Climate change is predicted to have far-reaching deleterious impacts worldwide; agriculture in particular is expected to be effected by significant loss of suitable land and crop yields in the world’s most populous and poorest regions. Crop wild relatives (CWR) are a rich source of underutilised genetic diversity which could help to mitigate climate change for agriculture through breeding new resilient varieties. However, CWR are under-conserved and threatened in the wild. This thesis researches and develops systematic methodologies to advance knowledge and support action on in situ CWR conservation at the global level. Methods included developing a global inventory of CWR associated with crops important for food security worldwide, species distribution modelling, climate change analysis, in situ gap analysis, reserve planning and prioritisation, and, examining the congruence of CWR distributions with regions of high biodiversity and crop diversity. The methods described here can be applied to CWR at both the national and regional level to ensure robust in situ CWR conservation. A principal success of this research is the global CWR inventory, which has been used in several national strategies and as the basis of a major ex situ germplasm collection mission worldwide.

Subsoil physical characteristics are often limiting to root growth, one of the major reasons being high density soil. However, deeper and more efficient root systems could help to explore a larger soil volume and reduce the input of nitrogen fertilisers if roots make more use of the nitrogen at depth. The first target was to develop a screening method which allowed barley root extension rates to be quantified after four days of growth in loose and compacted soils. Firstly, seed quality (loss of germination ability caused by poor conditions in storage and long storage time) was identified as a potential source of variation for root extension rate in seedlings. The screening showed that roots growing in compacted soil had a slower extension rate than roots growing in loose soil. In addition, there was an interaction between soil conditions and cultivars meaning that not all of them showed the same ability to overcome high soil density. Root architecture was characterized at days eight and 12 after planting for four selected divergent cultivars. Measurements were made using X-ray micro-computed tomography (µCT)-scanning. The differences between the four genotypes in root architecture (number of primary roots, root extension rate, root length, root area, root volume, convex hull, centre of mass, lateral density, lateral length) were significant at eight days after planting but disappeared at 12 days after planting for most of the traits measured (i.e. growth rate of primary roots). Soil density influenced the root system architecture at both two-time points, roots elongated less and explored less soil in the high compaction treatment. A third experiment was conducted to test the hypothesis that the differences in root architecture observed between the genotypes in response to the soil bulk density in the µCT-scanning would lead to different patterns of nutrient uptake from topsoil and subsoil. Layered soil columns of topsoil and subsoil were constructed with different subsoil physical parameters (loose, compacted and compacted with macropores) and a nitrogen tracer to measure nitrogen capture from the subsoil. Root length density and other traits determining root architecture differed between two barley cultivars and oat, but increased root length density in the subsoil did not improve nitrogen uptake from the subsoil. Hence showing that nitrogen uptake from the subsoil was not directly related with a greater presence of roots in the experiment.

