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RNA sequencing (RNA-seq) is now the standard methodology to explore gene and transcript expression changes in different conditions. RNA-seq has been extensively used in genomic and transcriptomics studies in multiple organisms for a wide range of analyses from developmental studies to diseases. Currently, there are state-of-the-art programs for accurate and rapid transcript quantifications and downstream differential expression analyses at the gene, transcript and isoform levels. However, the accuracy of these analyses are dependent on how comprehensive, diverse and accurate the reference transcriptome annotation is. In addition, to dissect gene expression, pre-mRNA processing and regulatory mechanisms correct translations and other transcript features are important for the correct interpretation of transcriptomic analysis. To address these issues, the RTDmaker and TranSuite programs were developed. RTDmaker is an automated, modular program that facilitates the creation of high-quality transcriptome annotations known as Reference Transcript Datasets (RTDs). RTDmaker was based on experience and principles used to create Arabidopsis AtRTD and AtRTD2. The transition to species with poorly annotated genomes/transcriptomes introduced a new set of issues that had to be addressed in the development of RTDmaker. TranSuite is a suite of programs for the accurate translation of transcripts (TransFix), and the determination of coding potential of transcripts and identification of transcript coding-related features such as premature termination codons and nonsense-mediated decay triggering signals (TransFeat). TransFix employs a novel method of identifying the authentic translation AUG start sites to generate realistic translations allowing the accurate determination of protein-coding capacity. In contrast, most translation programmes select the longest ORF present in transcripts of a gene as the representative coding sequence (CDS). Direct comparison with TransDecoder showed anomalous translations for around 25% of transcripts with TransDecoder. RTDmaker and TranSuite were used to create improved and fully characterized annotations for DM potato (StRTD) and barley (BaRT2.17) in comparison to previously annotations. The StRTD annotation was used to explore NMD in potato plants treated with cycloheximide and supported the prediction of NMD-sensitive transcripts by TranSuite. This analysis demonstrated the utility of RTDmaker and TranSuite to explore and interpret results for differential expression analysis. The BaRT2.17 annotation was generated using both short and long read sequencing (RNA-seq and Iso-seq). respectively). BaRT2.17 transcripts had well-defined 5' and 3' ends (from Iso-seq) and rich splice-junction diversity (from RNA-seq) and represents the most advanced and accurate barley transcriptome available.

Meiotic crossovers are skewed towards the chromosome ends in wheat and barley. As a result, ~30% of genes rarely recombine in the 'cold regions', resulting in linkage-drag, which can be problematic for plant breeders as beneficial traits co-segregate with undesirable traits. An increase in the number of COs, or a shift in their positions, may disrupt linkage blocks and consequently benefit breeders. Pachytene Checkpoint Protein 2 (PCH2) is a conserved AAA+ ATPase, required to remodel the chromosome axes during meiosis and ensure accurate crossover formation. Previous work in Arabidopsis thaliana pch2 mutants has shown that crossovers are more likely to cluster together on short regions of the synaptonemal complex, as well as lose the obligate crossover, in the absence of PCH2. By using different methods of gene interference (CRISPR/Cas9, TILLING and VIGS), pch2 knockouts have been generated in diploid barley and tetraploid wheat and hypomorphs have been produced in hexaploid wheat. A cytological analysis has shown that PCH2 is involved in the timing of meiosis in all three ploidies and is required for the formation of the obligate CO in tetraploid wheat, but not in diploid barley. Full synapsis is achieved in barley pch2 mutants, but not tetraploid wheat pch2 mutants, where only short ZYP1 stretches are observed, localising with class I crossover marker HEI10. In addition, ectopic recombination is observed at meiotic metaphase I in tetraploid wheat pch2 mutants, indicating loss of crossover control and possible homoeologous recombination. Immunolocalisation studies indicate that PCH2 is involved in maintaining positive interference in wheat and barley, as a shift from positive to negative interference is observed in the absence of PCH2. This thesis offers a substantial contribution of knowledge to the meiosis community, providing further understanding of the role of PCH2 in crops and, importantly, in PCH2-mediated interference in plants.

The thin layer of soil surrounding and influenced by plant roots, termed the rhizosphere, defines a distinct and selective microhabitat compared to that of the surrounding soil: the bulk soil. The microbial populations that reside in the rhizosphere, commonly referred to as the rhizosphere microbiota, participate in a variety of interactions with their host plant ranging from parasitic to mutualistic relationships. Due to the contribution of the microbiota to pathogen protection and nutrient uptake the rhizosphere microbiota has emerged as a determinant of crop yield. Consequently, a better understanding of plant-microbiota interactions in the rhizosphere can pave the way for novel applications enhancing sustainable crop yield and global food security. Experimental evidence indicates that the host plant is the driver of, at least in part, the rhizosphere microbiota, but genetic relationships underpinning these interactions are not fully understood. Filling this knowledge gap will allow plant breeders to select novel crops which better interact with the soil biota and, at the same time, biotechnologists can profit from microbial genetic information to develop a new generation of inoculants for agriculture. In this PhD project barley (Hordeum vulgare L.), the world's 4th and UK's 2nd most cultivated cereal, was used as an experimental model to dissect plant-bacteria interactions in the rhizosphere. The hypothesis that root hairs: the tubular outgrowths of the root epidermis, modulate the physical and chemical environment in the rhizosphere to facilitate the colonisation of members of the microbiota implicated in mineral uptake was tested. To test this hypothesis, three interconnected experimental approaches were developed: By using 16S rRNA gene amplicon sequencing, it was demonstrated that the presence and development of root hairs are a determinant of nearly one fifth of the barley rhizosphere microbiota, with a bias for members of the order Actinomycetales, Burkholderiales, Rhizobiales, Sphingomonadales, and Xanthomonadales. In order to gain further insights into the molecular basis of this differential recruitment, the project went on to investigate both the physical and chemical environment conditioned by root hairs. A pilot investigation of the rhizodeposition profiles using GC-MS of wild type and root hair mutants revealed that plant-genotype dependent patterns among the 69 amino and organic acids were detected and a further 23 sugars and sugar alcohols detected, pointing at root secretion as an additional selective layer in the barley rhizosphere. Additionally, an amplicon sequencing survey of soil cores with different density, mimicking presence/absence of plant root hairs, revealed that this physical parameter is capable of triggering the differential enrichment of bacteria associated with the orders Bacillales, Burkholderiales and Xanthomonadales but not Actinomycetales. Thus, the physical perturbation of the soil environment alone cannot be the sole recruitment cue for the barley microbiota. However, it does indicate that soil density contributes, in part, to microbial recruitment. Finally, to discern the full genetic potential of plant-associated bacteria, an indexed bacterial collection of the barley microbiota was constructed by isolating, on synthetic media, individual rhizosphere bacteria. A total of 85 isolates were further selected for full genome sequencing including members of the orders Actinomycetales, Flavobacteriales and Xanthomonadales. A comparative genomic approach was deployed to identify plant-growth promoting traits among 53 of these isolates. This experimental work was complemented with a critical appraisal of efforts to communicate to and increase the awareness of the general public on the importance of the plant microbiota for global food security. In the long term, the scientific outputs of this project can be deployed to devise novel strategies aimed at enhancing sustainable crop production in the UK and to globally and increase the awareness of the general public to global food security, particularly with the potential of the bacterial isolate library to be used in collaboration with multiple research groups investigating a range of microbial approaches to improve crop sustainability.

