Genetic architecture of inter-specific and -generic grass hybrids by network analysis on multi-omics data.

BACKGROUND: Understanding the mechanisms underlining forage production and its biomass nutritive quality at the omics level is crucial for boosting the output of high-quality dry matter per unit of land. Despite the advent of multiple omics integration for the study of biological systems in major crops, investigations on forage species are still scarce. RESULTS: Our results identified substantial changes in gene co-expression and metabolite-metabolite network topologies as a result of genetic perturbation by hybridizing L. perenne with another species within the genus (L. multiflorum) relative to across genera (F. pratensis). However, conserved hub genes and hub metabolomic features were detected between pedigree classes, some of which were highly heritable and displayed one or more significant edges with agronomic traits in a weighted omics-phenotype network. In spite of tagging relevant biological molecules as, for example, the light-induced rice 1 (LIR1), hub features were not necessarily better explanatory variables for omics-assisted prediction than features stochastically sampled and all available regressors. CONCLUSIONS: The utilization of computational techniques for the reconstruction of co-expression networks facilitates the identification of key omic features that serve as central nodes and demonstrate correlation with the manifestation of observed traits. Our results also indicate a robust association between early multi-omic traits measured in a greenhouse setting and phenotypic traits evaluated under field conditions.

Leveraging spatiotemporal genomic breeding value estimates of dry matter yield and herbage quality in ryegrass via random regression models.

Joint modeling of correlated multienvironment and multiharvest data of perennial crop species may offer advantages in prediction schemes and a better understanding of the underlying dynamics in space and time. The goal of the present study was to investigate the relevance of incorporating the longitudinal dimension of within-season multiple measurements of forage perennial ryegrass (Lolium perenne L.) traits in a reaction-norm model setup that additionally accounts for genotype × environment (G × E) interactions. Genetic parameters and accuracy of genomic estimated breeding value (gEBV) predictions were investigated by fitting three genomic random regression models (gRRMs) using Legendre polynomial functions to the data. Genomic DNA sequencing of family pools of diploid perennial ryegrass was performed using DNA nanoball-based technology and yielded 56,645 single-nucleotide polymorphisms, which were used to calculate the allele frequency-based genomic relationship matrix. Biomass yield's estimated additive genetic variance and heritability values were higher in later harvests. The additive genetic correlations were moderate to low in early measurements and peaked at intermediates with fairly stable values across the environmental gradient except for the initial harvest data collection. This led to the conclusion that complex (G × E) arises from spatial and temporal dimensions in the early season with lower reranking trends thereafter. In general, modeling the temporal dimension with a second-order orthogonal polynomial improved the accuracy of gEBV prediction for nutritive quality traits, but no gain in prediction accuracy was detected for dry matter yield (DMY). This study leverages the flexibility and usefulness of gRRM models for perennial ryegrass breeding and can be readily extended to other multiharvest crops.

Relative importance of genotype, gene expression, and DNA methylation on complex traits in perennial ryegrass.

The growing demand for food and feed crops in the world because of growing population and more extreme weather events requires high-yielding and resilient crops. Many agriculturally important traits are polygenic, controlled by multiple regulatory layers, and with a strong interaction with the environment. In this study, 120 F2 families of perennial ryegrass (Lolium perenne L.) were grown across a water gradient in a semifield facility with subsoil irrigation. Genomic (single-nucleotide polymorphism [SNP]), transcriptomic (gene expression [GE]), and DNA methylomic (MET) data were integrated with feed quality trait data collected from control and drought sections in the semifield facility, providing a treatment effect. Deep root length (DRL) below 110 cm was assessed with convolutional neural network image analysis. Bayesian prediction models were used to partition phenotypic variance into its components and evaluated the proportion of phenotypic variance in all traits captured by different regulatory layers (SNP, GE, and MET). The spatial effects and effects of SNP, GE, MET, the interaction between GE and MET (GE × MET) and GE × treatment (GEControl and GEDrought ) interaction were investigated. Gene expression explained a substantial part of the genetic and spatial variance for all the investigated phenotypes, whereas MET explained residual variance not accounted for by SNPs or GE. For DRL, MET also contributed to explaining spatial variance. The study provides a statistically elegant analytical paradigm that integrates genomic, transcriptomic, and MET information to understand the regulatory mechanisms of polygenic effects for complex traits.

Transcriptional and Metabolomic Analyses Indicate that Cell Wall Properties are Associated with Drought Tolerance in Brachypodium distachyon.

