Burkhard Steuernagel

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 4.98u2009Gb, with more than 3.90u2009Gb 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.

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.

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.

De novo 454 sequencing of barcoded BAC pools for comprehensive gene survey and genome analysis in the complex genome of barley

BackgroundDe novo sequencing the entire genome of a large complex plant genome like the one of barley (Hordeum vulgare L.) is a major challenge both in terms of experimental feasibility and costs. The emergence and breathtaking progress of next generation sequencing technologies has put this goal into focus and a clone based strategy combined with the 454/Roche technology is conceivable.ResultsTo test the feasibility, we sequenced 91 barcoded, pooled, gene containing barley BACs using the GS FLX platform and assembled the sequences under iterative change of parameters. The BAC assemblies were characterized by N50 of ~50 kb (N80 ~31 kb, N90 ~21 kb) and a Q40 of 94%. For ~80% of the clones, the best assemblies consisted of less than 10 contigs at 24-fold mean sequence coverage. Moreover we show that gene containing regions seem to assemble completely and uninterrupted thus making the approach suitable for detecting complete and positionally anchored genes.By comparing the assemblies of four clones to their complete reference sequences generated by the Sanger method, we evaluated the distribution, quality and representativeness of the 454 sequences as well as the consistency and reliability of the assemblies.ConclusionThe described multiplex 454 sequencing of barcoded BACs leads to sequence consensi highly representative for the clones. Assemblies are correct for the majority of contigs. Though the resolution of complex repetitive structures requires additional experimental efforts, our approach paves the way for a clone based strategy of sequencing the barley genome.

Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome

BackgroundThere is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys.ResultsA collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley.ConclusionsWe present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes.

PROTEIN DISULFIDE ISOMERASE LIKE 5-1 is a susceptibility factor to plant viruses

Significance This work describes a susceptibility factor to plant viruses that belongs to the conserved PROTEIN DISULFIDE ISOMERASE (PDI) gene family. We show that loss-of-function HvPDIL5-1 alleles at the recessive RESISTANCE TO YELLOW MOSAIC DISEASE 11 (rym11) resistance locus confer broad-spectrum resistance to multiple strains of Bymoviruses and could therefore play a central role in durable virus resistance breeding in barley. The geographic distribution of functional alleles of rym11 in East Asia suggests adaptive selection for resistance in this region. Orthologues of HvPDIL5-1 or related members of the PDI gene family potentially provide susceptibility factors to viruses across animal and plant kingdoms. Protein disulfide isomerases (PDIs) catalyze the correct folding of proteins and prevent the aggregation of unfolded or partially folded precursors. Whereas suppression of members of the PDI gene family can delay replication of several human and animal viruses (e.g., HIV), their role in interactions with plant viruses is largely unknown. Here, using a positional cloning strategy we identified variants of PROTEIN DISULFIDE ISOMERASE LIKE 5–1 (HvPDIL5-1) as the cause of naturally occurring resistance to multiple strains of Bymoviruses. The role of wild-type HvPDIL5-1 in conferring susceptibility was confirmed by targeting induced local lesions in genomes for induced mutant alleles, transgene-induced complementation, and allelism tests using different natural resistance alleles. The geographical distribution of natural genetic variants of HvPDIL5-1 revealed the origin of resistance conferring alleles in domesticated barley in Eastern Asia. Higher sequence diversity was correlated with areas with increased pathogen diversity suggesting adaptive selection for bymovirus resistance. HvPDIL5-1 homologs are highly conserved across species of the plant and animal kingdoms implying that orthologs of HvPDIL5-1 or other closely related members of the PDI gene family may be potential susceptibility factors to viruses in other eukaryotic species.

A distorted circadian clock causes early flowering and temperature-dependent variation in spike development in the Eps-3Am mutant of einkorn wheat.

