Expressed Sequence Tag Database Research Articles

Analyses of Expressed Sequence Tags from <i>Chironomus riparius</i> Using Pyrosequencing : Molecular Ecotoxicology Perspective

ObjectivesChironomus riparius, a non-biting midge (Chironomidae, Diptera), is extensively used as a model organism in aquatic ecotoxicological studies, and considering the potential of C. riparius larvae as a bio-monitoring species, little is known about its genome sequences. This study reports the results of an Expressed Sequence Tags (ESTs) sequencing project conducted on C. riparius larvae using 454 pyrosequencing.MethodsTo gain a better understanding of C. riparius transcriptome, we generated ESTs database of C. ripairus using pyrosequencing method.ResultsSequencing runs, using normalized cDNA collections from fourth instar larvae, yielded 20,020 expressed sequence tags, which were assembled into 8,565 contigs and 11,455 singletons. Sequence analysis was performed by BlastX search against the National Center for Biotechnology Information (NCBI) nucleotide (nr) and uniprot protein database. Based on the gene ontology classifications, 24% (E-value ≤1-5) of the sequences had known gene functions, 24% had unknown functions and 52% of sequences did not match any known sequences in the existing database. Sequence comparison revealed 81% of the genes have homologous genes among other insects belonging to the order Diptera providing tools for comparative genome analyses. Targeted searches using these annotations identified genes associated with essential metabolic pathways, signaling pathways, detoxification of toxic metabolites and stress response genes of ecotoxicological interest.ConclusionsThe results obtained from this study would eventually make ecotoxicogenomics possible in a truly environmentally relevant species, such as, C. riparius.

Conserved and Divergent Rhythms of Crassulacean Acid Metabolism-Related and Core Clock Gene Expression in the Cactus Opuntia ficus-indica

The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional levels.

Generation, analysis and functional annotation of expressed sequence tags from the ectoparasitic mite Psoroptes ovis

BackgroundSheep scab is caused by Psoroptes ovis and is arguably the most important ectoparasitic disease affecting sheep in the UK. The disease is highly contagious and causes and considerable pruritis and irritation and is therefore a major welfare concern. Current methods of treatment are unsustainable and in order to elucidate novel methods of disease control a more comprehensive understanding of the parasite is required. To date, no full genomic DNA sequence or large scale transcript datasets are available and prior to this study only 484 P. ovis expressed sequence tags (ESTs) were accessible in public databases.ResultsIn order to further expand upon the transcriptomic coverage of P. ovis thus facilitating novel insights into the mite biology we undertook a larger scale EST approach, incorporating newly generated and previously described P. ovis transcript data and representing the largest collection of P. ovis ESTs to date. We sequenced 1,574 ESTs and assembled these along with 484 previously generated P. ovis ESTs, which resulted in the identification of 1,545 unique P. ovis sequences. BLASTX searches identified 961 ESTs with significant hits (E-value < 1E-04) and 584 novel P. ovis ESTs. Gene Ontology (GO) analysis allowed the functional annotation of 880 ESTs and included predictions of signal peptide and transmembrane domains; allowing the identification of potential P. ovis excreted/secreted factors, and mapping of metabolic pathways.ConclusionsThis dataset currently represents the largest collection of P. ovis ESTs, all of which are publicly available in the GenBank EST database (dbEST) (accession numbers FR748230 - FR749648). Functional analysis of this dataset identified important homologues, including house dust mite allergens and tick salivary factors. These findings offer new insights into the underlying biology of P. ovis, facilitating further investigations into mite biology and the identification of novel methods of intervention.

