Nils Stein

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.

Map-based isolation of the leaf rust disease resistance gene Lr10 from the hexaploid wheat (Triticum aestivum L.) genome

More than 50 leaf rust resistance (Lr) genes against the fungal pathogen Puccinia triticina have been identified in the wheat gene pool, and a large number of them have been extensively used in breeding. Of the 50 Lr genes, all are known only from their phenotype and/or map position except for Lr21, which was cloned recently. For many years, the problems of molecular work in the large (1.6 × 1010 bp), highly repetitive (80%), and hexaploid bread wheat (Triticum aestivum L.) genome have hampered map-based cloning. Here, we report the isolation of the Lr gene Lr10 from hexaploid wheat by using a combination of subgenome map-based cloning and haplotype studies in the genus Triticum. Lr10 is a single-copy gene on chromosome 1AS. It encodes a CC-NBS-LRR type of protein with an N-terminal domain, which is under diversifying selection. When overexpressed in transgenic wheat plants, Lr10 confers enhanced resistance to leaf rust. Lr10 has similarities to RPM1 in Arabidopsis thaliana and to resistance gene analogs in rice and barley, but is not closely related to other wheat Lr genes based on Southern analysis. We conclude that map-based cloning of genes of agronomic importance in hexaploid wheat is now feasible, opening perspectives for molecular bread wheat improvement trough transgenic strategies and diagnostic allele detection.

Six-rowed barley originated from a mutation in a homeodomain-leucine zipper I-class homeobox gene

Increased seed production has been a common goal during the domestication of cereal crops, and early cultivators of barley (Hordeum vulgare ssp. vulgare) selected a phenotype with a six-rowed spike that stably produced three times the usual grain number. This improved yield established barley as a founder crop for the Near Eastern Neolithic civilization. The barley spike has one central and two lateral spikelets at each rachis node. The wild-type progenitor (H. vulgare ssp. spontaneum) has a two-rowed phenotype, with additional, strictly rudimentary, lateral rows; this natural adaptation is advantageous for seed dispersal after shattering. Until recently, the origin of the six-rowed phenotype remained unknown. In the present study, we isolated vrs1 (six-rowed spike 1), the gene responsible for the six-rowed spike in barley, by means of positional cloning. The wild-type Vrs1 allele (for two-rowed barley) encodes a transcription factor that includes a homeodomain with a closely linked leucine zipper motif. Expression of Vrs1 was strictly localized in the lateral-spikelet primordia of immature spikes, suggesting that the VRS1 protein suppresses development of the lateral rows. Loss of function of Vrs1 resulted in complete conversion of the rudimentary lateral spikelets in two-rowed barley into fully developed fertile spikelets in the six-rowed phenotype. Phylogenetic analysis demonstrated that the six-rowed phenotype originated repeatedly, at different times and in different regions, through independent mutations of Vrs1.

Recent history of artificial outcrossing facilitates whole-genome association mapping in elite inbred crop varieties

Genomewide association studies depend on the extent of linkage disequilibrium (LD), the number and distribution of markers, and the underlying structure in populations under study. Outbreeding species generally exhibit limited LD, and consequently, a very large number of markers are required for effective whole-genome association genetic scans. In contrast, several of the worlds major food crops are self-fertilizing inbreeding species with narrow genetic bases and theoretically extensive LD. Together these are predicted to result in a combination of low resolution and a high frequency of spurious associations in LD-based studies. However, inbred elite plant varieties represent a unique human-induced pseudooutbreeding population that has been subjected to strong selection for advantageous alleles. By assaying 1,524 genomewide SNPs we demonstrate that, after accounting for population substructure, the level of LD exhibited in elite northwest European barley, a typical inbred cereal crop, can be effectively exploited to map traits by using whole-genome association scans with several hundred to thousands of biallelic SNPs.

Natural variation in a homolog of Antirrhinum CENTRORADIALIS contributed to spring growth habit and environmental adaptation in cultivated barley

As early farming spread from the Fertile Crescent in the Near East around 10,000 years before the present, domesticated crops encountered considerable ecological and environmental change. Spring-sown crops that flowered without the need for an extended period of cold to promote flowering and day length–insensitive crops able to exploit the longer, cooler days of higher latitudes emerged and became established. To investigate the genetic consequences of adaptation to these new environments, we identified signatures of divergent selection in the highly differentiated modern-day spring and winter barleys. In one genetically divergent region, we identify a natural variant of the barley homolog of Antirrhinum CENTRORADIALIS (HvCEN) as a contributor to successful environmental adaptation. The distribution of HvCEN alleles in a large collection of wild and landrace accessions indicates that this involved selection and enrichment of preexisting genetic variants rather than the acquisition of mutations after domestication.

Agriculture: Feeding the future

Humanity depends on fewer than a dozen of the approximately 300,000 species of flowering plants for 80% of its caloric intake. And we capitalize on only a fraction of the genetic diversity that resides within each of these species. This is not enough to support our food system in the future. Food availability must double in the next 25 years to keep pace with population and income growth around the world. Already, food-production systems are precarious in the face of intensifying demand, climate change, soil degradation and water and land shortages. Farmers have saved the seeds of hundreds of crop species and hundreds of thousands of ‘primitive’ varieties (local domesticates called landraces), as well as the wild relatives of crop species and modern varieties no longer in use. These are stored in more than 1,700 gene banks worldwide. Maintaining the 11 international gene-bank collections alone costs about US

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

18 million a year.

Genome-wide association mapping to candidate polymorphism resolution in the unsequenced barley genome

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.

A chromosome conformation capture ordered sequence of the barley genome

Although commonplace in human disease genetics, genome-wide association (GWA) studies have only relatively recently been applied to plants. Using 32 phenotypes in the inbreeding crop barley, we report GWA mapping of 15 morphological traits across ∼500 cultivars genotyped with 1,536 SNPs. In contrast to the majority of human GWA studies, we observe high levels of linkage disequilibrium within and between chromosomes. Despite this, GWA analysis readily detected common alleles of high penetrance. To investigate the potential of combining GWA mapping with comparative analysis to resolve traits to candidate polymorphism level in unsequenced genomes, we fine-mapped a selected phenotype (anthocyanin pigmentation) within a 140-kb interval containing three genes. Of these, resequencing the putative anthocyanin pathway gene HvbHLH1 identified a deletion resulting in a premature stop codon upstream of the basic helix-loop-helix domain, which was diagnostic for lack of anthocyanin in our association and biparental mapping populations. The methodology described here is transferable to species with limited genomic resources, providing a paradigm for reducing the threshold of map-based cloning in unsequenced crops.