Genome-wide association of yield traits in a nested association mapping population of barley reveals new gene diversity for future breeding.

To explore wild barley as a source of useful alleles for yield improvement in breeding, we have carried out a genome-wide association scan using the nested association mapping population HEB-25, which contains 25 diverse exotic barley genomes superimposed on an ~70% genetic background of cultivated barley. A total of 1420 HEB-25 lines were trialled for nine yield-related grain traits for 2 years in Germany and Scotland, with varying N fertilizer application. The phenotypic data were related to genotype scores for 5398 gene-based single nucleotide polymorphism (SNP) markers. A total of 96 quantitative trait locus (QTL) regions were identified across all measured traits, the majority of which co-localize with known major genes controlling flowering time (Ppd-H2, HvCEN, HvGI, VRN-H1, and VRN-H3) and spike morphology (VRS3, VRS1, VRS4, and INT-C) in barley. Fourteen QTL hotspots, with at least three traits coinciding, were also identified, several of which co-localize with barley orthologues of genes controlling grain dimensions in rice. Most of the allele effects are specific to geographical location and/or exotic parental genotype. This study shows the existence of beneficial alleles for yield-related traits in exotic barley germplasm and provides candidate alleles for future improvement of these traits by the breeder.

Contrasting genetic regulation of plant development in wild barley grown in two European environments revealed by nested association mapping.

Barley is cultivated more widely than the other major world crops because it adapts well to environmental constraints, such as drought, heat, and day length. To better understand the genetic control of local adaptation in barley, we studied development in the nested association mapping population HEB-25, derived from crossing 25 wild barley accessions with the cultivar 'Barke'. HEB-25 was cultivated in replicated field trials in Dundee (Scotland) and Halle (Germany), differing in regard to day length, precipitation, and temperature. Applying a genome-wide association study, we located 60 and 66 quantitative trait locus (QTL) regions regulating eight plant development traits in Dundee and Halle, respectively. A number of QTLs could be explained by known major genes such as PHOTOPERIOD 1 (Ppd-H1) and FLOWERING LOCUS T (HvFT-1) that regulate plant development. In addition, we observed that developmental traits in HEB-25 were partly controlled via genotype × environment and genotype × donor interactions, defined as location-specific and family-specific QTL effects. Our findings indicate that QTL alleles are available in the wild barley gene pool that show contrasting effects on plant development, which may be deployed to improve adaptation of cultivated barley to future environmental changes.

Chromatin state analysis of the barley epigenome reveals a higher-order structure defined by H3K27me1 and H3K27me3 abundance.

Combinations of histones carrying different covalent modifications are a major component of epigenetic variation. We have mapped nine modified histones in the barley seedling epigenome by chromatin immunoprecipitation next-generation sequencing (ChIP-seq). The chromosomal distributions of the modifications group them into four different classes, and members of a given class also tend to coincide at the local DNA level, suggesting that global distribution patterns reflect local epigenetic environments. We used this peak sharing to define 10 chromatin states representing local epigenetic environments in the barley genome. Five states map mainly to genes and five to intergenic regions. Two genic states involving H3K36me3 are preferentially associated with constitutive gene expression, while an H3K27me3-containing genic state is associated with differentially expressed genes. The 10 states display striking distribution patterns that divide barley chromosomes into three distinct global environments. First, telomere-proximal regions contain high densities of H3K27me3 covering both genes and intergenic DNA, together with very low levels of the repressive H3K27me1 modification. Flanking these are gene-rich interior regions that are rich in active chromatin states and have greatly decreased levels of H3K27me3 and increasing amounts of H3K27me1 and H3K9me2. Lastly, H3K27me3-depleted pericentromeric regions contain gene islands with active chromatin states separated by extensive retrotransposon-rich regions that are associated with abundant H3K27me1 and H3K9me2 modifications. We propose an epigenomic framework for barley whereby intergenic H3K27me3 specifies facultative heterochromatin in the telomere-proximal regions and H3K27me1 is diagnostic for constitutive heterochromatin elsewhere in the barley genome.