To improve the sustainability of cereal production, fungicides should only be applied to crops where they are likely to result in an economic increase in yield or grain quality. In recent times, there has been increased utilisation by growers of hybrid six-row barley varieties due to their high yields in recommended list trials in both the UK and Ireland. Six- and two-row winter barley differ in their yield components, with studies showing that sixrow types produce fewer ears m-2 and more grains ear-1 (leading to an overall higher number of grains m-2), but a lower average (mean) grain weight when compared with two-row varieties. It was hypothesised that a six-row variety would require a different approach to the management of disease compared to a two-row. Six-rows might require greater protection post-anthesis to maximise assimilate supply for grain filling whereas current barley recommendations, developed for two-rows, emphasise protection pre-flowering to maximise the development of grain sink capacity. This is because yield formation in six row varieties, with their larger number of grains may be more source-limited (limited by the supply of assimilates) than in two-row varieties where yield is generally considered to be sink-limited (limited by the number and storage capacity of grains). The main objectives of this project were to 1) compare the responses of a two-row and a six-row winter barley variety to fungicide programmes with different timings of application; 2) determine the source-sink balance of a two-row and six-row barley variety grown with and without fungicide treatment and 3) determine the relative sensitivity of grain sink capacity to variations in pre-anthesis assimilate supply during ear development in both two- and six-row varieties. To investigate if the disease management strategy needs to be modified according to row- type a field experiment to investigate the yield response to fungicide application timing in a conventional two- (cv KWS Tower) and hybrid six-row (cv Volume) winter barley variety was carried out over three years (2014/2015, 2015/2016 and 2016/2017) at two sites, SRUC, Edinburgh, Scotland and Teagasc, Oak Park Carlow, Ireland. The fungicide applications were applied as part of programmes, ranging from untreated to a four spray programme. The results showed that despite the markedly different yield components of each variety there was no significant interaction between variety and fungicide application (p > 0.05) suggesting that disease management does not need to be tailored to ear type. Surprisingly there was a significant yield response to fungicide application at ear emergence, which had not been seen in barley previously. It was hypothesised that either this response resulted from a failure to effectively control ramularia at GS49 or that the response was unique to the two varieties selected for the current study. To test these hypotheses, a further field experiment was carried out at two sites in 2018. Two additional varieties were included to those used previously, a conventional two- (cv KWS Cassia) and six-row variety (cv KWS Kosmos). Fungicide treatments focused on late-season disease control. The results again indicated that there was no difference (p > 0.05) in how yield of the contrasting ear types responded to fungicide treatment. When ramularia was effectively controlled at GS49, there was no requirement for a later fungicide treatment at ear emergence. To investigate the source-sink balance during grain filling of cv KWS Tower and cv Volume, a field experiment was carried out at Teagasc, Oak Park in 2016 and 2017. The relative source-sink balance was determined in two ways; firstly by growth analysis and measurement of radiation interception, radiation use efficiency and utilisation of soluble carbohydrate reserves, and secondly by manipulation treatments to alter the assimilate supply per unit grain number 14 days after flowering by row opening, de-graining, and shading. The results indicated that despite the higher grain numbers and smaller MGW in the six-row variety Volume, the source-sink balance in fungicide treated and untreated crops were similar to that of the two-row variety Tower. For both ear types, when disease was allowed to develop in untreated crops, the sink capacity (grain number and potential grain weight) was reduced as well as the source capacity, such that the crops remained sink-limited during grain filling. The results from the above experiment indicated that the lower grain weight in the six-row variety was not the result of source limitation of grain filling associated with its higher grain number, but rather a smaller potential grain weight. This raises an important question as to the control of grain sink capacity (grain number and potential grain weight). Previous research suggests that the components of sink capacity may be determined pre-anthesis. Thus, the sensitivity of sink capacity to pre-anthesis assimilate supply was tested in both Tower, and Volume in a field experiment carried out in Teagasc, Oak Park in 2017 and 2018. Pre-anthesis assimilate supply was manipulated through shading and row-opening, with investigations of the effects of these treatments on sink capacity carried out on tagged main stems. The results showed that manipulations significantly affected pre-anthesis growth conditions with shoot growth rate and carpel weight being increased (p < 0.05) in both varieties when rows were opened and decreased when shaded. However, these changes in pre-anthesis growth conditions and carpel weight did not translate into effects on either MGW or grains ear-1. The results of this study raise important questions about the control of potential grain weight, as the previously held view of a direct relationship between MGW and carpel weight at anthesis is not supported. It is clear that further work into the mechanisms controlling potential grain weight in barley is required. If better understood, yield potential could be raised. The results of this research show that disease management strategies designed for two-row winter barley are also suitable for six-row varieties because the source-sink balance of the two variety types is comparable. The greatest yield response to fungicide application comes from applications made at the start of stem extension and during booting. There may be opportunities for omitting earlier applications during tiller production depending on the severity and type of the disease present in the crop.

