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Red algae, belonging to the phylum Rhodophyta, contain an abundance of useful chemicals including bioactive molecules and present opportunities for the production of different products through biorefinery cascades. The rhodophyte Palmaria palmata, commonly termed dulse or dillisk, grows predominantly on the northern coasts of the Atlantic and Pacific Oceans and is a well-known snack food. Due to its abundance, availability and cultivation capacity, P. palmata was selected for study as a potential candidate for a biorefinery process. In addition to studying juice and solid fractions of freshly harvested P. palmata, we have investigated the novel possibility of preserving algal biomass by ensilaging protocols similar to those employed for terrestrial forage crops. In the metabolite partitioning within the solid and liquid fractions following screw-pressing, the majority of the metabolites screened for—water soluble carbohydrates, proteins and amino acids, lipids, pigments, phenolics and antioxidant activity—remained in the solid fraction, though at differing proportions depending on the metabolite, from 70.8% soluble amino acids to 98.2% chlorophyll a and 98.1% total carotenoids. For the ensiling study, screw-pressed P. palmata, with comparative wilted and chopped, and chopped only samples, were ensiled at scale with and without Safesil silage additive. All samples were successfully ensiled after 90 days, with screw-pressing giving lower or equal pH before and after ensiling compared with the other preparations. Of particular note was the effluent volumes generated during ensiling: 26–49% of the fresh weight, containing 16–34% of the silage dry matter. This may be of advantage depending on the final use of the biomass.

Pyrolysis char residues from ensiled macroalgae were examined to determine their potential as growth promoters on germinating and transplanted seedlings. Macroalgae was harvested in May, July and August from beach collections, containing predominantly Laminaria digitata and Laminaria hyperborea; naturally seeded mussel lines dominated by Saccharina latissima; and lines seeded with cultivated L. digitata. Material was ensiled, pressed to pellets and underwent pyrolysis using a thermo‐catalytic reforming (TCR) process, with and without additional steam. The chars generated were then assessed through proximate and ultimate analysis. Seasonal changes had the prevalent impact on char composition, though using mixed beach‐harvested material gave a greater variability in elements than when using the offshore collections. Applying the char at 5% (v/v)/2% (w/w) into germination or seedling soils was universally negative for the plants, inhibiting or delaying all parameters assessed with no clear advantage in harvesting date, species or TCR processing methodology. In germinating lettuce seeds, soil containing the pyrolysis chars caused a longer germination time, poorer germination, fewer true leaves to be produced, a lower average plant health score and a lower final biomass yield. For transplanted ryegrass seedlings, there were lower plant survival rates, with surviving plants producing fewer leaves and tillers, lower biomass yields when cut and less regrowth after cutting. As water from the char‐contained plant pots inhibited the lettuce char control, one further observation was that run‐off water from the pyrolysis char released compounds which detrimentally affected cultivated plant growth. This study clearly shows that pyrolysed macroalgae char does not fit the standard assumption that chars can be used as soil amendments at 2% (w/w) addition levels. As the bioeconomy expands in the future, the end use of residues and wastes from bioprocessing will become a genuine global issue, requiring consideration and demonstration rather than hypothesized use., Lettuce seedlings 21 days after sowing with and without the addition of ensiled, thermo‐catalytic reforming macroalgae char and inorganic fertiliser to the growing medium. Orange square = SoilFixer control char.