Ramularia collo-cygni (Rcc) is a damaging fungal pathogen of barley (Hordeum vulgare). It is a causal agent of Ramularia Leaf Spot (RLS), which contributes to significant economic yield losses worldwide. Protection against the disease has been, and is currently, based on foliar fungicide applications as available seed treatments are not effective and there are no fully resistant barley varieties. Two groups of systemic fungicides, Succinate Dehydrogenase Inhibitors (SDHIs) and DeMethylation Inhibitors in sterol biosynthesis (DMIs), are used and in addition a protectant multisite inhibitor chlorothalonil has been used to minimise the damaging effects of RLS in barley. SDHI fungicides have been extensively used in cereals since 2005 in the UK. Resistance outbreaks to SDHIs have been reported in several plant pathogenic fungi. The risk of resistance in Rcc was unstudied and so this study aimed to develop the methodologies needed to screen populations, establish baseline sensitivity data, evaluate the risk of evolution of fungicide resistance to SDHIs in the population of Rcc and make recommendations on appropriate anti-resistance strategies to minimise the risk. A combined approach of SDHIs’ field performance under different application regimes, sensitivity testing in vitro, molecular analysis of resistance mutations and studies of the genetic structure of populations was used. The results in this study demonstrated that currently SDHI foliar applications remain effective in controlling RLS in barley. The most consistent control was observed when applying them in mixtures with DMIs. All of the tested field isolates were in the range of baseline sensitivity to SDHIs and no shifts between years and different applications regimes were observed. The molecular characterisation of Rcc resistant mutants developed in UV mutagenesis studies revealed that in four out of five mutants a single amino acid change in a target succinate dehydrogenase (Sdh) protein was associated with decrease in sensitivity to SDHIs. All of these mutations, with the exception of one mutant, were stable in the absence of SDHI fungicide. The analysis of fitness components indicated that mutated strains did not confer a fitness penalty associated with the mutation when measuring the growth in vitro. Additionally a detached leaf assay performed for two selected mutants with high or moderate resistant factors showed that the resistant phenotypes were able to colonise the leaf surface and reproduce successfully. Thus if the resistance was to develop due to these point mutations in the target Sdh gene, mutated phenotypes are likely to be fit enough to outcompete the sensitive population. Analysis of the genetic structure of the Rcc populations demonstrated that the pathogen is highly diverse, is likely to undergo sexual reproduction over the growing season (the sexual stage remains undiscovered) and has a potential for extensive spore dispersal across the field. Thus Rcc has a high evolutionary potential and could adapt to different control measures relatively quickly. In the 2012 growing season, two field phenotypes with reduced sensitivity to SDHIs were identified as a consequence of sensitivity monitoring in vitro. Neither of the isolates had any nucleotide or amino acid changes in the target Sdh gene and the mechanism responsible for the resistance in these two strains remains unknown. One of the field isolates was tested in planta and exhibited abnormal growth on the leaf surface. This could result either from fitness costs incurred due to resistance or the fact that this isolate does not belong to the Rcc species, a possibility which must be further investigated. In conclusion, SDHI fungicides remain effective in controlling RLS in barley, however this study demonstrated that there is a risk of measurable loss of efficacy in field conditions. Thus monitoring studies should be intensified and integrated crop management practices applied to prolong the life span of SDHI fungicide treatments.

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.

High temperature (HT) stress during wheat male reproductive development causes irreversible damage to the anther tapetum layer and the developing microspores it supports, resulting in reduced yield. With the frequency of pre-flowing temperature stress events likely to increase, a better understanding of the effects of high temperature stress on anther developmental regulation is required. Gibberellin (GA) signalling has been shown to regulate tapetum programmed cell death (PCD) and pollen coat formation via the transcription factor (TF) GAMYB. This project aimed to investigate the function of two putative GA-signalling components in wheat anther development and characterise the global hormonal and transcriptional anther responses to HT. RNAi and TILLInG mutants for TaGAMYB and a putative orthologue of a rice tapetum PCD component, TabHLH141, revealed that both are required for male fertility. Tagamyb mutants displayed stunted anther development with irregular tapetum vacuolisation and reduced pollen viability. An interaction between RHT-D1 and TabHLH141 suggests that GA may mediate anther development through regulation of DELLA-TF interactions. Having characterised and developed a non-destructive staging method for wheat anther development, RNA-Seq and global hormone analysis was used to investigate the response to HT stress around pollen mother cell meiosis. Significant changes in expression of tapetum metabolism and PCD annotated transcripts and anther GA, auxin and jasmonate concentrations indicates that hormonal regulation of HT-responsive transcription may contribute to defective anther development. The work in this project demonstrates that advanced functional genomics techniques can be now be applied to the dissection of complex signalling pathways in hexaploid wheat.

Take-all, caused by the soil-borne ascomycete fungus Gaeumannomyces graminis var. tritici, (Ggt), is a root disease that devastates wheat production worldwide. Current control measures consist of partially effective chemical seed dressings and cultural methods such as crop rotation. There is currently no genetic control of the disease. The first aim of this PhD project was to characterise a range of diploid and hexaploid wheat germplasm that possess a promising level of take-all resistance under field conditions. Both above and below ground phenotyping was carried out and soil moisture probes were used to evaluate upper root function for a range of hexaploid varieties. A diploid Triticum monococcum MDR037 (S) X MDR046 (R) mapping population was screened and revealed a good spread in susceptibility to take-all across two field seasons. The population has subsequently been genotyped and genetic analyses will be carried out to explore the genetic basis of resistance. Phialophora fungal species, belonging to the genus Gaeumannomyces, colonise wheat roots but do not destroy the vascular tissue and have previously been found to suppress take-all disease. In the second approach to control Ggt, winter wheat varieties on the AHDB Recommended List (RL) were screened for their ability to build-up natural populations of Phialophora fungi in the field. Differences were revealed in their potential to build-up Phialophora spp. under a first wheat crop. A Phialophora isolate collection was gathered and draft genomes were sequenced, assembled and annotated for the three Phialophora spp. found in UK soils. Preliminary analysis suggests that considerable polymorphism may exist between homologous genes found in all three species. These findings provide a novel contribution to the potential of these two differing control mechanisms against take-all disease.