Annotation and analysis of expressed sequence tags (ESTs) from Chinese sturgeon ( Acipenser sinensis) pituitary cDNA library

Chinese sturgeon Acipenser sinensis belongs to the family Acipenseridae, an ancient species of actinopterygian fishes. In order to advance molecular research on its reproduction, ontogenetic development, we were seeking for genomic information in the NCBI expressed sequence tag (EST database). We found 3384 indentified cDNA sequences which were assembled into 861 unigenes. Blast analysis revealed 301 unigenes shared high similarity with genes in the public databases, and these were classified into three groups: 202 known genes, 81 putative genes and 8 unknown genes. The remainder (560 genes) had no significant match to any protein sequence. Further, 255 unigenes and 333 unmatched unigenes were annotated with Gene Ontology (GO), which could be classified into cellular component, molecular function, and biological process. Among the known genes, the hormone genes pomc A (proopiomelanocortin), pomc B, GtH alpha I subunit (gonadotropin hormone), GtH alpha II subunit and GH (growth hormone) were present in this library. Comparison of the Chinese sturgeon proteins (GH, GtH alpha subunit and POMC) to proteins of other species showed higher levels of homology among sturgeon species. We performed five hormone related genes including GnRHRI (gonadotropin-releasing hormone receptor I), cpH (carboxypeptidase H), ppiB (peptidylprolyl isomerase B), stmn3 (stathmin-like 3), 7B2 (neuroendocrine protein 7B2), and four novel genes (contig 192, 177, 170 and 168) a semi-quantitative RT-PCR on different tissues from Chinese sturgeon.

Transcriptional interactions during barley susceptible genotype infection with Cochliobolus sativus

A systematic sequencing of expressed sequence tags (ESTs) was used to obtain a global picture of the assembly of barley genes differentially expressed during the hypersensitive reaction of a susceptible genotype in response to an incompatible Cochliobolus sativus pathovar. To identify a large number of plant ESTs, which are induced at different time points, an amplified fragment length polymorphism (AFLP) display of complementary DNA (cDNA) was ulilized. Significant transcriptional changes in the host plant occurred already 4 h post inoculation. Four hundred and fifty six ESTs have been generated, of which 17 (c. 53% up-regulated, 47% down-regulated) have no previously described function. On one hand, the majority of EST-annotations showed protein synthesis, but genes related to signal transduction pathway were also identified. This study provides novel global catalogue ofgene regulations involved in C. sativus-barley interaction not currently represented in EST databases.

The Littorina sequence database (LSD) – an online resource for genomic data

We present an interactive, searchable expressed sequence tag database for the periwinkle snail Littorina saxatilis, an upcoming model species in evolutionary biology. The database is the result of a hybrid assembly between Sanger and 454 sequences, 1290 and 147,491 sequences respectively. Normalized and non-normalized cDNA was obtained from different ecotypes of L. saxatilis collected in the UK and Sweden. The Littorina sequence database (LSD) contains 26,537 different contigs, of which 2453 showed similarity with annotated proteins in UniProt. Querying the LSD permits the selection of the taxonomic origin of blast hits for each contig, and the search can be restricted to particular taxonomic groups. The database allows access to UniProt annotations, blast output, protein family domains (PFAM) and Gene Ontology. The database will allow users to search for genetic markers and identifying candidate genes or genes for expression analyses. It is open for additional deposition of sequence information for L. saxatilis and other species of the genus Littorina. The LSD is available at http://mbio-serv2.mbioekol.lu.se/Littorina/.

The gamma-chain cytokine/receptor system in fish: More ligands and receptors

The mammalian gamma-chain (γC) cytokine family consists of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21. They signal through a receptor complex containing the common γC and a private alpha chain, and in the case of IL-2 and IL-15 an additional common IL-2/15Rβ chain. Deficiency of γC signalling in mammals prevents CD4 + T cells from developing effector functions and CD8 + T cells from developing immunological memory. Thus γC cytokines are critical for the generation and peripheral homeostasis of naïve and memory T cells. This review will give an update on the γC ligands and receptor subunits in fish, and also present some new data on the cloning and expression of a second γC and two IL-2Rβ chains in rainbow trout Oncorhynchus mykiss. In recent years, aided by the availability of sequenced fish genomes and expressed sequence tag databases, five of the six mammalian γC cytokines and their cognate receptors have been discovered in fish, with only the IL-9/IL-9R homologues apparently absent. Paralogues have been discovered in diploid fish and all the receptors described in the tetraploid rainbow trout, including γC itself, IL-2Rβ, IL-4Rα, IL-13Rα1, IL-13Rα2 and IL-2/15Rα, have duplicates. As a consequence of the teleost and salmonid whole genome duplications, even more paralogues may yet be discovered. Some of the paralogues have changes in domain structures and show differential expression and modulation, suggesting the potential for a change in function. Functional characterisation of fish γC cytokines is beginning but made more difficult by the co-existence of so many paralogues of the ligands and their receptors. Initial functional studies have shown that fish γC cytokines can modulate the expression of key cytokines (e.g. interferon-γ, IL-10 and IL-22) of the adaptive immune response, and may thus have promise as adjuvants to improve vaccination efficiency in fish.