Seaweeds can be a valuable resource for biorefinery and biotechnology applications, but their high water content is a recurrent problem and one of the key bottlenecks for their sustainable use. Treatments to increase dry matter content of the kelp Laminaria digitata were recently described by the authors. However macroalgae are an extremely diverse group of organisms and compositional variation between species may influence the effects of particular treatments. In this study, potential dewatering treatments including drying, osmotic media, and the application of both organic and mineral acids all followed by screw-pressing have been tested on two other species of kelp (Laminaria hyperborea and Saccharina latissima) and a red seaweed (Palmaria palmata). Conditions that dewatered these species were identified and the data have been combined with the previous results for L. digitata. There were significant differences between species across all the traits of interest. However dewatering was highly dependent on specific interactions with both treatment and season of collection. Nevertheless, the dry matter content of brown seaweeds was widely and successfully increased by air drying or acid treatment followed by screw-pressing. The results for P. palmata were quite different, particularly with regard to juice production. For this species, acid treatment did not result in dewatering, but dry matter content could be increased by screw-pressing immediately after harvest. Together the data presented here demonstrate that dewatering pre-treatments need to be specific for the type of seaweed to be processed; important knowledge for the future use of this sustainable biomass resource.

Leaf length is a key factor in the economic value of different grass species and cultivars in forage production. It is also important for the survival of individual plants within a sward. The objective of this paper is to discuss the basis of within-species variation in leaf length. Selection for leaf length has been highly efficient, with moderate to high narrow sense heritability. Nevertheless, the genetic regulation of leaf length is complex because it involves many genes with small individual effects. This could explain the low stability of QTL found in different studies. Leaf length has a strong response to environmental conditions. However, when significant genotype × environment interactions have been identified, their effects have been smaller than the main effects. Recent modelling-based research suggests that many of the reported environmental effects on leaf length and genotype × environment interactions could be biased. Indeed, it has been shown that leaf length is an emergent property strongly affected by the architectural state of the plant during significant periods prior to leaf emergence. This approach could lead to improved understanding of the factors affecting leaf length, as well as better estimates of the main genetic effects.

Plant carbohydrates are of increasing interest as renewable feedstocks to replace petrochemicals in the generation of fuels and production of high-value chemicals. Greater understanding of the genetic control of diversity in fructan synthesis and accumulation would facilitate more directed channelling of feedstock to process in a ryegrass biorefinery. Divergent populations produced by phenotypic selection for water-soluble carbohydrate content have been used to investigate relationships between traits, and to identify patterns of genetic differentiation which indicate genomic regions under high and low selection pressure. Selection for high water-soluble carbohydrate content was associated with increased synthesis of large fructan polymers and increased accumulation of above-ground plant biomass, particularly during spring. Three rounds of selection and two rounds of recombination resulted in widespread genetic differentiation across the whole genome, causing reduced allelic richness and increasing homozygosity at some loci. A smaller number of loci were shown to be subject to high selection pressure. Breeding material subjected to many years of selection for water-soluble carbohydrate also showed allelic differences which may reflect the consequences of high selection pressure at some of these same loci. However, some of the loci unaffected in the divergent selection experiment showed similar effects. This might arise from differences in linkage disequilibrium in these two sets of plant materials, but more likely from the different genetic background of the germplasm. This illustrates the complex nature of the water-soluble carbohydrate trait in perennial ryegrass.

Macroalgal water content is an on-going problem for the use of readily accessible seaweeds in sustainable biorefining, including fuel production. Silage is a reduced-water, compactable, easily stored, transportable material. Ensiling could establish a non-seasonal supply of preserved algal biomass, but requires high initial dry matter content to mitigate environmental pollution risks from effluent. This study investigated potential dewatering methods for kelp harvested throughout the year. Treatments included air-drying, osmotic media and acids. Significant interactions between treatment and harvest-time were observed for traits of interest. Fresh weight loss during treatment was composed of changes in water and dry matter content. Air-drying gave reliable increase in final dry matter content; in summer and autumn 30% dry matter content was reached after 24h. Dilute hydrochloric acid reduced stickiness and rendered material suitable for dewatering by screw-pressing; it may be possible to use the consequent pH reduction to promote efficient preservation.

