EST analysis in barley defines a unigene set comprising 4,000 genes

Abstract

We report the generation of 13,109 EST (Expressed Sequence Tag) sequences from barley as a first step towards the generation of a unigene set for this organism. Sequences were generated from three libraries encompassing 7,568 cDNA clones. Comparisons to nucleic acid and protein sequence databases enabled the assignment of putative functions to the mRNAs. The results of the searches against protein databases were parsed and built into a regularly updated database, available over the World Wide Web. The Stack_Pack clustering system has been applied to survey the level of redundancy, which was calculated to amount to 69%, thus we identified 4,000 different barley genes. To prove the usability of the results of the clustering process for further experiments, we subjected alignments with sequences similar to elongation factor 1 alpha to additional analysis. These sequences represented the largest group with identical putative functions (228 members) and clustering based on the analysis of 3; sequences subdivided the group into five different assemblies. Alignments of the consensus sequences facilitated the development of PCR assays suitable for genetic mapping of four of the different gene-family members, which reside on chromosomes 2H, 4H and 5H, thus demonstrating the suitability of the cluster-results as a basis for in-depth analyses of barley gene families.

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.)

“…ESTs comprising both 5′-and 3′-sequences were developed from cDNA libraries representing 16 different tissues or developmental stages, with the vast majority of sequences being derived from the cultivar 'Barke'. A detailed description of a subset of the ESTs used for this study is given by Michalek et al (2002). In a preliminary step, polyA and polyT stretches which correspond to polyA-tails in eucaryotic mRNA were removed with the help of a PERL5 script until no stretch of (T) 5 or (A) 5 was present in a window of 50 bp on the 5′-or 3′-end, respectively.…”

“…Possible explanations for this could be that primers extend across a splice site, the presence of large introns in the genomic sequence, the usage of questionable sequence information for primer development, or primers that were derived from chimeric cDNA clones. Certainly such failures can be minimized by rigorous quality checks of EST sequences and by careful cluster analysis, although there is evidence that the quality of the sequence data used in this study is good (Michalek et al 2002). To minimize primer failure due to low quality sequence reads, sequence information exceeding 700 bp was automatically rejected by the software used.…”

A global assembly of cotton ESTs

“…These projects have provided useful tools for intragenomic comparisons (Schlueter et al 2004) and intergenomic comparisons (Fulton et al 2002), gene discovery (Ewing et al 1999;Ronning et al 2003;Hughes and Friedman 2004), molecular marker identification (Michalek et al 2002), and microarray development (Wisman and Ohlrogge 2000;Kawasaki et al 2001;Alba et al 2004;Arpat et al 2004;Close et al 2004). An initial survey of ∼42,000 fiber ESTs based on a single fiber library from diploid G. arboreum (A genome) proved extremely useful for identifying genes, and led to the development of a 70-mer oligonucleotide cotton fiber microarray.…”

Large-Scale Identification of Expressed Sequence Tags Involved in Rice and Rice Blast Fungus Interaction

“…EST sequencing was the first method used for rapid identification of expressed genes (Adams et al, 1995). It has been employed to identify the genes that are expressed in various tissues, cell types, or developmental stages (Michalek et al, 2002;Ogihara et al, 2003;Ronning et al, 2003). In addition, the availability of cDNA sequences has accelerated further molecular characterization of interesting genes and provided sequence information for microarray design and genome annotation.…”