Open Access Highly Accessed Research article

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

Daniela Schulte16, Ruvini Ariyadasa1, Bujun Shi2, Delphine Fleury2, Chris Saski3, Michael Atkins3, Pieter deJong4, Cheng-Cang Wu5, Andreas Graner1, Peter Langridge2 and Nils Stein1*

Author Affiliations

1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany

2 Australian Centre of Plant Functional Genomics, University of Adelaide, PMB 1 Glen Osmond SA 5064, Australia

3 Clemson University Genomics Institute (CUGI), 51 New Cherry St. BRC 310, Clemson, SC 29634, USA

4 BACPAC Resources, Children's Hospital Oakland, 747 52nd St. Oakland, CA 94609, USA

5 Lucigen Corporation, 2120 West Greenview Dr., Middleton, WI 53562, USA

6 KWS SAAT AG, Grimsehlstr. 31, 37555 Einbeck, Germany

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BMC Genomics 2011, 12:247  doi:10.1186/1471-2164-12-247

Published: 19 May 2011

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.