Heritage genetics for adaptation to marginal soils in barley.

Future crops need to be sustainable in the face of climate change. Modern barley varieties have been bred for high productivity and quality; however, they have suffered considerable genetic erosion, losing crucial genetic diversity. This renders modern cultivars vulnerable to climate change and stressful environments. We highlight the potential to tailor crops to a specific environment by utilising diversity inherent in an adapted landrace population. Tapping into natural biodiversity, while incorporating information about local environmental and climatic conditions, allows targeting of key traits and genotypes, enabling crop production in marginal soils. We outline future directions for the utilisation of genetic resources maintained in landrace collections to support sustainable agriculture through germplasm development via the use of genomics technologies and big data.

Defining Composition and Function of the Rhizosphere Microbiota of Barley Genotypes Exposed to Growth-Limiting Nitrogen Supplies.

The microbiota populating the rhizosphere, the interface between roots and soil, can modulate plant growth, development, and health. These microbial communities are not stochastically assembled from the surrounding soil, but their composition and putative function are controlled, at least partially, by the host plant. Here, we use the staple cereal barley as a model to gain novel insights into the impact of differential applications of nitrogen, a rate-limiting step for global crop production, on the host genetic control of the rhizosphere microbiota. Using a high-throughput amplicon sequencing survey, we determined that nitrogen availability for plant uptake is a factor promoting the selective enrichment of individual taxa in the rhizosphere of wild and domesticated barley genotypes. Shotgun sequencing and metagenome-assembled genomes revealed that this taxonomic diversification is mirrored by a functional specialization, manifested by the differential enrichment of multiple Gene Ontology terms, of the microbiota of plants exposed to nitrogen conditions limiting barley growth. Finally, a plant soil feedback experiment revealed that host control of the barley microbiota underpins the assembly of a phylogenetically diverse group of bacteria putatively required to sustain plant performance under nitrogen-limiting supplies. Taken together, our observations indicate that under nitrogen conditions limiting plant growth, host-microbe and microbe-microbe interactions fine-tune the host genetic selection of the barley microbiota at both taxonomic and functional levels. The disruption of these recruitment cues negatively impacts plant growth. IMPORTANCE The microbiota inhabiting the rhizosphere, the thin layer of soil surrounding plant roots, can promote the growth, development, and health of their host plants. Previous research indicated that differences in the genetic composition of the host plant coincide with variations in the composition of the rhizosphere microbiota. This is particularly evident when looking at the microbiota associated with input-demanding modern cultivated varieties and their wild relatives, which have evolved under marginal conditions. However, the functional significance of these differences remains to be fully elucidated. We investigated the rhizosphere microbiota of wild and cultivated genotypes of the global crop barley and determined that nutrient conditions limiting plant growth amplify the host control on microbes at the root-soil interface. This is reflected in a plant- and genotype-dependent functional specialization of the rhizosphere microbiota, which appears to be required for optimal plant growth. These findings provide novel insights into the significance of the rhizosphere microbiota for plant growth and sustainable agriculture.

Identifying plant genes shaping microbiota composition in the barley rhizosphere.

A prerequisite to exploiting soil microbes for sustainable crop production is the identification of the plant genes shaping microbiota composition in the rhizosphere, the interface between roots and soil. Here, we use metagenomics information as an external quantitative phenotype to map the host genetic determinants of the rhizosphere microbiota in wild and domesticated genotypes of barley, the fourth most cultivated cereal globally. We identify a small number of loci with a major effect on the composition of rhizosphere communities. One of those, designated the QRMC-3HS, emerges as a major determinant of microbiota composition. We subject soil-grown sibling lines harbouring contrasting alleles at QRMC-3HS and hosting contrasting microbiotas to comparative root RNA-seq profiling. This allows us to identify three primary candidate genes, including a Nucleotide-Binding-Leucine-Rich-Repeat (NLR) gene in a region of structural variation of the barley genome. Our results provide insights into the footprint of crop improvement on the plant's capacity of shaping rhizosphere microbes.

Applications of the indole-alkaloid gramine modulate the assembly of individual members of the barley rhizosphere microbiota.

