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Abstract Background Brachypodium distachyon (Brachypodium) has been recognized as a new model species for comparative and functional genomics of cereal and bioenergy crops because it possesses many biological attributes desirable in a model, such as a small genome size, short stature, self-pollinating habit, and short generation cycle. To maximize the utility of Brachypodium as a model for basic and applied research it is necessary to develop genomic resources for it. A BAC-based physical map is one of them. A physical map will facilitate analysis of genome structure, comparative genomics, and assembly of the entire genome sequence. Results A total of 67,151 Brachypodium BAC clones were fingerprinted with the SNaPshot HICF fingerprinting method and a genome-wide physical map of the Brachypodium genome was constructed. The map consisted of 671 contigs and 2,161 clones remained as singletons. The contigs and singletons spanned 414 Mb. A total of 13,970 gene-related sequences were detected in the BAC end sequences (BES). These gene tags aligned 345 contigs with 336 Mb of rice genome sequence, showing that Brachypodium and rice genomes are generally highly colinear. Divergent regions were mainly in the rice centromeric regions. A dot-plot of Brachypodium contigs against the rice genome sequences revealed remnants of the whole-genome duplication caused by paleotetraploidy, which were previously found in rice and sorghum. Brachypodium contigs were anchored to the wheat deletion bin maps with the BES gene-tags, opening the door to Brachypodium-Triticeae comparative genomics. Conclusion The construction of the Brachypodium physical map, and its comparison with the rice genome sequence demonstrated the utility of the SNaPshot-HICF method in the construction of BAC-based physical maps. The map represents an important genomic resource for the completion of Brachypodium genome sequence and grass comparative genomics. A draft of the physical map and its comparisons with rice and wheat are available at http://phymap.ucdavis.edu/brachypodium/.

A comparative physical map of the AA genome (Ouza sativa) and the BB genome (O. punctata) was constructed by aligning a physical map of O. punctata, deduced from 63,942 BAC end sequences (BESs) and 34,224 fingerprints, onto the O. sativa genome sequence. The level of conservation of each chromosome between the two species was determined by calculating a ratio of BES alignments. The alignment result suggests more divergence of intergenic and repeat regions in comparison to gene-rich regions. Further, this characteristic enabled localization of heterochromatic and euchromatic regions for each chromosome of both species. The alignment identified 16 locations containing expansions, contractions, inversions, and transpositions. By aligning 40% of the punctata BES on the map, 87% of the punctata FPC map covered 98% of the O. saliva genome sequence. The genome size of O. punctata was estimated to be 8% larger than that of O. saliva with individual chromosome differences of 1.5-16.5%. The sum of expansions and contractions observed in regions >500 kb were similar, suggesting that most of the contractions/ expansions contributing to the genome size difference between the two species are small, thus preserving the macro-collinearity between these species, which diverged ~2 million years ago.

Author(s): The International Human Genome Mapping Consortium ; Washington University School of Medicine, Genome Sequencing Center: ; John D. McPherson (corresponding author) [1]; Marco Marra [1]; LaDeana Hillier [1]; Robert [...]

Abstract Background Plants can defend themselves against herbivorous insects prior to the onset of larval feeding by responding to the eggs laid on their leaves. In the European field elm (Ulmus minor), egg laying by the elm leaf beetle ( Xanthogaleruca luteola) activates the emission of volatiles that attract specialised egg parasitoids, which in turn kill the eggs. Little is known about the transcriptional changes that insect eggs trigger in plants and how such indirect defense mechanisms are orchestrated in the context of other biological processes. Results Here we present the first large scale study of egg-induced changes in the transcriptional profile of a tree. Five cDNA libraries were generated from leaves of (i) untreated control elms, and elms treated with (ii) egg laying and feeding by elm leaf beetles, (iii) feeding, (iv) artificial transfer of egg clutches, and (v) methyl jasmonate. A total of 361,196 ESTs expressed sequence tags (ESTs) were identified which clustered into 52,823 unique transcripts (Unitrans) and were stored in a database with a public web interface. Among the analyzed Unitrans, 73% could be annotated by homology to known genes in the UniProt (Plant) database, particularly to those from Vitis, Ricinus, Populus and Arabidopsis. Comparative in silico analysis among the different treatments revealed differences in Gene Ontology term abundances. Defense- and stress-related gene transcripts were present in high abundance in leaves after herbivore egg laying, but transcripts involved in photosynthesis showed decreased abundance. Many pathogen-related genes and genes involved in phytohormone signaling were expressed, indicative of jasmonic acid biosynthesis and activation of jasmonic acid responsive genes. Cross-comparisons between different libraries based on expression profiles allowed the identification of genes with a potential relevance in egg-induced defenses, as well as other biological processes, including signal transduction, transport and primary metabolism. Conclusion Here we present a dataset for a large-scale study of the mechanisms of plant defense against insect eggs in a co-evolved, natural ecological plant–insect system. The EST database analysis provided here is a first step in elucidating the transcriptional responses of elm to elm leaf beetle infestation, and adds further to our knowledge on insect egg-induced transcriptomic changes in plants. The sequences identified in our comparative analysis give many hints about novel defense mechanisms directed towards eggs.

