Uwe Scholz

A physical, genetic and functional sequence assembly of the barley genome

Barley (Hordeum vulgare L.) is among the world’s earliest domesticated and most important crop plants. It is diploid with a large haploid genome of 5.1 gigabases (Gb). Here we present an integrated and ordered physical, genetic and functional sequence resource that describes the barley gene-space in a structured whole-genome context. We developed a physical map of 4.98 Gb, with more than 3.90 Gb anchored to a high-resolution genetic map. Projecting a deep whole-genome shotgun assembly, complementary DNA and deep RNA sequence data onto this framework supports 79,379 transcript clusters, including 26,159 ‘high-confidence’ genes with homology support from other plant genomes. Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation. Our data provide a platform for both genome-assisted research and enabling contemporary crop improvement.

Unlocking the Barley Genome by Chromosomal and Comparative Genomics

Survey sequence and array hybridization data from flow-sorted barley chromosomes were integrated using a comparative genomics model to define an ordered gene map of the barley genome that contains approximately two-thirds of its estimated 32000 genes. The resulting high-resolution framework facilitated a genome-wide structural analysis of the barley genome and a detailed comparative analysis with wheat. We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley (Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum (Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.

Barley grain maturation and germination: Metabolic pathway and regulatory network commonalities and differences highlighted by new MapMan/PageMan profiling tools

Plant seeds prepare for germination already during seed maturation. We performed a detailed transcriptome analysis of barley (Hordeum vulgare) grain maturation, desiccation, and germination in two tissue fractions (starchy endosperm/aleurone and embryo/scutellum) using the Affymetrix Barley1 GeneChip. To aid data evaluation, Arabidopsis thaliana MapMan and PageMan tools were adapted to barley. The analyses allow a number of conclusions: (1) Cluster analysis revealed a smooth transition in transcription programs between late seed maturation and germination within embryo tissues, but not in the endosperm/aleurone. (2) More than 12,000 transcripts are stored in the embryo of dry barley grains, many of which are presumably activated during germination. (3) Transcriptional activation of storage reserve mobilization events occurs at an early stage of germination, well before radicle protrusion. (4) Key genes of gibberellin (GA) biosynthesis are already active during grain maturation at a time when abscisic acid peaks suggesting the formation of an endogenous store of GA in the aleurone. This GA probably acts later during germination in addition to newly synthesized GA. (5) Beside the well-known role of GA in gene activation during germination spatiotemporal expression data and cis-element searches in homologous rice promoters confirm an equally important gene-activating role of abscisic acid during this developmental period. The respective regulatory webs are linked to auxin and ethylene controlled networks. In summary, new bioinformatics PageMan and MapMan tools developed in barley have been successfully used to investigate in detail the transcriptome relationships between seed maturation and germination in an important crop plant.

Phosphate systemically inhibits development of arbuscular mycorrhiza in Petunia hybrida and represses genes involved in mycorrhizal functioning

Most terrestrial plants form arbuscular mycorrhiza (AM), mutualistic associations with soil fungi of the order Glomeromycota. The obligate biotrophic fungi trade mineral nutrients, mainly phosphate (P(i) ), for carbohydrates from the plants. Under conditions of high exogenous phosphate supply, when the plant can meet its own P requirements without the fungus, AM are suppressed, an effect which could be interpreted as an active strategy of the plant to limit carbohydrate consumption of the fungus by inhibiting its proliferation in the roots. However, the mechanisms involved in fungal inhibition are poorly understood. Here, we employ a transcriptomic approach to get insight into potential shifts in metabolic activity and symbiotic signalling, and in the defence status of plants exposed to high P(i) levels. We show that in mycorrhizal roots of petunia, a similar set of symbiosis-related genes is expressed as in mycorrhizal roots of Medicago, Lotus and rice. P(i) acts systemically to repress symbiotic gene expression and AM colonization in the root. In established mycorrhizal roots, P(i) repressed symbiotic gene expression rapidly, whereas the inhibition of colonization followed with a lag of more than a week. Taken together, these results suggest that P(i) acts by repressing essential symbiotic genes, in particular genes encoding enzymes of carotenoid and strigolactone biosynthesis, and symbiosis-associated phosphate transporters. The role of these effects in the suppression of symbiosis under high P(i) conditions is discussed.

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.