An investigation of causes of false positive single nucleotide polymorphisms using simulated reads from a small eukaryote genome.

BACKGROUND: Single Nucleotide Polymorphisms (SNPs) are widely used molecular markers, and their use has increased massively since the inception of Next Generation Sequencing (NGS) technologies, which allow detection of large numbers of SNPs at low cost. However, both NGS data and their analysis are error-prone, which can lead to the generation of false positive (FP) SNPs. We explored the relationship between FP SNPs and seven factors involved in mapping-based variant calling - quality of the reference sequence, read length, choice of mapper and variant caller, mapping stringency and filtering of SNPs by read mapping quality and read depth. This resulted in 576 possible factor level combinations. We used error- and variant-free simulated reads to ensure that every SNP found was indeed a false positive. RESULTS: The variation in the number of FP SNPs generated ranged from 0 to 36,621 for the 120 million base pairs (Mbp) genome. All of the experimental factors tested had statistically significant effects on the number of FP SNPs generated and there was a considerable amount of interaction between the different factors. Using a fragmented reference sequence led to a dramatic increase in the number of FP SNPs generated, as did relaxed read mapping and a lack of SNP filtering. The choice of reference assembler, mapper and variant caller also significantly affected the outcome. The effect of read length was more complex and suggests a possible interaction between mapping specificity and the potential for contributing more false positives as read length increases. CONCLUSIONS: The choice of tools and parameters involved in variant calling can have a dramatic effect on the number of FP SNPs produced, with particularly poor combinations of software and/or parameter settings yielding tens of thousands in this experiment. Between-factor interactions make simple recommendations difficult for a SNP discovery pipeline but the quality of the reference sequence is clearly of paramount importance. Our findings are also a stark reminder that it can be unwise to use the relaxed mismatch settings provided as defaults by some read mappers when reads are being mapped to a relatively unfinished reference sequence from e.g. a non-model organism in its early stages of genomic exploration.

The low-recombining pericentromeric region of barley restricts gene diversity and evolution but not gene expression.

The low-recombining pericentromeric region of the barley genome contains roughly a quarter of the genes of the species, embedded in low-recombining DNA that is rich in repeats and repressive chromatin signatures. We have investigated the effects of pericentromeric region residency upon the expression, diversity and evolution of these genes. We observe no significant difference in average transcript level or developmental RNA specificity between the barley pericentromeric region and the rest of the genome. In contrast, all of the evolutionary parameters studied here show evidence of compromised gene evolution in this region. First, genes within the pericentromeric region of wild barley show reduced diversity and significantly weakened purifying selection compared with the rest of the genome. Second, gene duplicates (ohnolog pairs) derived from the cereal whole-genome duplication event ca. 60MYa have been completely eliminated from the barley pericentromeric region. Third, local gene duplication in the pericentromeric region is reduced by 29% relative to the rest of the genome. Thus, the pericentromeric region of barley is a permissive environment for gene expression but has restricted gene evolution in a sizeable fraction of barley's genes.

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond.

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes.

Flapjack--graphical genotype visualization.

SUMMARY: New software tools for graphical genotyping are required that can routinely handle the large data volumes generated by the high-throughput single-nucleotide polymorphism (SNP) platforms, genotyping-by-sequencing and other comparable genotyping technologies. Flapjack has been developed to facilitate analysis of these data, providing real time rendering with rapid navigation and comparisons between lines, markers and chromosomes, with visualization, sorting and querying based on associated data, such as phenotypes, quantitative trait loci or other mappable features. AVAILABILITY: Flapjack is freely available for Microsoft Windows, Mac OS X, Linux and Solaris, and can be downloaded from http://bioinf.scri.ac.uk/flapjack .

Analysis of >1000 single nucleotide polymorphisms in geographically matched samples of landrace and wild barley indicates secondary contact and chromosome-level differences in diversity around domestication genes.

