454 sequencing put to the test using the complex genome of barley.

BACKGROUND: During the past decade, Sanger sequencing has been used to completely sequence hundreds of microbial and a few higher eukaryote genomes. In recent years, a number of alternative technologies became available, among them adaptations of the pyrosequencing procedure (i.e. "454 sequencing"), promising an approximately 100-fold increase in throughput over Sanger technology--an advancement which is needed to make large and complex genomes more amenable to full genome sequencing at affordable costs. Although several studies have demonstrated its potential usefulness for sequencing small and compact microbial genomes, it was unclear how the new technology would perform in large and highly repetitive genomes such as those of wheat or barley. RESULTS: To study its performance in complex genomes, we used 454 technology to sequence four barley Bacterial Artificial Chromosome (BAC) clones and compared the results to those from ABI-Sanger sequencing. All gene containing regions were covered efficiently and at high quality with 454 sequencing whereas repetitive sequences were more problematic with 454 sequencing than with ABI-Sanger sequencing. 454 sequencing provided a much more even coverage of the BAC clones than ABI-Sanger sequencing, resulting in almost complete assembly of all genic sequences even at only 9 to 10-fold coverage. To obtain highly advanced working draft sequences for the BACs, we developed a strategy to assemble large parts of the BAC sequences by combining comparative genomics, detailed repeat analysis and use of low-quality reads from 454 sequencing. Additionally, we describe an approach of including small numbers of ABI-Sanger sequences to produce hybrid assemblies to partly compensate the short read length of 454 sequences. CONCLUSION: Our data indicate that 454 pyrosequencing allows rapid and cost-effective sequencing of the gene-containing portions of large and complex genomes and that its combination with ABI-Sanger sequencing and targeted sequence analysis can result in large regions of high-quality finished genomic sequences.

Genetic subtraction profiling identifies genes essential for Arabidopsis reproduction and reveals interaction between the female gametophyte and the maternal sporophyte.

BACKGROUND: The embryo sac contains the haploid maternal cell types necessary for double fertilization and subsequent seed development in plants. Large-scale identification of genes expressed in the embryo sac remains cumbersome because of its inherent microscopic and inaccessible nature. We used genetic subtraction and comparative profiling by microarray between the Arabidopsis thaliana wild-type and a sporophytic mutant lacking an embryo sac in order to identify embryo sac expressed genes in this model organism. The influences of the embryo sac on the surrounding sporophytic tissues were previously thought to be negligible or nonexistent; we investigated the extent of these interactions by transcriptome analysis. RESULTS: We identified 1,260 genes as embryo sac expressed by analyzing both our dataset and a recently reported dataset, obtained by a similar approach, using three statistical procedures. Spatial expression of nine genes (for instance a central cell expressed trithorax-like gene, an egg cell expressed gene encoding a kinase, and a synergid expressed gene encoding a permease) validated our approach. We analyzed mutants in five of the newly identified genes that exhibited developmental anomalies during reproductive development. A total of 527 genes were identified for their expression in ovules of mutants lacking an embryo sac, at levels that were twofold higher than in the wild type. CONCLUSION: Identification of embryo sac expressed genes establishes a basis for the functional dissection of embryo sac development and function. Sporophytic gain of expression in mutants lacking an embryo sac suggests that a substantial portion of the sporophytic transcriptome involved in carpel and ovule development is, unexpectedly, under the indirect influence of the embryo sac.

Complex organization and evolution of the tomato pericentromeric region at the FER gene locus.

Tomato (Lycopersicon esculentum) is a model species for molecular biology research and a candidate for large-scale genome sequencing. Pericentromeric heterochromatin constitutes a large portion of the tomato chromosomes. However, the knowledge of the structure, organization, and evolution of such regions remains very limited. Here, we report the analysis of a 198-kb sequence near the FER gene, located in a distal part of pericentromeric heterochromatin on the long arm of tomato chromosome 6. Nine genes, one pseudogene, and 55 transposable elements (TEs) were identified, showing a low gene density (19.8 kb/gene) and a high content of transposable elements (>45% of the sequence). Six genes (56 B23_g3, g5, g7, g8, g9, and g10) have perfect matches (>98% identity) with tomato expressed sequence tags. Two genes (56 B23_g1 and g6), which share <98% sequence identity with expressed sequence tags, were confirmed for transcriptional activity by reverse transcription-PCR. The genes were not uniformly distributed along the sequence and grouped into gene islands separated by stretches of retrotransposons, forming a pattern similar to that found in the gene-rich regions of the large genomes of maize (Zea mays) and Triticeae. Long terminal repeat retrotransposons account for 60% of the TE sequence length. Sixteen of 55 TEs were completely new and remain unclassified. Surprisingly, five of the seven identified DNA transposons were closely associated with coding regions. The action of transposable elements and DNA rearrangements form the molecular basis of the dynamic genome evolution at the FER locus. Multiple rounds of genome duplication in Arabidopsis (Arabidopsis thaliana) and subsequent gene loss have generated a mosaic pattern of conservation between tomato and Arabidopsis orthologous sequences. Our data show that the distal parts of pericentromeric heterochromatin may contain many valuable genes and that these regions form an evolutionary active part of the tomato genome.

Rapid genome divergence at orthologous low molecular weight glutenin loci of the A and Am genomes of wheat.

To study genome evolution in wheat, we have sequenced and compared two large physical contigs of 285 and 142 kb covering orthologous low molecular weight (LMW) glutenin loci on chromosome 1AS of a diploid wheat species (Triticum monococcum subsp monococcum) and a tetraploid wheat species (Triticum turgidum subsp durum). Sequence conservation between the two species was restricted to small regions containing the orthologous LMW glutenin genes, whereas >90% of the compared sequences were not conserved. Dramatic sequence rearrangements occurred in the regions rich in repetitive elements. Dating of long terminal repeat retrotransposon insertions revealed different insertion events occurring during the last 5.5 million years in both species. These insertions are partially responsible for the lack of homology between the intergenic regions. In addition, the gene space was conserved only partially, because different predicted genes were identified on both contigs. Duplications and deletions of large fragments that might be attributable to illegitimate recombination also have contributed to the differentiation of this region in both species. The striking differences in the intergenic landscape between the A and A(m) genomes that diverged 1 to 3 million years ago provide evidence for a dynamic and rapid genome evolution in wheat species.