EST and transcriptome analysis of cephalochordate amphioxus--past, present and future

The cephalochordates, commonly known as amphioxus or lancelets, are now considered the most basal chordate group, and the studies of these organisms therefore offer important insights into various levels of evolutionary biology. In the past two decades, the investigation of amphioxus developmental biology has provided key knowledge for understanding the basic patterning mechanisms of chordates. Comparative genome studies of vertebrates and amphioxus have uncovered clear evidence supporting the hypothesis of two-round whole-genome duplication thought to have occurred early in vertebrate evolution and have shed light on the evolution of morphological novelties in the complex vertebrate body plan. Complementary to the amphioxus genome-sequencing project, a large collection of expressed sequence tags (ESTs) has been generated for amphioxus in recent years; this valuable collection represents a rich resource for gene discovery, expression profiling and molecular developmental studies in the amphioxus model. Here, we review previous EST analyses and available cDNA resources in amphioxus and discuss their value for use in evolutionary and developmental studies. We also discuss the potential advantages of applying high-throughput, next-generation sequencing (NGS) technologies to the field of amphioxus research.     

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

Background

Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management.

Results

We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org.

Conclusions

This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.     

An overview of the wcd EST clustering tool

The wcd system is an open source tool for clustering expressed sequence tags (EST) and other DNA and RNA sequences. wcd allows efficient all-versus-all comparison of ESTs using either the d(2) distance function or edit distance, improving existing implementations of d(2). It supports merging, refinement and reclustering of clusters. It is 'drop in' compatible with the StackPack clustering package. wcd supports parallelization under both shared memory and cluster architectures. It is distributed with an EMBOSS wrapper allowing wcd to be installed as part of an EMBOSS installation (and so provided by a web server). AVAILABILITY: wcd is distributed under a GPL licence and is available from http://code.google.com/p/wcdest. SUPPLEMENTARY INFORMATION: Additional experimental results. The wcd manual, a companion paper describing underlying algorithms, and all datasets used for experimentation can also be found at www.bioinf.wits.ac.za/~scott/wcdsupp.html.     

Abundance and diversity of resistance genes in the sugarcane transcriptome revealed by in silico analysis

Resistance genes (R-genes) are responsible for the first interaction of the plant with pathogens being responsible for the activation (or not) of the defense response. Despite their importance and abundance, no tools for their automatic annotation are available yet. The present study analyzed R-genes in the sugarcane expressed sequence tags database which includes 26 libraries of different tissues and development stages comprising 237,954 expressed sequence tags. A new annotation routine was used in order to avoid redundancies and overestimation of R-gene number, common mistakes in previous evaluations. After in silico screening, 280 R-genes were identified, with 196 bearing the complete domains expected. Regarding the alignments, most of the sugarcane's clusters yielded best matches with proteins from Oryza sativa, probably due to the prevalence of sequences of this monocot in data banks. All R-gene classes were found except the subclass LRR-NBS-TIR (leucine-rich repeats, nucleotide-binding site, including Toll interleukin-1 receptors), with prevalence of the kinase (Pto-like) class. R-genes were expressed in all libraries, but flowers, transition root to shoot, and roots were the most representative, suggesting that in sugarcane the expression of R-genes in non-induced conditions prevails in these tissues. In leaves, only low level of expression was found for some gene classes, while others were completely absent. A high allelic diversity was found in all classes of R-genes, sometimes showing best alignments with dicotyledons, despite the great number of genes from rice, maize and other grasses deposited in data banks. The results and future possibilities regarding R-genes in sugarcane research and breeding are further discussed.     

An EST database from saffron stigmas

Background  

Saffron (Crocus sativus L., Iridaceae) flowers have been used as a spice and medicinal plant ever since the Greek-Minoan civilization. The edible part – the stigmas – are commonly considered the most expensive spice in the world and are the site of a peculiar secondary metabolism, responsible for the characteristic color and flavor of saffron.     

A survey of genes in Eimeria tenella merozoites by EST sequencing

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.     

Comparative transcriptome profiling of resistant and susceptible sugarcane genotypes in response to the airborne pathogen Fusarium verticillioides

Molecular Biology Reports - Fusarium verticillioides is the pathogen associated with pokkah boeng disease (PBD), the most significant airborne disease of sugarcane. The molecular mechanisms that...     

A survey of genes in Eimeria tenella merozoites by EST sequencing

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.