Festulolium are grasses formed through interspecific hybridisation of ryegrass (Lolium) and fescue (Festuca) species. The Lolium-Festuca genome complex represents a vast array of heterogeneous and largely outbreeding grass species that have evolved, diverged, and adapted, allowing their world-wide colonisation of temperate grasslands. While strategies for grass improvement have focused primarily on intraspecific breeding and, in particular, on the agronomically desirable species Lolium perenne and Lolium multiflorum, a growing interest has emerged in interspecific hybrids as alternatives. The principal driver has been the increased appreciation of the capability of wide hybridisation to extend phenotypic variation beyond the ranges available within a single species. Lolium and Festuca species share complementary and desirable traits, and the prime aim in Festulolium (Festuca × Lolium) cultivar development has been to combine the agronomic performance of Lolium and the stress resistance of Festuca species. Advances in Festulolium development are timely, and support strategies aimed at delivering a more sustainable future for livestock agriculture, with grass cultivars that are persistent and productive. Festulolium hybrids occur naturally, including examples that demonstrate extreme heterosis with adaptations sufficient to sustain growth in harsh conditions. However, they are largely sterile and their perpetuity depends mainly on vegetative propagation. Synthetic Festulolium hybrids suitable for plant breeding require genome stability and fertility, sufficient for a cost-effective seed production. To this end, suitable amphiploid and introgression-breeding approaches have been developed. Herein, we provide detailed selected highlights in the research and breeding of Festulolium. In addition, recognising the multifunctional properties of grasslands and the development of enabling technologies that permit their study, we review additional benefits likely to accrue from Festulolium that may mitigate climate change effects and provide valuable ecosystem services.

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Festulolium, a century of research and breeding and its increased relevance in meeting the requirements for multifunctional grassland agriculture

Agricultural grass cultivars bred for increased forage yield commonly have extensive root systems. As roots are an important input of organic matter into the soil, it follows that such cultivars could lead to important increases in soil organic carbon (SOC), but this, and the mechanisms involved, are poorly understood with little empirical field evidence. We assessed the effect of two cultivars, Lolium perenne L. cv. ‘AberMagic’ and L. perenne L. × Festuca pratensis Huds. cv. ‘Prior’, in comparison with mixed-species permanent pasture (‘Pasture’) in a field experiment on temperate alluvial soil in the southwest UK. Root and SOC stocks, and specific plant-derived lipids as mechanistic biomarkers for shoot and root inputs of C, were measured on intact 1-m volumetric soil cores collected on four dates in 2013-2015. Total grass yields were greater for both grass cultivars (40-43 Mg ha–1) than Pasture (39 Mg ha–1), and by 2015 root stocks in the upper 0.1 m were significantly greater by more than 4.7 Mg ha–1. At 0.1-0.3 m depth, SOC stocks (per 0.1-m depth) were significantly greater under the two grass cultivars (24-50 Mg ha–1) compared to Pasture (18-32 Mg ha–1). In the full 1-m profile, mean SOC stocks were 137, 133 and 110 Mg ha–1 for AberMagic, Prior and Pasture, respectively, matching root biomass distribution. Odd-chain n-alkane concentrations were significantly greater for shoots than roots (274 vs. 64 mg kg–1, respectively), and roots had significantly greater concentrations than shoots of even-chain hydroxy fatty acids (1829 vs. 305 mg kg–1, respectively) and diacids (317 vs. 93 mg kg–1, respectively). This confirmed lipids as candidate biomarkers for distinguishing shoot and root inputs into the soil, with the latter being the dominant source of SOC. Based on the observations of increased forage yield and SOC stock, high-yielding forage grass cultivars can contribute to future strategies for climate-smart agriculture in grassland-dominated temperate regions.

