall fescue, respectively. In addition, a recent study identified two other putatively novel endophyte taxa from Mediterranean hexaploid and decaploid tall fescue accessions, which were designated as uncharacterised Neotyphodium species (UNS) and FaTG-3-like respectively. In contrast, diploid meadow fescue mainly forms associations with the endophyte taxon Neotyphodium uncinatum, although a second endophyte taxon, termed N. siegelii, has also been described. Results Multiple copies of the translation elongation factor 1-a (tefA) and β-tubulin (tub2) ‘house-keeping’ genes, as well as the endophyte-specific perA gene, were identified for each fescue-derived endophyte taxon from whole genome sequence data. The assembled gene sequences were used to reconstruct evolutionary relationships between the heteroploid fescue-derived endophytes and putative ancestral sub-genomes derived from known sexual Epichloë species. In addition to the nuclear genome-derived genes, the complete mitochondrial genome (mt genome) sequence was obtained for each of the sequenced endophyte, and phylogenetic relationships between the mt genome protein coding gene complements were also reconstructed. Conclusions Complex and highly reticulated evolutionary relationships between Epichloë-Neotyphodium endophytes have been predicted on the basis of multiple nuclear genes and entire mitochondrial protein-coding gene complements, derived from independent assembly of whole genome sequence reads. The results are consistent with previous studies while also providing novel phylogenetic insights, particularly through inclusion of data from the endophyte lineage-specific gene, as well as affording evidence for the origin of cytoplasmic genomes. In particular, the results obtained from the present study imply the possible occurrence of at least two distinct E. typhina progenitors for heteropoid taxa, as well the ancestral contribution of an endophyte species distinct from (although related to) contemporary E. baconii to the extant hybrid species. Furthermore, the present study confirmed the distinct taxonomic status of the newly identified fescue endophyte taxa, FaTG-3-like and UNS, which are consequently proposed to be renamed FaTG4 and FaTG5, respectively.

Background Variation of microorganism communities in the rumen of cattle (Bos taurus) is of great interest because of possible links to economically or environmentally important traits, such as feed conversion efficiency or methane emission levels. The resolution of studies investigating this variation may be improved by utilizing untargeted massively parallel sequencing (MPS), that is, sequencing without targeted amplification of genes. The objective of this study was to develop a method which used MPS to generate “rumen metagenome profiles”, and to investigate if these profiles were repeatable among samples taken from the same cow. Given faecal samples are much easier to obtain than rumen fluid samples; we also investigated whether rumen metagenome profiles were predictive of faecal metagenome profiles. Results Rather than focusing on individual organisms within the rumen, our method used MPS data to generate quantitative rumen micro-biome profiles, regardless of taxonomic classifications. The method requires a previously assembled reference metagenome. A number of such reference metagenomes were considered, including two rumen derived metagenomes, a human faecal microflora metagenome and a reference metagenome made up of publically available prokaryote sequences. Sequence reads from each test sample were aligned to these references. The “rumen metagenome profile” was generated from the number of the reads that aligned to each contig in the database. We used this method to test the hypothesis that rumen fluid microbial community profiles vary more between cows than within multiple samples from the same cow. Rumen fluid samples were taken from three cows, at three locations within the rumen. DNA from the samples was sequenced on the Illumina GAIIx. When the reads were aligned to a rumen metagenome reference, the rumen metagenome profiles were repeatable (P Conclusions We have presented a simple and high throughput method of metagenome profiling to assess the similarity of whole metagenomes, and illustrated its use on two novel datasets. This method utilises widely used freeware. The method should be useful in the exploration and comparison of metagenomes.

Fungal species of theNeotyphodiumandEpichloëgenera are endophytes of pasture grasses showing complex differences of life-cycle and genetic architecture. Simple sequence repeat (SSR) markers have been developed from endophyte-derived expressed sequence tag (EST) collections. Although SSR array size polymorphisms are appropriate for phenetic analysis to distinguish between taxa, the capacity to resolve phylogenetic relationships is limited by both homoplasy and heteroploidy effects. In contrast, nonrepetitive sequence regions that flank SSRs have been effectively implemented in this study to demonstrate a common evolutionary origin of grass fungal endophytes. Consistent patterns of relationships between specific taxa were apparent across multiple target loci, confirming previous studies of genome evolution based on variation of individual genes. Evidence was obtained for the definition of endophyte taxa not only through genomic affinities but also by relative gene content. Results were compatible with the current view that some asexualNeotyphodiumspecies arose following interspecific hybridisation between sexualEpichloëancestors. Phylogenetic analysis of SSR-flanking regions, in combination with the results of previous studies with other EST-derived SSR markers, further permitted characterisation ofNeotyphodiumisolates that could not be assigned to known taxa on the basis of morphological characteristics.