Frequent Gene Movement and Pseudogene Evolution Is Common to the Large and Complex Genomes of Wheat, Barley, and Their Relatives

This work uses survey sequence to examine the genic content of hexaploid wheat group 1 chromosomes, in comparison with barley, and other model grass genomes (rice, Brachypodium, and sorghum), finding that wheat and barley accumulate dramatically more nonsyntenic genes, many of which appear to be pseudogenes. All six arms of the group 1 chromosomes of hexaploid wheat (Triticum aestivum) were sequenced with Roche/454 to 1.3- to 2.2-fold coverage and compared with similar data sets from the homoeologous chromosome 1H of barley (Hordeum vulgare). Six to ten thousand gene sequences were sampled per chromosome. These were classified into genes that have their closest homologs in the Triticeae group 1 syntenic region in Brachypodium, rice (Oryza sativa), and/or sorghum (Sorghum bicolor) and genes that have their homologs elsewhere in these model grass genomes. Although the number of syntenic genes was similar between the homologous groups, the amount of nonsyntenic genes was found to be extremely diverse between wheat and barley and even between wheat subgenomes. Besides a small core group of genes that are nonsyntenic in other grasses but conserved among Triticeae, we found thousands of genic sequences that are specific to chromosomes of one single species or subgenome. By examining in detail 50 genes from chromosome 1H for which BAC sequences were available, we found that many represent pseudogenes that resulted from transposable element activity and double-strand break repair. Thus, Triticeae seem to accumulate nonsyntenic genes frequently. Since many of them are likely to be pseudogenes, total gene numbers in Triticeae are prone to pronounced overestimates.

Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ).

Next-generation whole-genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost-efficient establishment of powerful genomic information for many species.

ABA biosynthesis and degradation contributing to ABA homeostasis during barley seed development under control and terminal drought-stress conditions

Drought is one of the most severe environmental stress factors limiting crop yield especially when occurring during anthesis and seed filling. This terminal drought is characterized by an excess production of the phytohormone abscisic acid (ABA) which plays an important role during seed development and dormancy. All the genes putatively involved in ABA biosynthesis and inactivation in barley were identified and their expression studied during plant ontogeny under standard and drought-stress conditions to learn more about ABA homeostasis and the possible mode of cross-talk between source and sink tissues. Out of 41 genes related to ABA biosynthesis and inactivation 19 were found to be differentially regulated under drought stress in both flag leaves and developing seed during seed filling. Transcripts of plastid-located enzymes are regulated similarly in flag leaf and seed under terminal drought whereas transcripts of cytosolic enzymes are differentially regulated in the two tissues. Detailed information on the expression of defined gene family members is supplemented by measurements of ABA and its degradation and conjugation products, respectively. Under drought stress, flag leaves in particular contain high concentrations of both ABA and the ABA degradation products phaseic acid (PA) and diphaseic acid (DPA); whereas, in seeds, besides ABA, DPA was mainly found. The measurements also revealed a positive correlation between ABA level and starch content in developing seeds for the following reasons: (i) genes of the ABA controlled SnRK2.6 and RCAR/PP2C-mediated signal transduction pathway to the ABF transcription factor HvABI5 are activated in the developing grain under drought, (ii) novel ABA- and dehydration-responsive cis-elements have been found in the promoters of key genes of starch biosynthesis (HvSUS1, HvAGP-L1) and degradation (HvBAM1) and these transcripts/activity are prominently induced in developing seeds during 12 and 16 DAF, (iii) spraying of fluridone (an ABA biosynthesis inhibitor) to drought-stressed plants results in severely impaired starch content and thousand grain weight of mature seeds.

Gene Content and Virtual Gene Order of Barley Chromosome 1H

Chromosome 1H (approximately 622 Mb) of barley (Hordeum vulgare) was isolated by flow sorting and shotgun sequenced by GSFLX pyrosequencing to 1.3-fold coverage. Fluorescence in situ hybridization and stringent sequence comparison against genetically mapped barley genes revealed 95% purity of the sorted chromosome 1H fraction. Sequence comparison against the reference genomes of rice (Oryza sativa) and sorghum (Sorghum bicolor) and against wheat (Triticum aestivum) and barley expressed sequence tag datasets led to the estimation of 4,600 to 5,800 genes on chromosome 1H, and 38,000 to 48,000 genes in the whole barley genome. Conserved gene content between chromosome 1H and known syntenic regions of rice chromosomes 5 and 10, and of sorghum chromosomes 1 and 9 was detected on a per gene resolution. Informed by the syntenic relationships between the two reference genomes, genic barley sequence reads were integrated and ordered to deduce a virtual gene map of barley chromosome 1H. We demonstrate that synteny-based analysis of low-pass shotgun sequenced flow-sorted Triticeae chromosomes can deliver linearly ordered high-resolution gene inventories of individual chromosomes, which complement extensive Triticeae expressed sequence tag datasets. Thus, integration of genomic, transcriptomic, and synteny-derived information represents a major step toward developing reference sequences of chromosomes and complete genomes of the most important plant tribe for mankind.

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome

Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population.