A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.     

Testing the performance of automated annotation of ESTs with the Kegg Orthology (KO) database demonstrates lack of completeness of clusters

The KEGG Orthology (KO) database was tested as a source for automated annotation of expressed sequence tags (ESTs). We used a control experiment where every EST was assigned to its cognate protein, and an annotation experiment where the ESTs were annotated by proteins from other organisms. Analyzing the results, we could assign classes to the annotation: correct, changed and speculated. The correct annotation ranged from 57 (Caenorhabditis elegans) to 81% (Homo sapiens). In spite of the changed annotation being low (1 in H. sapiens to 9% in Arabidopsis thaliana), the speculation was very high (18 in H. sapiens to 38% in C. elegans). We propose eliminating part of the speculated annotation using the KEGG Genes database to enrich KO clusters, decreasing the speculation from 38 to 2% in C. elegans. Thus, the KO database still demands some effort for moving sequences from Kegg GENES to KO, to complement the annotation performance.     

A high-density consensus map of barley linking DArT markers to SSR,RFLP and STS loci and agricultural traits

Background

Wheat is an excellent species to study freezing tolerance and other abiotic stresses. However, the sequence of the wheat genome has not been completely characterized due to its complexity and large size. To circumvent this obstacle and identify genes involved in cold acclimation and associated stresses, a large scale EST sequencing approach was undertaken by the Functional Genomics of Abiotic Stress (FGAS) project.

Results

We generated 73,521 quality-filtered ESTs from eleven cDNA libraries constructed from wheat plants exposed to various abiotic stresses and at different developmental stages. In addition, 196,041 ESTs for which tracefiles were available from the National Science Foundation wheat EST sequencing program and DuPont were also quality-filtered and used in the analysis. Clustering of the combined ESTs with d2_cluster and TGICL yielded a few large clusters containing several thousand ESTs that were refractory to routine clustering techniques. To resolve this problem, the sequence proximity and "bridges" were identified by an e-value distance graph to manually break clusters into smaller groups. Assembly of the resolved ESTs generated a 75,488 unique sequence set (31,580 contigs and 43,908 singletons/singlets). Digital expression analyses indicated that the FGAS dataset is enriched in stress-regulated genes compared to the other public datasets. Over 43% of the unique sequence set was annotated and classified into functional categories according to Gene Ontology.

Conclusion

We have annotated 29,556 different sequences, an almost 5-fold increase in annotated sequences compared to the available wheat public databases. Digital expression analysis combined with gene annotation helped in the identification of several pathways associated with abiotic stress. The genomic resources and knowledge developed by this project will contribute to a better understanding of the different mechanisms that govern stress tolerance in wheat and other cereals.     

Analysis of expressed sequence tags from Brassica rapa L. ssp. pekinensis

Non-redundant expressed sequence tags (ESTs) were generated from six different organs at various developmental stages of Chinese cabbage, Brassica rapa L. ssp. pekinensis. Of the 1,295 ESTs, 915 (71%) showed significantly high homology in nucleotide or deduced amino acid sequences with other sequences deposited in databases, while 380 did not show similarity to any sequences. Briefly, 598 ESTs matched with proteins of identified biological function, 177 with hypothetical proteins or non-annotated Arabidopsis genome sequences, and 140 with other ESTs. About 82% of the top-scored matching sequences were from Arabidopsis or Brassica, but overall 558 (43%) ESTs matched with Arabidopsis ESTs at the nucleotide sequence level. This observation strongly supports the idea that gene-expression profiles of Chinese cabbage differ from that of Arabidopsis, despite their genome structures being similar to each other. Moreover, sequence analyses of 21 Brassica ESTs revealed that their primary structure is different from those of corresponding annotated sequences of Arabidopsis genes. Our data suggest that direct prediction of Brassica gene expression pattern based on the information from Arabidopsis genome research has some limitations. Thus, information obtained from the Brassica EST study is useful not only for understanding of unique developmental processes of the plant, but also for the study of Arabidopsis genome structure.     

The construction of Arabidopsis expressed sequence tag assemblies. A new resource to facilitate gene identification.

The generation of large numbers of partial cDNA sequences, or expressed sequence tags (ESTs), has provided a method with which to sample a large number of genes from an organism. More than 25,000 Arabidopsis thaliana ESTs have been deposited in public databases, producing the largest collection of ESTs for any plant species. We describe here the application of a method of reducing redundancy and increasing information content in this collection by grouping overlapping ESTs representing the same gene into a "contig" or assembly. The increased information content of these assemblies allows more putative identifications to be assigned based on the results of similarity searches with nucleotide and protein databases. The results of this analysis indicate that sequence information is available for approximately 12,600 nonoverlapping ESTs from Arabidopsis. Comparison of the assemblies with 953 Arabidopsis coding sequences indicates that up to 57% of all Arabidopsis genes are represented by an EST. Clustering analysis of these sequences suggests that between 300 and 700 gene families are represented by between 700 and 2000 sequences in the EST database. A database of the assembled sequences, their putative identifications, and cellular roles is available through the World Wide Web.     

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.