Microbial communities proliferating at the root-soil interface, collectively referred to as the rhizosphere microbiota, represent an untapped beneficial resource for plant growth, development and health. Integral to a rational manipulation of the microbiota for sustainable agriculture is the identification of the molecular determinants of these communities. In plants, biosynthesis of allelochemicals is centre stage in defining inter-organismal relationships in the environment. Intriguingly, this process has been moulded by domestication and breeding selection. The indole-alkaloid gramine, whose occurrence in barley (Hordeum vulgare L.) is widespread among wild genotypes but has been counter selected in several modern varieties, is a paradigmatic example of this phenomenon. This prompted us to investigate how exogenous applications of gramine impacted on the rhizosphere microbiota of two, gramine-free, elite barley varieties grown in a reference agricultural soil. High throughput 16S rRNA gene amplicon sequencing revealed that applications of gramine interfere with the proliferation of a subset of soil microbes with a relatively broad phylogenetic assignment. Strikingly, growth of these bacteria appeared to be rescued by barley plants in a genotype- and dosage-independent manner. In parallel, we discovered that host recruitment cues can interfere with the impact of gramine application in a host genotype-dependent manner. Interestingly, this latter effect displayed a bias for members of the phyla Proteobacteria. These initial observations indicate that gramine can act as a determinant of the prokaryotic communities inhabiting the root-soil interface.

Draft genome assemblies for tree pathogens Phytophthora pseudosyringae and Phytophthora boehmeriae.

Species of Phytophthora, plant pathogenic eukaryotic microbes, can cause disease on many tree species. Genome sequencing of species from this genus has helped to determine components of their pathogenicity arsenal. Here, we sequenced genomes for two widely distributed species, Phytophthora pseudosyringae and Phytophthora boehmeriae, yielding genome assemblies of 49 and 40 Mb, respectively. We identified more than 270 candidate disease promoting RXLR effector coding genes for each species, and hundreds of genes encoding candidate plant cell wall degrading carbohydrate active enzymes (CAZymes). These data boost genome sequence representation across the Phytophthora genus, and form resources for further study of Phytophthora pathogenesis.

Haustorium formation and a distinct biotrophic transcriptome characterize infection of Nicotiana benthamiana by the tree pathogen Phytophthora kernoviae.

Phytophthora species cause some of the most serious diseases of trees and threaten forests in many parts of the world. Despite the generation of genome sequence assemblies for over 10 tree-pathogenic Phytophthora species and improved detection methods, there are many gaps in our knowledge of how these pathogens interact with their hosts. To facilitate cell biology studies of the infection cycle we examined whether the tree pathogen Phytophthora kernoviae could infect the model plant Nicotiana benthamiana. We transformed P. kernoviae to express green fluorescent protein (GFP) and demonstrated that it forms haustoria within infected N. benthamiana cells. Haustoria were also formed in infected cells of natural hosts, Rhododendron ponticum and European beech (Fagus sylvatica). We analysed the transcriptome of P. kernoviae in cultured mycelia, spores, and during infection of N. benthamiana, and detected 12,559 transcripts. Of these, 1,052 were predicted to encode secreted proteins, some of which may function as effectors to facilitate disease development. From these, we identified 87 expressed candidate RXLR (Arg-any amino acid-Leu-Arg) effectors. We transiently expressed 12 of these as GFP fusions in N. benthamiana leaves and demonstrated that nine significantly enhanced P. kernoviae disease progression and diversely localized to the cytoplasm, nucleus, nucleolus, and plasma membrane. Our results show that N. benthamiana can be used as a model host plant for studying this tree pathogen, and that the interaction likely involves suppression of host immune responses by RXLR effectors. These results establish a platform to expand the understanding of Phytophthora tree diseases.

Chitosan primes plant defence mechanisms against Botrytis cinerea, including expression of Avr9/Cf-9 rapidly elicited genes.

