Novel exotic alleles of EARLY FLOWERING 3 determine plant development in barley.

EARLY FLOWERING 3 (ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be essential for future plant breeding. To expand the limited knowledge on barley ELF3 in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at the ELF3 locus, were compared for 10 developmental and yield-related traits. We determine novel exotic ELF3 alleles and show that HIF lines, carrying the exotic ELF3 allele, accelerated plant development compared with the cultivated ELF3 allele, depending on the genetic background. Remarkably, the most extreme effects on phenology could be attributed to one exotic ELF3 allele differing from the cultivated Barke ELF3 allele in only one single nucleotide polymorphism (SNP). This SNP causes an amino acid substitution (W669G), which as predicted has an impact on the protein structure of ELF3. Consequently, it may affect phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also its local cellular interactions causing significant trait differences between HIF sister lines.

Identifying genomic regions determining shoot and root traits related to nitrogen uptake efficiency in a multiparent advanced generation intercross (MAGIC) winter wheat population in a high-throughput phenotyping facility.

In the context of a continuously increasing human population that needs to be fed, with environmental protection in mind, nitrogen use efficiency (NUE) improvement is becoming very important. To understand the natural variation of traits linked to nitrogen uptake efficiency (UPE), one component of NUE, the multiparent advanced generation intercross (MAGIC) winter wheat population WM-800 was phenotyped under two contrasting nitrogen (N) levels in a high-throughput phenotyping facility for six weeks. Three biomass-related, three root-related, and two reflectance-related traits were measured weekly under each treatment. Subsequently, the population was genetically analysed using a total of 13,060 polymorphic haplotypes and singular SNPs for a genome-wide association study (GWAS). In total, we detected 543 quantitative trait loci (QTL) across all time points and traits, which were pooled into 42 stable QTL (sQTL; present in at least three of the six weeks). Besides Rht-B1 and Rht-D1, candidate genes playing a role in gibberellic acid-regulated growth and nitrate transporter genes from the NPF gene family, like NRT 1.1, were linked to sQTL. Two novel sQTL on chromosomes 5 A and 6D showed pleiotropic effects on several traits. The high number of N-specific sQTL indicates that selection for UPE is useful specifically under N-limited conditions.

A Haplotype-Based GWAS Identified Trait-Improving QTL Alleles Controlling Agronomic Traits under Contrasting Nitrogen Fertilization Treatments in the MAGIC Wheat Population WM-800.

The multi-parent-advanced-generation-intercross (MAGIC) population WM-800 was developed by intercrossing eight modern winter wheat cultivars to enhance the genetic diversity present in breeding populations. We cultivated WM-800 during two seasons in seven environments under two contrasting nitrogen fertilization treatments. WM-800 lines exhibited highly significant differences between treatments, as well as high heritabilities among the seven agronomic traits studied. The highest-yielding WM-line achieved an average yield increase of 4.40 dt/ha (5.2%) compared to the best founder cultivar Tobak. The subsequent genome-wide-association-study (GWAS), which was based on haplotypes, located QTL for seven agronomic traits including grain yield. In total, 40, 51, and 46 QTL were detected under low, high, and across nitrogen treatments, respectively. For example, the effect of QYLD_3A could be associated with the haplotype allele of cultivar Julius increasing yield by an average of 4.47 dt/ha (5.2%). A novel QTL on chromosome 2B exhibited pleiotropic effects, acting simultaneously on three-grain yield components (ears-per-square-meter, grains-per-ear, and thousand-grain-weight) and plant-height. These effects may be explained by a member of the nitrate-transporter-1 (NRT1)/peptide-family, TaNPF5.34, located 1.05 Mb apart. The WM-800 lines and favorable QTL haplotypes, associated with yield improvements, are currently implemented in wheat breeding programs to develop advanced nitrogen-use efficient wheat cultivars.

Genome-Wide Association Study of Salt Tolerance-Related Traits during Germination and Seedling Development in an Intermedium-Spike Barley Collection.

