Unlocking the genetic diversity and population structure of the newly introduced two-row spring European HerItage Barley collecTion (ExHIBiT).

In the last century, breeding programs have traditionally favoured yield-related traits, grown under high-input conditions, resulting in a loss of genetic diversity and an increased susceptibility to stresses in crops. Thus, exploiting understudied genetic resources, that potentially harbour tolerance genes, is vital for sustainable agriculture. Northern European barley germplasm has been relatively understudied despite its key role within the malting industry. The European Heritage Barley collection (ExHIBiT) was assembled to explore the genetic diversity in European barley focusing on Northern European accessions and further address environmental pressures. ExHIBiT consists of 363 spring-barley accessions, focusing on two-row type. The collection consists of landraces (~14%), old cultivars (~18%), elite cultivars (~67%) and accessions with unknown breeding history (~1%), with 70% of the collection from Northern Europe. The population structure of the ExHIBiT collection was subdivided into three main clusters primarily based on the accession's year of release using 26,585 informative SNPs based on 50k iSelect single nucleotide polymorphism (SNP) array data. Power analysis established a representative core collection of 230 genotypically and phenotypically diverse accessions. The effectiveness of this core collection for conducting statistical and association analysis was explored by undertaking genome-wide association studies (GWAS) using 24,876 SNPs for nine phenotypic traits, four of which were associated with SNPs. Genomic regions overlapping with previously characterised flowering genes (HvZTLb) were identified, demonstrating the utility of the ExHIBiT core collection for locating genetic regions that determine important traits. Overall, the ExHIBiT core collection represents the high level of untapped diversity within Northern European barley, providing a powerful resource for researchers and breeders to address future climate scenarios.

Genomic resources for a historical collection of cultivated two-row European spring barley genotypes.

Barley genomic resources are increasing rapidly, with the publication of a barley pangenome as one of the latest developments. Two-row spring barley cultivars are intensely studied as they are the source of high-quality grain for malting and distilling. Here we provide data from a European two-row spring barley population containing 209 different genotypes registered for the UK market between 1830 to 2014. The dataset encompasses RNA-sequencing data from six different tissues across a range of barley developmental stages, phenotypic datasets from two consecutive years of field-grown trials in the United Kingdom, Germany and the USA; and whole genome shotgun sequencing from all cultivars, which was used to complement the RNA-sequencing data for variant calling. The outcomes are a filtered SNP marker file, a phenotypic database and a large gene expression dataset providing a comprehensive resource which allows for downstream analyses like genome wide association studies or expression associations.

Phytosiderophore pathway response in barley exposed to iron, zinc or copper starvation.

Efficient micronutrient acquisition is a critical factor in selecting micronutrient dense crops for human consumption. Enhanced exudation and re-uptake of metal chelators, so-called phytosiderophores, by roots of graminaceous plants has been implicated in efficient micronutrient acquisition. We compared PS biosynthesis and exudation as a response mechanism to either Fe, Zn or Cu starvation. Two barley (Hordeum vulgare L.) lines with contrasting micronutrient grain yields were grown hydroponically and PS exudation (LC-MS) and root gene expression (RNAseq) were determined after either Fe, Zn, or Cu starvation. The response strength of the PS pathway was micronutrient dependent and decreased in the order Fe > Zn > Cu deficiency. We observed a stronger expression of PS pathway genes and greater PS exudation in the barley line with large micronutrient grain yield suggesting that a highly expressed PS pathway might be an important trait involved in high micronutrient accumulation. In addition to several metal specific transporters, we also found that the expression of IRO2 and bHLH156 transcription factors was not only induced under Fe but also under Zn and Cu deficiency. Our study delivers important insights into the role of the PS pathway in the acquisition of different micronutrients.

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.

Stability assessment of serum tumour markers: Calcitonin, chromogranin A, thyroglobulin and anti-thyroglobulin antibodies.