Current crop protection strategies against the fungal pathogen Botrytis cinerea rely on a combination of conventional fungicides and host genetic resistance. However, due to pathogen evolution and legislation in the use of fungicides, these strategies are not sufficient to protect plants against this pathogen. Defence elicitors can stimulate plant defence mechanisms through a phenomenon known as defence priming. Priming results in a faster and/or stronger expression of resistance upon pathogen recognition by the host. This work aims to study defence priming by a commercial formulation of the elicitor chitosan. Treatments with chitosan result in induced resistance (IR) in solanaceous and brassicaceous plants. In tomato plants, enhanced resistance has been linked with priming of callose deposition and accumulation of the plant hormone jasmonic acid (JA). Large-scale transcriptomic analysis revealed that chitosan primes gene expression at early time-points after infection. In addition, two novel tomato genes with a characteristic priming profile were identified, Avr9/Cf-9 rapidly elicited protein 75 (ACRE75) and 180 (ACRE180). Transient and stable over-expression of ACRE75, ACRE180 and their Nicotiana benthamiana homologs, revealed that they are positive regulators of plant resistance against B. cinerea. This provides valuable information in the search for strategies to protect Solanaceae plants against B. cinerea.

Light Regulation of Chlorophyll and Glycoalkaloid Biosynthesis During Tuber Greening of Potato S. tuberosum.

Potato, S. tuberosum, is one of the most important global crops, but has high levels of waste due to tuber greening under light, which is associated with the accumulation of neurotoxic glycoalkaloids. However, unlike the situation in de-etiolating seedlings, the mechanisms underlying tuber greening are not well understood. Here, we have investigated the effect of monochromatic blue, red, and far-red light on the regulation of chlorophyll and glycoalkaloid accumulation in potato tubers. Blue and red wavelengths were effective for induction and accumulation of chlorophyll, carotenoids and the two major potato glycoalkaloids, α-solanine and α-chaconine, whereas none of these accumulated in darkness or under far-red light. Key genes in chlorophyll biosynthesis (HEMA1, encoding the rate-limiting enzyme glutamyl-tRNA reductase, GSA, CHLH and GUN4) and six genes (HMG1, SQS, CAS1, SSR2, SGT1 and SGT2) required for glycoalkaloid synthesis were also induced under white, blue, and red light but not in darkness or under far-red light. These data suggest a role for both cryptochrome and phytochrome photoreceptors in chlorophyll and glycoalkaloid accumulation. The contribution of phytochrome was further supported by the observation that far-red light could inhibit white light-induced chlorophyll and glycoalkaloid accumulation and associated gene expression. Transcriptomic analysis of tubers exposed to white, blue, and red light showed that light induction of photosynthesis and tetrapyrrole-related genes grouped into three distinct groups with one group showing a generally progressive induction by light at both 6 h and 24 h, a second group showing induction at 6 h in all light treatments, but induction only by red and white light at 24 h and a third showing just a very moderate light induction at 6 h which was reduced to the dark control level at 24 h. All glycoalkaloid synthesis genes showed a group one profile consistent with what was seen for the most light regulated chlorophyll synthesis genes. Our data provide a molecular framework for developing new approaches to reducing waste due to potato greening.

A footprint of plant eco-geographic adaptation on the composition of the barley rhizosphere bacterial microbiota.

The microbiota thriving in the rhizosphere, the thin layer of soil surrounding plant roots, plays a critical role in plant's adaptation to the environment. Domestication and breeding selection have progressively differentiated the microbiota of modern crops from the ones of their wild ancestors. However, the impact of eco-geographical constraints faced by domesticated plants and crop wild relatives on recruitment and maintenance of the rhizosphere microbiota remains to be fully elucidated. Here we performed a comparative 16S rRNA gene survey of the rhizosphere of 4 domesticated and 20 wild barley (Hordeum vulgare) genotypes grown in an agricultural soil under controlled environmental conditions. We demonstrated the enrichment of individual bacteria mirrored the distinct eco-geographical constraints faced by their host plants. Unexpectedly, Elite varieties exerted a stronger genotype effect on the rhizosphere microbiota when compared with wild barley genotypes adapted to desert environments with a preferential enrichment for members of Actinobacteria. Finally, in wild barley genotypes, we discovered a limited, but significant, correlation between microbiota diversity and host genomic diversity. Our results revealed a footprint of the host's adaptation to the environment on the assembly of the bacteria thriving at the root-soil interface. In the tested conditions, this recruitment cue layered atop of the distinct evolutionary trajectories of wild and domesticated plants and, at least in part, is encoded by the barley genome. This knowledge will be critical to design experimental approaches aimed at elucidating the recruitment cues of the barley microbiota across a range of soil types.