Increased salinity is one of the major consequences of climatic change affecting global crop production. The early stages in the barley (Hordeum vulgare L.) life cycle are considered the most critical phases due to their contributions to final crop yield. Particularly, the germination and seedling development are sensitive to numerous environmental stresses, especially soil salinity. In this study, we aimed to identify SNP markers linked with germination and seedling development at 150 mM NaCl as a salinity treatment. We performed a genome-wide association study (GWAS) using a panel of 208 intermedium-spike barley (H. vulgare convar. intermedium (Körn.) Mansf.) accessions and their genotype data (i.e., 10,323 SNPs) using the genome reference sequence of "Morex". The phenotypic results showed that the 150 mM NaCl salinity treatment significantly reduced all recorded germination and seedling-related traits compared to the control treatment. Furthermore, six accessions (HOR 11747, HOR 11718, HOR 11640, HOR 11256, HOR 11275 and HOR 11291) were identified as the most salinity tolerant from the intermedium-spike barley collection. GWAS analysis indicated that a total of 38 highly significantly associated SNP markers under control and/or salinity traits were identified. Of these, two SNP markers on chromosome (chr) 1H, two on chr 3H, and one on chr 4H were significantly linked to seedling fresh and dry weight under salinity stress treatment. In addition, two SNP markers on chr 7H were also significantly associated with seedling fresh and dry weight but under control condition. Under salinity stress, one SNP marker on chr 1H, 5H and 7H were detected for more than one phenotypic trait. We found that in most of the accessions exhibiting the highest salinity tolerance, most of the salinity-related QTLs were presented. These results form the basis for detailed studies, leading to improved salt tolerance breeding programs in barley.

Initial Description of the Genome of Aeluropus littoralis, a Halophile Grass.

The use of wild plant species or their halophytic relatives has been considered in plant breeding programs to improve salt and drought tolerance in crop plants. Aeluropus littoralis serves as halophyte model for identification and isolation of novel stress adaptation genes. A. littoralis, a perennial monocot grass, grows in damp or arid areas, often salt-impregnated places and wasteland in cultivated areas, can survive periodically high water salinity, and tolerate high salt concentrations in the soil up to 1,100 mM sodium chloride. Therefore, it serves as valuable genetic resource to understand molecular mechanisms of stress-responses in monocots. The knowledge can potentially be used for improving tolerance to abiotic stresses in economically important crops. Several morphological, anatomical, ecological, and physiological traits of A. littoralis have been investigated so far. After watering with salt water the grass is able to excrete salt via its salt glands. Meanwhile, a number of ESTs (expressed sequence tag), genes and promoters induced by the salt and drought stresses were isolated, sequenced and annotated at a molecular level. Transfer of stress related genes to other species resulted in enhanced stress resistance. Here we describe the genome sequence and structure of A. littoralis analyzed by whole genome sequencing and histological analysis. The chromosome number was determined to be 20 (2n = 2x = 20). The genome size was calculated to be 354 Mb. This genomic information provided here, will support the functional investigation and application of novel genes improving salt stress resistance in crop plants. The utility of the sequence information is exemplified by the analysis of the DREB-transcription factor family.

Genome-wide association study on metabolite accumulation in a wild barley NAM population reveals natural variation in sugar metabolism.

Metabolites play a key role in plants as they are routing plant developmental processes and are involved in biotic and abiotic stress responses. Their analysis can offer important information on the underlying processes. Regarding plant breeding, metabolite concentrations can be used as biomarkers instead of or in addition to genetic markers to predict important phenotypic traits (metabolic prediction). In this study, we applied a genome-wide association study (GWAS) in a wild barley nested association mapping (NAM) population to identify metabolic quantitative trait loci (mQTL). A set of approximately 130 metabolites, measured at early and late sampling dates, was analysed. For four metabolites from the early and six metabolites from the late sampling date significant mQTL (grouped as 19 mQTL for the early and 25 mQTL for the late sampling date) were found. Interestingly, all of those metabolites could be classified as sugars. Sugars are known to be involved in signalling, plant growth and plant development. Sugar-related genes, encoding mainly sugar transporters, have been identified as candidate genes for most of the mQTL. Moreover, several of them co-localized with known flowering time genes like Ppd-H1, HvELF3, Vrn-H1, Vrn-H2 and Vrn-H3, hinting on the known role of sugars in flowering. Furthermore, numerous disease resistance-related genes were detected, pointing to the signalling function of sugars in plant resistance. An mQTL on chromosome 1H in the region of 13 Mbp to 20 Mbp stood out, that alone explained up to 65% of the phenotypic variation of a single metabolite. Analysis of family-specific effects within the diverse NAM population showed the available natural genetic variation regarding sugar metabolites due to different wild alleles. The study represents a step towards a better understanding of the genetic components of metabolite accumulation, especially sugars, thereby linking them to biological functions in barley.