Barley (Hordeum vulgare L.) is a crop of prime importance to the malting, brewing and distilling sectors. Modern varieties are susceptible to a defect called “skinning”, a condition in which the adhesion of the husk to the caryopsis has been weakened, and partial or complete loss of the husk occurs. Malting of skinned grains is inefficient and results in production of poor quality malt and financial losses to the farmers and maltsters. Although the malting industry recognises that grain skinning causes inefficiencies and loss of production, to date there has been no quantitative assessment of these effects. Furthermore, there is little understanding of how the condition of harvested grain (e.g. moisture content) influences the occurrence of skinning, and thus its potential value in the malting process. The overall aim of this research was to investigate how grain condition at harvest influences the quality of husk adhesion and how skinning severity and husk-loss type impacts the malting process and quality of the malt produced. This thesis details an investigation into the effects of moisture content (MC) to determine whether grain or tissue moisture content affects grain skinning. In the three varieties examined in the field, MCs were between 22% and 45% and skinning was positively correlated with MC; in the four varieties examined in the glasshouse, MCs were between 11% and 25% and skinning was negatively correlated with MC. Light microscopy examination of the grains has confirmed that two distinctly different mechanisms of skinning: in the glasshouse, parenchyma cells were damaged whereas in the field tissue has separated along the cementing layer, without causing cell damage. Micromalting studies were conducted on two varieties (Concerto and Chronicle), with four skinning severities: intact (0% skinned), mild (16% skinned), skinned (50% skinned) and severe (90% skinned), to examine the effect of skinning severity on the quality of the malt produced. Quality was determined by measuring friability and homogeneity, hot water extract (HWE) and α-amylase content. Under- and over-modification of the samples was also investigated at a single grain level, using the Carlsberg Malt Modification Method, thin layer chromatography and scanning electron microscopy. Hot water extract increased with the increase in severity of skinning, however when it was adjusted to account for lost husk biomass HWE then decreased with skinning severity. Friability, homogeneity and the α amylase content also decreased with higher skinning severity. Detailed investigation into modification indicated that malt quality declines with increase in skinning due to a large proportion of undermodified grains in skinned malt. No signs of overmodification were detected. Lastly, two most common husk-loss types in the bulk (missing lemma and huskless) were investigated and compared to grain with attached husk to determine if the type of husk damage is of importance during malting. The difference in germination rate and vigour, production of α-amylase and the rate of water uptake were investigated. Huskless grains had significantly lower levels of germination rate and α-amylase production, compared to the grains with lemma missing and unbroken husks. The main findings from this project are that grain skinning reduces malting efficiency and the quality of the malt produced due to undermodification of the grains and lack of homogeneity in the malted bulk. For the farmer, reduction in the skinning risk could be achieved through careful observation of the moisture during harvest. Maltsters would be able to achieve improved homogeneity and efficiency through careful examination of the bulks for the types of skinning and alterations in the malting regimes employed. On the methods currently used in micro-malting tests and variety evaluation, it is evident that malting of samples with high levels of skinned grain will give a HWE compared to samples with intact grain. Therefore, a corrected measure of HWE is required to take into account the proportion of skinned grain, or mass of husk-loss, in order to determine the true malting efficiency of the sample, or variety.

To keep pace with the increasing demand for new and improved barley varieties, commercial interest in novel breeding techniques which improve efficiency is high. With a growing population and a range of evolving demands placed on crop production by a more variable climate, breeders need new methods to breed barley faster, more accurately and where possible with fewer resources. Genomic selection (GS) is a breeding tool that uses genome-wide marker information to make selections/predictions in a breeding program. GS uses both genotypic data and phenotypic data to assign breeding values (BVs) for a specific trait to cultivars or breeding lines for use in crossing or selection. Progeny can then be selected and taken forward in a breeding program based on genotype alone, saving the cost, time and effort of phenotypic selection. This project aims to test a multi-environment (MET) GS model to breed elite 2-row spring barley for high straw saccharification (digestibility to yield simple sugars). Aligning with future climate policy, an increase in biofuel production will be useful as we reduce our reliance on fossil-based fuels. Second generation biofuels (produced from non-food sources) are promising for meeting sustainability targets as they need not result in land/resource competition with food crops. In comparison with first generation biofuels (produced from food sources), second generation biofuels should have much less impact on food security and food prices. Second generation biofuels currently have a higher production cost associated with them in comparison to first generation biofuels. To date there has not been a focus on breeding barley to develop varieties with high straw saccharification yield which would increase the amount of biofuel produced, reducing costs. The main objective of this thesis was to test a previously developed MET GS model to assess the potential of using GS to breed barley more efficiently for improved straw saccharification. In addition to saccharification, other agronomically important traits were also assessed to test the GS model predictions. Alongside the GS model, other methods to breed barley more rapidly were implemented.

(1,3;1,4)-β-Glucan is the most abundant non-cellulosic polysaccharide that accumulates in the cell walls of barley grain. This polysaccharide is of importance because barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals. Grain (1,3;1,4)-β-glucan concentration influences the use of barley, having undesirable effects on brewing and distilling and beneficial effects linked to human health. These health benefits are because (1,3;1,4)-β-glucan is a soluble dietary fibre, reducing the risk of developing cardiovascular disease, colorectal cancer and type II diabetes. Previous research identified members of the Cellulose synthase-like HvCslF/H gene families as (1,3;1,4)-β-glucan synthases differentially expressed across barley tissues (Burton et al., 2006; Doblin et al., 2009). Amongst them, HvCslF6 is thought to be the main driver of (1,3;1,4)-β-glucan biosynthesis in the grain (Burton et al., 2011) with other putative (1,3;1,4)-β-glucan synthases, remodelling and hydrolytic enzymes potentially affecting variation in (1,3;1,4)-β-glucan content. Despite limited sequence variation in the HvCslF6 CDS and promoter, this gene and HvGlbI, a putative (1,3;1,4)-β-glucan endohydrolase, were found to be differentially expressed across elite barleys exhibiting different levels of grain (1,3;1,4)-β-glucan. The transcriptional regulation of HvCslF6 was investigated by its putative promoter region in a barley protoplast transient expression system. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the essential promoter fragment driving HvCslF6 expression. This finding was combined with an in silico analysis of putative TF binding sites which allowed the identification of three candidate MYB TFs. The transient overexpression of HvMyb61 in barley protoplasts suggested positive regulatory effect on HvCslF6 expression. In addition, the functional characterization of CRISPR/Cas9 knockout mutants of putative (1,3;1,4)-β-glucan synthases revealed that HvCslF9 is not essential for (1,3;1,4)-β-glucan synthesis in the grain, whereas HvCslF6 disruption led to decreased (1,3;1,4)-β-glucan content and altered grain size and shape. The collection of CRISPR/Cas9-induced mutants generated for HvCslF/H demonstrated the effectiveness of genome editing technology in barley and constitute a useful genetic resource to study barley cell walls.

With an increasing population it is important to increase crop yield. However, there is a low priority in breeding for increased tolerance to low input or marginal environments. A potential source of viable resilience and resource use efficiency traits are landraces local to areas of marginal land, such as the Scots Bere from the highlands and islands of Scotland. Bere barley is a deeply historically rooted landrace of barley which has been grown on predominately marginal land for the last half millennia, allowing them to yield well in these conditions. The project aim was to assess and genetically characterise traits associated with enhanced resistance/tolerance, and to identify contributing genomic regions. In this project the JHI spring barley collection, consisting of a number of Bere lines, was screened for biotic stress resistance to Rhynchosporium commune and abiotic stress resistance to the conditions of Mn deficiency and salt stress, additionally the interaction of these stresses was assessed. The results identify a number of Bere lines that show an increased resistance/tolerance to each of the three stresses, compared to elite cultivars. The Bere population, as a whole, showed an inherent enhanced Mn use efficiency correlating to increased accumulation of Mn in their shoot. These results suggest that Bere landraces have unique abilities to cope with stress. Interaction studies revealed complex line-specific interactions, along with an overall adverse effect of salt on rhynchosporium symptoms. Several genomic regions for Mn use efficiency, salt tolerance, and rhynchosporium resistance traits originating from the Bere lines were identified, along with potential candidate genes. Further examination and validation of these regions should be undertaken for future breeding for marginal lands. By introgression into elite cultivar backgrounds they may contribute biotic and abiotic stress-tolerance genes creating novel cultivars to efficiently and resiliently yield under low input and marginal environments.

