Abstract
Chromosome 1H (approximately 622 Mb) of barley (Hordeum vulgare) was isolated by flow sorting and shotgun sequenced by GSFLX pyrosequencing to 1.3-fold coverage. Fluorescence in situ hybridization and stringent sequence comparison against genetically mapped barley genes revealed 95% purity of the sorted chromosome 1H fraction. Sequence comparison against the reference genomes of rice (Oryza sativa) and sorghum (Sorghum bicolor) and against wheat (Triticum aestivum) and barley expressed sequence tag datasets led to the estimation of 4,600 to 5,800 genes on chromosome 1H, and 38,000 to 48,000 genes in the whole barley genome. Conserved gene content between chromosome 1H and known syntenic regions of rice chromosomes 5 and 10, and of sorghum chromosomes 1 and 9 was detected on a per gene resolution. Informed by the syntenic relationships between the two reference genomes, genic barley sequence reads were integrated and ordered to deduce a virtual gene map of barley chromosome 1H. We demonstrate that synteny-based analysis of low-pass shotgun sequenced flow-sorted Triticeae chromosomes can deliver linearly ordered high-resolution gene inventories of individual chromosomes, which complement extensive Triticeae expressed sequence tag datasets. Thus, integration of genomic, transcriptomic, and synteny-derived information represents a major step toward developing reference sequences of chromosomes and complete genomes of the most important plant tribe for mankind.
Highlights
Based on the number of genes detected by 454 sequence reads in the genome reference datasets of rice and sorghum and EST datasets of wheat and barley and a 95% probability of chromosome 1H origin, this translated into a gene content of roughly 5,400 genes for chromosome 1H
Our results indicated that roughly one-fifth of the genes of barley chromosome
In our study we focused on barley chromosome 1H
Summary
Purification and Amplification of Chromosomal DNAIntact mitotic chromosomes were isolated by flow cytometric sorting and the purity of the obtained chromosome suspension was determined by FISH essentially as described previously (Suchankova et al, 2006). The DNA of sorted chromosomes was purified and amplified by MDA as described by Simkovaet al. DNA amplified from sorted chromosome 1H (WCA1H) and from sorted chromosomes 1H to 7H (WCAall) was used for 454 shotgun sequencing. Five micrograms of MDA DNA was used to prepare the 454 sequencing library using the GS FLX DNA library preparation kit, following the manufacturer’s instructions (Roche Diagnostics). For WCA1H, six complete GS FLX sequencer runs (70 3 75 picotiter plates) resulted in 3,046,327 reads with a median read length of 258 bp, yielding 799,343,261 bp of raw sequence data (675,561,265 high-quality bases). Two runs with DNA from pooled chromosomes 1H to 7H (WCAall) using half of a 70 3 75 picotiter plate resulted in overall 381,617 reads (median read length = 259 bp), yielding 99,401,554 bp raw sequence data (90,536,939 high-quality bases). All sequence information generated in this study was submitted to the National Center for Biotechnology Information short read archive under accession number SRP001030
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