Disease tolerance is defined as the ability to maintain grain yield in the presence of disease and could be a potential defence mechanism to be incorporated into breeding programmes. It is an attractive goal, as disease tolerance has the potential to be a broad-spectrum, durable defence mechanism while exerting little selection pressure on pathogen populations. Relatively little is known about how disease tolerance is conferred, but most of the hypotheses suggest resource capture and resource-use traits such as large green canopy area, increased light extinction coefficient and a high source to sink balance. Disease tolerance in current wheat genotypes is generally associated with low yield potential, and for disease tolerance to be incorporated into commercial breeding it is important to determine whether this link can be disassociated. In this study, an attempt was made to identify physiological traits conferring disease tolerance to Septoria tritici blotch (STB) in winter wheat. Wheat genotypes contrasting in disease tolerance were selected for in-depth phenotyping of selected physiological traits to determine their association with disease tolerance. A number of publications have attempted to link disease tolerance to physiological traits in wheat, based on their yield loss to disease symptom relationship. However, in this study it was proposed that variation in non-symptomatic disease could influence the appearance of disease tolerance which has not previously been investigated. The ratio of in-leaf pathogen biomass to visual disease symptoms was studied in both controlled-environment experiments and in field experiments to determine whether a high in-leaf pathogen biomass was associated with disease tolerance. Two field experiments were conducted during the field seasons 2011/12 and 2013/14 at Teagasc Oak Park, Carlow, Ireland and ADAS Rosemaund, Herefordshire, UK, respectively. A field experiment was also conducted in 2012/13 at Teagasc Oak Park, but due to dry conditions and little disease presence this field experiment was excluded from nearly all experimental analyses. In each experiment, there were two fungicide treatments, non-target disease control and full disease control. In order to increase genetic variability, 38 selected lines from a L14 x Rialto doubled-haploid (DH) mapping population developed by the International Maize and Wheat Improvement Centre (CIMMYT) were screened alongside 10 UK-adapted reference genotypes for contrasting disease tolerance in 2012. Tolerance was quantified as yield loss per unit of green lamina area index (GLAI) loss to disease. L14 is a CIMMYT spring wheat large-ear phenotype advanced line and Rialto is a UK winter wheat which has high radiation-use efficiency and stem soluble carbohydrate. The DH lines displayed an increased range of disease tolerance compared to the UK-adapted reference genotypes. Selected genotypes were subjected to in-depth phenotyping for an extended range of physiological traits in 2014 to identify traits associated with increased disease tolerance. The traits measured included pre- and post- anthesis radiation interception, light extinction coefficient at anthesis, pre- and post anthesis radiation-use efficiency and stem water soluble carbohydrate accumulation at ear emergence + 7 days. In general, there was a wide range of physiological traits displaying weak associations with disease tolerance. The main traits associated with disease tolerance were related to large and/or maintained source capacity in the presence of disease, such as increased GLAI at anthesis and increased post-anthesis light interception. There was also a general association with low grain yield in the absence of disease and decreased harvest index. Increased disease tolerance was associated with high source capacity and low sink capacity, and there was an association between a high source to sink balance, measured as increased Healthy Area Duration (HAD) per grain, and disease tolerance. The impact of genotype variation on the amount of non-symptomatic disease to visual disease expression was investigated in controlled-environment (CE) experiments. In-leaf Zymoseptoria tritici fungal biomass (pathogen load) was quantified by a Real Time qPCR assay targeting the β-tubulin gene (Accession no. AY547264) and compared to visual disease expression. In the first CE experiment, two wheat genotypes were exposed to increasing concentrations of Z. tritici inoculum. There were differences in rates of pathogen development and pathogen presence between inoculum concentrations in both visual disease symptoms and pathogen loads. In the following CE experiment, a wider range of genotypes exposed to a high inoculum level were shown to differ significantly in the relationship between visual disease symptoms and pathogen loads. In order to determine the impact of genotype variation on the visual disease symptoms to pathogen load ratio, flag leaves of genotypes screened for in-field disease tolerance in 2012 and 2014 were analysed. Large variations in the disease symptoms to pathogen load ratio were identified, which has not previously been shown in wheat experiments. An attempt was made to relate the visual symptoms – pathogen load ratio to non-lesion green area loss as a measure of a potential metabolic cost of increased pathogen pressure, but no such relationship was found. An increased pathogen load per unit visual symptoms did not account for larger yield losses than predicted for a given disease level and there was no direct relationship between symptom expression - pathogen load ratios and disease tolerance. The consistency of high/low displays of disease tolerance calculated by different disease measures was investigated using three different ways of measuring disease; HAD, area under disease progress curve (AUDPC) and pathogen DNA quantified by qPCR. In general, the two measures of pathogen presence (AUDPC and pathogen load) tended to quantify disease tolerance similarly, while the HAD-based tolerance contrasted. There were also differences in which traits were associated with disease tolerance for the different methods of calculating tolerance; the calculations based on AUDPC and pathogen DNA tended to associate a decreased source capacity to disease tolerance while the HAD-based tolerance indicated an association with increased source capacity. All methods, however, indicated that a low yield potential was associated with disease tolerance. In conclusion, there was a large range of disease tolerance found in the field experiments compared to previous investigations. The HAD-based disease tolerance seems to be mainly related to a large source capacity and a low sink capacity. However, the genotype ratings of high/low disease tolerance and associated physiological traits seem to vary according to the method of calculating tolerance. There were large differences in the ratio of visual symptoms-pathogen load between genotypes; even though this did not have a direct impact on disease tolerance or yield loss it could potentially be associated with increased metabolic costs.