Aphids are phloem-feeding insects that cause severe yield losses on crops, including cereals such as barley. While most aphid species are limited to one or few host species, some are able to reproduce on many plants belonging to different families. Interestingly, aphid probing-behaviour can be observed on both host and non-host plants indicating that interactions take place at the molecular level and may impact host range. We found that the barley specialist Rhopalosiphum padi and the broad host range Myzus persicae showed differences in colonization, settlement and probing-behaviour on barley. Analyses of barley transcriptional responses revealed gene sets differentially regulated upon the different barley-aphid interactions, where M. persicae induced the most pronounced barley transcriptional response. Interestingly, we identified several genes strongly up-regulated upon interaction with M. persicae, and to a lesser extent upon R. padi interaction, including thionins and the Late Embryogenesis Abundant gene 14 (LEA14). Ectopic expression of two barley thionins in Nicotiana benthamiana reduced host susceptibility to M. persicae, potentially reflecting a role in defence against aphids. We have generated barley mutant lines, using CRISPR-Cas9 technology, to investigate its role in plant-aphid interactions. We characterized also the R. padi and M. persicae responses upon being exposed to different plant environments. We characterized the aphid probing and feeding in different host and poor-/non-host plants, revealing that plant resistance to aphids can be based in different plant cell layers. Aphids only showed limited transcriptional responses upon being moved from host plants to either a host-, poor-host/non-host plant, or to an artificial diet, and predicted aphid effector genes, essential for parasitism, were expressed regardless of the type of interaction. Focusing on aphid effectors, we characterized 4 putative orthologous effector pairs from R. padi and M. persicae with regards to their subcellular localization, expression, and contribution to plant susceptibility. We demonstrate that ectopic expression of two R. padi effectors in barley enhances plant susceptibility to R. padi but not M. persicae, highlighting the importance of these two effectors in barley colonization in an aphid species-specific manner.

Grain nitrogen (N) concentration is a major quality criterion of malting barley for which there is a narrow range that producers must meet to satisfy market requirements (1.52 – 1.84 %). In recent years growers in Ireland have had difficulty producing grain with a high enough N concentration to meet these requirements using standard recommended agronomic regimes. The reasons for the lower than expected grain N concentrations are not known. There is interest from growers and maltsters in the development of a system to forecast likely grain N concentration from crop measurements made at or before flowering. A forecasting system would allow growers to identify crops at risk of falling below specification and to apply late N fertiliser if needed. It would also enable maltsters to plan grain intake and malting operations in advance of harvest. The aim of this project was to investigate the potential for predicting grain N concentrations of spring barley from crop and soil measurements made at ear emergence. The main objectives were to 1) investigate the relationships between measurements made at ear emergence and grain N concentration at harvest in order to identify which characteristics should be included as variables in multiple regression models to explain variation in grain N concentration, 2) to use the models with independent data sets to predict grain N concentration and test the accuracy of the predictions, 3) to quantify the recovery by the crop of fertiliser N applied at anthesis and its effects on grain N concentration and 4) to determine whether non-destructive techniques can provide estimates of crop growth and N content for use in the prediction models. Field experiments were established with plots of spring barley (Hordeum vulgare cv. SY Taberna) at one site in 2013 and two sites in 2014 representative of those employed in malting barley production in Ireland. Fertiliser N applications were varied over the range 0 – 210 kg N/ha (with dressings split between sowing and mid-tillering) to provide a range of crop growth and grain N concentrations. In some experiments additional applications of N were made at anthesis to quantify effects on grain N concentration and seed rate treatments (150, 300 and 600 seeds per m-2) imposed to test the accuracy of predictions of grain N concentration in crops of varying canopy structure. Destructive samples were taken to determine total crop N content and canopy N distribution at ear emergence and harvest. Measurements of soil mineral N availability, ear numbers per m-2 and the number of spikelets per ear were made at ear emergence. Final grain yield and quality were also determined at harvest. Grain N concentration is the quotient of grain N content and grain yield. Both grain N content and yield explained a significant amount of the variation in grain N concentration observed across sites and fertiliser N treatments indicating that estimates of both must be included in models to predict N concentration. Grain N content was strongly related to total crop N content at harvest (P < 0.001; R2 = 0.96), which in turn was related to canopy N content at ear emergence (P < 0.001; R2 = 0.94). Similarly, grain yield was strongly related to total crop biomass at harvest (P < 0.001; R2 = 0.83), which in turn was related to crop biomass at ear emergence (P < 0.001; R2 = 0.88). These results indicated that predictions of grain N concentration might be possible from measurements of crop N content and biomass at ear emergence and that the effects of variation in harvest index, nitrogen harvest index and post-anthesis N uptake on grain N concentration are likely to be negligible under normal agronomic conditions in Ireland. Weather conditions in 2013 were unusually dry and estimates of soil moisture deficit and available water capacity indicated that the crop was water stressed. In 2014 weather conditions were close to the long term averages for the sites. Multiple regression models using canopy N content and biomass at ear emergence as explanatory variables accounted for 91% of the variation in grain N concentration when data from 2014 were used and 80% when data from both 2013 and 2014 were combined. The models developed using data from plots sown at 300 seed per m-2 in 2014 were tested against independent data from plots sown at 150 seeds per m-2 in the same year and at the same sites to test the accuracy of predictions across plant populations and canopy structures. The models were also tested using data from experimental plots and commercial fields collected in 2015 to test the accuracy of predictions in a different year across a range of sites and varieties. Values of grain N concentration predicted from measurements at ear emergence were compared with actual grain N concentrations measured at harvest. The accuracy of predictions was good with an R2 of 0.80 and RMSE of 0.114 %N for the test across seed rates and R2 of 0.80 and RMSE 0.220 %N for the validation in 2015 across sites and varieties. In 2014, grain N concentrations were increased significantly by applications of additional N fertiliser at anthesis with apparent recoveries (increase in N content (kg) /kg fertiliser N applied) in grain averaging 50% over the range of application rates indicating scope for increasing grain N concentration in crops predicted to be at risk of not meeting malting specifications Non-destructive measurements displayed significant relationships with N content and biomass at GS 59 across a combination of sites and seasons. However, issues in performance relating to instrument saturation were obvious and estimates never produced more accurate predictions of grain N concentration than destructive sampling. The results show that grain N concentration of spring barley can be predicted with good accuracy from measurements of canopy N and crop biomass made at ear emergence when the weather conditions are comparable to the long term average for the region. As conditions of drought are rare in Ireland, the prediction models are a potentially valuable tool to aid crop management and post-harvest operations by growers and maltsters. Further testing will be needed before users can be confident in the reliability of predictions over years and a larger set of varieties.