Brachypodium distachyon is an established model for drought tolerance. We previously identified accessions exhibiting high tolerance, susceptibility and intermediate tolerance to drought; respectively, ABR8, KOZ1 and ABR4. Transcriptomics and metabolomic approaches were used to define tolerance mechanisms. Transcriptional analyses suggested relatively few drought responsive genes in ABR8 compared to KOZ1. Linking these to gene ontology (GO) terms indicated enrichment for "regulated stress response", "plant cell wall" and "oxidative stress" associated genes. Further, tolerance correlated with pre-existing differences in cell wall-associated gene expression including glycoside hydrolases, pectin methylesterases, expansins and a pectin acetylesterase. Metabolomic assessments of the same samples also indicated few significant changes in ABR8 with drought. Instead, pre-existing differences in the cell wall-associated metabolites correlated with drought tolerance. Although other features, e.g., jasmonate signaling were suggested in our study, cell wall-focused events appeared to be predominant. Our data suggests two different modes through which the cell wall could confer drought tolerance: (i) An active response mode linked to stress induced changes in cell wall features, and (ii) an intrinsic mode where innate differences in cell wall composition and architecture are important. Both modes seem to contribute to ABR8 drought tolerance. Identification of the exact mechanisms through which the cell wall confers drought tolerance will be important in order to inform development of drought tolerant crops.

Accuracy of Genomic Prediction in a Commercial Perennial Ryegrass Breeding Program.

The implementation of genomic selection (GS) in plant breeding, so far, has been mainly evaluated in crops farmed as homogeneous varieties, and the results have been generally positive. Fewer results are available for species, such as forage grasses, that are grown as heterogenous families (developed from multiparent crosses) in which the control of the genetic variation is far more complex. Here we test the potential for implementing GS in the breeding of perennial ryegrass ( L.) using empirical data from a commercial forage breeding program. Biparental F and multiparental synthetic (SYN) families of diploid perennial ryegrass were genotyped using genotyping-by-sequencing, and phenotypes for five different traits were analyzed. Genotypes were expressed as family allele frequencies, and phenotypes were recorded as family means. Different models for genomic prediction were compared by using practically relevant cross-validation strategies. All traits showed a highly significant level of genetic variance, which could be traced using the genotyping assay. While there was significant genotype × environment (G × E) interaction for some traits, accuracies were high among F families and between biparental F and multiparental SYN families. We have demonstrated that the implementation of GS in grass breeding is now possible and presents an opportunity to make significant gains for various traits.

Linking Dynamic Phenotyping with Metabolite Analysis to Study Natural Variation in Drought Responses of Brachypodium distachyon.

Drought is an important environmental stress limiting the productivity of major crops worldwide. Understanding drought tolerance and possible mechanisms for improving drought resistance is therefore a prerequisite to develop drought-tolerant crops that produce significant yields with reduced amounts of water. Brachypodium distachyon (Brachypodium) is a key model species for cereals, forage grasses, and energy grasses. In this study, initial screening of a Brachypodium germplasm collection consisting of 138 different ecotypes exposed to progressive drought, highlighted the natural variation in morphology, biomass accumulation, and responses to drought stress. A core set of ten ecotypes, classified as being either tolerant, susceptible or intermediate, in response to drought stress, were exposed to mild or severe (respectively, 15 and 0% soil water content) drought stress and phenomic parameters linked to growth and color changes were assessed. When exposed to severe drought stress, phenotypic data and metabolite profiling combined with multivariate analysis revealed a remarkable consistency in separating the selected ecotypes into their different pre-defined drought tolerance groups. Increases in several metabolites, including for the phytohormones jasmonic acid and salicylic acid, and TCA-cycle intermediates, were positively correlated with biomass yield and with reduced yellow pixel counts; suggestive of delayed senescence, both key target traits for crop improvement to drought stress. While metabolite analysis also separated ecotypes into the distinct tolerance groupings after exposure to mild drought stress, similar analysis of the phenotypic data failed to do so, confirming the value of metabolomics to investigate early responses to drought stress. The results highlight the potential of combining the analyses of phenotypic and metabolic responses to identify key mechanisms and markers associated with drought tolerance in both the Brachypodium model plant as well as agronomically important crops.

Estimating genomic heritabilities at the level of family-pool samples of perennial ryegrass using genotyping-by-sequencing.