High-yielding forage grass cultivars increase root biomass and soil organic carbon stocks compared with mixed-species permanent pasture in temperate soil

Hybrid vigor (or heterosis) is the over-performance of a hybrid over its parents in a specific trait or a set of traits. As such, hybrid breeding serves as a tool to efficiently trigger gains in breeding programs. Moreover, hybrids of genetically distant landraces, varieties or even species may become evolutionary successful. In Swiss alpine swards, we observed frequent prevalence of triploid hybrids of Festuca pratensis × F. apennina with outstanding competitiveness relative to their parental species in the sites of sympatric occurrence. Observations of these highly vigorous hybrids prompted the study on their heterosis across various environmental conditions. Phenotypic observations during 3 years at four locations at different altitudes (from 200 to 1850 m a.s.l.) have shown significant heterosis for dry biomass production at all sites during the first and second year, and at the mid- and high altitude sites also in the third year. At mid-altitude (1000 m a.s.l.), heterosis increased steadily and reached a maximum of + 508% for annual yield (+ 626% for a single cut) in the third year. This is by far the highest value of heterosis ever reported for annual dry matter yield of a forage grass. Further utilization of triploid hybrids in forage grass breeding is hampered by their sterility. Hence there is a need for vegetative propagation. However, artificial chromosome doubling of triploids to create fertile hexaploids, or seeking ways to propagate them vegetatively at an industrial scale might overcome this limitation.

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Triploid forage grass hybrids Festuca apennina × F. pratensis display extraordinary heterosis for yield characteristics

Microdochium nivale causes pink snow mould - a destructive disease of seedlings, stem bases, and ears of winter grasses and cereals. Glucanase and chitinase belong to pathogenesis-related proteins and exhibit a specific activity in response to pathogens. The aim of this study was to investigate changes in the activity of these enzymes in the leaves of chosen forage grasses during Microdochium nivale infection. Different cultivars of Festuca arudinacea, F. pratensis, Festulolium brauni, Lolium multiflorum, and L. perenne were prehardened at 12 °C and hardened at 2 °C and then inoculated with M. nivale mycelium. Leaf samples were collected before infection, as well as two, four, and eight days after inoculation. Each cultivar showed a specific pattern of changes in the enzymatic activities in response to M. nivale infection. We conclude that a rapid increase in glucanase and chitinase activities after M. nivale attack enhanced resistance to this pathogen and may be recognized as a physiological marker of grass resistance to this snow mould.

Rapid increases in β-1,3-glucanase and chitinase activities are markers of resistance to Microdochium nivale in grasses of the Lolium‑Festuca complex

In order to introduce drought tolerance and improved cell wall digestibility from fescue in fodder ryegrasses, we developed two amphiploid Festulolium synthetics. One is a synthetic composed of three selected drought tolerant F1 hybrid genotypes from a cross between tetraploid Lolium multiflorum and hexaploid Festuca arundinacea, further on called LMFA. The other is a synthetic composed of five selected genotypes with soft leaves from a cross between tetraploid Lolium perenne and tetraploid Festuca pratensis, further on called LPFP. We produced seeds in polycrosses of two generations of both amphiploids, i.e., syn1 and syn2, and tested them in plot trials to determine the yield and fodder quality. The syn1 of both Festulolium populations had a higher annual dry matter yield than the reference Lolium cultivars and Festulolium cultivars composed of the same parental species. However, the syn2 of LMFA did not show an improved drought tolerance during a dry growing season compared to other Festulolium cultivars, and the seed yield of LMFA syn1 was low and dropped extremely in syn2. The number of chromosomes of LMFA also decreased gradually from F1 to syn2, and there was a clear shift in chromosome composition towards the Lolium genome. The LPFP synthetic performed better. Although the sugar content was significantly lower than the sugar content of the perennial ryegrass cultivars, organic matter digestibility (OMD) of LPFP was as high as OMD of the tetraploid perennial ryegrass cultivars. The cell wall digestibility (NDFD) of LPFP was significantly higher than the NDFD of both parental species and higher than the NDFD of all tested Festulolium cultivars. The seed yield of LPFP was the same in syn1 and syn2. The chromosome number remained on average the same and no clear shift of the chromosome composition to one of the composing genomes was observed. Overall, chromosome analysis revealed a high number of aneuploidy in syn1 and syn2 generations of both LMFA and LPFP and a lot of variation in number of Lolium, Festuca and recombinant chromosomes, and in the Lolium:Festuca genome ratio was observed among different genotypes of the same population. Therefore, selection for genotypes with a more stable genome composition will be a prerequisite for a sufficient seed yield and a broader exploitation of these new Festulolium synthetics.