In silico mining for simple sequence repeat loci in a pineapple expressed sequence tag database and cross-species amplification of EST-SSR markers across Bromeliaceae

A collection of 5,659 expressed sequence tags (ESTs) from pineapple [Ananas comosus (L.) Merr.] was screened for simple sequence repeats (EST-SSRs) with motif lengths between 1 and 6bp. Lower thresholds of 15, 7 and 5 repeat units were used to define microsatellites of the mono-, di-, and tri- to hexanucleotide repeat type, respectively. Based on these criteria, 696 SSRs were identified among 3,389 EST unigenes, together representing 2,840kb. This corresponds to an average density of one SSR every 4.1kb of non-redundant EST sequences. Dinucleotide repeats were most abundant (38.4% of all SSRs) followed by trinucleotide repeats (38.1%). Flanking primer pairs were designed for 537 EST-SSR loci, and 49 of these were screened for their functionality in 12 accessions of A. comosus, 14 accessions of 5 additional Ananas species and 1 species of Pseudananas. Distinct PCR products of the expected size range were obtained with 36 primer pairs. Eighteen loci analyzed in more detail were all polymorphic in pineapple, and primer pairs flanking these loci also generated PCR products from a wide range of genera and species from six subfamilies of the Bromeliaceae. The potential to reveal polymorphism in a heterologous target species was demonstrated in Deuterocohnia brevifolia (subfamily Pitcairnioideae).

Exploiting Expressed Sequence Tag Databases for the Development and Characterization of Gene-Derived Simple Sequence Repeat Markers in the Opium Poppy (Papaver somniferum L.) for Forensic Applications

Simple sequence repeat (SSR) markers in the opium poppy (Papaver somniferum L.) were identified from an expressed sequence tag (EST) database comprised of 20,340 sequences. In total, 2780 SSR-containing sequences were identified. The most frequent microsatellite had an AT/TA motif (37%). Twenty-two opium poppy EST-SSR markers were presently developed and polymorphisms of six markers (psom 2, 4, 12, 13, 17, and 22) were utilized in 135 individuals under narcotic control investigation. An average of three alleles per locus (range: 2-5 alleles) with a mean heterozygosity of 0.167 was detected. Six loci identified 29 unique profiles in 135 individuals. The EST-SSR markers exhibited small degrees of genetic differentiation (fixation index = 0.727, p < 0.001). Other variable markers will be needed to facilitate the forensic identification of the opium poppy for future cases. To determine the potential for cross-species amplification, six markers were tested in five Papaver genera species and two Eschscholzia genera. The psom 4 and psom 17 primer pair was transferable. This is the first study to report SSR markers of the opium poppy.

Exploiting expressed sequence tag databases for mapping markers associated with fruit development and fruit quality in apple

Apple (Malus × domestica Borkh.) is one of the most important fruit trees grown in Europe and around the world for human consumption, and therefore plant breeders aim at producing new apple varieties with high fruit quality. The availability of molecular markers suitable for marker-assisted selection could greatly increase the efficiency and power of breeding. The recent release of the cv. Golden Delicious genome sequence contributed to an exponential increase in the number of Malus sequences publicly available, while the successful achievement of two apple expressed sequence tag (EST) projects permitted the development of molecular markers from coding sequences. Here we present the setting up and mapping of new EST-based markers specific for fruit development and fruit quality traits. Since a large proportion of the ESTs used in our work were transcriptionally characterized and some of them co-localize within quantitative trait locus regions controlling fruit quality traits, the data reported will be effective in the identification of candidate genes. Due to the high level of polymorphism present in the Malus genome, 70% of the entire set of ESTs analyzed were polymorphic and 80% of them were successfully located using the conventional map-based approach. Fifty new EST-based markers were placed on the apple reference genetic map Fiesta × Discovery, thus enhancing the saturation of some regions, and 17 on Prima × Fiesta. Finally, another 17 markers were located on the Golden Delicious genome sequence using an in-silico approach.