Increasing the extractable sugar yield from perennial crops is one strategy to generate renewable fuels such as bio-ethanol. Lolium perenne L. (perennial ryegrass) can contain significant (>30% dry matter) water-soluble sugars in the form of polymeric fructan which is readily extracted, broken down and fermented to bio-ethanol. A population of L. perenne generated from four parents which differed in water-soluble carbohydrate (WSC) content was subjected to multiple rounds of selection and recombination on the basis of early spring WSC content to produce a high WSC, and a low WSC population. A control population was generated by selecting the same number of plants at random. The alleles present at six candidate gene loci were analysed before and after selection and correlated to WSC content. Significant differences in the allele frequency of L. perenne soluble-acid invertase1:4 were observed between the three populations with one haplotype significantly associated with the high WSC C2S+ population (after three rounds of selection and two rounds of recombination). Moreover, WSC content was also associated with biomass accumulation. Thus, in addition to a 2.84-fold increase in WSC yield, the C2S+ population also had 1.48-fold more biomass per plant, resulting in 3.9-fold higher WSC yield per plant than the control population. Electronic supplementary material The online version of this article (doi:10.1007/s12155-011-9156-0) contains supplementary material, which is available to authorized users.

Gregory, A.S., Webster, C.W., Watts, C.W., Whalley, W.R., Macleod, C.J.A., Joynes, A., Papadopoulos, A., Haygarth, P.M., Binley, A., Humphreys, M.W., Turner, L.B., Skot, L., Matthews, G.P. (2010). Soil management and grass species effects on the hydraulic properties of shrinking soils. Soil Science Society of America Journal, 74, 753-761.

Water-soluble carbohydrates (WSC) are an important factor determining the nutritional value of grass forage and development of genetic markers for selection of WSC traits in perennial ryegrass would benefit future breeding programmes. Quantitative trait loci (QTLs) for WSC have been published for an F(2) ryegrass mapping family. Markers showing significant associations with these QTLs were used to design narrow-based populations with homozygosity for target QTLs. Founders were selected from within the mapping family. The divergent populations produced were analysed for WSC content in the glasshouse and the field. There was evidence of complex interactions between WSC content and other factors and traits, including the scale of assessment, time/degree of sward establishment and other forage quality parameters. Differences between the divergent pairs of the various populations were small. However, differences observed between the founder selection groups were maintained and the roles of the QTL regions in regulating forage WSC content were confirmed. In general, the individual divergent populations exploited only a limited extent of the large phenotypic variation available within the mapping family. However, this study sets the scene for exploring the opportunities for marker-assisted breeding strategies for complex traits in obligate out-breeding species, and the challenges of doing this are discussed.

Mutational load and resource allocation factors and their effects on limiting seed set were investigated in ryegrass by comparative mapping genomics and quantitative trait loci (QTL) analysis in two perennial ryegrass (Lolium perenne) mapping families sharing common genetic markers. Quantitative trait loci for seed-set were identified on chromosome (LG) 7 in both families and on LG4 of the F2/WSC family. On LG7, seed-set and heading date QTLs colocalized in both families and cannot be unequivocally resolved. Comparative genomics suggests that the LG7 region is syntenous to a region of rice LG6 which contains both fertility (S5(n)) and heading date (Hd1, Hd3a) candidate genes. The LG4 region is syntenous to a region of rice LG3 which contains a fertility (S33) candidate gene. QTL maxima for seed-set and heading date on LG4 in the F2/WSC family are separated by c. 8 cm, indicating distinct genetic control. Low seed set is under the control of recessive genes at both LG4 and LG7 locations. The identification of QTLs associated with seed set, a major component of seed yield in perennial ryegrass, indicates that mutational load associated with these genomic regions can be mitigated through marker-assisted selection.