TERMINAL FLOWER-1/CENTRORADIALIS inhibits tuberisation via protein interaction with the tuberigen activation complex.

Potato tuber formation is a secondary developmental programme by which cells in the subapical stolon region divide and radially expand to further differentiate into starch-accumulating parenchyma. Although some details of the molecular pathway that signals tuberisation are known, important gaps in our knowledge persist. Here, the role of a member of the TERMINAL FLOWER 1/CENTRORADIALIS gene family (termed StCEN) in the negative control of tuberisation is demonstrated for what is thought to be the first time. It is shown that reduced expression of StCEN accelerates tuber formation whereas transgenic lines overexpressing this gene display delayed tuberisation and reduced tuber yield. Protein-protein interaction studies (yeast two-hybrid and bimolecular fluorescence complementation) demonstrate that StCEN binds components of the recently described tuberigen activation complex. Using transient transactivation assays, we show that the StSP6A tuberisation signal is an activation target of the tuberigen activation complex, and that co-expression of StCEN blocks activation of the StSP6A gene by StFD-Like-1. Transcriptomic analysis of transgenic lines misexpressing StCEN identifies early transcriptional events in tuber formation. These results demonstrate that StCEN suppresses tuberisation by directly antagonising the function of StSP6A in stolons, identifying StCEN as a breeding marker to improve tuber initiation and yield through the selection of genotypes with reduced StCEN expression.

TRA1: A Locus Responsible for Controlling Agrobacterium-Mediated Transformability in Barley.

In barley (Hordeum vulgare L.), Agrobacterium-mediated transformation efficiency is highly dependent on genotype with very few cultivars being amenable to transformation. Golden Promise is the cultivar most widely used for barley transformation and developing embryos are the most common donor tissue. We tested whether barley mutants with abnormally large embryos were more or less amenable to transformation and discovered that mutant M1460 had a transformation efficiency similar to that of Golden Promise. The large-embryo phenotype of M1460 is due to mutation at the LYS3 locus. There are three other barley lines with independent mutations at the same LYS3 locus, and one of these, Risø1508 has an identical missense mutation to that in M1460. However, none of the lys3 mutants except M1460 were transformable showing that the locus responsible for transformation efficiency, TRA1, was not LYS3 but another locus unique to M1460. To identify TRA1, we generated a segregating population by crossing M1460 to the cultivar Optic, which is recalcitrant to transformation. After four rounds of backcrossing to Optic, plants were genotyped and their progeny were tested for transformability. Some of the progeny lines were transformable at high efficiencies similar to those seen for the parent M1460 and some were not transformable, like Optic. A region on chromosome 2H inherited from M1460 is present in transformable lines only. We propose that one of the 225 genes in this region is TRA1.

Physiological, Biochemical, and Transcriptional Responses to Single and Combined Abiotic Stress in Stress-Tolerant and Stress-Sensitive Potato Genotypes.

Potato production is often constrained by abiotic stresses such as drought and high temperatures which are often present in combination. In the present work, we aimed to identify key mechanisms and processes underlying single and combined abiotic stress tolerance by comparative analysis of tolerant and susceptible cultivars. Physiological data indicated that the cultivars Desiree and Unica were stress tolerant while Agria and Russett Burbank were stress susceptible. Abiotic stress caused a greater reduction of photosynthetic carbon assimilation in the susceptible cultivars which was associated with a lower leaf transpiration rate. Oxidative stress, as estimated by the accumulation of malondialdehyde was not induced by stress treatments in any of the genotypes with the exception of drought stress in Russett Burbank. Stress treatment resulted in increases in ascorbate peroxidase activity in all cultivars except Agria which increased catalase activity in response to stress. Transcript profiling highlighted a decrease in the abundance of transcripts encoding proteins associated with PSII light harvesting complex in stress tolerant cultivars. Furthermore, stress tolerant cultivars accumulated fewer transcripts encoding a type-1 metacaspase implicated in programmed cell death. Stress tolerant cultivars exhibited stronger expression of genes associated with plant growth and development, hormone metabolism and primary and secondary metabolism than stress susceptible cultivars. Metabolite profiling revealed accumulation of proline in all genotypes following drought stress that was partially suppressed in combined heat and drought. On the contrary, the sugar alcohols inositol and mannitol were strongly accumulated under heat and combined heat and drought stress while galactinol was most strongly accumulated under drought. Combined heat and drought also resulted in the accumulation of Valine, isoleucine, and lysine in all genotypes. These data indicate that single and multiple abiotic stress tolerance in potato is associated with a maintenance of CO2 assimilation and protection of PSII by a reduction of light harvesting capacity. The data further suggests that stress tolerant cultivars suppress cell death and maintain growth and development via fine tuning of hormone signaling, and primary and secondary metabolism. This study highlights potential targets for the development of stress tolerant potato cultivars.

