Abstract
BackgroundWheat 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.ResultsWe 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.ConclusionWe 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.
Highlights
Wheat is an excellent species to study freezing tolerance and other abiotic stresses
The large-scale Functional Genomics of Abiotic Stress (FGAS) wheat EST sequencing project was undertaken to identify new genes associated with abiotic stress and to provide physical resources for functional studies
We have developed a unique wheat EST resource from eleven cDNA libraries prepared from tissues at different developmental stages and exposed to different stress conditions (Table 1)
Summary
Wheat is an excellent species to study freezing tolerance and other abiotic stresses. 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. Cold acclimation (CA) allows hardy plants to develop the efficient freezing tolerance (FT) mechanisms needed for winter survival. The availability of wheat genotypes with varying degree of FT makes this species an excellent model to study freezing tolerance and other abiotic stresses. The identification of new genes involved in the cold response will provide invaluable tools to further our understanding of the metabolic pathways of cold acclimation and the acquisition of superior freezing tolerance of hardy genotypes
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