Abstract
BackgroundGenome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp). Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H.ResultsWe sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1.ConclusionsWe demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.
Highlights
Genome sequencing of barley has been delayed due to its large genome size
Our goal is to contribute to this deeper understanding of allelic variation in barley by developing, characterizing, and providing the full complement of genomics tools (ESTs, transcript map, sequenced full-length (FL) cDNAs and bacterial artificial chromosome (BAC) library) developed for Haruna Nijo at Okayama University
Of the 444 expressed sequence tag (EST) sequences used for the BAC clone selection, 393 ESTs allowed the selection of 400 BAC clones, as seven ESTs were used to select first and second BAC clones due to an insufficient insert size of first BAC clone in the initial pooled library
Summary
Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp). Morex traces to barley germplasm of Manchurian origin and was used as a parent of mapping populations used for extensive linkage and QTL mapping [14] Another high quality BAC library was developed using the Japanese malting barley cultivar “Haruna Nijo” [15]. Haruna Nijo traces to barley germplasm of European origin and may have a haplotype for brewing-related genes similar to European malting barleys. Since these two representative barley cultivars have different origins, the BAC libraries developed from them should contain the sequence variation that leads to phenotypic variation for quality and agronomic characters
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