Marty J. Faville

A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne.

The endophytic fungus Neotyphodium lolii forms symbiotic associations with perennial ryegrass (Lolium perenne) and infection is typically described as asymptomatic. Here we describe a naturally occurring New Zealand N. lolii isolate that can induce dwarfing of L. perenne and suppress floral meristem development in the dwarfed plants. Further to this we demonstrate that the observed host dwarfing correlates with a reversible morphological change in the endophyte that appears associated with colony age. Mycelium isolated from normally growing plants had a typical cottony appearance in culture whereas mycelium from dwarfed plants appeared mucoid. Cottony colonies could be induced to turn mucoid after prolonged incubation and seedlings inoculated with this mucoid mycelium formed dwarfed plants. Mucoid colonies on the other hand could be induced to form cottony colonies through additional further incubation and these did not induce dwarfing. The reversibility of colony morphology indicates that the mucoid dwarfing phenotype is not the result of mutation. Ten isolates from other locations in New Zealand could also undergo the reversible morphological changes in culture, induce dwarfing and had the same microsatellite genotype as the original isolate, indicating that a N. lolii genotype with the ability to dwarf host plants is common in New Zealand.

Semi-quantitative and structural metabolic phenotyping by direct infusion ion trap mass spectrometry and its application in genetical metabolomics

The identification of quantitative trait loci (QTL) for plant metabolites requires the quantitation of these metabolites across a large range of progeny. We developed a rapid metabolic profiling method using both untargeted and targeted direct infusion tandem mass spectrometry (DIMSMS) with a linear ion trap mass spectrometer yielding sufficient precision and accuracy for the quantification of a large number of metabolites in a high-throughput environment. The untargeted DIMSMS method uses top-down data-dependent fragmentation yielding MS2 and MS3 spectra. We have developed software tools to assess the structural homogeneity of the MS2 and MS3 spectra hence their utility for phenotyping and genetical metabolomics. In addition we used a targeted DIMS(MS) method for rapid quantitation of specific compounds. This method was compared with targeted LC/MS/MS methods for these compounds. The DIMSMS methods showed sufficient precision and accuracy for QTL discovery. We phenotyped 200 individual Lolium perenne genotypes from a mapping population harvested in two consecutive years. Computational and statistical analyses identified 246 nominal m/z bins with sufficient precision and homogeneity for QTL discovery. Comparison of the data for specific metabolites obtained by DIMSMS with the results from targeted LC/MS/MS analysis showed that quantitation by this metabolic profiling method is reasonably accurate. Of the top 100 MS1 bins, 22 ions gave one or more reproducible QTL across the 2 years. Copyright

Mutualistic fungal endophytes in the Triticeae – survey and description

Grasses of the tribe Triticeae were screened to determine the presence of mutualistic epichloae fungal endophytes. Over 1500 accessions, from more than 250 species, encompassing 22 genera within the Triticeae were screened using immunodetection and direct staining/microscopy techniques. Only two genera, Elymus and Hordeum, were identified as harbouring epichloae endophytes with accessions native to a range of countries including Canada, China, Iran, Kazakhstan, Kyrgyzstan, Mongolia, Russia and the USA. Genetic analysis based on simple sequence repeat data revealed that the majority of endophytes cluster according to geographical regions rather than to host species; many strains isolated from Hordeum grouped with those derived from Elymus, and amongst the Elymus-derived strains, there was no clear correspondence between clustering topology and host species. This is the first detailed survey demonstrating the genetic diversity of epichloae endophytes within the Triticeae and highlights the importance of germplasm centres for not only preserving the genetic diversity of plant species but also the beneficial microorganisms they may contain.