In the past two decades a new barley threat has emerged as the disease Ramularia leaf spot (RLS) became more prevalent in temperate regions worldwide. This disease, first identified in the late 19th century, is caused by the filamentous fungus Ramularia collo-cygni (Rcc) and can cause substantial yield losses as well as reduce grain quality. RLS typically occurs late in the growing season and characteristic disease symptoms are usually seen after the crop has flowered. Expression of RLS lesions is thought to be associated with the action of fungal secondary metabolism products. The one group of secondary metabolites (SMs) characterised to date from Rcc, the anthraquinone toxins rubellins, are known to cause necrosis to plant tissues in a non-host specific manner. Therefore, it appears that fungal secondary metabolism might be a key component in understanding the interaction between Rcc and its host. In this study, more than 23 core genes involved in the biosynthesis of SMs belonging to the polyketide and non-ribosomal peptide pathways were identified in the genome of Rcc. Putative clusters containing genes with a predicted function relating to secondary metabolism were identified by in silico genome walking in the genetic loci adjacent to Rcc SM core genes. Two gene clusters containing no SM core gene were also identified. Five of the putative SM clusters exhibited similarity to the known fungal SM biochemical pathways involved in gliotoxin, monodictyphenone, ferricrocin, betaenone and chaetoglobosins biosynthesis. Several gene clusters exhibited similarity to SM clusters from fungal species where the SM pathway is uncharacterised. Changes in transcript abundance of selected SM core genes during RLS development in artificially inoculated barley seedlings were tested. Transcript levels were found to be the highest at an early stage of disease development, typically during the asymptomatic and early lesions formation stages and declined over time, suggesting that the associated SMs in Rcc, may not necessarily be involved in symptoms appearance. The in planta mode of action of the non-host specific photoactivated toxin rubellin D was studied in the model plant Arabidopsis thaliana. Rubellin-induced cell death appeared phenotypically reminiscent of programmed cell death (PCD). Full expression of rubellin D-induced cell death required the host salicylic acid (SA) pathway and the host proteasome supporting the PCD response to this fungal SM. However, a clear correlation between toxin sensitivity and disease susceptibility could not be found, suggesting a potential alternative role for rubellin in disease symptom development.

Rhynchosporium commune is one of the most destructive fungal pathogens of barley worldwide. It causes scald, responsible for reduced grain quality and yield losses of up to 40%. This project aimed to identify genetic resistance in barley using two different approaches: an effector approach through the identification of important pathogen virulence factors and their barley targets, and a genomics association approach. Numerous secreted effectors have been identified in many phytopathogens including R. commune. Rrs1 resistance, recognising the R. commune avirulence protein - AvrRrs1 (NIP1) has been deployed in the field to prevent infection but has soon proven ineffective. R. commune has managed to overcome this resistance by alteration or deletion of the NIP1 gene as it is not essential for pathogenicity. However, our field trial data suggests that Rrs1 remains an important component of resistance to R. commune in the field. Resistance genes recognising more essential Avr genes are likely to be more durable and as a consequence, the discovery of novel R. commune Avr genes is fundamental for the implementation of an integrated pest management approach to prevent this disease. Recent sequencing of the R. commune genome allowed identification of putative effectors. Expression of 26 potential effectors with low sequence variability in 9 sequenced R. commune strains have been analysed during barley infection. The best genes were selected for gene disruption and individual expression in barley cultivars and landraces using the Barley Stripe Mosaic Virus (BSMV) – based expression system to see if they are recognised by the plant. The work also focused on candidate effectors with putative functions. A putative protease inhibitor was chosen for functional characterisation but its function and importance for pathogenicity could not be confirmed. In addition, high amount of the candidate protein appeared to be toxic for barley and Nicotinana benthamiana. Two SA (salicylic acid)-related putative effectors were also chosen for further characterisation and revealed a direct link between the SA pathway of barley and R. commune. The results of this project suggest that R. commune might be able to manipulate the SA pathway of the host confirming the existence of a biotrophic phase of the fungus. The genomics association approach to identify resistance genes against R. commune in barley used a Genome Wide Association Scan (GWAS) using a combination of three years of disease nursery field trial data for a collection of over 500 elite spring barley cultivars. This analysis identified a number of quantitative trait loci (QTL) in barley genome regions previously shown to contain major resistance genes such as Rrs1 on chromosome 3H, Rrs2 on chromosome 7H, Rrs3 on chromosome 4H, Rrs4 on chromosome 3H, Rrs13 on chromosome 6H, Rrs14 on chromosome 1H and Rrs16 on chromosome 4H, as well as novel QTL. The work was focused on Rrs1 resistance. R. commune strains producing a type of NIP1 effector, recognised by barley lines containing Rrs1, were used to confirm the resistance in predicted Rrs1 barley cultivars. The Rrs1 interval has been narrowed down to 3 Mbp, and high resolution mapping led to the identification of 3 SNP markers which perfectly discriminated Rrs1Rh4 lines from susceptible lines. These diagnostic markers will provide a useful breeding tool for improving the design of new varieties allowing the incorporation of the Rrs1 resistance. This research takes us a step closer towards cloning the first barley major resistance (R) gene against R. commune, which is likely to be present only in Rrs1 lines and have a kinase domain very similar to the one in a putative wall associated kinase found within the Rrs1 interval in the genome assembly of susceptible cultivar Morex. It will also help us to better understand R. commune-barley pathosystem and to identify further R genes.

Major changes in expression occur in Arabidopsis in response to cold. It is now clear that genome-wide changes in alternative splicing (AS) also occur in Arabidopsis during the cold-response and many of the genes which undergo cold-induced AS have been linked to roles in either the regulation of the cold-response or regulation of AS. Mutations in splicing factors in Arabidopsis, such as STA1 and SRL1 have been shown to lead to both changes in AS and changes in cold-sensitivity/tolerance, suggesting an important the role for AS in regulating the cold-response. Less is known about the effect of cold on AS in barley or how AS impacts the barley cold-response. There are only a few studied examples of cold-induced AS changes in barley genes, although this is rapidly changing due to both the publication of the barley genome and next generation sequencing of the transcriptome. To investigate AS in the barley cold response, 11 barley genes with cold-induced AS changes were identified and the AS change was analyzed in detail. The barley genes FRY2 and SUA change AS after 30 minutes exposure to cold and are both genes have been linked to roles in regulating AS, indicating a complex role for AS in the earliest stages in the cold-response. The Serine Arginine (SR)-Rich protein genes have been shown to change AS and affect AS under stress conditions in Arabidopsis, rice and other plant species but little is known about the SR protein genes in barley or how the genes change splicing/expression in response to cold. The 16 members of the barley SR protein gene family were identified and analyzed for cold-induced expression changes using available microarray and RNA-Seq data. The HvRS41 gene showed a >2 fold increase in expression after 3h exposure to 6°C in a cold-based microarray experiment. A cold-based microarray experiment in Arabidopsis showed a similar cold-induced expression of the AtRS40 gene, a RS-type SR protein gene with high homology to HvRS41. The cold-induced expression of HvRS41 and AtRS40 indicate a role for the RS-type SR protein genes in the cold response. The RS-type SR proteins form a splicing complex with FRY2 which could potentially be regulated through both AS and expression change during the cold-response. The Barley SR Proteins were dived into six sub-groups previously established for plant SR protein genes. Five out of the six sub-groups of the barley SR protein genes contained AS which could be validated through RT-PCR based methods. The SR-type SR protein genes contained was shown to contain three genes (HvSR34, HvSR30a and HvSR34) within barley. All 3 barley SR-type protein genes showed AS change in response to low temperatures, indicating a role for the barley SR-type SR protein genes in regulating AS during the cold-response. The role for SR-type SR protein genes in regulating AS was tested through a creation of a barley transgenic line over-expressing gene the HvSR34. The HvSR34 overexpression lines are in the process of being tested for changes in AS and cold tolerance.