The plant hormone gibberellin (GA) is known to influence grain size and flour quality, flowering, development and germination in wheat. GA also induces the production of α-amylase by the aleurone layer and premature production of this enzyme during development results in degraded starch in the mature grain. While GA is proposed to have a negative effect on flour quality, it is essential for early grain development and these effects are separated both temporarily and spatially in the grain. It was the aim of this project to further understand the role GA plays in wheat grain development and in order to achieve this constructs were designed to alter GA metabolism or signalling in the seed-coat, endosperm, embryo or aleurone of developing wheat grains. In plants where GA content was manipulated in the developing endosperm it was shown that GA produced by this tissue is involved in regulating grain size and morphology. This was demonstrated by the differences observed between the transgenics and their nulls in grain size, hardness index and moisture content. Additionally, in these lines no differences were observed in the α-amylase levels, implying that GA produced by the endosperm might not be influencing the production of this enzyme. However, GA insensitivity introduced in the embryo and aleurone layers did not display the hypothesised phenotypes and was inconclusive in determining the role of GA signalling in grain development. During this project a reliable qPCR based method using TaqMan assays was also developed to determine zygosity of transgenic plants in the T1 generation. This method was successful in reducing the number of generations required to select homozygous material compared to more conventional methods.

Cold conditions during imbibition can result in slow or no germination in some maize seed, leading to sub-optimal crop density and uniformity and loss of yield. A novel seed treatment is required that restores germination in seed batches that perform poorly under cold conditions. Germination of seed batches from different varieties was characterised following imbibition under cold conditions which permit no or slow germination. Hydroxamic acid inhibitors of 9-cis-epoxycarotenoid dioxygenase (NCED) stimulate germination through ABA biosynthesis inhibition in other species and had a small significant effect in increasing the proportion of normal seedlings after cold imbibition. This indicated that normal germination of maize may be inhibited by dormancy-related mechanisms during or after imbibition in cold conditions. The maize NCED (ZmNCED) family was characterised. D2 and D4 inhibit other enzymes in the carotenoid cleavage dioxygenase family and exhibit relatively weak inhibition of NCED. ZmNCEDs were cloned for in vitro enzyme inhibition studies to aid identification of NCED-specific inhibitors. An RT-qPCR assay for measuring ZmNCED expression was developed. Seed ZmNCED expression and ABA concentration was elevated under cold conditions, compared to optimal germination conditions. An assay was developed to screen for germination stimulating compounds. 965 of a diverse library of 5074 compounds were identified as potential germination stimulators. Germination stimulating activity was replicated in 171 of these compounds, with some more efficacious than D4. Germination stimulating activity of 88 compounds related to the current lead compound, D4, was assessed at concentrations of 10 ppb to 10 ppm. Compounds were identified that, at less than 10 ppm stimulated germination more than D4 at 312 ppm. The mode-of-action of these compounds will need to be determined and may yield novel targets for germination stimulation. Thus novel seed treatments for improving germination of low vigour maize seed lots under cold conditions could be based on NCED inhibition or the action of the newly identified compounds.