BACKGROUND: There is limited published data on the stability of calcitonin, chromogranin A, thyroglobulin and anti-thyroglobulin antibodies in serum. The aim of this study was to determine stability at three temperature conditions over 7 days, reflecting current laboratory practices. METHODS: Surplus serum was stored at room temperature, refrigerated and in the freezer; for 1, 3, 5 and 7 days. Samples were analysed in batch and analyte concentrations compared to that of a baseline sample. Measurement Uncertainty of the assay was used to determine the Maximal Permissible Difference and thus the stability of the analyte. RESULTS: Calcitonin was found to be stable for at least 7 days in the freezer but only 24 h refrigerated. Chromogranin A was stable for 3 days when refrigerated and only 24 h at room temperature. Thyroglobulin and anti-thyroglobulin antibodies were stable under all conditions for 7 days. CONCLUSION: This study has enabled the laboratory to increase the add-on time limit of Chromogranin A to 3 days, and up to 60 min for calcitonin and inform optimal storage and transportation conditions for referring specimens.

Multi-environment genome -wide association mapping of culm morphology traits in barley.

In cereals with hollow internodes, lodging resistance is influenced by morphological characteristics such as internode diameter and culm wall thickness. Despite their relevance, knowledge of the genetic control of these traits and their relationship with lodging is lacking in temperate cereals such as barley. To fill this gap, we developed an image analysis-based protocol to accurately phenotype culm diameters and culm wall thickness across 261 barley accessions. Analysis of culm trait data collected from field trials in seven different environments revealed high heritability values (>50%) for most traits except thickness and stiffness, as well as genotype-by-environment interactions. The collection was structured mainly according to row-type, which had a confounding effect on culm traits as evidenced by phenotypic correlations. Within both row-type subsets, outer diameter and section modulus showed significant negative correlations with lodging (<-0.52 and <-0.45, respectively), but no correlation with plant height, indicating the possibility of improving lodging resistance independent of plant height. Using 50k iSelect SNP genotyping data, we conducted multi-environment genome-wide association studies using mixed model approach across the whole panel and row-type subsets: we identified a total of 192 quantitative trait loci (QTLs) for the studied traits, including subpopulation-specific QTLs and 21 main effect loci for culm diameter and/or section modulus showing effects on lodging without impacting plant height. Providing insights into the genetic architecture of culm morphology in barley and the possible role of candidate genes involved in hormone and cell wall-related pathways, this work supports the potential of loci underpinning culm features to improve lodging resistance and increase barley yield stability under changing environments.

The evolutionary patterns of barley pericentromeric chromosome regions, as shaped by linkage disequilibrium and domestication.

The distribution of recombination events along large cereal chromosomes is uneven and is generally restricted to gene-rich telomeric ends. To understand how the lack of recombination affects diversity in the large pericentromeric regions, we analysed deep exome capture data from a final panel of 815 Hordeum vulgare (barley) cultivars, landraces and wild barleys, sampled from across their eco-geographical ranges. We defined and compared variant data across the pericentromeric and non-pericentromeric regions, observing a clear partitioning of diversity both within and between chromosomes and germplasm groups. Dramatically reduced diversity was found in the pericentromeres of both cultivars and landraces when compared with wild barley. We observed a mixture of completely and partially differentiated single-nucleotide polymorphisms (SNPs) between domesticated and wild gene pools, suggesting that domesticated gene pools were derived from multiple wild ancestors. Patterns of genome-wide linkage disequilibrium, haplotype block size and number, and variant frequency within blocks showed clear contrasts among individual chromosomes and between cultivars and wild barleys. Although most cultivar chromosomes shared a single major pericentromeric haplotype, chromosome 7H clearly differentiated the two-row and six-row types associated with different geographical origins. Within the pericentromeric regions we identified 22 387 non-synonymous SNPs, 92 of which were fixed for alternative alleles in cultivar versus wild accessions. Surprisingly, only 29 SNPs found exclusively in the cultivars were predicted to be 'highly deleterious'. Overall, our data reveal an unconventional pericentromeric genetic landscape among distinct barley gene pools, with different evolutionary processes driving domestication and diversification.