Genomic Dissection of Peduncle Morphology in Barley through Nested Association Mapping.

Straw biomass and stability are crucial for stable yields. Moreover, straw harbors the potential to serve as a valuable raw material for bio-economic processes. The peduncle is the top part of the last shoot internode and carries the spike. This study investigates the genetic control of barley peduncle morphology. Therefore, 1411 BC1S3 lines of the nested association mapping (NAM) population "Halle Exotic Barley 25" (HEB-25), generated by crossing the spring barley elite cultivar Barke with an assortment of 25 exotic barley accessions, were used. Applying 50k Illumina Infinium iSelect SNP genotyping yielded new insights and a better understanding of the quantitative trait loci (QTL) involved in controlling the peduncle diameter traits, we found the total thickness of peduncle tissues and the area of the peduncle cross-section. We identified three major QTL regions on chromosomes 2H and 3H mainly impacting the traits. Remarkably, the exotic allele at the QTL on chromosome 3H improved all three traits investigated in this work. Introgressing this QTL in elite cultivars might facilitate to adjust peduncle morphology for improved plant stability or enlarged straw biomass production independent of flowering time and without detrimental effects on grain yield.

COMPOSITUM 1 contributes to the architectural simplification of barley inflorescence via meristem identity signals.

Grasses have varying inflorescence shapes; however, little is known about the genetic mechanisms specifying such shapes among tribes. Here, we identify the grass-specific TCP transcription factor COMPOSITUM 1 (COM1) expressing in inflorescence meristematic boundaries of different grasses. COM1 specifies branch-inhibition in barley (Triticeae) versus branch-formation in non-Triticeae grasses. Analyses of cell size, cell walls and transcripts reveal barley COM1 regulates cell growth, thereby affecting cell wall properties and signaling specifically in meristematic boundaries to establish identity of adjacent meristems. COM1 acts upstream of the boundary gene Liguleless1 and confers meristem identity partially independent of the COM2 pathway. Furthermore, COM1 is subject to purifying natural selection, thereby contributing to specification of the spike inflorescence shape. This meristem identity pathway has conceptual implications for both inflorescence evolution and molecular breeding in Triticeae.

Natural variation in meiotic recombination rate shapes introgression patterns in intraspecific hybrids between wild and domesticated barley.

Meiotic recombination rates vary considerably between species, populations and individuals. The genetic exchange between homologous chromosomes plays a major role in evolution by breaking linkage between advantageous and deleterious alleles in the case of introgressions. Identifying recombination rate modifiers is thus of both fundamental and practical interest to understand and utilize variation in meiotic recombination rates. We investigated recombination rate variation in a large intraspecific hybrid population (named HEB-25) derived from a cross between domesticated barley and 25 wild barley accessions. We observed quantitative variation in total crossover number with a maximum of a 1.4-fold difference between subpopulations and increased recombination rates across pericentromeric regions. The meiosis-specific α-kleisin cohesin subunit REC8 was identified as a candidate gene influencing crossover number and patterning. Furthermore, we quantified wild barley introgression patterns and revealed how local and genome-wide recombination rate variation shapes patterns of introgression. The identification of allelic variation in REC8 in combination with the observed changes in crossover patterning suggest a difference in how chromatin loops are tethered to the chromosome axis, resulting in reduced crossover suppression across pericentromeric regions. Local and genome-wide recombination rate variation is shaping patterns of introgressions and thereby directly influences the consequences of linkage drag.

Can metabolic prediction be an alternative to genomic prediction in barley?

Like other crop species, barley, the fourth most important crop worldwide, suffers from the genetic bottleneck effect, where further improvements in performance through classical breeding methods become difficult. Therefore, indirect selection methods are of great interest. Here, genomic prediction (GP) based on 33,005 SNP markers and, alternatively, metabolic prediction (MP) based on 128 metabolites with sampling at two different time points in one year, were applied to predict multi-year agronomic traits in the nested association mapping (NAM) population HEB-25. We found prediction abilities of up to 0.93 for plant height with SNP markers and of up to 0.61 for flowering time with metabolites. Interestingly, prediction abilities in GP increased after reducing the number of incorporated SNP markers. The estimated effects of GP and MP were highly concordant, indicating MP as an interesting alternative to GP, being able to reflect a stable genotype-specific metabolite profile. In MP, sampling at an early developmental stage outperformed sampling at a later stage. The results confirm the value of GP for future breeding. With MP, an interesting alternative was also applied successfully. However, based on our results, usage of MP alone cannot be recommended in barley. Nevertheless, MP can assist in unravelling physiological pathways for the expression of agronomically important traits.