Barley Anther and Meiocyte Transcriptome Dynamics in Meiotic Prophase I.

In flowering plants, successful germinal cell development and meiotic recombination depend upon a combination of environmental and genetic factors. To gain insights into this specialized reproductive development program we used short- and long-read RNA-sequencing (RNA-seq) to study the temporal dynamics of transcript abundance in immuno-cytologically staged barley (Hordeum vulgare) anthers and meiocytes. We show that the most significant transcriptional changes in anthers occur at the transition from pre-meiosis to leptotene-zygotene, which is followed by increasingly stable transcript abundance throughout prophase I into metaphase I-tetrad. Our analysis reveals that the pre-meiotic anthers are enriched in long non-coding RNAs (lncRNAs) and that entry to meiosis is characterized by their robust and significant down regulation. Intriguingly, only 24% of a collection of putative meiotic gene orthologs showed differential transcript abundance in at least one stage or tissue comparison. Argonautes, E3 ubiquitin ligases, and lys48 specific de-ubiquitinating enzymes were enriched in prophase I meiocyte samples. These developmental, time-resolved transcriptomes demonstrate remarkable stability in transcript abundance in meiocytes throughout prophase I after the initial and substantial reprogramming at meiosis entry and the complexity of the regulatory networks involved in early meiotic processes.

BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq.

BACKGROUND: The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. RESULTS: A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts - BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427-433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20-28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5' and 3' UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. CONCLUSION: A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.

A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing.

BACKGROUND: We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates. RESULTS: Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations. CONCLUSIONS: We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.

Identification of TIMING OF CAB EXPRESSION 1 as a temperature-sensitive negative regulator of tuberization in potato.

For many potato cultivars, tuber yield is optimal at average daytime temperatures in the range 14-22 °C. Above this range, tuber yield is reduced for most cultivars. We previously reported that moderately elevated temperature increases steady-state expression of the core circadian clock gene TIMING OF CAB EXPRESSION 1 (StTOC1) in developing tubers, whereas expression of the StSP6A tuberization signal is reduced, along with tuber yield. In this study we provide evidence that StTOC1 links environmental signalling with potato tuberization by suppressing StSP6A autoactivation in the stolons. We show that transgenic lines silenced in StTOC1 expression exhibit enhanced StSP6A transcript levels and changes in gene expression in developing tubers that are indicative of an elevated sink strength. Nodal cuttings of StTOC1 antisense lines displayed increased tuber yields at moderately elevated temperatures, whereas tuber yield and StSP6A expression were reduced in StTOC1 overexpressor lines. Here we identify a number of StTOC1 binding partners and demonstrate that suppression of StSP6A expression is independent of StTOC1 complex formation with the potato homolog StPIF3. Down-regulation of StTOC1 thus provides a strategy to mitigate the effects of elevated temperature on tuber yield.

Development and Quality of Barley Husk Adhesion Correlates With Changes in Caryopsis Cuticle Biosynthesis and Composition.