Abstract Background Brachypodium distachyon (Brachypodium) has been recognized as a new model species for comparative and functional genomics of cereal and bioenergy crops because it possesses many biological attributes desirable in a model, such as a small genome size, short stature, self-pollinating habit, and short generation cycle. To maximize the utility of Brachypodium as a model for basic and applied research it is necessary to develop genomic resources for it. A BAC-based physical map is one of them. A physical map will facilitate analysis of genome structure, comparative genomics, and assembly of the entire genome sequence. Results A total of 67,151 Brachypodium BAC clones were fingerprinted with the SNaPshot HICF fingerprinting method and a genome-wide physical map of the Brachypodium genome was constructed. The map consisted of 671 contigs and 2,161 clones remained as singletons. The contigs and singletons spanned 414 Mb. A total of 13,970 gene-related sequences were detected in the BAC end sequences (BES). These gene tags aligned 345 contigs with 336 Mb of rice genome sequence, showing that Brachypodium and rice genomes are generally highly colinear. Divergent regions were mainly in the rice centromeric regions. A dot-plot of Brachypodium contigs against the rice genome sequences revealed remnants of the whole-genome duplication caused by paleotetraploidy, which were previously found in rice and sorghum. Brachypodium contigs were anchored to the wheat deletion bin maps with the BES gene-tags, opening the door to Brachypodium-Triticeae comparative genomics. Conclusion The construction of the Brachypodium physical map, and its comparison with the rice genome sequence demonstrated the utility of the SNaPshot-HICF method in the construction of BAC-based physical maps. The map represents an important genomic resource for the completion of Brachypodium genome sequence and grass comparative genomics. A draft of the physical map and its comparisons with rice and wheat are available at http://phymap.ucdavis.edu/brachypodium/.

Abstract Background New sequencing technologies are rapidly emerging. Many laboratories are simultaneously working with the traditional Sanger ESTs and experimenting with ESTs generated by the 454 Life Science sequencers. Though Sanger ESTs have been used to generate contigs for many years, no program takes full advantage of the 5' and 3' mate-pair information, hence, many tentative transcripts are assembled into two separate contigs. The new 454 technology has the benefit of high-throughput expression profiling, but introduces time and space problems for assembling large contigs. Results The PAVE (Program for Assembling and Viewing ESTs) assembler takes advantage of the 5' and 3' mate-pair information by requiring that the mate-pairs be assembled into the same contig and joined by n's if the two sub-contigs do not overlap. It handles the depth of 454 data sets by "burying" similar ESTs during assembly, which retains the expression level information while circumventing time and space problems. PAVE uses MegaBLAST for the clustering step and CAP3 for assembly, however it assembles incrementally to enforce the mate-pair constraint, bury ESTs, and reduce incorrect joins and splits. The PAVE data management system uses a MySQL database to store multiple libraries of ESTs along with their metadata; the management system allows multiple assemblies with variations on libraries and parameters. Analysis routines provide standard annotation for the contigs including a measure of differentially expressed genes across the libraries. A Java viewer program is provided for display and analysis of the results. Our results clearly show the benefit of using the PAVE assembler to explicitly use mate-pair information and bury ESTs for large contigs. Conclusion The PAVE assembler provides a software package for assembling Sanger and/or 454 ESTs. The assembly software, data management software, Java viewer and user's guide are freely available.

Abstract Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely understood at this time, MSLL technology flags both approximate boundaries and methylated genes that deserve additional investigation. MSLL and HMPR sequences provide a valuable resource for maize genome annotation, and are a uniquely valuable complement to any plant genome sequencing project. In order to make these results fully accessible to the community, a web display was developed that shows the alignment of MSLL, HMPR, and other gene-rich sequences to the BACs; this display is continually updated with the latest ESTs and BAC sequences.