KEYMESSAGE: By using the genotyping-by-sequencing method, it is feasible to characterize genomic relationships directly at the level of family pools and to estimate genomic heritabilities from phenotypes scored on family-pools in outbreeding species. Genotyping-by-sequencing (GBS) has recently become a promising approach for characterizing plant genetic diversity on a genome-wide scale. We use GBS to extend the concept of heritability beyond individuals by genotyping family-pool samples by GBS and computing genomic relationship matrices (GRMs) and genomic heritabilities directly at the level of family-pools from pool-frequencies obtained by sequencing. The concept is of interest for species where breeding and phenotyping is not done at the individual level but operates uniquely at the level of (multi-parent) families. As an example we demonstrate the approach using a set of 990 two-parent F2 families of perennial ryegrass (Lolium Perenne). The families were phenotyped as a family-unit in field plots for heading date and crown rust resistance. A total of 728 K single nucleotide polymorphism (SNP) variants were available and were divided in groups of different sequencing depths. GRMs based on GBS data showed diagonal values biased upwards at low sequencing depth, while off-diagonals were little affected by the sequencing depth. Using variants with high sequencing depth, genomic heritability for crown rust resistance was 0.33, and for heading date 0.22, and these genomic heritabilities were biased downwards when using variants with lower sequencing depth. Broad sense heritabilities were 0.61 and 0.66, respectively. Underestimation of genomic heritability at lower sequencing depth was confirmed with simulated data. We conclude that it is feasible to use GBS to describe relationships between family-pools and to estimate genomic heritability directly at the level of F2 family-pool samples, but estimates are biased at low sequencing depth.

Ensiling and hydrothermal pretreatment of grass: consequences for enzymatic biomass conversion and total monosaccharide yields.

BACKGROUND: Ensiling may act as a pretreatment of fresh grass biomass and increase the enzymatic conversion of structural carbohydrates to fermentable sugars. However, ensiling does not provide sufficient severity to be a standalone pretreatment method. Here, ensiling of grass is combined with hydrothermal treatment (HTT) with the aim of improving the enzymatic biomass convertibility and decrease the required temperature of the HTT. RESULTS: Grass silage (Festulolium Hykor) was hydrothermally treated at temperatures of 170, 180, and 190°C for 10 minutes. Relative to HTT treated dry grass, ensiling increased the solubilization of dry matter (DM) during HTT and gave increased glucan content, but lower lignin in the insoluble fiber fraction. Ensiling improved glucose yields in the enzymatic hydrolysis of the washed solid fiber fraction at the lower HTT temperatures. At 170°C glucose yield improved from 17 to 24 (w/w)% (45 to 57% cellulose convertibility), and at 180°C glucose yield improved from 22 to 29 (w/w)% (54 to 69% cellulose convertibility). Direct HTT of grass at 190°C gave the same high glucose yield as for grass silage (35 (w/w)% (77% cellulose convertibility)) and improved xylan yields (27% xylan convertibility). The effect of ensiling of grass prior to HTT improved the enzymatic conversion of cellulose for HTT at 170 and 180°C, but the increased glucose release did not make up for the loss of water soluble carbohydrates (WSC) during ensiling. Overall, sugar yields (C6 + C5) were similar for HTT of grass and grass silage at both 170 and 180°C, but at 190°C the overall sugar yield was better for HTT of dry grass. CONCLUSIONS: This study unequivocally establishes that ensiling of grass as a biomass pretreatment method comes with a loss of WSC. The loss of WSC by ensiling is not necessarily compensated for by providing a lower temperature requirement for HTT for high enzymatic monosaccharide release. However, ensiling can be an advantageous storage method prior to grass processing.

Comparative exomics of Phalaris cultivars under salt stress.

BACKGROUND: Reed canary grass (Phalaris arundinacea) is an economically important forage and bioenergy grass of the temperate regions of the world. Despite its economic importance, it is lacking in public genomic data. We explore comparative exomics of the grass cultivars in the context of response to salt exposure. The limited data set poses challenges to the computational pipeline. METHODS: As a prerequisite for the comparative study, we generate the Phalaris reference transcriptome sequence, one of the first steps in addressing the issue of paucity of processed genomic data in this species. In addition, the differential expression (DE) and active-but-stable genes for salt stress conditions were analyzed by a novel method that was experimentally verified on human RNA-seq data. For the comparative exomics, we focus on the DE and stable genic regions, with respect to salt stress, of the genome. RESULTS AND CONCLUSIONS: In our comparative study, we find that phylogeny of the DE and stable genic regions of the Phalaris cultivars are distinct. At the same time we find the phylogeny of the entire expressed reference transcriptome matches the phylogeny of only the stable genes. Thus the behavior of the different cultivars is distinguished by the salt stress response. This is also reflected in the genomic distinctions in the DE genic regions. These observations have important implications in the choice of cultivars, and their breeding, for bio-energy fuels. Further, we identified genes that are representative of DE under salt stress and could provide vital clues in our understanding of the stress handling mechanisms in general.

