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Here, we report the transcript profile of wheat egg cells and proembryos, just after the first cell division. Microdissected female gametophytes of wheat were used to isolate eggs and two-celled proembryos to construct cell type-specific cDNA libraries. In total, 1197 expressed sequence tags (ESTs) were generated. Analysis of these ESTs revealed numerous novel transcripts. In egg cells, 17.6% of the clustered ESTs represented novel transcripts, while 11.4% novel clusters were identified in the two-celled proembryo. Functional classification of sequences with similarity to previously characterized proteins indicates that the unfertilized egg cell has a higher metabolic activity and protein turnover than previously thought. Transcript composition of two-celled proembryos was significantly distinct from egg cells, reflecting DNA replication as well as high transcriptional and translational activity. Several novel transcripts of the egg cell are specific for this cell. In contrast, some fertilization induced novel mRNAs are abundant also in sporophytic tissues indicating a more general role in plant growth and development. The potential functions of genes based on similarity to known genes involved in developmental processes are discussed. Our analysis has identified numerous genes with potential roles in embryo sac function such as signaling, fertilization or induction of embryogenesis.

Summary: GOSLING is a web-based protein function annotator that uses a decision tree-derived rule set to quickly predict Gene Ontology terms for a protein. A score is assigned to each term prediction that is indicative of the accuracy of the prediction. Due to its speed and accuracy GOSLING is ideally suited for high-throughput annotation tasks. Availability: https://www.sapac.edu.au/gosling Contact: craig@cs.adelaide.edu.au

Tolerance to boron (B) toxicity in barley (Hordeum vulgare L.) is partially attributable to HvNIP2;1, an aquaporin with permeability to B, as well as to silicon, arsenic and germanium (Ge). In this study, we mapped leaf symptoms of Ge toxicity in a doubled-haploid barley population (Clipper × Sahara 3771). Two quantitative trait loci (QTL) associated with Ge toxicity symptoms were identified, located on Chromosomes 6H and 2H. These QTL co-located with two of four B toxicity tolerance loci previously mapped in the same population. The B toxicity tolerance gene underlying the 6H locus encodes HvNIP2;1, whereas the gene(s) and mechanisms underlying the 2H locus are as yet unknown. We provide examples of the application of Ge in studying specific aspects of B toxicity tolerance in plants, including screening of wheat (Triticum aestivum L.) and barley populations for altered function of HvNIP2;1 and related proteins. In particular, Ge may facilitate elucidation of the mechanism and gene(s) underlying the barley Chromosome 2H B tolerance locus.

Background Annotations that describe the function of sequences are enormously important to researchers during laboratory investigations and when making computational inferences. However, there has been little investigation into the data quality of sequence function annotations. Here we have developed a new method of estimating the error rate of curated sequence annotations, and applied this to the Gene Ontology (GO) sequence database (GOSeqLite). This method involved artificially adding errors to sequence annotations at known rates, and used regression to model the impact on the precision of annotations based on BLAST matched sequences. Results We estimated the error rate of curated GO sequence annotations in the GOSeqLite database (March 2006) at between 28% and 30%. Annotations made without use of sequence similarity based methods (non-ISS) had an estimated error rate of between 13% and 18%. Annotations made with the use of sequence similarity methodology (ISS) had an estimated error rate of 49%. Conclusion While the overall error rate is reasonably low, it would be prudent to treat all ISS annotations with caution. Electronic annotators that use ISS annotations as the basis of predictions are likely to have higher false prediction rates, and for this reason designers of these systems should consider avoiding ISS annotations where possible. Electronic annotators that use ISS annotations to make predictions should be viewed sceptically. We recommend that curators thoroughly review ISS annotations before accepting them as valid. Overall, users of curated sequence annotations from the GO database should feel assured that they are using a comparatively high quality source of information.