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Breeding and genetics of two new amphiploid Festulolium synthetics with improved yield and digestibility

A comprehensive set of Festulolium cultivars from on-going field trials in the Eucarpia network was characterised at the chromosome level using genomic in situ hybridization (GISH) and by Diversity Array Technology (DArT) markers. Both technologies were found to be complementary in describing the breeding history of the plant material. The genomic composition of the Lolium × Festuca cultivars varied from those that comprised equivalent proportions of their parental genomes to introgression lines where small chromosome segments of Festuca had been translocated onto Lolium chromosomes. The breadth of genotype combinations found within the grass cultivars described represents an important resource of genetic variations necessary to combat the diverse abiotic stresses encountered within Europe, including safeguards against prolonged exposure to harsh weather conditions. It is likely that in future plant breeding, genotyping will contribute to precision-transfers of targeted Festuca genes into Lolium germplasm in order to enhance resilience to climate change.

Genotyping of Festulolium Cultivars Involved in EUCARPIA Multi-site Trial Using DArT Markers and GISH

Ryegrass x fescue interspecific hybridization (so-called Festulolium) has great potential for forage diversification because of its many amphiploid and introgression-bred forms. Festulolium breeding efforts aim to improve adaptation to mainly winter cold and summer drought through higher abiotic stress tolerance. This goal must be met with a minimum of compromise regarding productivity in the first years of full exploitation. To compare field performances, a 21-entry trial of Festulolium and controls of pure species has been ongoing since 2012 in eight European countries. The yield data collected in 2012 and 2013 in nine locations showed that the Festulolium cultivars performed on average quite well compared to pure species controls. In amphiploids, the annual yield appeared to be mainly driven by the Lolium sp. parent combined with F. pratensis (Fp); the Lm x Fp amphiploids performed thus far better on average than the Lp x Fp equivalents. The only amphiploid from F. glaucescens included in the study had an intermediate response over locations, which was closer to tall fescue than to F. pratensis. Interestingly, a broad variability for yield among the Lm x Fp cultivars appeared to be associated with climate interactions when cold, wet locations were contrasted with more temperate locations with early spring growth.

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Enhancing the Productivity in Forage Grasses on the European Scale using Interspecific Hybridization

DArTFest is a DNA microarray developed for the Festuca-Lolium complex, consisting of 7,680 probes. As it offers high-throughput and low-cost screening of thousands of genomic loci, it has been successfully used in a number of projects. These include the analysis of interspecific, intraspecific and intravarietal genetic variation, genetic mapping, assigning markers to chromosomal segments, comparative analysis of the Festuca and Lolium genomes against model plant species rice and Brachypodium, an analysis of genomic constitution of Festulolium cultivars and QTL mapping of agronomically important traits such as resistance against crown rust and frost tolerance. In order to facilitate the use of DArT markers linked to traits of interests, a large number of them has been already sequenced and conversion to PCR-based markers is in progress.

V. Cernoch

Papers

Genotyping of Festulolium Cultivars Involved in EUCARPIA Multi-site Trial Using DArT Markers and GISH

Enhancing the Productivity in Forage Grasses on the European Scale using Interspecific Hybridization

DArTFest DNA Array—Applications and Perspectives for Grass Genetics, Genomics and Breeding