Differences have been shown between ryegrass and fescue within the Festulolium subline introgression family for fructan synthesis, metabolism and polymer-size traits. It is well-established that there is considerable variation for water-soluble carbohydrate and fructan content within perennial ryegrass. However there is much still to be discovered about the fructan polymer pool in this species, especially in regard to its composition and regulation. It is postulated that similar considerable variation for polymer traits may exist, providing useful polymers for biorefining applications. Seasonal effects on fructan content together with fructan synthesis and polymer-size traits have been examined in diverse perennial ryegrass material comprising contrasting plants from a perennial ryegrass F2 mapping family and from populations produced by three rounds of phenotypic selection. Relationships with copy number variation in candidate genes have been investigated. There was little evidence of any variation in fructan metabolism across this diverse germplasm under these conditions that resulted in substantial differences in the complement of fructan polymers present in leaf tissue at high water-soluble carbohydrate concentrations. The importance of fructan synthesis during fructan accumulation was unclear as fructan content and polymer characteristics in intact plants during the growing season did not reflect the capacity for de novo synthesis. However, the retention of fructan in environmental conditions favouring high sink / low source demand may be an important component of the high sugar trait and the roles of breakdown and turnover are discussed.

The fructans found as storage carbohydrates in temperate forage grasses have a physiological role in regrowth and stress tolerance. They are also important for the nutritional value of fresh and preserved livestock feeds, and are potentially useful as feedstocks for biorefining. Seasonal variation in fructan content and the capacity for de novo fructan synthesis have been examined in a Festulolium monosomic substitution line family to investigate variation in the polymers produced by grasses in the ryegrass-fescue complex. There were significant differences between ryegrass and fescue. Fescue had low polymeric fructan content and a high oligomer/polymer ratio; synthesis of polymers longer than degree of polymerisation 6 (DP6) from oligomers was slow. However, extension of polymer length from DP10/DP20 upwards appeared to occur relatively freely, and, unlike ryegrass, fescue had a relatively even spread of polymer chain lengths above DP20. This included the presence of some very large polymers. Additionally fescue retained high concentrations of fructan, both polymeric and oligomeric, during conditions of low source/high sink demand. There were indications that major genes involved in the control of some of these traits might be located on fescue chromosome 3 opening the possibility to develop grasses optimised for specific applications.

Perennial ryegrass (Lolium perenne L.) is the most important grass species used in temperate grassland agriculture. Our objective was to obtain an overview of the genetic relationships between 20 individual genotypes of perennial ryegrass of diverse origins, using amplified fragment length polymorphism (AFLP), inter-simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD) and two sets of simple sequence repeat (SSR) markers. All 20 individuals were uniquely fingerprinted by all four marker systems and comparisons were made on the basis of 85 markers each. Mean genetic similarities were estimated at 0.31, 0.43, 0.23 and 0.15 for AFLPs, ISSRs, RAPDs and SSRs, respectively. Cophenetic values resulted in good (AFLP and SSR-B = 0.88) to moderately good fits (ISSR = 0.76, RAPD = 0.70, and SSR-A = 0.79). Comparing the four marker systems to each other, AFLP and SSR-A were correlated best ( r = 0.57). All other comparisons revealed rather low correlation coefficients in the Mantel Z test. With twice as many markers cophenetic values increased to a very good fit for AFLPs (0.90) and SSRs (0.92).

Crown rust (Puccinia coronata) resistance (CRres), which had been introgressed from meadow fescue (Festuca pratensis) into the Italian ryegrass (Lolium multiflorum) background, was genetically mapped with amplified fragment length polymorphism (AFLP) and sequence tagged site (STS) markers to a terminal segment of chromosome 5. Comparative mapping had previously shown that this region of the Lolium/Festuca genome has a degree of conserved genetic synteny with chromosomes 11 and 12 of rice. Sequences from rice chromosome 12 were used as templates for identifying further STS markers that cosegregated with CRres. The relative genomic positions of molecular markers associated with CRres in L. multiflorum, L. perenne, F. pratensis and oats is discussed, along with their relationships to physical positions on rice chromosomes C11 and C12.