Epichloë fungal endophytes and the formation of synthetic symbioses in Hordeeae (=Triticeae) grasses

This review examines two classes of organism that live in symbiosis; grasses, and fungi. Specifically it deals with grasses of the tribe Hordeeae (formerly Triticeae) of the subfamily Poöideae and the Epichloë fungi of family Clavicipitaceae. Epichloë endophytes, particularly asexual forms, have important roles in pastoral agricultural systems in the Americas, Australia, and New Zealand. Selected strains add value to some grass‐based forage systems by providing both biotic and abiotic stress resistance. The importance of cereal grasses such as wheat, barley, rye, and oats to human and animal nutrition and indeed to the foundation and maintenance of human civilization is well documented. Both organism classes, Epichloë endophytes and cereal grasses, are of great importance in their own contexts. Here, we seek to review these two classes of organism and examine the possibility of bringing them together in symbiosis with the ultimate goal of improving cereal production systems.

Breaking through the feed barrier: options for improving forage genetics

Pasture based on perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) is the foundation for production and profit in the Australasian pastoral sectors. The improvement of these species offers direct opportunities to enhance sector performance, provided there is good alignment with industry priorities as quantified by means such as the forage value index. However, the rate of forage genetic improvement must increase to sustain industry competitiveness. New forage technologies and breeding strategies that can complement and enhance traditional approaches are required to achieve this. We highlight current and future research in plant breeding, including genomic and gene technology approaches to improve rate of genetic gain. Genomic diversity is the basis of breeding and improvement. Recent advances in the range and focus of introgression from wild Trifolium species have created additional specific options to improve production and resource-use-efficiency traits. Symbiont genetic resources, especially advances in grass fungal endophytes, make a critical contribution to forage, supporting pastoral productivity, with benefits to both pastures and animals in some dairy regions. Genomic selection, now widely used in animal breeding, offers an opportunity to lift the rate of genetic gain in forages as well. Accuracy and relevance of trait data are paramount, it is essential that genomic breeding approaches be linked with robust field evaluation strategies including advanced phenotyping technologies. This requires excellent data management and integration with decision-support systems to deliver improved effectiveness from forage breeding. Novel traits being developed through genetic modification include increased energy content and potential increased biomass in ryegrass, and expression of condensed tannins in forage legumes. These examples from the wider set of research emphasise forage adaptation, yield and energy content, while covering the spectrum from exotic germplasm and symbionts through to advanced breeding strategies and gene technologies. To ensure that these opportunities are realised on farm, continuity of industry-relevant delivery of forage-improvement research is essential, as is sustained research input from the supporting pasture and plant sciences.

Plant vigour at establishment and following defoliation are both associated with responses to drought in perennial ryegrass ( Lolium perenne L.)

Highlight text We have developed a new methodology to assess individual perennial ryegrass plant performance under moisture stress and identified QTLs associated with improved performance during drought in this important forage species.

Comparative Genomics and Functional Characterisation of the GIGANTEA Gene from the Temperate Forage Perennial Ryegrass Lolium perenne

Lolium perenne (ryegrass) is a perennial forage grass of worldwide importance. It is a temperate climate grass and flowering is induced by long day photoperiods. The agronomic productivity of ryegrass is strongly influenced by flowering time, but less is known about ryegrass flowering time regulators in other temperate Poaceae like wheat, barley and the model grass Brachypodium. The GIGANTEA (GI) gene was first identified in Arabidopsis and is an important regulator of photoperiodic flowering in angiosperms. GI usually exists as a conserved, single-copy gene. However, recently, genome sequencing has revealed that some plants, including the tropical grass maize, carry more than one full-length copy of GI. Here we describe the isolation and characterisation of the ryegrass L. perenne GIGANTEA gene (LpGI) gene. Comparative genomic analysis indicates that LpGI gene structure is very well conserved overall with GI genes from monocots and eudicots. LpGI protein clusters with GI proteins from other temperate grasses and is most closely related to the GI protein from meadow fescue. Genetic mapping locates LpGI to a chromosomal region that is syntenic with rice and Brachypodium GI. Our functional characterisation shows that LpGI shows a diurnal pattern of expression, continues to oscillate under constant light and adjusts its phase in response to changes in day length as seen in other plants. Constitutive expression of LpGI completely rescues the Arabidopsis gigantea-3 allele (gi-3) mutation, confirming that the isolated ryegrass gene is fully functional. Taken together, it is highly likely that LpGI is orthologous to Arabidopsis GI and involved in photoperiodic flowering time control in ryegrass.