Expressed sequence tags reveal genetic diversity and putative virulence factors of the pathogenic oomycete Pythium insidiosum

Oomycetes are unique eukaryotic microorganisms that share a mycelial morphology with fungi. Many oomycetes are pathogenic to plants, and a more limited number are pathogenic to animals. Pythium insidiosum is the only oomycete that is capable of infecting both humans and animals, and causes a life-threatening infectious disease, called “pythiosis”. In the majority of pythiosis patients life-long handicaps result from the inevitable radical excision of infected organs, and many die from advanced infection. Better understanding P. insidiosum pathogenesis at molecular levels could lead to new forms of treatment. Genetic and genomic information is lacking for P. insidiosum, so we have undertaken an expressed sequence tag (EST) study, and report on the first dataset of 486 ESTs, assembled into 217 unigenes. Of these, 144 had significant sequence similarity with known genes, including 47 with ribosomal protein homology. Potential virulence factors included genes involved in antioxidation, thermal adaptation, immunomodulation, and iron and sterol binding. Effectors resembling pathogenicity factors of plant-pathogenic oomycetes were also discovered, such as, a CBEL-like protein (possible involvement in host cell adhesion and hemagglutination), a putative RXLR effector (possibly involved in host cell modulation) and elicitin-like (ELL) proteins. Phylogenetic analysis mapped P. insidiosum ELLs to several novel clades of oomycete elicitins (ELIs), and homology modeling predicted that P. insidiosum ELLs should bind sterols. Most of the P. insidiosum ESTs showed homology to sequences in the genome or EST databases of other oomycetes, but one putative gene, with unknown function, was found to be unique to P. insidiosum. The EST dataset reported here represents the first steps in identifying genes of P. insidiosum and beginning transcriptome analysis. This genetic information will facilitate understanding of pathogenic mechanisms of this devastating pathogen.

The characterization of a new set of EST-derived simple sequence repeat (SSR) markers as a resource for the genetic analysis of Phaseolus vulgaris

BackgroundOver recent years, a growing effort has been made to develop microsatellite markers for the genomic analysis of the common bean (Phaseolus vulgaris) to broaden the knowledge of the molecular genetic basis of this species. The availability of large sets of expressed sequence tags (ESTs) in public databases has given rise to an expedient approach for the identification of SSRs (Simple Sequence Repeats), specifically EST-derived SSRs. In the present work, a battery of new microsatellite markers was obtained from a search of the Phaseolus vulgaris EST database. The diversity, degree of transferability and polymorphism of these markers were tested.ResultsFrom 9,583 valid ESTs, 4,764 had microsatellite motifs, from which 377 were used to design primers, and 302 (80.11%) showed good amplification quality. To analyze transferability, a group of 167 SSRs were tested, and the results showed that they were 82% transferable across at least one species. The highest amplification rates were observed between the species from the Phaseolus (63.7%), Vigna (25.9%), Glycine (19.8%), Medicago (10.2%), Dipterix (6%) and Arachis (1.8%) genera. The average PIC (Polymorphism Information Content) varied from 0.53 for genomic SSRs to 0.47 for EST-SSRs, and the average number of alleles per locus was 4 and 3, respectively. Among the 315 newly tested SSRs in the BJ (BAT93 X Jalo EEP558) population, 24% (76) were polymorphic. The integration of these segregant loci into a framework map composed of 123 previously obtained SSR markers yielded a total of 199 segregant loci, of which 182 (91.5%) were mapped to 14 linkage groups, resulting in a map length of 1,157 cM.ConclusionsA total of 302 newly developed EST-SSR markers, showing good amplification quality, are available for the genetic analysis of Phaseolus vulgaris. These markers showed satisfactory rates of transferability, especially between species that have great economic and genomic values. Their diversity was comparable to genomic SSRs, and they were incorporated in the common bean reference genetic map, which constitutes an important contribution to and advance in Phaseolus vulgaris genomic research.

