Bio-processing of macroalgae Palmaria palmata: metabolite fractionation from pressed fresh material and ensiling considerations for long-term storage.

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.

Testing water-soluble carbohydrate QTL effects in perennial ryegrass (Lolium perenne L.) by marker selection.

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.

Pen studies on the control of cattle tick (Rhipicephalus (Boophilus) microplus) with Metarhizium anisopliae (Sorokin).

Three field trials were conducted over 12 months to assess the pathogenicity of Metarhizium anisopliae to parasitic stages of Rhipicephalus (Boophilus) microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30 degrees C), good spore production characteristics and ability to quickly kill adult engorged ticks in the laboratory. Spores were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated in the laboratory to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. In each trial the formulation rapidly caused 100% mortality in unengorged ticks that were removed from cattle and cultured in the laboratory. A significant reduction in egg production was recorded for engorged ticks collected in the 3 days post-treatment. However, there was little effect of the formulation on the survival of ticks on cattle, indicating that there is an interaction between the environment of the ticks on the cattle and the biopesticide, which reduces its efficacy against ticks.

Does fructan have a functional role in physiological traits? Investigation by quantitative trait locus mapping.

* 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.

Identifying genetic components controlling fertility in the outcrossing grass species perennial ryegrass (Lolium perenne) by quantitative trait loci analysis and comparative genetics.

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.

A changing climate for grassland research.

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.

Dissecting the regulation of fructan metabolism in perennial ryegrass (Lolium perenne) with quantitative trait locus mapping.

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.

Identification of perennial ryegrass (Lolium perenne (L.)) and meadow fescue (Festuca pratensis (Huds.)) candidate orthologous sequences to the rice Hd1(Se1) and barley HvCO1 CONSTANS-like genes through comparative mapping and microsynteny.

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.

Identification and mode of action of self-compatibility loci in Lolium perenne L.

The two-locus gametophytic incompatibility system in perennial ryegrass (Lolium perenne L.) is not always fully effective: obligate selfing of plants sieves self-compatible pollen mutants, and self-fertility becomes fixed in subsequent generations. Self-compatibility (SC) was investigated in an F2 family. In vitro self-pollinations were analysed and recorded and plants were classified as being either partially or fully compatible. Distorted segregation ratios of markers on linkage group (LG) 5 were found, which indicate the possible presence of a gametophytic SC locus. Interval linkage analysis of pollen compatibility after selfing confirmed that this distortion was due to a locus (T) analogous to the S5 locus of rye. However, even though markers in this region were, on average, less than 1 cM apart, the minimum number of plants possessing the unfavoured allele was never less than 6% for any marker locus. We proved that this was because of the presence of another SC locus, exhibiting gametophytic selection, segregating in this population and identified by interval mapping analysis of compatibility classes of in vitro self-pollinations. This locus was located on LG1, and probably corresponds to the S locus. We show that the T locus, a relic of a multilocus system, functions through interaction with the S locus: F2 segregation of incompatibility phenotypes and linked markers demonstrated that the S/t pollen genotype combination, expected to be compatible on selfing, was sometimes incompatible. Further evidence is presented to show that this interaction must be dependent on yet another locus located on LG2. A prime candidate would be the Z incompatibility locus.

Synteny between a major heading-date QTL in perennial ryegrass (Lolium perenne L.) and the Hd3 heading-date locus in rice.

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.