Wild plants can contribute valuable genes to their domesticated relatives1. Fertility barriers and a lack of genomic resources have hindered the effective use of crop-wild introgressions. Decades of research into barley's closest wild relative, Hordeum bulbosum, a grass native to the Mediterranean basin and Western Asia, have yet to manifest themselves in the release of a cultivar bearing alien genes2. Here we construct a pangenome of bulbous barley comprising 10 phased genome sequence assemblies amounting to 32 distinct haplotypes. Autotetraploid cytotypes, among which the donors of resistance-conferring introgressions are found, arose at least twice, and are connected among each other and to diploid forms through gene flow. The differential amplification of transposable elements after barley and H. bulbosum diverged from each other is responsible for genome size differences between them. We illustrate the translational value of our resource by mapping non-host resistance to a viral pathogen to a structurally diverse multigene cluster that has been implicated in diverse immune responses in wheat and barley.

• Publication type
• Journal Article MeSH

Optical mapping-a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size-has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purification of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting-assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.

• Keywords
• Bionano genome map, Chromosomes, Flow cytometry and sorting, HMW DNA preparation, Nuclei, Optical mapping, ultralong high-molecular-weight DNA,
• MeSH
• Chromosomes, Plant * genetics   MeSH
• Genome, Plant MeSH
• Flow Cytometry methods   MeSH
• Restriction Mapping MeSH
• Plants * genetics   MeSH
• Sequence Analysis, DNA methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The first gapless, telomere-to-telomere (T2T) sequence assemblies of plant chromosomes were reported recently. However, sequence assemblies of most plant genomes remain fragmented. Only recent breakthroughs in accurate long-read sequencing have made it possible to achieve highly contiguous sequence assemblies with a few tens of contigs per chromosome, that is a number small enough to allow for a systematic inquiry into the causes of the remaining sequence gaps and the approaches and resources needed to close them. Here, we analyse sequence gaps in the current reference genome sequence of barley cv. Morex (MorexV3). Optical map and sequence raw data, complemented by ChIP-seq data for centromeric histone variant CENH3, were used to estimate the abundance of centromeric, ribosomal DNA, and subtelomeric repeats in the barley genome. These estimates were compared with copy numbers in the MorexV3 pseudomolecule sequence. We found that almost all centromeric sequences and 45S ribosomal DNA repeat arrays were absent from the MorexV3 pseudomolecules and that the majority of sequence gaps can be attributed to assembly breakdown in long stretches of satellite repeats. However, missing sequences cannot fully account for the difference between assembly size and flow cytometric genome size estimates. We discuss the prospects of gap closure with ultra-long sequence reads.

• Keywords
• CenH3, Cereba, ChIP-seq, PacBio HiFi reads, flow cytometry, nanopore, ribosomal DNA, satellite, telomeric repeats,
• MeSH
• Chromosomes, Plant genetics   MeSH
• Genome, Plant genetics   MeSH
• Hordeum * genetics   MeSH
• DNA, Ribosomal genetics   MeSH
• Sequence Analysis, DNA MeSH
• Telomere genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Ribosomal MeSH

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool. To further enhance the agronomic potential of rye, we produced a chromosome-scale annotated assembly of the 7.9-gigabase rye genome and extensively validated its quality by using a suite of molecular genetic resources. We demonstrate applications of this resource with a broad range of investigations. We present findings on cultivated rye's incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution, pathogen resistance, low-temperature tolerance, fertility control systems for hybrid breeding and the yield benefits of rye-wheat introgressions.

• MeSH
• Adaptation, Physiological genetics   MeSH
• Stress, Physiological MeSH
• Genome, Plant * MeSH
• Genetic Introgression MeSH
• Plant Immunity genetics   MeSH
• Karyotype MeSH
• Chromosome Mapping methods   MeSH
• Triticum genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Proteins genetics   metabolism   MeSH
• Plant Breeding methods   MeSH
• Crops, Agricultural genetics   immunology   MeSH
• Secale genetics   immunology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Plant Proteins MeSH

Rapid plant genome evolution is crucial to adapt to environmental changes. Chromosomal rearrangements and gene copy number variation (CNV) are two important tools for genome evolution and sources for the creation of new genes. However, their emergence takes many generations. In this study, we show that in Arabidopsis thaliana, a significant loss of ribosomal RNA (rRNA) genes with a past history of a mutation for the chromatin assembly factor 1 (CAF1) complex causes rapid changes in the genome structure. Using long-read sequencing and microscopic approaches, we have identified up to 15 independent large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. Our data suggest that these TDDOs appeared within a few generations, leading to the duplication of hundreds of genes. By subsequently focusing on a line only containing 20% of rRNA gene copies (20rDNA line), we investigated the impact of TDDOs on 3D genome organization, gene expression, and cytosine methylation. We found that duplicated genes often accumulate more transcripts. Among them, several are involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and discuss their potential implications for the evolution of plant genomes.

• MeSH
• Arabidopsis genetics   MeSH
• Gene Duplication * MeSH
• Gene Expression MeSH
• Genome, Plant MeSH
• Genes, rRNA * MeSH
• Genomic Instability MeSH
• Disease Resistance genetics   MeSH
• Gene Expression Regulation, Plant * MeSH
• Genes, Plant MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH