Development of New Placental and Fetal Expressed Sequence Tags (EST) for Gene Discovery in Pig Reproduction

One major problem that has high economic impact on pig reproduction is the unexplained loss of potential porcine conceptuses during the first month of gestation. To better understand when and how these losses occur, it is imperative to investigate the underlying genetic regulatory mechanisms. We have recently initiated a large-scale cDNA sequencing project to provide molecular information regarding the genes expressed in female reproductive tissues. cDNA libraries are planned for ovary, hypothalamus, pituitary, placenta, uterus, and several stages of embryonic development. Sequence information will also be highly useful in developing sequence-tagged sites for physical mapping and developing comparative links between the human, mouse, and pig genome maps. We have previously reported the creation of two cDNA libraries, porcine fetal (day 20), and conceptus (day 17). Sequencing of these libraries produced 220 Expressed Sequence Tags (ESTs), with 180 sequences analyzed by clustering algorithms, and 139 clusters identified within these sequences. We now report the creation of two more libraries from porcine fetal (day 45) and placental tissues. The day 45 fetal library has 971,150 independent clones (average insert: 1.4 kb), whereas the placental library has 1,320,000 independent clones. Initial sequencing of the fetal library has produced 119 ESTs (81 clusters), whereas we have obtained 1411 ESTs (1056 clusters) from the placental library. After clustering all sequences thus far obtained, we have identified 1,233 unique clusters. Sequences obtained in this project will be deposited into Genbank dbEST, and all comparative homolog

Development of 30 Novel Polymorphic Expressed Sequence Tags (EST)-Derived Microsatellite Markers for the Miiuy Croaker, Miichthys miiuy

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 30 polymorphic microsatellite markers from 5053 ESTs of the Miichthys miiuy. Out of 123 EST derived microsatellites for which PCR primers were designed, 30 loci were polymorphic in 30 individuals from a single natural population with 2–13 alleles per locus. The observed and expected heterozygosities were from 0.1024 to 0.7917 and from 0.2732 to 0.8845, respectively. Nine loci deviated from the Hardy-Weinberg equilibrium, and linkage disequilibrium was significant between 22 pairs of loci. These polymorphic microsatellite loci will be useful for genetic diversity analysis and molecule-assisted breeding for M. miiuy

An overview of the wcd EST clustering tool

Summary: 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)

SoyXpress: A database for exploring the soybean transcriptome

Abstract Background Experiments using whole transcriptome microarrays produce massive amounts of data. To gain a comprehensive understanding of this gene expression data it needs to be integrated with other available information such as gene function and metabolic pathways. Bioinformatics tools are essential to handle, organize and interpret the results. To date, no database provides whole transcriptome analysis capabilities integrated with terms describing biological functions for soybean (Glycine max (L) Merr.). To this end we have developed SoyXpress, a relational database with a suite of web interfaces to allow users to easily retrieve data and results of the microarray experiment with cross-referenced annotations of expressed sequence tags (EST) and hyperlinks to external public databases. This environment makes it possible to explore differences in gene expression, if any, between for instance transgenic and non-transgenic soybean cultivars and to interpret the results based on gene functional annotations to determine any changes that could potentially alter biological processes. Results SoyXpress is a database designed for exploring the soybean transcriptome. Currently SoyXpress houses 380,095 soybean Expressed Sequence Tags (EST), linked with metabolic pathways, Gene Ontology terms, SwissProt identifiers and Affymetrix gene expression data. Array data is presently available from an experiment profiling global gene expression of three conventional and two genetically engineered soybean cultivars. The microarray data is linked with the sequence data, for maximum knowledge extraction. SoyXpress is implemented in MySQL and uses a Perl CGI interface. Conclusion SoyXpress is designed for the purpose of exploring potential transcriptome differences in different plant genotypes, including genetically modified crops. Soybean EST sequences, microarray and pathway data as well as searchable and browsable gene ontology are integrated and presented. SoyXpress is publicly accessible at http://soyxpress.agrenv.mcgill.ca.</p

Development and Characterization of Polymorphic Microsatellite Markers (SSRs) for an Endemic Plant, Pseudolarix amabilis (Nelson) Rehd. (Pinaceae)