Supernumerary B chromosomes of Aegilops speltoides undergo precise elimination in roots early in embryo development.

Not necessarily all cells of an organism contain the same genome. Some eukaryotes exhibit dramatic differences between cells of different organs, resulting from programmed elimination of chromosomes or their fragments. Here, we present a detailed analysis of programmed B chromosome elimination in plants. Using goatgrass Aegilops speltoides as a model, we demonstrate that the elimination of B chromosomes is a strictly controlled and highly efficient root-specific process. At the onset of embryo differentiation B chromosomes undergo elimination in proto-root cells. Independent of centromere activity, B chromosomes demonstrate nondisjunction of chromatids and lagging in anaphase, leading to micronucleation. Chromatin structure and DNA replication differ between micronuclei and primary nuclei and degradation of micronucleated DNA is the final step of B chromosome elimination. This process might allow root tissues to survive the detrimental expression, or overexpression of B chromosome-located root-specific genes with paralogs located on standard chromosomes.

Kmasker plants - a tool for assessing complex sequence space in plant species.

Many plant genomes display high levels of repetitive sequences. The assembly of these complex genomes using short high-throughput sequence reads is still a challenging task. Underestimation or disregard of repeat complexity in these datasets can easily misguide downstream analysis. Detection of repetitive regions by k-mer counting methods has proved to be reliable. Easy-to-use applications utilizing k-mer counting are in high demand, especially in the domain of plants. We present Kmasker plants, a tool that uses k-mer count information as an assistant throughout the analytical workflow of genome data that is provided as a command-line and web-based solution. Beside its core competence to screen and mask repetitive sequences, we have integrated features that enable comparative studies between different cultivars or closely related species and methods that estimate target specificity of guide RNAs for application of site-directed mutagenesis using Cas9 endonuclease. In addition, we have set up a web service for Kmasker plants that maintains pre-computed indices for 10 of the economically most important cultivated plants. Source code for Kmasker plants has been made publically available at https://github.com/tschmutzer/kmasker. The web service is accessible at https://kmasker.ipk-gatersleben.de.

Evolutionarily conserved partial gene duplication in the Triticeae tribe of grasses confers pathogen resistance.

BACKGROUND: The large and highly repetitive genomes of the cultivated species Hordeum vulgare (barley), Triticum aestivum (wheat), and Secale cereale (rye) belonging to the Triticeae tribe of grasses appear to be particularly rich in gene-like sequences including partial duplicates. Most of them have been classified as putative pseudogenes. In this study we employ transient and stable gene silencing- and over-expression systems in barley to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15). RESULTS: The partial ARM1 gene is derived from a gene-duplication event in a common ancestor of the Triticeae and contributes to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis. In barley, allelic variants of HvARM1 but not of HvPUB15 are significantly associated with levels of powdery mildew infection. Both HvPUB15 and HvARM1 proteins interact in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. A genome-wide scan for partial gene duplicates reveals further events in barley resulting in stress-regulated, potentially neo-functionalized, genes. CONCLUSION: The results suggest neo-functionalization of the partial gene copy HvARM1 increases resistance against powdery mildew infection. It further links plastid function with susceptibility to biotrophic pathogen attack. These findings shed new light on a novel mechanism to employ partial duplication of protein-protein interaction domains to facilitate the expansion of immune signaling networks.

How Next-Generation Sequencing Has Aided Our Understanding of the Sequence Composition and Origin of B Chromosomes.

Accessory, supernumerary, or-most simply-B chromosomes, are found in many eukaryotic karyotypes. These small chromosomes do not follow the usual pattern of segregation, but rather are transmitted in a higher than expected frequency. As increasingly being demonstrated by next-generation sequencing (NGS), their structure comprises fragments of standard (A) chromosomes, although in some plant species, their sequence also includes contributions from organellar genomes. Transcriptomic analyses of various animal and plant species have revealed that, contrary to what used to be the common belief, some of the B chromosome DNA is protein-encoding. This review summarizes the progress in understanding B chromosome biology enabled by the application of next-generation sequencing technology and state-of-the-art bioinformatics. In particular, a contrast is drawn between a direct sequencing approach and a strategy based on a comparative genomics as alternative routes that can be taken towards the identification of B chromosome sequences.