SUMMARY Artificial selection experiments provide insights into the evolutionary factors that can shape adaptive responses and have previously been utilized to examine the physiological adaptations that can improve survival to desiccation in Drosophila melanogaster. While such studies demonstrate that multiple resistance mechanisms may arise via different base populations and selection regimes, water retention emerges as a key mechanism for desiccation survival. Here, we present the physiological, correlated response and life history data for a new set of selection lines designed for the genetic dissection of desiccation resistance. After 26 generations of selection for desiccation resistance, female survival increased twofold. In contrast to previous studies, the altered resistance was associated primarily with enhanced dehydration tolerance and increased mass and less consistently with decreased rates of water loss. Life history tradeoffs and correlated selection responses were examined and overlap with previously published data. We crossed the resistant selected lines to desiccation-sensitive lines from the same control background to examine how each heterozygous resistant chromosome (excluding four) may improve desiccation resistance and observed that most of the resistance was due to genes on the third and first chromosomes, although interaction effects with the second chromosome were also detected. Results are compared to other selection responses and highlight the multiple evolutionary solutions that can arise when organisms are faced with a common selection pressure, although water loss rate remains a common mechanism in all studies.

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST-RFLP loci in the F(1)(NA(6) x AU(6)) population. A comprehensive set of EST-SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA(6) genetic map contains 88 EST-RFLP and 71 EST-SSR loci with a total map length of 963 cM, while the AU(6) genetic map contains 67 EST-RFLP and 58 EST-SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

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.

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.

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.

The temperate pasture grass Lolium perenne L. is commonly found in association with the fungal endophyte Neotyphodium lolii. Viability of both seed and endophyte was evaluated by inoculation of individual genotypes from the host cultivar Bronsyn with six distinct endophyte strains. The resulting populations were subjected to either long-term storage under various temperature conditions, or accelerated ageing (AA) treatments. High temperature storage was detrimental to endophyte viability. The AA variables (relative humidity [RH] and time) and subsequent temperature and duration of storage significantly (P

The temperate pasture grass Lolium perenne L. is commonly found in symbiotic association with the asexual fungal endophyte Neotyphodium lolii. Levels of endophyte colonisation and alkaloid content were evaluated in associations formed by plant genotypes from cv. Bronsyn with the standard endophyte (SE) and five distinct commercial endophyte strains. Bronsyn–SE produced all of the measured alkaloids (lolitrem B, peramine, and ergovaline). Bronsyn–AR1 produced only peramine, while Bronsyn–AR37 produced none of the tested alkaloids. Bronsyn–NEA2, Bronsyn–NEA3, and Bronsyn–NEA6 produced both ergovaline and peramine. Both endophyte strain and host genotype exerted significant effects on alkaloid production. Analysis of endophyte colonisation using qPCR revealed differences between each association. With the exception of Bronsyn–AR1 and Bronsyn–NEA3, host genotype also significantly affected colonisation levels. Phenotypic performance of each association was also assessed, based on measurement of morphological traits under glasshouse conditions in hydroponic culture. Significant variation due to different endophyte and host genotypes was observed. Collectively, these studies confirm that differences in both endophyte and host genotypes contribute to host–endophyte performance in a complex interactive manner.

Background Perennial ryegrass (Lolium perenne L.) is one of the most important species for temperate pastoral agriculture, forming associations with genetically diverse groups of mutualistic fungal endophytes. However, only two taxonomic groups (E. festucae var. lolii and LpTG-2) have so far been described. In addition to these two well-characterised taxa, a third distinct group of previously unclassified perennial ryegrass-associated endophytes was identified as belonging to a putative novel taxon (or taxa) (PNT) in a previous analysis based on simple sequence repeat (SSR) marker diversity. As well as genotypic differences, distinctive alkaloid production profiles were observed for members of the PNT group. Results A detailed phylogenetic analysis of perennial ryegrass-associated endophytes using components of whole genome sequence data was performed using complete sequences of 7 nuclear protein-encoding genes. Three independently selected genes (encoding a DEAD/DEAH box helicase [Sbp4], a glycosyl hydrolase [family 92 protein] and a MEAB protein), none of which have been previously used for taxonomic studies of endophytes, were selected together with the frequently used ‘house-keeping’ genes tefA and tubB (encoding translation elongation factor 1-α and β-tubulin, respectively). In addition, an endophyte-specific gene (perA for peramine biosynthesis) and the fungal-specific MT genes for mating-type control were included. The results supported previous phylogenomic inferences for the known species, but revealed distinctive patterns of diversity for the previously unclassified endophyte strains, which were further proposed to belong to not one but two distinct novel taxa. Potential progenitor genomes for the asexual endophytes among contemporary teleomorphic (sexual Epichloë) species were also identified from the phylogenetic analysis. Conclusions Unique taxonomic status for the PNT was confirmed through comparison of multiple nuclear gene sequences, and also supported by evidence from chemotypic diversity. Analysis of MT gene idiomorphs further supported a predicted independent origin of two distinct perennial ryegrass-associated novel taxa, designated LpTG-3 and LpTG-4, from different members of a similar founder population related to contemporary E. festucae. The analysis also provided higher resolution to the known progenitor contributions of previously characterised perennial ryegrass-associated endophyte taxa. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0349-6) contains supplementary material, which is available to authorized users.