New genes for 'sustainability' traits are being incorporated into ryegrass (Lolium sp.) breeding populations using marker-assisted intra- and inter- specific introgession from a wide range of genetic resources. Mapping families, substitution and introgression lines have been produced to facilitate gene transfer between Festuca and Lolium species. Advanced metabolomic phenotyping techniques (e.g. NIRS and FT-IR) can help close genotype-phenotype gaps to increase the environmental sustainability of grassland and improve the nutritional value of genotypes for forage or their chemical suitability for biofermentation/biofuel. Use of genetic markers Marker Assisted Selection (MAS) improves the efficiency of breeding particularly where traits are difficult or expensive to evaluate; are expressed late in crop development; or where several components need to be combined. QTL mapping which identifies trait-associated genetic markers has been carried out for a wide range of agronomic 'sustainability' traits including seasonal patterns of production, regrowth, root characteristics and forage quality including components of water-soluble carbohydrate (WSC) content (Turner et al. 2005). Linkage disequilibrium-based association mapping offers potential to identify functional SNP markers within candidate gene sequences or extremely closely linked markers. Candidate genes have been identified for flowering time, WSC and tolerance to abiotic stresses and SNPs are being detected. For example a 68 base-pair indel has been identified in the 5' upstream region of an alkaline invertase gene, which is located on LG 6 in L. perenne, underlying a QTL for glucose and fructose content (Skot, unpublished). Gene introgression in Lolium/Festuca The Lolium/Festuca species complex is unique in that high levels of recombination are combined with the ability to distinguish homoeologous chromosomes through genomic in situ hybridisation (GISH) (King et al. 2002). Thus Lolium-Festuca hybrids are highly amenable to introgression-mapping to identify gene complexes involved in trait expression (Humphreys et al 2003) and major advances in breeding for stress resistance have been achieved. QTL for survival and recovery following severe drought were found along the entire length of Festuca chromosome 3 (Alm et al. 2005) which provides a genetic resource for improving drought tolerance in Lolium (Humphreys et al. 2005). F. arundinacea and F. glaucescens genes for drought resistance, introgressed onto different sites on Lolium chromosome 3 were also found to confer improved salinity tolerance (Lattore, unpublished). Chromosome 3 in Lolium and Festuca shares considerable synteny with rice chromosome 1 (Jones et al. 2002) which has QTL for traits known to contribute to drought resistance such as root development and osmotic adjustment. F. glaucescens genes for drought resistance have also been introgressed into

Quantitative trait locus (QTL) mapping, which can be a useful tool for dissecting complex traits, has been used here to study the regulation of fructan metabolism in temperate forage grasses. An F2 mapping family, derived from a high water-soluble carbohydrate (WSC) x low WSC cross, was used to map fructans and the other components of WSC (sucrose, glucose and fructose) in leaves and tiller bases of perennial ryegrass (Lolium perenne) in spring and autumn. To characterize regions of the genome that control basic carbohydrate metabolism, a strategy to minimize the impact of genotype (G) x environment (E), and E-effects on the characterization of G-effects, was adopted. Most traits were highly variable within the family. There was also considerable year-to-year environmental variation. However, significant genetic effects were detected, and several traits had high broad-sense heritability. QTL were identified on chromosomes 1, 2, 5 and 6. Leaf and tiller base QTL did not coincide. Individual QTL explained between 8 and 59% of the total phenotypic variation in the traits. Fructan turnover, metabolism and their genetic control, and the effect of environment, are discussed in the context of the results.

Microsynteny with rice and comparative genetic mapping were used to identify candidate orthologous sequences to the rice Hd1(Se1) gene in Lolium perenne and Festuca pratensis. A F. pratensis bacterial artificial chromosome (BAC) library was screened with a marker (S2539) physically close to Hd1 in rice to identify the equivalent genomic region in F. pratensis. The BAC sequence was used to identify and map the same region in L. perenne. Predicted protein sequences for L. perenne and F. pratensis Hd1 candidates (LpHd1 and FpHd1) indicated they were CONSTANS-like zinc finger proteins with 61-62% sequence identity with rice Hd1 and 72% identity with barley HvCO1. LpHd1 and FpHd1 were physically linked in their respective genomes (< 4 kb) to marker S2539, which was mapped to L. perenne chromosome 7. The identified candidate orthologues of rice Hd1 and barley HvCO1 in L. perenne and F. pratensis map to chromosome 7, a region of the L. perenne genome which has a degree of conserved genetic synteny both with rice chromosome 6, which contains Hd1, and barley chromosome 7H, which contains HvCO1.