The caryopses of barley become firmly adhered to the husk during grain development through a cuticular cementing layer on the caryopsis surface. The degree of this attachment varies among cultivars, with poor quality adhesion causing "skinning", an economically significant grain quality defect for the malting industry. Malting cultivars encompassing a range of husk adhesion qualities were grown under a misting treatment known to induce skinning. Development of the cementing layer was examined by electron microscopy and compositional changes of the cementing layer were investigated with gas-chromatography followed by mass spectroscopy. Changes in gene expression during adhesion development were examined with a custom barley microarray. The abundance of transcripts involved early in cuticular lipid biosynthesis, including those encoding acetyl-CoA carboxylase, and all four members of the fatty acid elongase complex of enzymes, was significantly higher earlier in caryopsis development than later. Genes associated with subsequent cuticular lipid biosynthetic pathways were also expressed higher early in development, including the decarbonylation and reductive pathways, and sterol biosynthesis. Changes in cuticular composition indicate that lowered proportions of alkanes and higher proportions of fatty acids are associated with development of good quality husk adhesion, in addition to higher proportions of sterols.

A Comparison of Mainstream Genotyping Platforms for the Evaluation and Use of Barley Genetic Resources.

We compared the performance of two commonly used genotyping platforms, genotyping-by-sequencing (GBS) and single nucleotide polymorphism-arrays (SNP), to investigate the extent and pattern of genetic variation within a collection of 1,000 diverse barley genotypes selected from the German Federal ex situ GenBank hosted at IPK Gatersleben. Each platform revealed equivalent numbers of robust bi-allelic SNPs (39,733 and 37,930 SNPs for the 50K SNP-array and GBS datasets respectively). A small overlap of 464 SNPs was common to both platforms, indicating that the methodologies we used selectively access informative polymorphism in different portions of the barley genome. Approximately half of the GBS dataset was comprised of SNPs with minor allele frequencies (MAFs) below 1%, illustrating the power of GBS to detect rare alleles in diverse germplasm collections. While desired for certain applications, the highly robust calling of alleles at the same SNPs across multiple populations is an advantage of the SNP-array, allowing direct comparisons of data from related or unrelated studies. Overall MAFs and diversity statistics (π) were higher for the SNP-array data, potentially reflecting the conscious removal of markers with a low MAF in the ascertainment population. A comparison of similarity matrices revealed a positive correlation between both approaches, supporting the validity of using either for entire GenBank characterization. To explore the potential of each dataset for focused genetic analyses we explored the outcomes of their use in genome-wide association scans for row type, growth habit and non-adhering hull, and discriminant analysis of principal components for the drivers of sub-population differentiation. Interpretation of the results from both types of analysis yielded broadly similar conclusions indicating that choice of platform used for such analyses should be determined by the research question being asked, group preferences and their capabilities to extract and interpret the different types of output data easily and quickly. Access to the requisite infrastructure for running, processing, analyzing, querying, storing, and displaying either datatype is an additional consideration. Our investigations reveal that for barley the cost per genotyping assay is less for SNP-arrays than GBS, which translates to a cost per informative datapoint being significantly lower for the SNP-array.

Metabarcoding reveals a high diversity of woody host-associated Phytophthora spp. in soils at public gardens and amenity woodlands in Britain.

Forests and woodlands worldwide are being severely impacted by invasive Phytophthora species, with initial outbreaks in some cases occurring on host trees located in public parks and gardens. These highly disturbed sites with diverse planting practices may indeed act as harbours for invasive Phytophthora pathogens which are particularly well adapted to surviving in soil. High throughput Illumina sequencing was used to analyse Phytophthora species diversity in soil samples collected from 14 public garden/amenity woodland sites in northern Britain. Bioinformatic analyses revealed some limitations to using internal transcribed spacer as the barcode region; namely reporting of false positives and ambiguous species matches. Taking this into account, 35 distinct sequences were amplified across the sites, corresponding to 23 known Phytophthora species as well as twelve oomycete sequences with no match to any known Phytophthora species. Phytophthora pseudosyringae and P. austrocedri, both of which cause serious damage to trees and are regarded as fairly recent introductions to Britain, were the two most abundant Phytophthora species detected. There was no evidence that any of the detected Phytophthora species were more associated with any one type of host, healthy or otherwise. This study has demonstrated the ubiquity and diversity of Phytophthora species endemic in highly managed, extensively planted soil environments in Britain. Suggested improvements to the methodology and the practical implications of the findings in terms of mitigating Phytophthora spread and impact are discussed.