Trends of genetic changes uncovered by Env- and Eigen-GWAS in wheat and barley.

KEY MESSAGE: Variety age and population structure detect novel QTL for yield and adaptation in wheat and barley without the need to phenotype. The process of crop breeding over the last century has delivered new varieties with increased genetic gains, resulting in higher crop performance and yield. However, in many cases, the alleles and genomic regions underpinning this success remain unknown. This is partly due to the difficulty of generating sufficient phenotypic data on large numbers of historical varieties to enable such analyses. Here we demonstrate the ability to circumvent such bottlenecks by identifying genomic regions selected over 100 years of crop breeding using age of a variety as a surrogate for yield. Rather than collecting phenotype data, we deployed 'environmental genome-wide association scans' (EnvGWAS) based on variety age in two of the world's most important crops, wheat and barley, and detected strong signals of selection across both genomes. EnvGWAS identified 16 genomic regions in barley and 10 in wheat with contrasting patterns between spring and winter types of the two crops. To further examine changes in genome structure, we used the genomic relationship matrix of the genotypic data to derive eigenvectors for analysis in EigenGWAS. This detected seven major chromosomal introgressions that contributed to adaptation in wheat. EigenGWAS and EnvGWAS based on variety age avoid costly phenotyping and facilitate the identification of genomic tracts that have been under selection during breeding. Our results demonstrate the potential of using historical cultivar collections coupled with genomic data to identify chromosomal regions under selection and may help guide future plant breeding strategies to maximise the rate of genetic gain and adaptation.

Overcoming barriers to the registration of new plant varieties under the DUS system.

Distinctness, Uniformity and Stability (DUS) is an intellectual property system introduced in 1961 by the International Union for the Protection of New Varieties of Plants (UPOV) for safeguarding the investment and rewarding innovation in developing new plant varieties. Despite the rapid advancement in our understanding of crop biology over the past 60 years, the DUS system has changed little and is still largely dependent upon a set of morphological traits for testing candidate varieties. As the demand for more plant varieties increases, the barriers to registration of new varieties become more acute and thus require urgent review to the system. To highlight the challenges and remedies in the current system, we evaluated a comprehensive panel of 805 UK barley varieties that span the entire history of DUS testing. Our findings reveal the system deficiencies such as inconsistencies in DUS traits across environments, limitations in DUS trait combinatorial space, and inadequacies in currently available DUS markers. We advocate the concept of genomic DUS and provide evidence for a shift towards a robust genomics-enabled registration system for new crop varieties.

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.

Dissecting new genetic components of salinity tolerance in two-row spring barley at the vegetative and reproductive stages.

Soil salinity imposes an agricultural and economic burden that may be alleviated by identifying the components of salinity tolerance in barley, a major crop and the most salt tolerant cereal. To improve our understanding of these components, we evaluated a diversity panel of 377 two-row spring barley cultivars during both the vegetative, in a controlled environment, and the reproductive stages, in the field. In the controlled environment, a high-throughput phenotyping platform was used to assess the growth-related traits under both control and saline conditions. In the field, the agronomic traits were measured from plots irrigated with either fresh or saline water. Association mapping for the different components of salinity tolerance enabled us to detect previously known associations, such as HvHKT1;5. Using an "interaction model", which took into account the interaction between treatment (control and salt) and genetic markers, we identified several loci associated with yield components related to salinity tolerance. We also observed that the two developmental stages did not share genetic regions associated with the components of salinity tolerance, suggesting that different mechanisms play distinct roles throughout the barley life cycle. Our association analysis revealed that genetically defined regions containing known flowering genes (Vrn-H3, Vrn-H1, and HvNAM-1) were responsive to salt stress. We identified a salt-responsive locus (7H, 128.35 cM) that was associated with grain number per ear, and suggest a gene encoding a vacuolar H+-translocating pyrophosphatase, HVP1, as a candidate. We also found a new QTL on chromosome 3H (139.22 cM), which was significant for ear number per plant, and a locus on chromosome 2H (141.87 cM), previously identified using a nested association mapping population, which associated with a yield component and interacted with salinity stress. Our study is the first to evaluate a barley diversity panel for salinity stress under both controlled and field conditions, allowing us to identify contributions from new components of salinity tolerance which could be used for marker-assisted selection when breeding for marginal and saline regions.