In the world today there is a massive dependency on fossil fuels as they are currently used to provide around 80% of the world’s energy. This is hugely detrimental to the environment and is a major contributory factor in climate change. Biofuel is a renewable energy source that is already being used to lessen some of the fossil fuel dependency. 2nd generation biofuels, by using non-food parts of plants, circumvent the food vs fuel argument, and by using farming waste or surplus can also avoid changing land use problems. Additionally liquid biofuels can use existing infrastructure for storage and delivery, and also fit into current lifestyles. Cost-effective 2nd generation biofuel production is directly affected by the presence of the polymer lignin in plant biomass, as it has been shown to impede enzymatic sugar release (saccharification) that is used for biofuel production. The work undertaken in this project developed a high-throughput methodology for the assessment of straw lignin content and composition across a large population of elite varieties in the economically important cereal crop, barley. Saccharification yield was also measured across the same population along with a number of other agronomically important traits, such as thousand-grain weight, biomass, mechanical stem properties and height. The data provided by these measurements allowed correlations between traits to be identified and their strength gauged. Genome wide association studies (GWAS) were also carried out and identified influential regions of the genome for each trait. The results revealed varying levels of association between measured traits and lignin content and monomeric constituents. Importantly a negative connection was shown between lignin content and saccharification yield, with lignin content being responsible for approximately 1/5th of the variation seen. Interestingly there was no correlation between lignin content and mechanical stem properties, an important factor in the agronomically important trait, lodging. GWAS results revealed a number of genomic regions that were influential across several traits indicating regions that would be difficult to separate through breeding due to their close proximities. However, unique QTL were identified for saccharification yield and lignin content providing candidates for breeding or genetic manipulation to improve the crop for biofuel production.

Biochar is the solid, carbon-rich by-product obtained from pyrolysis. It offers the prospect of long-term carbon sequestration and soil conditioning with agronomic benefits, often referred to as the ‘biochar effect‘. These multiple direct or indirect changes in the soil plant interface have also been associated with the control of plant diseases by influencing the host’s systemic induced resistance. The biological impact of biochar on the phytopathology of a major cereal grain has not yet been investigated. The most damaging foliar disease of barley in the UK is Rhynchosporium leaf blotch caused by the hemibiotrophic fungal pathogen Rhynchosporium commune. The aim of this project was to evaluate biochar performance and effectiveness as a disease control agent in the barley – R. commune – pathosystem, and attempted to identify underlying mechanisms by which biochar may function in the interaction between barley and the causal pathogen. Therefore, a unique set of nine slow pyrolysis biochars were established along a 350 to 800°C pyrolysis temperature gradient, with eight of these made from pelleted softwoods and a single one made from Miscanthus straw. A comprehensive biochar quality assessment was undertaken and established that the biochars differed in their chemical composition, which largely depended on production parameters, predominantly temperature (P < 0.05). The analysis proposed that biochar 9, made from Miscanthus at 800°C, showed added value as a soil conditioner over softwood biochars, due to higher pH, mineral ash and macronutrient recoveries, which pointed towards a possible liming potential. Regardless of the feedstock, biochars pyrolysed above 600°C indicated potential use for carbon sequestration purposes, due to higher carbon stability. Short-term controlled bioassays showed significant restricted growth of R. commune mycelium on defined medium to direct (1.0% w/w) and indirect volatile exposure from certain biochars (P < 0.001). The findings suggested a synergistic effect of the softwood biochars acidic nature and presence of fungicidal compounds, with observed inhibition of 100% attributed to re-condensation of tarry vapours onto biochar surfaces during pyrolysis. Qualitative biochar volatile organic compound analysis was conducted and identified biocide active phenolic and organic acid compounds, similar to those commonly found in smoke, bio-oils or wood vinegars. These findings proposed possible application for mitigation of inoculum pressure in field-grown barley, but the toxic nature of volatiles raised concerns over risks to human and environmental health, as also evidenced by detrimental barley growth effects. Subsequent controlled in vivo and in planta experiments revealed significant (P < 0.05) symptomatic barley leaf blotch reduction effects of up to 100%, following 5% (w/w) application of biochars 4, 5, 8 and 9. Barley plants transcriptional changes in ISR-dependent LOX2 and SAR-dependent PR1-b expression in planta verified systemic induced resistance as mechanisms behind the significant disease suppression of barley plants grown in soil amended with biochar 5 and 8. Disease reduction and biochar mediated induced resistance was attributed to either low concentrations of phytotoxic compounds, a direct toxicity effect from fungicidal compounds or indirect promotion of beneficial microbes. The results provided evidence, that in the case of the studied pathosystem, there is potential for biochar with specific characteristics to be considered as a soil amendment, offering not only carbon sequestration, but also possible improved disease resistance.

