Abstract
Whole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (∼4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.
Highlights
Field pea (Pisum sativum L.) is an important grain legume crop, which was domesticated ∼7000 years ago (Ambrose, 1995; Abbo et al, 2010)
These Whole genome profiling (WGP) tags were tagged to 220,013 bacterial artificial chromosome (BAC) (Supplementary Table S2) with an average of 28.7 tags generated per BAC
The two major steps involved in traditional physical map construction, restriction digestion-based fingerprinting severalfold genome equivalents of BAC clones, and their assembly into contigs, are highly intensive and error prone for a genome as large as pea
Summary
Field pea (Pisum sativum L.) is an important grain legume crop, which was domesticated ∼7000 years ago (Ambrose, 1995; Abbo et al, 2010). Whole genome profiling (WGP) is a sequence-based physical mapping technology for construction of bacterial artificial chromosome (BAC) contigs of complex genomes (van Oeveren et al, 2011).
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