Armstead, I. P., Turner, L. B., King, I. P., Cairns, A. J., Humphreys, M. O. (2002). Comparison and integration of genetic maps generated from F2 and BC1-type mapping populations in perennial ryegrass (Lolium perenne L.). Plant Breeding, 121, (6), 501-507. Sponsorship: BBSRC RASP Initiative; EC FAIR CT98-4063

Summary Excised and illuminated leaves of temperate gramineae have previously provided a powerful system for the analysis of the regulation of partitioning of photosynthate between sucrose and fructan. This paper complements previous data by describing the concomitant accumulation of transitory starch, an assimilate which has been ignored in this system. Leaves of Lolium temulentum were depleted to near-zero reserve carbohydrate content. In this condition, the tissue contained no detectable fructan or starch and only a trace of sucrose. When subsequently excised and illuminated continuously, the accumulation of sucrose and starch commenced immediately and occurred simultaneously. Starch anabolism was constitutive and the polymer was apparently synthesised de novo . The maximal rate of starch accumulation, at 0.6 mg g −1 fresh mass, was 5-10-fold lower than that of sucrose. Fructan was also synthesised de novo and was induced after a lag of 8 h, after which rates of net sucrose and starch accumulation slowed, ceasing completely by 14-16 h. Whilst total non-structural carbohydrate concentration increased continuously, reaching 56 mg g −1 at 30 h, starch concentration was asymptotic and was limited to a maximum of only 7 mg g −1 reached at 14-16 h. Hence, net starch accumulation was not restricted by the continued production of photosynthate. The illumination period spanned 1.25 diurnal cycles. Despite continued photoassimilation, there was no resumption of net starch synthesis at the beginning of the second cycle and hence the limitation of net starch accumulation was not imposed by an endogenous circadian rhythmicity. When assimilate partitioning into fructan was abolished by transpirational feeding of cycloheximide, sucrose concentration increased 2.7-fold to 49 mg g −1 . This abnormally high concentration did not feedback to enhance starch accumulation. Transpirational feeding of 10 mmol/L mannose inhibited sucrose accumulation by c .90 %, but this inhibition was not accompanied by an increase net starch accumulation, rather, starch accumulation was inhibited by 60 %. Mannose caused the formation of maltose in the tissue. Starch synthesis, in common with sucrose and fructan syntheses, was shown to be localised predominantly in mesophyll cells where presumably the control of partitioning between these three assimilates is exercised. The low rates, restricted tissue concentration, sucrose-insensitivity and mannose-inhibition of starch synthesis differ from many species where starch is the primary reserve. Although the available information is limited, some of these features are in common with other fructan grasses. The low capacity for starch accumulation provides a possible explanation for the evolution of fructan synthesis in these species.

Turner, L. B., Humphreys, M. O., Cairns, A. J., Pollock, C. J. (2001). Comparison of growth and carbohydrate accumulation in seedlings of two varieties of Lolium perenne. Journal of Plant Physiology, 158, (7), 891-897. Sponsorship: BBSRC RASP Initiative

We report on the evaluation of a novel grass hybrid that provides efficient forage production and could help mitigate flooding. Perennial ryegrass (Lolium perenne) is the grass species of choice for most farmers, but lacks resilience against extremes of climate. We hybridised L. perenne onto a closely related and more stress-resistant grass species, meadow fescue Festuca pratensis. We demonstrate that the L. perenne × F. pratensis cultivar can reduce runoff during the events by 51% compared to a leading UK nationally recommended L. perenne cultivar and by 43% compared to F. pratensis over a two year field experiment. We present evidence that the reduced runoff from this Festulolium cultivar was due to intense initial root growth followed by rapid senescence, especially at depth. Hybrid grasses of this type show potential for reducing the likelihood of flooding, whilst providing food production under conditions of changing climate.