Identification of SSR loci in Betula luminifera using birch EST data

Expressed sequence tags (ESTs) are generated from single-pass sequencing of randomly picked cDNA clones and can be used for development of simple sequence repeat (SSR) markers or microsatellites. However, EST databases have been developed for only a small number of species. This paper provides a case study of the utility of freely available birch EST resources for the development of markers necessary for the genetic analysis of Betula luminifera. Based on birch EST data, primers for 80 EST-SSR candidate loci were developed and tested in birch. Of these, 59 EST-SSR loci yielded single, stable and clear PCR products. We then tested the utility of those 59 markers in B. luminifera. The results showed 28 (47.6%) yielded stable and clear PCR products for at least one B. luminifera genotype. In addition, this study describes a rapid and inexpensive alternative for the development of SSRs in species with scarce available sequence data.

Localization of antimicrobial peptides in the tunic of Ciona intestinalis (Ascidiacea, Tunicata) and their involvement in local inflammatory-like reactions

Tunicates comprising a wide variety of different species synthesize antimicrobial peptides as important effector molecules of the innate immune system. Recently, two putative gene families coding for antimicrobial peptides were identified in the expressed sequence tag database of the tunicate Ciona intestinalis. Two synthetic peptides representing the cationic core region of one member of each of the families displayed potent antibacterial and antifungal activities. Moreover, the natural peptides were demonstrated to be synthesized and stored in distinct hemocyte types. Here, we investigated the presence of these natural peptides, namely Ci-MAM-A and Ci-PAP-A, in the tunic of C. intestinalis considering that the ascidian tunic is a body surface barrier exposed to constant microbial assault. Furthermore, as the tunic may represent a major route of entry for pathogen invasion after its damage we monitored the location of these peptides upon a local inflammatory-like reaction induced by injection of foreign cells. Using immunocytochemistry and electron microscopy both peptides were localized to the tunic and were massively present in granulocytes of inflamed tissue. Conclusively, antimicrobial peptides may constitute a chemical barrier within the tunic of urochordates.

Post-transcriptional Control of Na,K-ATPase Activity and Cell Growth by a Splice Variant of FXYD2 Protein with Modified mRNA

In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na(+) affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172C→T sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,K-ATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172C→T mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore, the error-prone PCR alone introduced the mutation with high frequency, implying a structural peculiarity. The data confirm truncation of FXYD2b as a potential mechanism to regulate the amount of FXYD2 at the cell surface to control activity of Na,K-ATPase and cell growth.

Development of new microsatellite loci for the genus Polistes from publicly available expressed sequence tag sequences

Over the last 20 years, microsatellites have revolutionized the study of cooperation in the social insects. The Polistes paper wasps have been an important model system for investigations of cooperative behavior. Recently, an expressed sequence tag (EST) library has been developed for P. metricus, allowing researchers to investigate the genetic basis of cooperative behavior in primitive social insect societies for the first time. We searched these freely available EST sequences for microsatellite motifs. This represents a relatively new approach to the development of microsatellite loci that allows for the development of a greater number of loci at less expense. We designed 32 PCR primer pairs, of which 23 amplified PCR products and 18 were polymorphic. These loci exhibited high levels of polymorphism, comparable to anonymous loci isolated via screens of partial genomic libraries. Thus, they are appropriate for population genetic studies as well as the reconstruction of colony genetic structure. A screen of the entire EST database found a total of 708 di-, tri-, tetra- and penta-nucleotide repeats with large repeat units typical of polymorphic loci and at least 30 bp of flanking sequence for primer design. This pool of potential loci represents a new genetic tool for P. metricus, as well as Polistes more generally, as there is great promise for cross amplification in other species.

Cabomba as a model for studies of early angiosperm evolution.

