The transcriptome of Spodoptera exigua larvae exposed to different types of microbes.

We have obtained and characterized the transcriptome of Spodoptera exigua larvae with special emphasis on pathogen-induced genes. In order to obtain a highly representative transcriptome, we have pooled RNA from diverse insect colonies, conditions and tissues. Sequenced cDNA included samples from 3 geographically different colonies. Enrichment of RNA from pathogen-related genes was accomplished by exposing larvae to different pathogenic and non-pathogenic microbial agents such as the bacteria Bacillus thuringiensis, Micrococcus luteus, and Escherichia coli, the yeast Saccharomyces cerevisiae, and the S. exigua nucleopolyhedrovirus (SeMNPV). In addition, to avoid the loss of tissue-specific genes we included cDNA from the midgut, fat body, hemocytes and integument derived from pathogen exposed insects. RNA obtained from the different types of samples was pooled, normalized and sequenced. Analysis of the sequences obtained using the Roche 454 FLX and Sanger methods has allowed the generation of the largest public set of ESTs from S. exigua, including a large group of immune genes, and the identification of an important number of SSR (simple sequence repeats) and SNVs (single nucleotide variants: SNPs and INDELs) with potential use as genetic markers. Moreover, data mining has allowed the discovery of novel RNA viruses with potential influence in the insect population dynamics and the larval interactions with the microbial pesticides that are currently in use for the biological control of this pest.

ngs_backbone: a pipeline for read cleaning, mapping and SNP calling using next generation sequence.

BACKGROUND: The possibilities offered by next generation sequencing (NGS) platforms are revolutionizing biotechnological laboratories. Moreover, the combination of NGS sequencing and affordable high-throughput genotyping technologies is facilitating the rapid discovery and use of SNPs in non-model species. However, this abundance of sequences and polymorphisms creates new software needs. To fulfill these needs, we have developed a powerful, yet easy-to-use application. RESULTS: The ngs_backbone software is a parallel pipeline capable of analyzing Sanger, 454, Illumina and SOLiD (Sequencing by Oligonucleotide Ligation and Detection) sequence reads. Its main supported analyses are: read cleaning, transcriptome assembly and annotation, read mapping and single nucleotide polymorphism (SNP) calling and selection. In order to build a truly useful tool, the software development was paired with a laboratory experiment. All public tomato Sanger EST reads plus 14.2 million Illumina reads were employed to test the tool and predict polymorphism in tomato. The cleaned reads were mapped to the SGN tomato transcriptome obtaining a coverage of 4.2 for Sanger and 8.5 for Illumina. 23,360 single nucleotide variations (SNVs) were predicted. A total of 76 SNVs were experimentally validated, and 85% were found to be real. CONCLUSIONS: ngs_backbone is a new software package capable of analyzing sequences produced by NGS technologies and predicting SNVs with great accuracy. In our tomato example, we created a highly polymorphic collection of SNVs that will be a useful resource for tomato researchers and breeders. The software developed along with its documentation is freely available under the AGPL license and can be downloaded from http://bioinf.comav.upv.es/ngs_backbone/ or http://github.com/JoseBlanca/franklin.

Transcriptomic analysis of tomato carpel development reveals alterations in ethylene and gibberellin synthesis during pat3/pat4 parthenocarpic fruit set.

BACKGROUND: Tomato fruit set is a key process that has a great economic impact on crop production. We employed the Affymetrix GeneChip Tomato Genome Array to compare the transcriptome of a non-parthenocarpic line, UC82, with that of the parthenocarpic line RP75/59 (pat3/pat4 mutant). We analyzed the transcriptome under normal conditions as well as with forced parthenocarpic development in RP75/59, emasculating the flowers 2 days before anthesis. This analysis helps to understand the fruit set in tomato. RESULTS: Differentially expressed genes were extracted with maSigPro, which is designed for the analysis of single and multiseries time course microarray experiments. 2842 genes showed changes throughout normal carpel development and fruit set. Most of them showed a change of expression at or after anthesis. The main differences between lines were concentrated at the anthesis stage. We found 758 genes differentially expressed in parthenocarpic fruit set. Among these genes we detected cell cycle-related genes that were still activated at anthesis in the parthenocarpic line, which shows the lack of arrest in the parthenocarpic line at anthesis. Key genes for the synthesis of gibberellins and ethylene, which were up-regulated in the parthenocarpic line were also detected. CONCLUSION: Comparisons between array experiments determined that anthesis was the most different stage and the key point at which most of the genes were modulated. In the parthenocarpic line, anthesis seemed to be a short transitional stage to fruit set. In this line, the high GAs contends leads to the development of a parthenocarpic fruit, and ethylene may mimic pollination signals, inducing auxin synthesis in the ovary and the development of a jelly fruit.

EST2uni: an open, parallel tool for automated EST analysis and database creation, with a data mining web interface and microarray expression data integration.