Pseudolarix (Pinaceae) is a vulnerable (sensu IUCN) monotypic genus restricted to southeastern China. To better understand levels of genetic diversity, population structure and gene flow among populations of P. amabilis, we developed five compound SSR markers and ten novel polymorphic expressed sequence tags (EST) derived microsatellites. The results showed that all 15 loci were polymorphic with the number of alleles per locus ranging from two to seven. The expected and observed heterozygosities varied from 0.169 to 0.752, and 0.000 to 1.000, respectively. The inbreeding coefficient ranged from −0.833 to 1.000. These markers will contribute to research on genetic diversity and population genetic structure of P. amabilis, which in turn will contribute to the species conservation

Expressed sequence tag analysis of genes expressed during development of the tropical abalone Haliotis asinina

The tropical abalone. Haliotis asinina. is,in ideal species to investigate the molecular mechanisms that control development. growth, reproduction and shell formation in all cultured haliotids. Here we describe the analysis of 232 expressed sequence tags (EST) obtained front a developmental H. asinina cDNA library intended for future microarray studies. From this data set we identified 183 unique gene Clusters. Of these, 90 clusters showed significant homology with sequences lodged in GenBank, ranging in function from general housekeeping to signal transduction, gene regulation and cell-cell communication. Seventy-one clusters possessed completely novel ORFs greater than 50 codons in length, highlighting the paucity of sequence data from molluscs and other lophotrochozoans. This study of developmental gene expression in H. asinina provides the foundation for further detailed analyses of abalone growth, development and reproduction

An In Silico DNA Sequence Comparison of the Perennial Ryegrass and Rice Genomes

Comparative mapping studies in the family Poaceae, which includes rice (a model species for this family) and perennial ryegrass (PRG) have indicated macro-colinearity of genes is generally conserved across different genomes. Genome mapping of simple sequence repeat markers derived from expressed sequence tags (EST-SSRs) for PRG (Faville et al., 2004) provides a vehicle for DNA sequence-based matching of mapped PRG genes to orthologous positions in the rice genome, which can be used to establish comparative relationships between these species’ genomes. We have initiated such an analysis using an EST-SSR-based PRG genome map. Our objective was to assess this in silico approach as a tool for candidate gene identification from rice, and for targeting markers to specified PRG genome map regions based on rice genome position

Expression analysis of Clavata1-like and Nodulin21-like genes from Pinus sylvestris during ectomycorrhiza formation

The ecology and physiology of ectomycorrhizal (EcM) symbiosis with conifer trees are well documented. In comparison, however, very little is known about the molecular regulation of these associations. In an earlier study, we identified three EcM-regulated Pinus expressed sequence tags (EST), two of which were identified as homologous to the Medicago truncatula nodulin MtN21. The third EST was a homologue to the receptor-like kinase Clavata1. We have characterized the expression patterns of these genes and of auxin- and mycorrhiza-regulated genes after induction with indole-3-butyric acid in Pinus sylvestris and in a time course experiment during ectomycorrhizal initiation with the co-inoculation of 2,3,5-triiodobenzoic acid, an auxin transport inhibitor. Our results suggest that different P. sylvestris nodulin homologues are associated with diverse processes in the root. The results also suggest a potential role of the Clv1-like gene in lateral root initiation by the ectomycorrhizal fungus

Bursaphelenchus xylophilus: opportunities in comparative genomics and molecular host–parasite interactions.

Bursaphelenchus xylophilus, the pinewood nematode (PWN), is the causal agent of the pine wilt disease (PWD), which is a major threat to European and Far East forestlands. Knowledge concerning various aspects of the PWN ecology and interaction with different species of the insect-vector, a broad range of susceptibility/resistance of the tree hosts, coupled with associated bacteria, play a decisive role in the development of the disease. However, these mechanisms are not totally understood. The first insights into PWN genomics were based upon expressed sequence tags (EST) providing the identification of putative parasitism genes, or effectors. Moreover, the recent release of the whole-genome sequence of B. xylophilus will open new opportunities to further studies on nematode interactions, in particular with ecto-mutualistic bacteria that may contribute synergetically for PWN pathogenicity. A review of the current know-how on B. xylophilus is presented with recent developments in molecular biology of PWNwhich may bring new insights into its pathogenicity and interactions with the other factors governing PWD