Barley is a model species for the investigation of the evolution, adaptation and spread of the world's important crops. In this article, we describe the first application of an oligonucleotide pool assay single nucleotide polymorphism (SNP) platform to assess the evolution of barley in a portion of the Fertile Crescent, a key region in the development of farming. A large collection of >1000 genetically mapped, genome-wide SNPs was assayed in geographically matched landrace and wild barley accessions (N=448) from Jordan and Syria. Landrace and wild barley categories were clearly genetically differentiated, but a limited degree of secondary contact was evident. Significant chromosome-level differences in diversity between barley types were observed around genes known to be involved in the evolution of cultivars. The region of Jordan and southern Syria, compared with the north of Syria, was supported by SNP data as a more likely domestication origin. Our data provide evidence for hybridization as a possible mechanism for the continued adaptation of landrace barley under cultivation, indicate regions of the genome that may be subject to selection processes and suggest limited origins for the development of the cultivated crop.

The genetic diversity and evolution of field pea (Pisum) studied by high throughput retrotransposon based insertion polymorphism (RBIP) marker analysis.

BACKGROUND: The genetic diversity of crop species is the result of natural selection on the wild progenitor and human intervention by ancient and modern farmers and breeders. The genomes of modern cultivars, old cultivated landraces, ecotypes and wild relatives reflect the effects of these forces and provide insights into germplasm structural diversity, the geographical dimension to species diversity and the process of domestication of wild organisms. This issue is also of great practical importance for crop improvement because wild germplasm represents a rich potential source of useful under-exploited alleles or allele combinations. The aim of the present study was to analyse a major Pisum germplasm collection to gain a broad understanding of the diversity and evolution of Pisum and provide a new rational framework for designing germplasm core collections of the genus. RESULTS: 3020 Pisum germplasm samples from the John Innes Pisum germplasm collection were genotyped for 45 retrotransposon based insertion polymorphism (RBIP) markers by the Tagged Array Marker (TAM) method. The data set was stored in a purpose-built Germinate relational database and analysed by both principal coordinate analysis and a nested application of the Structure program which yielded substantially similar but complementary views of the diversity of the genus Pisum. Structure revealed three Groups (1-3) corresponding approximately to landrace, cultivar and wild Pisum respectively, which were resolved by nested Structure analysis into 14 Sub-Groups, many of which correlate with taxonomic sub-divisions of Pisum, domestication related phenotypic traits and/or restricted geographical locations. Genetic distances calculated between these Sub-Groups are broadly supported by principal coordinate analysis and these, together with the trait and geographical data, were used to infer a detailed model for the domestication of Pisum. CONCLUSIONS: These data provide a clear picture of the major distinct gene pools into which the genus Pisum is partitioned and their geographical distribution. The data strongly support the model of independent domestications for P. sativum ssp abyssinicum and P. sativum. The relationships between these two cultivated germplasms and the various sub-divisions of wild Pisum have been clarified and the most likely ancestral wild gene pools for domesticated P. sativum identified. Lastly, this study provides a framework for defining global Pisum germplasm which will be useful for designing core collections.

"Wild barley serves as a source for biofortification of barley grains".

The continuing growth of the human population creates an inevitable necessity for higher crop yields, which are mandatory for the supply with adequate amounts of food. However, increasing grain yield may lead to a reduction of grain quality, such as a decline in protein and mineral nutrient concentrations causing the so-called hidden hunger. To assess the interdependence between quantity and quality and to evaluate the biofortification potential of wild barley, we conducted field studies, examining the interplay between plant development, yield, and nutrient concentrations, using HEB-YIELD, a subset of the wild barley nested association mapping population HEB-25. A huge variation of nutrient concentration in grains was obtained, since we identified lines with a more than 50% higher grain protein, iron, and zinc concentration in comparison to the recurrent parent 'Barke'. We observed a negative relationship between grain yield and nutritional value in barley, indicated by predominantly negative correlations between yield and nutrient concentrations. Analyzing the genetic control of nutrient concentration in mature grains indicated that numerous genomic regions determine the final nutritional value of grains and wild alleles were frequently associated with higher nutrient concentrations. The targeted introgression of wild barley alleles may enable biofortification in future barley breeding.