Seasonal variation of provitamin D2 and vitamin D2 in perennial ryegrass (Lolium perenne L.).

Ergosterol (provitamin D(2)) is converted to vitamin D(2) in grass by exposure to UV light. Six varieties of perennial ryegrass (Lolium perenne L.) were harvested four times during the season, and the contents of vitamin D(2) and ergosterol were analyzed by a sensitive and selective liquid chromatography tandem mass spectrometry method. Weather factors were recorded, and a principal component analysis was performed to study which factors were important for the formation of vitamin D(2). The results suggest that a combination of weather factors is involved and that the contents of ergosterol and vitamin D(2) change more than a factor of 10 during the season. These results demonstrate that grass potentially can be a significant source of vitamin D for grazing animals and animals fed on silage and hay.

Seasonal changes in the metabolic fingerprint of 21 grass and legume cultivars studied by nuclear magnetic resonance-based metabolomics.

A nuclear magnetic resonance (NMR)-based approach was introduced for metabolic fingerprinting of 21 grass and legume cultivars in the present study. Applying principal component analysis (PCA) on the fingerprints obtained on water extracts, it was possible to elucidate the variation between cultivars and the magnitude of changes in the metabolic fingerprint between the spring growth and the second regrowth. Consequently, the potential of the method for tracking differences and changes related to cultivar and season was demonstrated. In addition, partial least-squares (PLS) regressions revealed correlations between the NMR fingerprints and the value of the grasses as animal feed evaluated as concentration of sugars, neutral detergent fibres (NDF) (R = 0.82), indigestible neutral detergent fibres (iNDF) (R = 0.90), and in vitro organic matter digestibility (IVOMD) (R = 0.75). The correlations between these parameters and the NMR fingerprint could mainly be ascribed to differences in spectral intensities from signals assigned to malic acid (2.40 and 4.70 ppm), choline (3.27 ppm), and glucose (5.24 ppm), and the biochemical rationale for this relation is discussed.

Improved fructan accumulation in perennial ryegrass transformed with the onion fructosyltransferase genes 1-SST and 6G-FFT.

Carbohydrate limitation has been identified as a main cause of inefficient nitrogen use in ruminant animals, which feed mainly on fresh forage, hay and silage. This inefficiency results in suboptimal meat and milk productivity. One important molecular breeding strategy is to improve the nutritional value of ryegrass (Lolium perenne) by increasing the fructan content through expression of heterologous fructan biosynthetic genes. We developed perennial ryegrass lines expressing sucrose:sucrose 1-fructosyltransferase and fructan:fructan 6G-fructosyltransferase genes from onion (Allium cepa) which exhibited up to a 3-fold increased fructan content. Further, the high fructan content was stable during the growth period, whereas the fructan content in an elite variety, marketed as a high sugar variety, dropped rapidly after reaching its maximum and subsequently remained low.

Frequency, type, and distribution of EST-SSRs from three genotypes of Lolium perenne, and their conservation across orthologous sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa.

BACKGROUND: Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies in several plant species. They are used for cultivar identification, variety protection, as anchor markers in genetic mapping, and in marker-assisted breeding. Currently, a limited number of SSR markers are publicly available for perennial ryegrass (Lolium perenne). We report on the exploitation of a comprehensive EST collection in L. perenne for SSR identification. The objectives of this study were 1) to analyse the frequency, type, and distribution of SSR motifs in ESTs derived from three genotypes of L. perenne, 2) to perform a comparative analysis of SSR motif polymorphisms between allelic sequences, 3) to conduct a comparative analysis of SSR motif polymorphisms between orthologous sequences of L. perenne, Festuca arundinacea, Brachypodium distachyon, and O. sativa, 4) to identify functionally associated EST-SSR markers for application in comparative genomics and breeding. RESULTS: From 25,744 ESTs, representing 8.53 megabases of nucleotide information from three genotypes of L. perenne, 1,458 ESTs (5.7%) contained one or more SSRs. Of these SSRs, 955 (3.7%) were non-redundant. Tri-nucleotide repeats were the most abundant type of repeats followed by di- and tetra-nucleotide repeats. The EST-SSRs from the three genotypes were analysed for allelic- and/or genotypic SSR motif polymorphisms. Most of the SSR motifs (97.7%) showed no polymorphisms, whereas 22 EST-SSRs showed allelic- and/or genotypic polymorphisms. All polymorphisms identified were changes in the number of repeat units. Comparative analysis of the L. perenne EST-SSRs with sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa identified 19 clusters of orthologous sequences between these four species. Analysis of the clusters showed that the SSR motif generally is conserved in the closely related species F. arundinacea, but often differs in length of the SSR motif. In contrast, SSR motifs are often lost in the more distant related species B. distachyon and O. sativa. CONCLUSION: The results indicate that the L. perenne EST-SSR markers are a valuable resource for genetic mapping, as well as evaluation of co-location between QTLs and functionally associated markers.