In recent years, a new threat to barley crops has emerged and gained substantial attention due to its rising economic importance. Relatively little is known about the infection strategy and development of Ramularia leaf spot (RLS) disease on barley. Therefore the overall aim of this project was to increase the understanding of fundamental biology and life cycle of the causal agent, R. collo-cygni. Chapter 2 describes the horizontal transmission of the fungus on barley. Both field and transgenic R. collo-cygni isolates expressing GFP and dsRed fluorescent reporter markers were utilised to visualise the infection progression in living host tissues by various light and confocal microscopy. The existence of a previously unknown structure called stomatopodium (infection peg), involved in the penetration of stomata, was demonstrated. The fungus initially exhibited symptomless epiphytic growth, extending above epidermis and connecting the hyphal aggregates inside substomatal cavities and subsequent initial sporulation. However, during the transition into symptomatic phase, the organised intercellular growth of hyphae into the mesophyll was observed. This hyphal network was involved in the production of asexual spores demonstrating that the raprture of epidermal layer was responsible for local necrosis observed for RLS. In addition to barley, several other speculated R. collo-cygni hosts have been used to verify their compatibility to the pathogen. In chapter 3, a whole plant inoculation assay was developed to investigate the mode of the fungal seed-borne transmission by using GFP expressing strain of the fungus. It is shown here for the first time that the vertical transmission is systemic, involving symptomless colonisation of embryo and closely resembled the mode of dissemination observed for Neotyphodium species, mutualistic fungal endosymbionts on grasses. The impact of fungal infection on seed germination ability was also examined that revealed no significant difference between clean, moderate and high levels of R. collo-cygni DNA. Chapter 4 represents an attempt to discover and analyse the sexual development in R. collo-cygni. As a first step to understand the sexual reproduction cycle in this apparently asexual species, the genetic structure of the mating system was characterised by using PCR-based techniques which demonstrated the heterothallic nature of the fungus. The defined population of R. collo-cygni field isolates was then screened for the presence of the discovered mating type idiomorphs (mat) to determine the frequencies of the mating types in the defined R. collo-cygni populations. The segregation ratio of mat1 and mat2 close to 1:1 indicated a frequent sexual reproduction. In order to verify the existence of functional sexual stage in R. collo-cygni, potential sexual development was induced using the potentially compatible isolates and a comprehensive analysis was undertaken by correlative use of light-, confocal- and low temperature scanning electron microscopy. Two types of multicellular bodies were observed and described. First was the speculated Asteromella stage (male donor) that carries spore-like spermatia. The second structure initially resembled sclerotia that in only a few instances developed into perythecium/ pseudothecium that appeared to carry the sexual spores, ascospores enveloped in asci. Chapter 5 demonstrates the role of rubellin toxin in symptom development by using autofluorescence phenomenon. The structure of putative molecular machinery involved in rubellin biosynthesis was addressed by using bioinformatics approaches and the complete R. collo-cygni genome sequence. A gene cluster encompassing several components of other known secondary metabolite biosynthesis pathways, such as that of dothistromin and aflatoxin, was found and putative protein function of the genes is hypothesised.

Barley row-type describes the number of grains present at a node on the barley spike. Two forms exist amongst cultivated barley: two-rowed with only the central of three spikelets fertile producing a single grain at a node and six-rowed with all three spikelets fertile, producing three grain at a node. Twelve regions of the barley genome have been associated with the row-type character with specific genes identified at three loci, VRS1, VRS4 and INT-C. Advancements in the understanding of the genetic control underpinning barley row-type enables the identification of potential mechanisms for improving yield and yield architecture within the cereals.  This study used genetic linkage mapping in segregating F2 populations to refine the genetic location of the row-type locus, VRS3, to 16 candidate genes on barley chromosome 1H. Sequencing candidate loci in 32 vrs3 induced mutant alleles identified VRS3 to be a highly conserved JmjC histone demethylase, with two natural alleles within European cultivated barley. VRS3 was further characterised as a potential means of improving grain uniformity within cultivated six-rowed barley, through phenotypic assessment of grain size in varying allele combinations of VRS3, VRS1 and INT-C. The addition of six-rowed alleles at these loci was found to improve balance between central and lateral grain parameters, resulting in a more uniform grain sample. Analysis of gene expression found Vrs3 to be constitutively expressed across a diverse panel of barley tissues. Moreover, detailed study within the developing inflorescence suggests a role for Vrs3 in the regulation of the row-type genes VRS1 and INT-C.

Barley (Hordeum vulgare) is an economically important crop species with a large diploid genome. Around a half of the barley genome and a fifth of the genes are constrained within a low-recombining pericentromeric (LR-PC) region. I explored the LR-PC gene component with a genomic investigation of gene expression, diversity and evolution. Chromatin environments were also explored in the LR and high recombining (HR) regions by surveying the genic and genomic distributions of nine histone modifications. Firstly, regions of HR and LR were identified and compared for gene evolution, expression and diversity. LR regions of the barley genome were found to be restrictive for gene evolution and diversity, but not gene expression. I employed a bioinformatics approach to identify ancient gene pairs in barley to determine the long-term effects of residency in those regions upon gene evolution. Gene pair loss in LR regions was found to be elevated relative to the HR regions. Applying the same method to rice and Brachypodium distachyon revealed the same situation, suggesting a universal process in the grasses for loss of gene pairs in LR regions. The chromosomal distributions of transposable elements (TEs) were also explored and examined for correlations with recombination rate. Secondly, I developed a chromatin immunoprecipitation followed by Next Generation Sequencing (ChIP-seq) protocol for the investigation of histone modifications in barley seedlings. A protocol was optimised for the fixation, extraction and sonication of barley chromatin. The protocol was applied using antibodies against 13 different histone modifications. Following DNA library construction and Illumina sequencing, a bioinformatics pipeline was devised to analyse the sequence data. NGS reads were mapped to a custom assembly of the barley cultivar Morex reference genome sequence before peak calling. Genomic and genic locations were determined for the covalently modified histones. Four modifications were discarded from further study on the basis of low peak numbers or unexpected chromosomal locations. The remaining nine modifications were classified into four groups based on chromosomal distributions. Groupings were closely mirrored by peak sharing relationships between the modifications except histone H3 lysine-27 tri-methylation (H3K27me3). In addition, chromatin states representing local chromatin environments were defined in the barley genome using the peak sharing data. Mapping the states onto the genome revealed a striking chromatin structure of the gene-rich chromosome arms. A telomere-proximal region bearing high levels of H3K27me3-containing states was found adjacent to an interior gene-rich region characterised by active chromatin states lacking H3K27me3. The LTR retroelement-rich interior was found to be associated with repressive chromatin states. The histone modification status of TE classes were also probed revealing unexpected differences relating to the genomic and genic distributions of these elements. Finally, a genome browser was created to host the information publicly.

To prove the post-natal depression model, the antidepressant sertraline, was assessed in rat mothers (n=14) divided into Prenatally Stressed (PS) and Non-Stressed (NS) groups. The data failed to support the hypothesis that ‘the progeny of 10mg of sertraline-treated PS mothers displayed less anxiety than the progeny of vehicle-treated PS mothers’. The forced swim test (FST) was used to examine depressive-like behaviour in mice. Barley successfully increased mobility in mice exposed to the FST. Barley was antidepressant at low doses (0.8g/kg and upwards) if used subchronic; and at high doses(6.4g/kg and 12.8g/kg) if used acutely;(n=113,56acute,57 subchronic- treated). Barley (6.4g/kg) was also able to alleviate the depressive-behaviour in mice induced by the Reserpine Test (n=114, 58 reserpinised, 56 non-reserpinised) and Social ‘Defeat’ Test (n=24, 8 vehicle undefeated, 8 barley defeated, 8 vehicle defeated mice). To confirm that the anti-depressant effects of barley(6.4g/kg) were not simply due to increased locomotor activity in the FST, an Open Field Test(OFT) was undertaken (n=14,7 vehicle, 7 barley). Barley had no effect on locomotor activity and also caused no significant changes in weight (n=16, 8vehicle, 8 barley). In mice,Barley(6.4g/kg) significantly delayed the tremorogenic effects of Physostigmine (n=18, 6 control,6 Physostigmine, 6 Physostigmine with barley); reduced bradykinesia induced by reserpine (n=18,6 control, 6 vehicle, 6 barley treated);and was analgesic in nociception tests (n =20, 5 control, 5 barley, 5 pain, 5 pain with barley). Overall, barley was seen to have many useful properties, though its effect in PND remains to be assessed.