International audience; Temperate productive grasslands are often located in areas of high rainfall prone to flooding, but even here moderate summer droughts occur with regularity causing significant yield reductions. Grasslands capable of resisting both water excess and deficit are required. Alternative breeding technologies are employed to combine as Festulolium cultivars the desirable traits of Lolium and Festuca species, and also through their enhanced root systems, improve soil structures and hydrology. An amphiploid L. perenne × F. pratensis cultivar can significantly reduce rainfall runoff compared to either its parental species. Evidence suggests this was due to an initial intensive root growth followed by extensive root senescence. This appears to alter soil structure and increase soil porosity and moisture retention providing an ecosystem service by both combating run-off subsequent to heavy rainfall and increasing soil water supply during dry periods.

The role of light in the isomerization and cyclodimerization of the cinnamic acid component of plant cell walls has been examined in a number of ways. Cinnamic acid derivatives were extracted from neutral detergent fibre cell walls by alkaline hydrolysis and quantified by GC. Amounts of these acids in leaf and stem tissues of barley straw which had naturally been exposed to very different levels of radiation were compared. Leaf cell walls contained less p-coumaric acid than stem cell walls, but a higher proportion of this was found as the (Z)-isomer and cyclodimer (4,4′-dihydroxy-α-truxillic acid). Samples of wheat straw from a sequence of 10 consecutive years, grown under conditions where meteorological conditions were the major variable, showed considerable year-to-year differences; the (Z)-/(E)-ratio and dimer/monomer ratio varied three-fold. Additional studies compared the direct effect of light on the cinnamic acid derivatives in growing barley seedlings (live), freeze-dried seedlings (dead) and freshly harvested straw samples (dead). There were no significant changes in the cinnamic acid derivatives extracted from the cell walls of dead tissue which had been stored in the dark. Storage in light resulted in increased (Z)-isomer and cyclodimer contents. These changes occurred whether the tissue was alive or dead. They can, therefore, be directly attributed to the effect of light, and do not appear to be mediated by any enzymic reactions.

Water-soluble carbohydrate provides an easily fermentable energy source in perennial ryegrass fodder. Previously the considerable genetic variation available within the species has been characterised by quantitative trait locus mapping and regions of the genome with significant control over trait expression validated by individual test crosses. However, only limited extents of the large variation available within the source population were recovered. Here a quantitative trait locus marker index has been devised to test the potential of this strategy to exploit the available variation more efficiently. Parents for polycrossing were chosen on the basis of an index weighted for the size of leaf water-soluble carbohydrate effects. Populations of one hundred plants from the crosses were maintained in a glasshouse and the water-soluble carbohydrate content of tiller bases and leaves was measured. The fructan and total water-soluble carbohydrate content of the high populations were always greater than in the low populations. However the differences were not always significant and were generally lower than for the parental populations. This highlights the confounding effects of genome-wide, uncontrolled, segregation for carbohydrate content outside marker-selection regions and the challenges ahead for marker selection of polygenic, complex traits in outbreeding species.

Summary • Valid models are the foundation of systems biology. However, even well-established models may warrant reassessment. A testable feature of the currently accepted vacuolar model for fructan biosynthesis is its independence from metabolic energy at substrate level. The effects of limiting energy provision on fructan biosynthesis in grass leaves were determined. • It was found that, in darkness in air, the rate of fructan accumulation was reduced to half relative to a light control. In darkness under anoxia the process was immediately abolished. In the light, the leaf sucrose concentration remained high, but in darkness ± O2, 40% of this sucrose was rapidly degraded. The constant rate of dark-aerobic fructan accumulation was independent of the decrease in sucrose concentration. • Constant rates of aerobic fructan synthesis were independent of marked changes in extractable polymerase rates. In the dark under anoxia, fructan accumulation was abolished but leaves maintained ≥ 80% of the extractable polymerase. Extractable polymerase rates cannot explain the rates of fructan accumulation observed in vivo, if the process is vacuolar. • It was shown that the results were inconsistent with a vacuolar site for fructan synthesis. Six inhibitors of endomembrane function were shown to abolish fructan synthesis in vivo.