Association mapping of malting quality traits in UK spring and winter barley cultivar collections.

KEY MESSAGE: Historical malting quality data was collated from UK national and recommended list trial data and used in a GWAS. 25 QTL were identified, with the majority from spring barley cultivar sets. In Europe, the most economically significant use of barley is the production of malt for use in the brewing and distilling industries. As such, selection for traits related to malting quality is of great commercial interest. In order to study the genetic basis of variation for malting quality traits in UK cultivars, a historical set of trial data was collated from national and recommended list trials from the period 1988 to 2016. This data was used to estimate variety means for 20 quality related traits in 451 spring barley cultivars, and 407 winter cultivars. Genotypes for these cultivars were generated using iSelect 9k and 50k genotyping platforms, and a genome wide association scan performed to identify malting quality quantitative trait loci (QTL). 24 QTL were identified in spring barley cultivars, and 2 from the winter set. A number of these correspond to known malting quality related genes but the remainder represents novel genetic variation that is accessible to breeders for the genetic improvement of new cultivars.

Barley sodium content is regulated by natural variants of the Na+ transporter HvHKT1;5.

During plant growth, sodium (Na+) in the soil is transported via the xylem from the root to the shoot. While excess Na+ is toxic to most plants, non-toxic concentrations have been shown to improve crop yields under certain conditions, such as when soil K+ is low. We quantified grain Na+ across a barley genome-wide association study panel grown under non-saline conditions and identified variants of a Class 1 HIGH-AFFINITY-POTASSIUM-TRANSPORTER (HvHKT1;5)-encoding gene responsible for Na+ content variation under these conditions. A leucine to proline substitution at position 189 (L189P) in HvHKT1;5 disturbs its characteristic plasma membrane localisation and disrupts Na+ transport. Under low and moderate soil Na+, genotypes containing HvHKT1:5P189 accumulate high concentrations of Na+ but exhibit no evidence of toxicity. As the frequency of HvHKT1:5P189 increases significantly in cultivated European germplasm, we cautiously speculate that this non-functional variant may enhance yield potential in non-saline environments, possibly by offsetting limitations of low available K+.

Segmental duplications are hot spots of copy number variants affecting barley gene content.

Copy number variants (CNVs) are pervasive in several animal and plant genomes and contribute to shaping genetic diversity. In barley, there is evidence that changes in gene copy number underlie important agronomic traits. The recently released reference sequence of barley represents a valuable genomic resource for unveiling the incidence of CNVs that affect gene content and for identifying sequence features associated with CNV formation. Using exome sequencing and read count data, we detected 16 605 deletions and duplications that affect barley gene content by surveying a diverse panel of 172 cultivars, 171 landraces, 22 wild relatives and other 32 uncategorized domesticated accessions. The quest for segmental duplications (SDs) in the reference sequence revealed many low-copy repeats, most of which overlap predicted coding sequences. Statistical analyses revealed that the incidence of CNVs increases significantly in SD-rich regions, indicating that these sequence elements act as hot spots for the formation of CNVs. The present study delivers a comprehensive genome-wide study of CNVs affecting barley gene content and implicates SDs in the molecular mechanisms that lead to the formation of this class of CNVs.

Assessing the variation in manganese use efficiency traits in Scottish barley landrace Bere (Hordeum vulgare L.).