Optimizing the procedure of grain nutrient predictions in barley via hyperspectral imaging.

Hyperspectral imaging enables researchers and plant breeders to analyze various traits of interest like nutritional value in high throughput. In order to achieve this, the optimal design of a reliable calibration model, linking the measured spectra with the investigated traits, is necessary. In the present study we investigated the impact of different regression models, calibration set sizes and calibration set compositions on prediction performance. For this purpose, we analyzed concentrations of six globally relevant grain nutrients of the wild barley population HEB-YIELD as case study. The data comprised 1,593 plots, grown in 2015 and 2016 at the locations Dundee and Halle, which have been entirely analyzed through traditional laboratory methods and hyperspectral imaging. The results indicated that a linear regression model based on partial least squares outperformed neural networks in this particular data modelling task. There existed a positive relationship between the number of samples in a calibration model and prediction performance, with a local optimum at a calibration set size of ~40% of the total data. The inclusion of samples from several years and locations could clearly improve the predictions of the investigated nutrient traits at small calibration set sizes. It should be stated that the expansion of calibration models with additional samples is only useful as long as they are able to increase trait variability. Models obtained in a certain environment were only to a limited extent transferable to other environments. They should therefore be successively upgraded with new calibration data to enable a reliable prediction of the desired traits. The presented results will assist the design and conceptualization of future hyperspectral imaging projects in order to achieve reliable predictions. It will in general help to establish practical applications of hyperspectral imaging systems, for instance in plant breeding concepts.

Barley yield formation under abiotic stress depends on the interplay between flowering time genes and environmental cues.

Since the dawn of agriculture, crop yield has always been impaired through abiotic stresses. In a field trial across five locations worldwide, we tested three abiotic stresses, nitrogen deficiency, drought and salinity, using HEB-YIELD, a selected subset of the wild barley nested association mapping population HEB-25. We show that barley flowering time genes Ppd-H1, Sdw1, Vrn-H1 and Vrn-H3 exert pleiotropic effects on plant development and grain yield. Under field conditions, these effects are strongly influenced by environmental cues like day length and temperature. For example, in Al-Karak, Jordan, the day length-sensitive wild barley allele of Ppd-H1 was associated with an increase of grain yield by up to 30% compared to the insensitive elite barley allele. The observed yield increase is accompanied by pleiotropic effects of Ppd-H1 resulting in shorter life cycle, extended grain filling period and increased grain size. Our study indicates that the adequate timing of plant development is crucial to maximize yield formation under harsh environmental conditions. We provide evidence that wild barley alleles, introgressed into elite barley cultivars, can be utilized to support grain yield formation. The presented knowledge may be transferred to related crop species like wheat and rice securing the rising global food demand for cereals.

Gene-based sequence diversity analysis of field pea (Pisum).

Sequence diversity of 39 dispersed gene loci was analyzed in 48 diverse individuals representative of the genus Pisum. The different genes show large variation in diversity parameters, suggesting widely differing levels of selection and a high overall diversity level for the species. The data set yields a genetic diversity tree whose deep branches, involving wild samples, are preserved in a tree derived from a polymorphic retrotransposon insertions in an identical sample set. Thus, gene regions and intergenic "junk DNA" share a consistent picture for the genomic diversity of Pisum, despite low linkage disequilibrium in wild and landrace germplasm, which might be expected to allow independent evolution of these very different DNA classes. Additional lines of evidence indicate that recombination has shuffled gene haplotypes efficiently within Pisum, despite its high level of inbreeding and widespread geographic distribution. Trees derived from individual gene loci show marked differences from each other, and genetic distance values between sample pairs show high standard deviations. Sequence mosaic analysis of aligned sequences identifies nine loci showing evidence for intragenic recombination. Lastly, phylogenetic network analysis confirms the non-treelike structure of Pisum diversity and indicates the major germplasm classes involved. Overall, these data emphasize the artificiality of simple tree structures for representing genomic sequence variation within Pisum and emphasize the need for fine structure haplotype analysis to accurately define the genetic structure of the species.