The angiosperms, or flowering plants, diversified in the Cretaceous to dominate almost all terrestrial environments. Molecular phylogenetic studies indicate that the orders Amborellales, Nymphaeales and Austrobaileyales, collectively termed the ANA grade, diverged as separate lineages from a remaining angiosperm clade at a very early stage in flowering plant evolution. By comparing these early diverging lineages, it is possible to infer the possible morphology and ecology of the last common ancestor of the extant angiosperms, and this analysis can now be extended to try to deduce the developmental mechanisms that were present in early flowering plants. However, not all species in the ANA grade form convenient molecular-genetic models. The present study reviews the genus Cabomba (Nymphaeales), which shows a range of features that make it potentially useful as a genetic model. We focus on characters that have probably been conserved since the last common ancestor of the extant flowering plants. To facilitate the use of Cabomba as a molecular model, we describe methods for its cultivation to flowering in the laboratory, a novel Cabomba flower expressed sequence tag database, a well-adapted in situ hybridization protocol and a measurement of the nuclear genome size of C. caroliniana. We discuss the features required for species to become tractable models, and discuss the relative merits of Cabomba and other ANA-grade angiosperms in molecular-genetic studies aimed at understanding the origin of the flowering plants.

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

BackgroundRust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology.ResultsTo support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs.ConclusionsThe current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.

Transcriptome analysis of the desert locust central nervous system: production and annotation of a Schistocerca gregaria EST database.

BackgroundThe desert locust (Schistocerca gregaria) displays a fascinating type of phenotypic plasticity, designated as ‘phase polyphenism’. Depending on environmental conditions, one genome can be translated into two highly divergent phenotypes, termed the solitarious and gregarious (swarming) phase. Although many of the underlying molecular events remain elusive, the central nervous system (CNS) is expected to play a crucial role in the phase transition process. Locusts have also proven to be interesting model organisms in a physiological and neurobiological research context. However, molecular studies in locusts are hampered by the fact that genome/transcriptome sequence information available for this branch of insects is still limited.MethodologyWe have generated 34,672 raw expressed sequence tags (EST) from the CNS of desert locusts in both phases. These ESTs were assembled in 12,709 unique transcript sequences and nearly 4,000 sequences were functionally annotated. Moreover, the obtained S. gregaria EST information is highly complementary to the existing orthopteran transcriptomic data. Since many novel transcripts encode neuronal signaling and signal transduction components, this paper includes an overview of these sequences. Furthermore, several transcripts being differentially represented in solitarious and gregarious locusts were retrieved from this EST database. The findings highlight the involvement of the CNS in the phase transition process and indicate that this novel annotated database may also add to the emerging knowledge of concomitant neuronal signaling and neuroplasticity events.ConclusionsIn summary, we met the need for novel sequence data from desert locust CNS. To our knowledge, we hereby also present the first insect EST database that is derived from the complete CNS. The obtained S. gregaria EST data constitute an important new source of information that will be instrumental in further unraveling the molecular principles of phase polyphenism, in further establishing locusts as valuable research model organisms and in molecular evolutionary and comparative entomology.

Stanniocalcin Has Deep Evolutionary Roots in Eukaryotes

Vertebrates have a large glycoprotein hormone, stanniocalcin, which originally was shown to inhibit calcium uptake from the environment in teleost fish gills. Later, humans, other mammals, and teleost fish were shown to have two forms of stanniocalcin (STC1 and STC2) that were widely distributed in many tissues. STC1 is associated with calcium and phosphate homeostasis and STC2 with phosphate, but their receptors and signaling pathways have not been elucidated. We undertook a phylogenetic investigation of stanniocalcin beyond the vertebrates using a combination of BLAST and HMMER homology searches in protein, genomic, and expressed sequence tag databases. We identified novel STC homologs in a diverse array of multicellular and unicellular organisms. Within the eukaryotes, almost all major taxonomic groups except plants and algae have STC homologs, although some groups like echinoderms and arthropods lack STC genes. The critical structural feature for recognition of stanniocalcins was the conserved pattern of ten cysteines, even though the amino acid sequence identity was low. Signal peptides in STC sequences suggest they are secreted from the cell of synthesis. The role of glycosylation signals and additional cysteines is not yet clear, although the 11th cysteine, if present, has been shown to form homodimers in some vertebrates. We predict that large secreted stanniocalcin homologs appeared in evolution as early as single-celled eukaryotes. Stanniocalcin's tertiary structure with five disulfide bonds and its primary structure with modest amino acid conservation currently lack an established receptor-signaling system, although we suggest possible alternatives.