Viable circadian clocks help organisms to synchronize their development with daily and seasonal changes, thereby providing both evolutionary fitness and advantage from an agricultural perspective. A high-resolution mapping approach combined with mutant analysis revealed a cereal ortholog of Arabidopsis thaliana LUX ARRHYTHMO/PHYTOCLOCK 1 (LUX/PCL1) as a promising candidate for the earliness per se 3 (Eps-3Am) locus in einkorn wheat (Triticum monococcum L.). Using delayed fluorescence measurements it was shown that Eps-3Am containing einkorn wheat accession KT3-5 had a distorted circadian clock. The hypothesis was subsequently confirmed by performing a time course study on central and output circadian clock genes, which showed arrhythmic transcript patterns in KT3-5 under constant ambient conditions, i.e., constant light and temperature. It was also demonstrated that variation in spikelet number between wild-type and mutants is sensitive to temperature, becoming negligible at 25°. These observations lead us to propose that the distorted clock is causative for both early flowering and variation in spike size and spikelet number, and that having a dysfunctional LUX could have neutral, or even positive, effects in warmer climates. To test the latter hypothesis we ascertained sequence variation of LUX in a range of wheat germplasm. We observed a higher variation in the LUX sequence among accessions coming from the warmer climate and a unique in-frame mutation in early-flowering Chinese T. turgidum cultivar ‘Tsing Hua no. 559.’ Our results emphasize the importance of the circadian clock in temperate cereals as a promising target for adaptation to new environments.

Genome Dynamics Explain the Evolution of Flowering Time CCT Domain Gene Families in the Poaceae

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ∼200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken.

Molecular, phylogenetic and comparative genomic analysis of the cytokinin oxidase/dehydrogenase gene family in the Poaceae

The genomes of cereals such as wheat (Triticum aestivum) and barley (Hordeum vulgare) are large and therefore problematic for the map-based cloning of agronomicaly important traits. However, comparative approaches within the Poaceae permit transfer of molecular knowledge between species, despite their divergence from a common ancestor sixty million years ago. The finding that null variants of the rice gene cytokinin oxidase/dehydrogenase 2 (OsCKX2) result in large yield increases provides an opportunity to explore whether similar gains could be achieved in other Poaceae members. Here, phylogenetic, molecular and comparative analyses of CKX families in the sequenced grass species rice, brachypodium, sorghum, maize and foxtail millet, as well as members identified from the transcriptomes/genomes of wheat and barley, are presented. Phylogenetic analyses define four Poaceae CKX clades. Comparative analyses showed that CKX phylogenetic groupings can largely be explained by a combination of local gene duplication, and the whole-genome duplication event that predates their speciation. Full-length OsCKX2 homologues in barley (HvCKX2.1, HvCKX2.2) and wheat (TaCKX2.3, TaCKX2.4, TaCKX2.5) are characterized, with comparative analysis at the DNA, protein and genetic/physical map levels suggesting that true CKX2 orthologs have been identified. Furthermore, our analysis shows CKX2 genes in barley and wheat have undergone a Triticeae-specific gene-duplication event. Finally, by identifying ten of the eleven CKX genes predicted to be present in barley by comparative analyses, we show that next-generation sequencing approaches can efficiently determine the gene space of large-genome crops. Together, this work provides the foundation for future functional investigation of CKX family members within the Poaceae.

The Barley Uniculme4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning

A transcriptional coactivator acts at developmental boundaries to control vegetative branching and leaf patterning. Tillers are vegetative branches that develop from axillary buds located in the leaf axils at the base of many grasses. Genetic manipulation of tillering is a major objective in breeding for improved cereal yields and competition with weeds. Despite this, very little is known about the molecular genetic bases of tiller development in important Triticeae crops such as barley (Hordeum vulgare) and wheat (Triticum aestivum). Recessive mutations at the barley Uniculme4 (Cul4) locus cause reduced tillering, deregulation of the number of axillary buds in an axil, and alterations in leaf proximal-distal patterning. We isolated the Cul4 gene by positional cloning and showed that it encodes a BROAD-COMPLEX, TRAMTRACK, BRIC-À-BRAC-ankyrin protein closely related to Arabidopsis (Arabidopsis thaliana) BLADE-ON-PETIOLE1 (BOP1) and BOP2. Morphological, histological, and in situ RNA expression analyses indicate that Cul4 acts at axil and leaf boundary regions to control axillary bud differentiation as well as the development of the ligule, which separates the distal blade and proximal sheath of the leaf. As, to our knowledge, the first functionally characterized BOP gene in monocots, Cul4 suggests the partial conservation of BOP gene function between dicots and monocots, while phylogenetic analyses highlight distinct evolutionary patterns in the two lineages.