Genome-Wide Association Analysis of Grain Yield-Associated Traits in a Pan-European Barley Cultivar Collection.

A collection of 379 Hordeum vulgare cultivars, comprising all combinations of spring and winter growth habits with two and six row ear type, was screened by genome wide association analysis to discover alleles controlling traits related to grain yield. Genotypes were obtained at 6,810 segregating gene-based single nucleotide polymorphism (SNP) loci and corresponding field trial data were obtained for eight traits related to grain yield at four European sites in three countries over two growth years. The combined data were analyzed and statistically significant associations between the traits and regions of the barley genomes were obtained. Combining this information with the high resolution gene map for barley allowed the identification of candidate genes underlying all scored traits and superposition of this information with the known genomics of grain trait genes in rice resulted in the assignation of 13 putative barley genes controlling grain traits in European cultivated barley. Several of these genes are associated with grain traits in both winter and spring barley.

Barley SIX-ROWED SPIKE3 encodes a putative Jumonji C-type H3K9me2/me3 demethylase that represses lateral spikelet fertility.

The barley inflorescence (spike) comprises a multi-noded central stalk (rachis) with tri-partite clusters of uni-floretted spikelets attached alternately along its length. Relative fertility of lateral spikelets within each cluster leads to spikes with two or six rows of grain, or an intermediate morphology. Understanding the mechanisms controlling this key developmental step could provide novel solutions to enhanced grain yield. Classical genetic studies identified five major SIX-ROWED SPIKE (VRS) genes, with four now known to encode transcription factors. Here we identify and characterise the remaining major VRS gene, VRS3, as encoding a putative Jumonji C-type H3K9me2/me3 demethylase, a regulator of chromatin state. Exploring the expression network modulated by VRS3 reveals specific interactions, both with other VRS genes and genes involved in stress, hormone and sugar metabolism. We show that combining a vrs3 mutant allele with natural six-rowed alleles of VRS1 and VRS5 leads to increased lateral grain size and greater grain uniformity.The VRS genes of barley control the fertility of the lateral spikelets on the barley inflorescence. Here, Bull et al. show that VRS3 encodes a putative Jumonji C-type histone demethylase that regulates expression of other VRS genes, and genes involved in stress, hormone and sugar metabolism.

Insertional polymorphism and antiquity of PDR1 retrotransposon insertions in pisum species.

Sequences flanking 73 insertions of the retrotransposon PDR1 have been characterized, together with an additional 270 flanking regions from one side alone, from a diverse collection of Pisum germ plasm. Most of the identified flanking sequences are repetitious DNAs but more than expected (7%) lie within nuclear gene protein-coding regions. The approximate age of 52 of the PDR1 insertions has been determined by measuring sequence divergence among LTR pairs. These data show that PDR1 transpositions occurred within the last 5 MY, with a peak at 1-2.5 MYA. The insertional polymorphism of 68 insertions has been assessed across 47 selected Pisum accessions, representing the diversity of the genus. None of the insertions are fixed, showing that PDR1 insertions can persist in a polymorphic state for millions of years in Pisum. The insertional polymorphism data have been compared with the age estimations to ask what rules control the proliferation of PDR1 insertions in Pisum. Relatively recent insertions (< approximately 1.5 MYA) tend to be found in small subsets of the Pisum accessions set, "middle-aged" insertions (between approximately 1.5 and 2.5 MYA) vary greatly in their occurrence, and older insertions (> approximately 2.5 MYA) are mostly found in small subsets of Pisum. Finally, the average age estimate for PDR1 insertions, together with an existing data set for PDR1 retrotransposon SSAP markers, has been used to derive an estimate of the effective population size for Pisum of approximately 7.5 x 10(5).