* The role of fructan in growth and drought-stress responses of perennial ryegrass (Lolium perenne) was investigated in an F(2) mapping family that segregates for carbohydrate metabolism. * A quantitative trait locus approach was used to compare the genetic control of traits. * Growth and drought-stress traits were extremely variable within the family. Most traits had high broad-sense heritability. Quantitative trait loci (QTL) were identified for most traits; the maximum number of QTL per trait was four. Between 11% and 75% of total phenotypic variation was explained. Few growth-trait QTL coincided with previously identified fructan QTL. A cluster of drought-trait QTL was close to two previously identified regions of the genome with tiller base fructan QTL in repulsion. * The high sugar parent contributed few alleles that increased 'reserve-driven' growth or performance during drought-stress. Correlation of growth and drought-stress traits with fructan content was low and increasing fructan content per se would not appear to improve drought resistance. Complex patterns of carbohydrate partitioning and metabolism within the cell may explain contradictory relationships between carbohydrate content and growth/stress-resistance traits.

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses. Udgivelsesdato: May An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses.

Ryegrasses and fescues of the Lolium/Festuca complex form the basis of many temperate grassland agricultural and turf systems and the ability to manipulate flowering and fertility within these grasses is of considerable importance. Genetic studies can identify multiple quantitative trait loci affecting these traits which can be used in the development of marker-assisted selection protocols for combining favourable alleles. By applying a knowledge of plant comparative genetics and genomics, it is possible to increase the information content of these studies by ‘cross-referencing’ between the Lolium/Festuca grasses and other crop and model species (e.g. rice, Triticeae cereals, Arabidopsis). Syntenic genomic regions, new genetic markers and candidate genes can, thus, be identified.

Here, we review the current genetic approaches for grass improvement and their potential for the enhanced breeding of new varieties appropriate for a sustainable agriculture in a changing global climate. These generally out-breeding, perennial, self-incompatible species present unique challenges and opportunities for genetic analysis. We emphasise their distinctiveness from model species and from the in-breeding, annual cereals. We describe the modern genetic approaches appropriate for their analysis, including association mapping. Sustainability traits discussed here include stress resistance (drought, cold and pathogeneses) and favourable agronomic characters (nutrient use efficiency, carbohydrate content, fatty acid content, winter survival, flowering time and biomass yield). Global warming will predictably affect temperature-sensitive traits such as vernalisation, and these traits are under investigation. Grass biomass utilisation for carbon-neutral energy generation may contribute to reduced atmospheric carbon emissions. Because the wider potential outcomes of climate change are unpredictable, breeders must be reactive to events and have a range of well-characterised germplasm available for new applications.

The genetic control of induction to flowering has been studied extensively in both model and crop species because of its fundamental biological and economic significance. An ultimate aim of many of these studies has been the application of the understanding of control of flowering that can be gained from the study of model species, to the improvement of crop species. The present study identifies a region of genetic synteny between rice and Lolium perenne, which contains the Hd3 heading-date QTL in rice and a major QTL, accounting for up to 70% of the variance associated with heading date in L. perenne. The identification of synteny between rice and L. perenne in this region demonstrates the direct applicability of the rice genome to the understanding of biological processes in other species. Specifically, this syntenic relationship will greatly facilitate the genetic dissection of aspects of heading-date induction by enabling the magnitude of the genetic component of the heading-date QTL in L. perenne to be combined with the sequencing and annotation information from the rice genome.