Quantitative trait locus mapping of genomic regions controlling herbage yield in perennial ryegrass

Abstract Genetic control of herbage yield—a complex trait of fundamental importance to agricultural productivity—was characterized in a perennial ryegrass biparental mapping population (I×S). Seasonal and combined growth score estimates of herbage yield (GS) were obtained from a trial conducted at three sites in New Zealand, over 2 years. Twenty-nine quantitative trait loci (QTL) for GS were identified on an I×S genetic linkage map constructed using simple sequence repeat (SSR) markers. Most QTL explained less than 15% of the phenotypic variation. QTL consistency was low among and within trial sites and years but 17 QTL were observed in more than one environment or season. QTL on linkage groups (LG) 1, 2, 4 and 6 were most consistent, based on numbers of QTL co-located at these positions and persistence of their allelic effects. SSR markers from consistent QTL positions may be beneficially applied in marker-assisted selection to enhance genetic gain for herbage yield in ryegrass breeding.

Development of Genomic Selection for Perennial Ryegrass

Mixed-species pasture based on perennial ryegrass (Lolium perenne) and white clover (Trifolium repens) is the foundation for profitable production from temperate grasslands. In theory, genomic selection (GS) offers an opportunity to lift the rate of genetic gain in these species. Critical research questions include to what extent theoretical expectations for GS can be realized in practice, and understanding the genetic and economic implications of GS in breeding programs and on farm. We describe a limited experiment to derive and cross-validate genomic estimated breeding values (GEBVs) from 211 perennial ryegrass plants evaluated for plant herbage dry weight (DW) and days-to-heading (DTH), using field phenotypic data and up to 10,885 markers typed via genotyping-by-sequencing (GBS). Using Ridge Regression-BLUP and Random Forest regression, cross validation prediction accuracies ranged from r = 0.16–0.34 (DW) and r = 0.52–0.56 (DTH). Accuracy was not influenced by marker density, but there was an interaction between statistical model and trait. The data indicate that, in these elite breeding populations, low marker densities in a limited training population dataset may be viable for generation of GEBVs in perennial ryegrass. Variance attributable to population structure, rather than linkage disequilibrium, is likely the primary basis of GEBV accuracy in this study. Generation of a training set of increased size, scope and greater relevance for key economic traits is outlined in the context of developing a GS capability with an Australasian focus.

Untargeted Metabotyping Lolium perenne Reveals Population-Level Variation in Plant Flavonoids and Alkaloids

Metabolomics provides a powerful platform to characterize plants at the biochemical level, allowing a search for underlying genes and associations with higher level complex traits such as yield and nutritional value. Efficient and reliable methods to characterize metabolic variation in economically important species are considered of high value to the evaluation and prioritization of germplasm and breeding lines. In this investigation, a large-scale metabolomic survey was performed on a collection of diverse perennial ryegrass (Lolium perenne L.) plants. A total of 2,708 data files, derived from liquid chromatography coupled to high resolution mass spectrometry (LCMS), were selected to assess the effectiveness and efficiency of applying high throughput metabolomics to survey chemical diversity in plant populations. The data set was generated from 23 ryegrass populations, with 3–25 genotypes per population, and five clonal replicates per genotype. We demonstrate an integrated approach to rapidly mine and analyze metabolic variation from this large, multi-batch LCMS data set. After performing quality control, statistical data mining and peak annotation, a wide range of variation for flavonoid glycosides and plant alkaloids was discovered among the populations. Structural variation of flavonoids occurs both in aglycone structures and acetylated/malonylated/feruloylated sugar moieties. The discovery of comprehensive metabolic variation among the plant populations offers opportunities to probe into the genetic basis of the variation, and provides a valuable resource to gain insight into biochemical functions and to relate metabolic variation with higher level traits in the species.