BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.)

Abstract

Background: Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place.

Result: Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with <30 or >250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X.

Conclusion: BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.

Figure 1

Insert size estimation of barley BAC clones by pulsed field gel electrophoresis (PFGE). A sample set of 8 clones is visualized for all five new libraries. Each set of eight clones is preceeded by a lane showing low range PFG marker (New England Biolabs).Arrowheads indicate in each panel the position of the 97, 48.5 and 6.55 kb fragments of the PFG marker.

Figure 2

Example of a BAC clone characterized by High Information Content Fingerprinting (HICF). After restriction digestion, SNaPShot labeling and separation of labeled fragments (red, blue, green, black) the sample was loaded together with the size standard GS1200LIZ (orange). Image is a screenshot taken from FPMiner 2.0 software

Figure 3

From all BAC libraries random subsamples between10,279-10,685 clones (Table 2) were fingerprinted (HICF). The total number of fragments per analyzed clone was plotted in ascending order for each library. The legend shows the color-coding for the investigated libraries

Figure 4

Hybridization results of EST-derived probes on different BAC-filters. Probe details are provided in additional file 2. A: Hybridization with GBR-probes on 5 BAC libraries. The probe average including the standard deviation is given in the last column B: Hybridization result on HVVMRXALLhC library with 17 EST-derived probes assigned to chromosome 3H