Cost effective production of biofuel from plant biomass (second generation biofuels) is currently a key challenge. To achieve this, accessibility of plant cell wall polysaccharides to chemical, enzymatic and microbial digestion could be improved by altering lignin structure and composition or by reducing lignin content, as lignin is one cell wall component that has already been shown to contribute to biomass recalcitrance. Therefore, this thesis reports the genetic manipulation of lignin biosynthesis through down-regulation of cinnamyl alcohol dehydrogenase (CAD) genes in barley (Hordeum vulgare L.). Barley has been chosen as the target plant for lignin manipulation for a few reasons: it is a major cereal crop that produces large amounts of lignocellulosic plant biomass that can potentially be used as animal feed or to produce second generation biofuels and also because it is a model grass for other bioenergy crops. CAD, as the final enzyme in the lignin pathway, is a perfect target for lignin manipulation. Characterised CAD mutants and transgenics have shown that down-regulation of CAD improves digestibility and does not influence plant growth and fertility. Due to the difficulty and complexity of transformation of monocot species, there are only a few reports describing down-regulation of CAD in monocots, and none in barley. Here, in this thesis, lignin was altered by down-regulating CAD genes using an RNAi construct with part of the HvCAD2 gene, the gene which has the highest expression level of all CAD genes. Transgenic barley plants showed reduced enzyme activity in the T0 generation (31% compared to EV plants) and enzyme activity was reduced even more in the T1 (to 3%) and T2 (to 2%) generations. The HvCAD2 RNAi barley lines had similar or slightly reduced Klason total lignin contents relative to control plants, but lignin structure and composition were altered. The RNAi plants had lower thioacidolysis yields, S/G ratio was reduced (1.59 in the empty vector controls versus 0.96–1.21 in the transgenic barley plants), the relative frequency of S units was reduced by 11–20%, the proportion of G units was increased by 17–32%, there was increased sinapaldehyde accumulation in lignin and ferulic acid abundance was reduced relative to control plants. Analysed transgenic barley plants had an orange stem phenotype. Growth season and conditions hugely affected the intensity of the phenotype. Because lignin plays a major role in culm strength and pathogen resistance, the influence of down-regulation of CAD on these features was characterised. The changed physicochemical nature of cell walls in HvCAD2 RNAi lines does not decrease the strength of the straw and does not decrease the resistance to the biotrophic Blumeria graminis and to the hemibiotrophic Rhynchosporium commune pathogens. The modified cell walls in the HvCAD2 RNAi lines had moderately improved sugar release for biofuel production. This study proves that it is possible to down-regulate CAD in cereal crops in order to change lignin structure and composition in plants without a negative impact on plant growth, fertility or pathogen resistance.

Demand for both food and water are projected to increase substantially in the next four decades. Water scarcity is also projected to increase in scale and complexity. Climate change is projected to increase temperatures, spatio-temporal variability in rainfall, frequency and severity of droughts and soil water stresses to crops. Due to the crucial role of water in crop growth and yield formation, prolonged or severe soil water deficits in crop producing areas can result in substantial yield penalties. The potential of food trade to help address food insecurity as a result of insufficient water availability for crop production has been rationalized in the virtual water concept. The aim of this thesis was to improve the evidence base for understanding and evaluating the relationships between future water availability for crop production and food trade (or virtual water flows), and the utility of the virtual water concept to inform policy and management decisions on water-food security. The UK and barley were used as a model country and crop, respectively. Three crop growth simulation models (AquaCrop, CropWat and WaSim) were evaluated for their abilities to estimate the water use of 10 barley genotypes. Subsequently, the effect of projected climate change on UK barley yields in the 2030s, 2040s and 2050s was simulated using the high, medium and low emission scenarios data from the UK Climate Projections 2009 (UKCP09). Projections of total UK feed barley supply and demand were performed to quantify potential virtual water flows and to analyse the implications for food security and policy. The results show that the predicted water use of barley differed between the models but not among the genotypes. Predicted seasonal water use of the barley genotypes ranged from 241.4 to 319.2 mm. Based on the root mean square error (RMSE) and the index of agreement (D-Stat) values, CropWat performed poorly while AquaCrop and WaSim performed excellently. Barley yields under projected climate change increased substantially over baseline yields in all UK regions. Projected mean barley yields for the UK ranged from 6.04 tons ha-1 (2030s) to 7.77 tons ha-1 (2050s). In spite of the projected increase in yields, the UK faces the risk of large deficits in feed barley and meat supply from the 2030s to the 2050s due to a combination of population growth, increased per capita meat demand and reductions in land area allocated to barley production. Finally, current water scarcity concepts were found to be incompatible with water availability and consumption in crop producing areas, a situation that diminishes the usefulness of the virtual water concept for policy. To address this deficiency, a framework for making water scarcity compatible with crop production was proposed. In conclusion, the poor performance of CropWat has implications for its wider use in quantifying global virtual water flows associated with crop trade. Even though UK barley yields are projected to increase under projected climate change, the projected deficits in feed barley and meat supply threatens to destabilize future UK food security. The UK can rely on import to offset the large deficits in feed barley and meat supply but can use the proposed framework to reduce the effect of its imports on water scarcity in the exporting countries. The proposed framework improves understanding and evaluation of the role and usefulness of the virtual water concept in water-food security policy and management decisions.

One of the challenges in the 21st Century is to overcome the recalcitrance of lignocellulose for the production of liquid biofuels. Lignin is one of the key factors in this recalcitrance. Grasses such as Miscanthus and switchgrass could become major sources of lignocellulose. Barley has potential as a genetically-tractable research model for such novel bioenergy crops and also as a bioenergy crop itself. This thesis concerns the 4CL and the CCR enzymes on the lignin pathway which were chosen as the targets to manipulate lignin in barley. They were selected because there is evidence that suppression of each of them in dicot species can lead to increased saccharification. The 4CL and CCR genes constitute multigene families where members have different expression patterns. RNAi was used to down-regulate 4CL1 and CCR1 using a constitutive promoter via Agrobacterium-mediated transformation of barley. From an extensive screen of the primary transformants for changes in protein level and lignin content, six CCR and four 4CL lines were taken forward for detailed analysis. Antibodies were also raised against barley 4CL and CCR recombinant proteins and these showed substantial reductions in the respective target protein levels in the RNAi lines. Both 4CL and CCR transgenic lines had significant reductions in lignin content, and CCR lines had changes in lignin structure due to changes in the proportions of acid soluble and acid insoluble lignin. No substantial consistent adverse effects on key agronomic traits were apparent in the 4CL and CCR transgenics. Selected 4CL and CCR transgenics had improved saccharification yield after using three different pretreatment methods, which is a desirable feature for biofuel production.