A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach.

A microarray-based method has been developed for scoring thousands of DNAs for a co-dominant molecular marker on a glass slide. The approach was developed to detect insertional polymorphism of transposons and works well with single nucleotide polymorphism (SNP) markers. Biotin- terminated allele-specific PCR products are spotted unpurified onto streptavidin-coated glass slides and visualised by hybridisation of fluorescent detector oligonucleotides to tags attached to the allele- specific PCR primers. Two tagged primer oligonucleotides are used per locus and each tag is detected by hybridisation to a concatameric DNA probe labelled with multiple fluorochromes.

The application of LTR retrotransposons as molecular markers in plants.

Retrotransposons are major, dispersed components of most eukaryotic genomes. They replicate by a cycle of transcription, reverse transcription, and integration of new copies, without excising from the genome in the process. Because they represent a major share of the genome, cause easily detectable genetic changes having known ancestral and derived states, and contain conserved regions for which polymerase chain reaction (PCR) primers may be designed, retrotransposon insertions can be exploited as powerful molecular marker systems. Here, we describe the background and strategies, as well as give detailed laboratory protocols, for four key retrotransposon-based methods: SSAP, IRAP, REMAP, and RBIP. The SSAP, IRAP, and REMAP methods are multiplex and generate anonymous marker bands; RBIP scores individual loci, much as microsatellite-based marker systems do. The methods are variously suited to marker detection on agarose and polyacrylamide slab gels, slab and capillary sequencing devices, and arrays on solid supports. The different strengths and weaknesses of these approaches and their performance relative to conventional marker methods are discussed, together with their applicability to marker-assisted breeding, phylogenetic analyses, biodiversity determinations, and evolutionary studies.

The application of LTR retrotransposons as molecular markers in plants.

Retrotransposons are a major agent of genome evolution. Various molecular marker systems have been developed that exploit the ubiquitous nature of these genetic elements and their property of stable integration into dispersed chromosomal loci that are polymorphic within species. The key methods, SSAP, IRAP, REMAP, RBIP, and ISBP, all detect the sites at which the retrotransposon DNA, which is conserved between families of elements, is integrated into the genome. Marker systems exploiting these methods can be easily developed and inexpensively deployed in the absence of extensive genome sequence data. They offer access to the dynamic and polymorphic, nongenic portion of the genome and thereby complement methods, such as gene-derived SNPs, that target primarily the genic fraction.

Locus-dependent selection in crop-wild hybrids of lettuce under field conditions and its implication for GM crop development.

Gene escape from crops has gained much attention in the last two decades, as transgenes introgressing into wild populations could affect the latter's ecological characteristics. However, different genes have different likelihoods of introgression. The mixture of selective forces provided by natural conditions creates an adaptive mosaic of alleles from both parental species. We investigated segregation patterns after hybridization between lettuce (Lactuca sativa) and its wild relative, L. serriola. Three generations of hybrids (S1, BC1, and BC1S1) were grown in habitats mimicking the wild parent's habitat. As control, we harvested S1 seedlings grown under controlled conditions, providing very limited possibility for selection. We used 89 AFLP loci, as well as more recently developed dominant markers, 115 retrotransposon markers (SSAP), and 28 NBS loci linked to resistance genes. For many loci, allele frequencies were biased in plants exposed to natural field conditions, including over-representation of crop alleles for various loci. Furthermore, Linkage disequilibrium was locally changed, allegedly by selection caused by the natural field conditions, providing ample opportunity for genetic hitchhiking. Our study indicates that when developing genetically modified crops, a judicious selection of insertion sites, based on knowledge of selective (dis)advantages of the surrounding crop genome under field conditions, could diminish transgene persistence.