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

Abstract

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).

Figure 1. Assembly workflow.

(a) Assembly of individual BAC clones from paired-end and mate-pair read data. (b) Data integration procedures for pseudomolecule construction.

Figure 2. Collinearity between the Hi-C map and two genetic maps.

The positions of genetic markers (x-axis) are plotted against their genetic positions (y-axis) in a GBS map (top row) and a POPSEQ map (bottom row) of the Morex x Barke recombinant inbred lines.

Figure 3. Collinearity between the Hi-C map and a cytogenetic map of chromosome 3H.

Dots mark the positions of probes in the cytogenetic map (x-axis) and the Hi-C-derived pseudomolecule (y-axis). A linear regression line (red) was fitted with the R function lm(). Note that cytogenetic data is not available for distal regions because probes were designed only for non-recombining peri-centromeric regions.

Figure 4. Accessing sequence and positional information with the barley genome explorer (BARLEX).

The barley pseudomolecule data was imported into BARLEX, where it is directly linked to the IPK Barley BLAST server. Users can paste a nucleotide or amino acid sequence (1) into the BARLEX input query form and select reference database such as pseudomolecules sequence, the set of all BAC assemblies or annotated genes (2). The sequence is then transferred to the IPK barley BLAST Server (3). The web page with the BLAST results (4) contains references to BARLEX information pages for different structural units (BAC sequence contigs, BAC, BAC cluster, chromosomal Hi-C map). For example, the pages of BAC sequence contigs visualize the repeat content based on genome-wide k-mer histograms (5) and are linked to a graph-based visualization (6) of the entire BAC assembly. Summary statistics and positional information of BAC clusters are presented in tables that can be searched, sorted and subsetted using user-defined criteria (7). Users can convert pseudomolecule coordinates (AGP positions) to intervals in the underlying BAC sequence assemblies (8).