Iain S. Donnison

Seasonal variation in Laminaria digitata and its impact on biochemical conversion routes to biofuels.

Laminaria digitata is a highly prevalent kelp growing off the coast of the UK but has rarely been considered as a source of biomass to date. This study shows it can be used as a feedstock in both ethanol fermentation and anaerobic digestion for methane production. The study optimised several parameters in the fermentation of L. digitata and investigated the suitability of the macroalgae through the year using samples harvested every month. For both methane and ethanol production, minimum yields were seen in material harvested in March when the carbohydrates laminarin and mannitol were lowest. July material contained the highest combined laminarin and mannitol content and maximum yields of 167 mL ethanol and 0.219 m(3) kg(-1)L. digitata.

Alignment of the Genomes of Brachypodium distachyon and Temperate Cereals and Grasses Using Bacterial Artificial Chromosome Landing With Fluorescence in Situ Hybridization

As part of an initiative to develop Brachypodium distachyon as a genomic “bridge” species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice.

Variation in Miscanthus chemical composition and implications for conversion by pyrolysis and thermo-chemical bio-refining for fuels and chemicals

Different species and genotypes of Miscanthus were analysed to determine the influence of genotypic variation and harvest time on cell wall composition and the products which may be refined via pyrolysis. Wet chemical, thermo-gravimetric (TGA) and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) methods were used to identify the main pyrolysis products and determine the extent to which genotypic differences in cell wall composition influence the range and yield of pyrolysis products. Significant genotypic variation in composition was identified between species and genotypes, and a clear relationship was observed between the biomass composition, yields of pyrolysis products, and the composition of the volatile fraction. Results indicated that genotypes other than the commercially cultivated Miscanthus x giganteus may have greater potential for use in bio-refining of fuels and chemicals and several genotypes were identified as excellent candidates for the generation of genetic mapping families and the breeding of new genotypes with improved conversion quality characteristics.

High resolution genetic mapping by genome sequencing reveals genome duplication and tetraploid genetic structure of the diploid Miscanthus sinensis.

We have created a high-resolution linkage map of Miscanthus sinensis, using genotyping-by-sequencing (GBS), identifying all 19 linkage groups for the first time. The result is technically significant since Miscanthus has a very large and highly heterozygous genome, but has no or limited genomics information to date. The composite linkage map containing markers from both parental linkage maps is composed of 3,745 SNP markers spanning 2,396 cM on 19 linkage groups with a 0.64 cM average resolution. Comparative genomics analyses of the M. sinensis composite linkage map to the genomes of sorghum, maize, rice, and Brachypodium distachyon indicate that sorghum has the closest syntenic relationship to Miscanthus compared to other species. The comparative results revealed that each pair of the 19 M. sinensis linkages aligned to one sorghum chromosome, except for LG8, which mapped to two sorghum chromosomes (4 and 7), presumably due to a chromosome fusion event after genome duplication. The data also revealed several other chromosome rearrangements relative to sorghum, including two telomere-centromere inversions of the sorghum syntenic chromosome 7 in LG8 of M. sinensis and two paracentric inversions of sorghum syntenic chromosome 4 in LG7 and LG8 of M. sinensis. The results clearly demonstrate, for the first time, that the diploid M. sinensis is tetraploid origin consisting of two sub-genomes. This complete and high resolution composite linkage map will not only serve as a useful resource for novel QTL discoveries, but also enable informed deployment of the wealth of existing genomics resources of other species to the improvement of Miscanthus as a high biomass energy crop. In addition, it has utility as a reference for genome sequence assembly for the forthcoming whole genome sequencing of the Miscanthus genus.

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.

Identification of genes involved in cell wall biogenesis in grasses by differential gene expression profiling of elongating and non-elongating maize internodes

Despite the economic importance of grasses as food, feed, and energy crops, little is known about the genes that control their cell wall synthesis, assembly, and remodelling. Here a detailed transcriptome analysis that allowed the identification of genes involved in grass cell wall biogenesis is provided. Differential gene expression profiling, using maize oligonucleotide arrays, was used to identify genes differentially expressed between an elongating internode, containing cells exhibiting primary cell wall synthesis, and an internode that had just ceased elongation and in which many cells were depositing secondary cell wall material. This is one of only a few studies specifically aimed at the identification of cell wall-related genes in grasses. Analysis identified new candidate genes for a role in primary and secondary cell wall biogenesis in grasses. The results suggest that many proteins involved in cell wall processes during normal development are also recruited during defence-related cell wall remodelling events. This work provides a platform for studies in which candidate genes will be functionally tested for involvement in cell wall-related processes, increasing our knowledge of cell wall biogenesis and its regulation in grasses. Since several grasses are currently being developed as lignocellulosic feedstocks for biofuel production, this improved understanding of grass cell wall biogenesis is timely, as it will facilitate the manipulation of traits favourable for sustainable food and biofuel production.

Miscanthus as a feedstock for fast-pyrolysis: does agronomic treatment affect quality?

The objectives of the experiment were to assess the impact of nitrogen (N) and potassium (K) fertiliser application on the cell wall composition and fast-pyrolysis conversion quality of the commercially cultivated hybrid Miscanthus x giganteus. Five different fertiliser treatments were applied to mature Miscanthus plants which were sampled at five intervals over a growing season. The different fertiliser treatments produced significant variation in concentrations of cell wall components and ash within the biomass and affected the composition and quality of the resulting fast-pyrolysis liquids. The results indicated that application of high rates of N fertiliser had a negative effect on feedstock quality for this conversion pathway: reducing the proportion of cell wall components and increasing accumulation of ash in the harvested biomass. No exclusive effect of potassium fertiliser was observed. The low-N fertiliser treatment produced high quality, low ash-high lignin biomass most suitable as a feedstock for thermo-chemical conversion.

Characterization of flowering time diversity in Miscanthus species

Miscanthus sinensis, M. sacchariflorus and their hybrids have been identified as leading candidates for the provision of bioenergy production across several continents. Flowering time is an important trait affecting biomass yield as well as certain quality attributes, such as moisture content at harvest. The aim of this study was to ascertain the level of diversity available to breeders and potential for hybridisation of different accessions in a large collection of Miscanthus. We also sought to determine trends in flowering time within and between species with respect to environment and origin of collection data (where known), whether flowering order was maintained across years, and the extent of uniformity of flowering in different genotypes. Flowering time was observed weekly in 244 genotypes of two Miscanthus species (M. sinensis, M. sacchariflorus) and inter‐specific hybrids including M. x giganteus over 3 years and using 4 clonal replicates of each genotype on a trial planted near Aberystwyth (Wales, UK). Differences in flowering time across the entire collection ranged from 160 to 334 days (June to November) and photoperiods between 7.8 and 16.6 h, with associated accumulated temperatures of 161 to 865oCd. More than two thirds of the collection flowered by the end of each growing season. M. sinensis individuals were the earliest genotypes to flower and showed the greatest diversity with respect to the onset of flowering. Flowering times in genotypes of known origin in Asia could be partially explained by growing season rain fall, degree days and mean temperature. Uniform flowering was identified in some genotypes. This will be important for the development of genetically diverse seed‐based crops. Rank order of flowering was shown to be consistent across Western Europe, thereby justifying single site trials as the basis of germplasm characterisation for wider geographical deployment.

Genome-wide association studies and prediction of 17 traits related to phenology, biomass and cell wall composition in the energy grass Miscanthus sinensis

Increasing demands for food and energy require a step change in the effectiveness, speed and flexibility of crop breeding. Therefore, the aim of this study was to assess the potential of genome-wide association studies (GWASs) and genomic selection (i.e. phenotype prediction from a genome-wide set of markers) to guide fundamental plant science and to accelerate breeding in the energy grass Miscanthus. We generated over 100 000 single-nucleotide variants (SNVs) by sequencing restriction site-associated DNA (RAD) tags in 138 Micanthus sinensis genotypes, and related SNVs to phenotypic data for 17 traits measured in a field trial. Confounding by population structure and relatedness was severe in naïve GWAS analyses, but mixed-linear models robustly controlled for these effects and allowed us to detect multiple associations that reached genome-wide significance. Genome-wide prediction accuracies tended to be moderate to high (average of 0.57), but varied dramatically across traits. As expected, predictive abilities increased linearly with the size of the mapping population, but reached a plateau when the number of markers used for prediction exceeded 10 000–20 000, and tended to decline, but remain significant, when cross-validations were performed across subpopulations. Our results suggest that the immediate implementation of genomic selection in Miscanthus breeding programs may be feasible.

Direct fermentation of fodder maize, chicory fructans and perennial ryegrass to hydrogen using mixed microflora

This study examined the feasibility of producing hydrogen by direct fermentation of fodder maize, chicory fructooligosaccharides and perennial ryegrass (Lolium perenne) in batch culture (pH 5.2-5.3, 35 degrees C, heat-treated anaerobically digested sludge inoculum). Gas was produced from each substrate and contained up to 50-80% hydrogen during the peak periods of gas production with the remainder carbon dioxide. Hydrogen yields obtained were 62.4+/-6.1mL/g dry matter added for fodder maize, 218+/-28mL/g chicory fructooligosaccharides added, 75.6+/-8.8mL H(2)/g dry matter added for wilted perennial ryegrass and 21.8+/-8mL H(2)/g dry matter added for fresh perennial ryegrass. Butyrate, acetate and ethanol were the main soluble fermentation products. Hydrogen yields of 392-501m(3)/hectare of perennial ryegrass per year and 1060-1309m(3)/hectare of fodder maize per year can be obtained based on the UK annual yield per hectare of these crops. These results significantly extend the range of substrates that can be used for hydrogen production without pre-treatment.