The News and Views article by Gray and Raybould1 paints a rosy picture of the future of agricultural crops engineered with resistance to the herbicide glyphosate. It is true that the presence of the glyphosate resistance transgene (the EPSPS gene) in the chloroplast genome of crop plants would eliminate pollen transfer of this gene to weedy relatives. But there have already been large-scale releases of transgenic, glyphosate-resistant (‘Roundup Ready’) crops, including glyphosate-resistant Brassica napus (Canola, varieties Quest and LG3295), which is disturbing as there are many Brassica weeds that could potentially hybridize with the engineered B. napus (for example, see ref. 2).

Genes expressed by the male gametophyte have been divided into two classes, the early genes, which are expressed shortly after meiosis, and the late genes, which accumulate after microspore mitosis and are thought to play a role in germination, pollen-stigma interactions and early pollen tube growth (Mascarenhas 1989). Among the best-studied examples of late genes are Zm13 of maize (Hamilton et al. 1989: Hanson et al. 1989) and its homologs PS1 from rice (Zou et al. 1994) and LAT52 of tomato (Twell et al. 1989). Here we report the cDNA sequence of a Zm13 homologue from Phalaris coerulescens, a mediterranean grass which is used as a model system for the study of self-incompatibility in the Poaceae (Li et al. 1994). A cDNA library was constructed from pollen of Phalaris coerulescens in bacteriophage vector Lambda gt10. In order to isolate pollen-specific clones, this library was differentially screened with cDNA probes from leaves, roots, seeds, 7-day-old seedlings and young anthers around meiosis. AWPC-C7c was selected on the basis that it did not show hybridization with cDNA from any of the non-pollen tissues. A developmental RNA blot confirmed that AWPC-C7c expression is restricted to Nucleotide sequence data will appear in the EMBL, GenBank and DDBJ databases under the accession number U85499.

In the grasses, self-incompatibility is controlled by two unlinked loci, S and Z. Previously, Li et al. ([1994][1]) presented a differential screening technique to identify a putative S gene clone from Phalaris coerulescens. In this approach, a cDNA library made from mature pollen of P. coerulescens

Background Upstream open reading frames (uORFs) can down-regulate the translation of the main open reading frame (mORF) through two broad mechanisms: ribosomal stalling and reducing reinitiation efficiency. In distantly related plants, such as rice and Arabidopsis, it has been found that conserved uORFs are rare in these transcriptomes with approximately 100 loci. It is unclear how prevalent conserved uORFs are in closely related plants. Results We used a homology-based approach to identify conserved uORFs in five cereals (monocots) that could potentially regulate translation. Our approach used a modified reciprocal best hit method to identify putative orthologous sequences that were then analysed by a comparative R-nomics program called uORFSCAN to find conserved uORFs. Conclusion This research identified new genes that may be controlled at the level of translation by conserved uORFs. We report that conserved uORFs are rare (

Background Bread wheat is one of the world’s most important food crops and considerable efforts have been made to develop genomic resources for this species. This includes an on-going project by the International Wheat Genome Sequencing Consortium to assemble its large and complex genome, which is hexaploid and contains three closely related ‘homoeologous’ copies for each chromosome. This multi-national effort avoids the complications polyploidy entails for correct assembly of the genome by sequencing flow-sorted chromosome arms one at a time. Here we report on an alternate approach, a direct homoeolog-specific assembly of the expressed portion of the genome, the transcriptome. Results After assessment of the ability of various assemblers to generate homoeolog-specific assemblies, we employed a two-stage assembly process to produce a high-quality assembly of the transcriptome of hexaploid wheat from Roche-454 and Illumina GAIIx paired-end sequence reads. The assembly process made use of a rapid partitioning of expressed sequences into homoeologous clusters, followed by a parallel high-fidelity assembly of each cluster on a 1150-processor compute cloud. We assessed assembly quality through comparison to known wheat gene sequences and found that in ca. 98.5% of cases the assembly was sufficiently accurate for homoeologous triplets to be cleanly separated into either two or three separate contigs. Comparison to publicly available transcript collections suggests that the assembly covers ~75-80% of the complete transcriptome. Conclusions This work therefore describes the first homoeolog-specific sequence assembly of the wheat transcriptome and provides a reference transcriptome for future wheat research. Furthermore, our assembly methodology is transferable to other polyploid organisms.