Bioinformatics in the plant genomic and phenomic domain: The German contribution to resources, services and perspectives.

Plant genetic resources are a substantial opportunity for plant breeding, preservation and maintenance of biological diversity. As part of the German Network for Bioinformatics Infrastructure (de.NBI) the German Crop BioGreenformatics Network (GCBN) focuses mainly on crop plants and provides both data and software infrastructure which are tailored to the needs of the plant research community. Our mission and key objectives include: (1) provision of transparent access to germplasm seeds, (2) the delivery of improved workflows for plant gene annotation, and (3) implementation of bioinformatics services that link genotypes and phenotypes. This review introduces the GCBN's spectrum of web-services and integrated data resources that address common research problems in the plant genomics community.

From plant genomes to phenotypes.

Recent advances in sequencing technologies have greatly accelerated the rate of plant genome and applied breeding research. Despite this advancing trend, plant genomes continue to present numerous difficulties to the standard tools and pipelines not only for genome assembly but also gene annotation and downstream analysis. Here we give a perspective on tools, resources and services necessary to assemble and analyze plant genomes and link them to plant phenotypes.

Cross-talk of the biotrophic pathogen Claviceps purpurea and its host Secale cereale.

BACKGROUND: The economically important Ergot fungus Claviceps purpurea is an interesting biotrophic model system because of its strict organ specificity (grass ovaries) and the lack of any detectable plant defense reactions. Though several virulence factors were identified, the exact infection mechanisms are unknown, e.g. how the fungus masks its attack and if the host detects the infection at all. RESULTS: We present a first dual transcriptome analysis using an RNA-Seq approach. We studied both, fungal and plant gene expression in young ovaries infected by the wild-type and two virulence-attenuated mutants. We can show that the plant recognizes the fungus, since defense related genes are upregulated, especially several phytohormone genes. We present a survey of in planta expressed fungal genes, among them several confirmed virulence genes. Interestingly, the set of most highly expressed genes includes a high proportion of genes encoding putative effectors, small secreted proteins which might be involved in masking the fungal attack or interfering with host defense reactions. As known from several other phytopathogens, the C. purpurea genome contains more than 400 of such genes, many of them clustered and probably highly redundant. Since the lack of effective defense reactions in spite of recognition of the fungus could very well be achieved by effectors, we started a functional analysis of some of the most highly expressed candidates. However, the redundancy of the system made the identification of a drastic effect of a single gene most unlikely. We can show that at least one candidate accumulates in the plant apoplast. Deletion of some candidates led to a reduced virulence of C. purpurea on rye, indicating a role of the respective proteins during the infection process. CONCLUSIONS: We show for the first time that- despite the absence of effective plant defense reactions- the biotrophic pathogen C. purpurea is detected by its host. This points to a role of effectors in modulation of the effective plant response. Indeed, several putative effector genes are among the highest expressed genes in planta.

A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

Construction of a map-based reference genome sequence for barley, Hordeum vulgare L.

Barley (Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. 'Morex' was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX).

Towards a whole-genome sequence for rye (Secale cereale L.).

We report on a whole-genome draft sequence of rye (Secale cereale L.). Rye is a diploid Triticeae species closely related to wheat and barley, and an important crop for food and feed in Central and Eastern Europe. Through whole-genome shotgun sequencing of the 7.9-Gbp genome of the winter rye inbred line Lo7 we obtained a de novo assembly represented by 1.29 million scaffolds covering a total length of 2.8 Gbp. Our reference sequence represents nearly the entire low-copy portion of the rye genome. This genome assembly was used to predict 27 784 rye gene models based on homology to sequenced grass genomes. Through resequencing of 10 rye inbred lines and one accession of the wild relative S. vavilovii, we discovered more than 90 million single nucleotide variants and short insertions/deletions in the rye genome. From these variants, we developed the high-density Rye600k genotyping array with 600 843 markers, which enabled anchoring the sequence contigs along a high-density genetic map and establishing a synteny-based virtual gene order. Genotyping data were used to characterize the diversity of rye breeding pools and genetic resources, and to obtain a genome-wide map of selection signals differentiating the divergent gene pools. This rye whole-genome sequence closes a gap in Triticeae genome research, and will be highly valuable for comparative genomics, functional studies and genome-based breeding in rye.