A rapid and efficient transformation protocol for the grass Brachypodium distachyon.

A fast and efficient microprojectile bombardment-mediated transformation protocol is reported for the grass species Brachypodium distachyon, a proposed alternative model plant to Oryza sativa for functional genomics in grasses. Embryogenic calli derived from immature embryos were transformed by a construct containing the uidA (coding for beta-glucuronidase) and bar (coding for phosphinothricin acetyl transferase) genes, and bialaphos, a non-selective herbicide, was used as the selection agent throughout all phases of the tissue culture. Average transformation efficiencies of 5.3% were achieved, and for single bombardments transformation efficiencies of up to 14% were observed. The time frame from the bombardment of embryogenic callus to the harvesting of transgenic T1 seeds was 29 weeks and 25 weeks for the diploid and two tetraploid accessions used, respectively. Since the seed-to-seed life cycle is 19 weeks for the diploid and 15 weeks for the tetraploid accessions, our B. distachyon transformation system allows testing of both the T0 and the T1 generation as well as production of T2 seeds within 1 year.

MADS-box genes from perennial ryegrass differentially expressed during transition from vegetative to reproductive growth.

In contrast to well-studied dicot plants like Arabidopsis and Antirrhinum, relatively few genes controlling the transition to flowering and flower development of agronomically important monocot species have been identified. In perennial ryegrass (Lolium perenne) the transition from vegetative to reproductive growth is triggered by an obligate vernalization period (primary induction) of at least 12 weeks at temperatures below 5 degrees C under short days, followed by increased temperature and day length (secondary induction). Here we report the isolation of nine ryegrass MADS-box (LpMADS) genes by a differential display method specific to this family of transcription factors. Three of the nine MADS-box genes show homology to the APETALA 1 (AP1) subfamily, two to the SEPALLATA (SEP) subfamily, one to the AGAMOUS-LIKE 6 (AGL6) subfamily, and three show homology to the newly identified OsMADS1 subfamily. The three AP1 homologues are up-regulated, both in the shoot apex and in leaves, in response to vernalization, while expression of the other six are increased by secondary induction during inflorescence development, although not in leaves. Differences in the sequence and hierarchy of flowering gene expression patterns indicate that the Arabidopsis-based flowering model is not completely applicable to explain the molecular events leading to the floral transition in grasses.

Expression of a 434:VP16 chimeric activator leads to high-level activation of gene expression in stable transformants of Arabidopsis.

The performance of an expression system based on a fusion of the bacteriophage 434-repressor to the VP16 activation domain of Herpes simplex virus type 1 (434:VP16) was tested after stable integration into Arabidopsis. A special feature of this system was the use of the monocot maize ubiquitin1 and rice actin1 promoters to drive the expression of the 434:VP16 activator and 434-repressor, respectively. Our results demonstrate that the maize ubiquitin1 and the rice actin1 promoters, each of which contain introns, are active in Arabidopsis and can be used to express genes in this dicot species. Activation of gene expression after co-integration of the activator and reporter cassettes into the same genomic locus resulted in a higher activation level (84-fold activation) compared to crossing individual lines expressing only the activator or the operator reporter cassette alone (9-fold activation). Increasing the number of operator elements in the reporter cassette from 1 to 4 increased the activation level in cross-activated lines to an average of 281-fold with one combination of parental lines giving a 900-fold activation. Simultaneous expression of the 434-repressor protein driven by the rice actin promoter resulted in a significant decrease in the 434:VP16 mediated reporter gene expression. Nevertheless, an overall induction via 434:VP16 was possible even in the presence of the 434-repressor protein. This feature is important for genes which need to be absolutely repressed except under activating conditions. To our knowledge this investigation is the first report on the use of the 434:VP16 chimeric activator in an expression system in stably transformed plant lines.