The response of plants to abiotic stress factors is a major determinant of the growth and yields of crops. In this study the effects of two separate but related abiotic stress factors on elite spring barley cultivars (Horedeum vulgare ) were studied; the effects of high leaf temperatures (Tleaf) on photosynthesis rates, and the effects of high nitrogen supply on photosynthesis rates and flowering. A novel method was developed for precisely controlling Tleaf within ± 0.2ºC of the set temperature. These experiments confirmed the results of others that increasing Tleaf above 36.0ºC for 3 hours severely impaired light saturated CO2 assimilation rates (Asat) by irreversibly suppressing the activity of the C3 cycle by >80%. This suppression was not attributable to stomatal closure, the generation of ROS, or an increase in photorespiration; instead the data were consistent with the hypothesis that limitations imposed by low chloroplast ATP levels. Measurements on whole leaf ATP levels in the light and dark of control and heat stressed leaves, however, were equivocal. Whole leaf ATP levels of light adapted leaves increased with Tleaf whereas the levels in dark adapted leaves initially decreased but increased again above 38ºC; most importantly, the difference – an estimate of chloroplast ATP levels - increased with Tleaf, an observation that is not consistent with the hypothesis. The effects of high Tleaf was assessed on plants grown in soil and hydroponic solutions over a range of N-supply. Similar responses were observed regardless of the nitrogen status of the plants. Surprisingly, the unit leaf area (ULA) photosynthesis rates of control (not heat stressed) attached leaves doubled when plants were grown in 16 mM N compared with 0.6 mM N (commensurate with arable soils); detailed analysis of CO2 Assimilation vs. Internal CO2 concentration (A/Ci) curves showed the carboxylation coefficient (ϕCO2) increased suggesting the ULA capacity of the C3 cycle had been boosted and this correlated well with ULA protein levels. It seems there is a good prospect, therefore, for boosting ULA photosynthesis rates, and hence grain yield, by increasing plant N-status above that currently used in arable production. Increasing N-supply to these levels, however, has detrimental effects on the morphology and development of barley. Increasing N-supply above 0.3 mM induced tillering (increased resource sink strength) as well as yield; above 1.6 mM, however, yields began to decline, flowering was delayed, although tillering (vegetative growth) continued to proliferate. At the highest levels used (16 mM) floweing was completely suppressed; the crown meristem underwent a vegetative to reproductive transition but stem elongation was incomplete – plants rarely progress beyond the 3 node stage. A series of transcript profiling experiments were conducted to establish the mechanisms by which high N-status suppressed flowering. Analysis of the transcriptional activity of key components of the flowering pathway in leaves, coupled with observations on floral spike development suggested flowering was triggered an initiated the development of the inflorescence at the crown meristem, but high N inhibits the development of the floral primordia. A RNA-Seq experiment was undertaken to determine the transcriptome profiles of 2-3 node stage floral primordia in plants grown in 16 mM and 0.64 mM N-supply. These studies were hampered by the poor level of annotation of published barley sequences, but none-the-less several interesting candidate sequences, including a homologue of the Arabisopsis flowering gene AtAPETELLA2, were strongly down regulated; these results from these experiments are discussed in detail. Studies were also undertaken to manipulate sink strength in barley plants by reducing the number of tillers either mechanically (removal) or using ‘uniculm’ mutants from the Bowman barley accession lines. These experiments have proved to be challenging and progress has been slow; a discussion is provided on how these experiments may be completed. The ultimate goal of this project is to develop barley lines with an optimized sink strength (tiller number) that will not trigger excessive vegetative growth when plant N-status is high. This should lead to the retention of N in the leaves of the main culm leading to higher ULA photosynthesis rates and hence higher yields. To achieve this, however, these plants will have to be further manipulated so that high plant N-status does not suppress flower development.