A sequence-ready physical map of barley anchored genetically by two million single-nucleotide polymorphisms

Abstract

Barley (Hordeum vulgare) is an important cereal crop and a model species for Triticeae genomics. To lay the foundation for hierarchical map-based sequencing, a genome-wide physical map of its large and complex 5.1 billion-bp genome was constructed by high-information content fingerprinting of almost 600,000 bacterial artificial chromosomes representing 14-fold haploid genome coverage. The resultant physical map comprises 9,265 contigs with a cumulative size of 4.9 Gb representing 96% of the physical length of the barley genome. The reliability of the map was verified through extensive genetic marker information and the analysis of topological networks of clone overlaps. A minimum tiling path of 66,772 minimally overlapping clones was defined that will serve as a template for hierarchical clone-by-clone map-based shotgun sequencing. We integrated whole-genome shotgun sequence data from the individuals of two mapping populations with published bacterial artificial chromosome survey sequence information to genetically anchor the physical map. This novel approach in combination with the comprehensive whole-genome shotgun sequence data sets allowed us to independently validate and improve a previously reported physical and genetic framework. The resources developed in this study will underpin fine-mapping and cloning of agronomically important genes and the assembly of a draft genome sequence.

Figure 1.

BAC contig example and visualization. A, Visualization of FPcontig_619 and associated clones in Gbrowse (http://seacow.helmholtz-muenchen.de/cgi-bin/gb2/gbrowse/Barley_PhysMap). B, The topology of FPcontig_619 shown as a graph structure with the LTC tool.

Figure 2.

Normalized distribution of clones from different libraries along chromosomes. The distribution of clones from different libraries was plotted along the seven barley chromosomes. The counts of clones from different libraries were normalized by calculating the ratio between observed and expected clone number in a sliding window. Contigs toward telomeres were enriched for clones of library HVVMRX83khA, containing BAC clones identified for the presence of genes (Madishetty et al., 2007).

Figure 3.

Agreement between different anchoring methods. Dot-plot comparisons of different genetic anchoring methods are shown. We anchored BAC contigs by POPSEQ to the Morex × Barke and OWB maps. A, Collinearity between the two genetic maps. B, Combined POPSEQ anchoring and the previously reported anchoring to various genetic maps (The International Barley Genome Sequencing Consortium, 2012).

Figure 4.

Anchored sequence in 5-cM bins. The length of anchored physical contigs was calculated in 5-cM bins and plotted along the genetic map of each chromosome. Note that the y axis has a logarithmic scale.

Figure 5.

Positions of cloned barley genes in the physical map. The positions of 14 previously isolated barley genes on BAC contigs in the physical and genetic framework are visualized along chromosome ideograms. Contigs of the genome-wide physical map (pink bars) are compared with the contigs of the local physical maps from the original publications (blue bars). Centromere positions (marked yellow) are taken from Comadran et al. (2012).