BACKGROUND AND AIMS: Manganese (Mn) deficiency in barley is a global problem. It is difficult to detect in the early stages of symptom development and is commonly pre-emptively corrected by Mn foliar sprays that can be costly. Landraces adapted to marginal lands around the world represent a genetic resource for potential sustainability traits including mineral use efficiency. This research aims to confirm novel Mn use efficiency traits from the Scottish landrace Bere and use an association mapping approach to identify genetic loci associated with the trait. METHODS: A hydroponic system was developed to identify and characterize the Mn deficiency tolerance traits in a collection of landraces, including a large number of Scottish Bere barleys, a group of six-rowed heritage landraces grown in the highlands and islands of Scotland. Measuring chlorophyll fluorescence, the effect of Mn deficiency was identified in the early stages of development. Genotypic data, generated using the 50k Illumina iSelect genotyping array, were coupled with the Mn phenotypic data to create a genome-wide association study (GWAS) identifying candidate loci associated with Mn use efficiency. KEY RESULTS: The Bere lines generally had good Mn use efficiency traits. Individual Bere lines showed large efficiencies, with some Bere lines recording almost double chlorophyll fluorescence readings in limited Mn conditions compared with the elite cultivar Scholar. The Mn-efficient Bere lines had increased accumulation of Mn in their shoot biomass compared with elite cultivars, which was highly correlated to the chlorophyll fluorescence. Several candidate genes were identified as being associated with Mn use efficiency in the GWAS. CONCLUSIONS: Several genomic regions for Mn use efficiency traits originating from the Bere lines were identified. Further examination and validation of these regions should be undertaken to identify candidate genes for future breeding for marginal lands.

Barley grain (1,3;1,4)-ß-glucan content: effects of transcript and sequence variation in genes encoding the corresponding synthase and endohydrolase enzymes.

The composition of plant cell walls is important in determining cereal end uses. Unlike other widely consumed cereal grains barley is comparatively rich in (1,3;1,4)-ß-glucan, a source of dietary fibre. Previous work showed Cellulose synthase-like genes synthesise (1,3;1,4)-ß-glucan in several tissues. HvCslF6 encodes a grain (1,3;1,4)-ß-glucan synthase, whereas the function of HvCslF9 is unknown. Here, the relationship between mRNA levels of HvCslF6, HvCslF9, HvGlbI (1,3;1,4)-ß-glucan endohydrolase, and (1,3;1,4)-ß-glucan content was studied in developing grains of four barley cultivars. HvCslF6 was differentially expressed during mid (8-15 DPA) and late (38 DPA) grain development stages while HvCslF9 transcript was only clearly detected at 8-10 DPA. A peak of HvGlbI expression was detected at 15 DPA. Differences in transcript abundance across the three genes could partially explain variation in grain (1,3;1,4)-ß-glucan content in these genotypes. Remarkably narrow sequence variation was found within the HvCslF6 promoter and coding sequence and does not explain variation in (1,3;1,4)-ß-glucan content. Our data emphasise the genotype-dependent accumulation of (1,3;1,4)-ß-glucan during barley grain development and a role for the balance between hydrolysis and synthesis in determining (1,3;1,4)-ß-glucan content, and suggests that other regulatory sequences or proteins are likely to be involved in this trait in developing grain.

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.

Exome sequences and multi-environment field trials elucidate the genetic basis of adaptation in barley.

Broadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi-environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype-by-environment (G×E) modelling. Sub-populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock-related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large-effect alleles. Our analysis supports a gene-level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.

Tracing the ancestry of modern bread wheats.

For more than 10,000 years, the selection of plant and animal traits that are better tailored for human use has shaped the development of civilizations. During this period, bread wheat (Triticum aestivum) emerged as one of the world's most important crops. We use exome sequencing of a worldwide panel of almost 500 genotypes selected from across the geographical range of the wheat species complex to explore how 10,000 years of hybridization, selection, adaptation and plant breeding has shaped the genetic makeup of modern bread wheats. We observe considerable genetic variation at the genic, chromosomal and subgenomic levels, and use this information to decipher the likely origins of modern day wheats, the consequences of range expansion and the allelic variants selected since its domestication. Our data support a reconciled model of wheat evolution and provide novel avenues for future breeding improvement.