BACKGROUND: Expressed sequence tag (EST) collections are composed of a high number of single-pass, redundant, partial sequences, which need to be processed, clustered, and annotated to remove low-quality and vector regions, eliminate redundancy and sequencing errors, and provide biologically relevant information. In order to provide a suitable way of performing the different steps in the analysis of the ESTs, flexible computation pipelines adapted to the local needs of specific EST projects have to be developed. Furthermore, EST collections must be stored in highly structured relational databases available to researchers through user-friendly interfaces which allow efficient and complex data mining, thus offering maximum capabilities for their full exploitation. RESULTS: We have created EST2uni, an integrated, highly-configurable EST analysis pipeline and data mining software package that automates the pre-processing, clustering, annotation, database creation, and data mining of EST collections. The pipeline uses standard EST analysis tools and the software has a modular design to facilitate the addition of new analytical methods and their configuration. Currently implemented analyses include functional and structural annotation, SNP and microsatellite discovery, integration of previously known genetic marker data and gene expression results, and assistance in cDNA microarray design. It can be run in parallel in a PC cluster in order to reduce the time necessary for the analysis. It also creates a web site linked to the database, showing collection statistics, with complex query capabilities and tools for data mining and retrieval. CONCLUSION: The software package presented here provides an efficient and complete bioinformatics tool for the management of EST collections which is very easy to adapt to the local needs of different EST projects. The code is freely available under the GPL license and can be obtained at http://bioinf.comav.upv.es/est2uni. This site also provides detailed instructions for installation and configuration of the software package. The code is under active development to incorporate new analyses, methods, and algorithms as they are released by the bioinformatics community.

AFLP and DNA sequence variation in an Andean domesticate, pepino (Solanum muricatum, Solanaceae): implications for evolution and domestication.

The pepino (Solanum muricatum) is a vegetatively propagated, domesticated native of the Andes, where it grows with wild relatives. We used AFLPs and a 1-kb sequence of the 3-methylcrotonyl-CoA carboxylase gene to study variation of 27 accessions of S. muricatum and 35 collections of 10 species of wild relatives (Solanum section Basarthrum). A total of 298 AFLP fragments and 29 DNA sequence haplotypes were detected. Cluster and principal coordinate analyses and other genetic parameters estimated from both types of markers, show that S. muricatum is closely related to the species from one of the series (Caripensia) of section Basarthrum and that >90% of the variation of the cultigen is also represented in that series. Pepino is highly diverse, either because it is not monophyletic or it has been subjected to regular introgression with wild species, or both. Although a continuous distribution of the genetic variation occurred within the cultivated species, three genetic clusters were recognized. Cluster 1 is mostly centered in Ecuador, cluster 2 in Ecuador and Peru, and cluster 3 in Colombia and Ecuador. Cluster 3 also includes all modern cultivars studied. These results and other evidence suggest that northern Ecuador/southern Colombia is the main center of pepino diversity and the center of origin. The high genetic variation of this cultigen indicates that domestication does not always produce a genetic bottleneck.

Relationships, origin, and diversity of Galapagos tomatoes: implications for the conservation of natural populations.

Endemic Galápagos tomatoes (Lycopersicon cheesmanii) are of great value for cultivated tomato (L. esculentum) breeding, and therefore their conservation is of significance. Although within L. cheesmanii there is heterogeneity for many traits and formal infraspecific classification is not justified, here we distinguish three forms, without taxonomic significance, of L. cheesmanii that are of interest to breeders because of their distinctive morphology and habitat preferences: L. cheesmanii 'short' (one- to two-pinnate leaves, short internodes, and coastal habitats), L. cheesmanii 'long' (one- to two-pinnate leaves, long internodes, and inland habitats), and L. cheesmanii forma minor (three- to four-pinnate leaves, short internodes, and coastal habitats). In a recent survey of tomato populations in the Galápagos Islands, we found that several populations of L. cheesmanii reported 30-50 years earlier had disappeared, mostly as a consequence of human activity. In addition, a previously unreported invasive wild red-fruited form, which we named L. esculentum 'Gal cer,' was found on the island of Santa Cruz. The total diversity (estimated with amplified fragment length polymorphisms [AFLPs]) within L. cheesmanii (H(T) = 0.051) is almost as high as that for the mainland wild species L. pimpinellifolium (H(T) = 0.072). Lycopersicon esculentum 'Gal cer,' on the other hand, has a much lower diversity (H(T) = 0.014). Comparison of AFLP fragments shared by L. esculentum 'Gal cer' with other species showed that it is closely related to weedy tomato L. esculentum var. cerasiforme and, therefore, likely of recent origin. Genetic differentiation among the three native L. cheesmanii forms is low (G(ST) = 0.235), indicating that they share a common genetic background. Nonetheless, L. cheesmanii 'short' is about twice as diverse as L. cheesmanii 'long' or L. cheesmanii f. minor. UPGMA cluster and principal components analysis distinguish four groups within Eulycopersicon: L. pimpinellifolium, cultivated L. esculentum, L. esculentum var. cerasiforme including L. esculentum 'Gal cer,' and L. cheesmanii. The geographic distance and genetic distance in the wild forms of Galápagos tomatoes were not correlated. Apart from the pressure of humans, some native L. cheesmanii populations, especially L. cheesmanii 'long,' might be displaced by invasive L. esculentum 'Gal cer' because they share a similar habitat. We did not find evidence of intercrossing of L. cheesmanii with introduced L. esculentum, but occasional hybridization that contributes to loss of genetic integrity of L. cheesmanii cannot be ruled out. Establishment of reserves of L. cheesmanii to protect this species from introduced herbivorous animals and from hybridization with L. esculentum 'Gal cer' would help to conserve L. cheesmanii. Furthermore, accessions collected by C. M. Rick and others in the 1950s-1970s and now stored in germplasm banks could be used to reinstate some extinct populations.