Even if Petunia has been used as a model plant to enrich our knowledge on several physiological processes, the magnitude of unexplored sequence space is still large. Here, we report the use of RNA-Seq to characterize the transcriptome of both Petunia inflata and Petunia axillaris and to develop affordable tools for functional genomics. RNA samples from five different plant organs were extracted, pooled and used to generate normalized cDNA libraries. Then, 3’ cDNA fragments were sequenced using the Roche GS-FLX platform. The 454 reads were pre-processed, clustered, assembled and annotated in order to obtain a catalogue of unique transcripts (tentative consensus sequences plus singletons) for each species. The subsequent analyses were restricted to tentative consensus sequences (TCs) since they represent a more reliable and reduced set of sequences with respect to singletons. Firstly, we combined P. inflata and P. axillaris TC sequences into a unique collection and organized them into ‘mixed super-assemblies’ in order to compare the transcriptomes and investigate the extent to which they overlap. Secondly, we selected a well-annotated reference proteome and evaluated if proteins encoded by both the transcriptomes were equally represented with respect to the reference proteome. Lastly, oligonucleotide probes were designed based on TCs and two CombiMatrix custom array chips were synthesized per species. These arrays were used for large scale analysis of organ-specific gene expression in both the species. In addition, RNA from seeds of the partially-characterized an11 mutant from Petunia x hybrida was hybridized on both the arrays in order to obtain information about the molecular mechanisms responsible of the observed seed coat morphological alteration. At the moment, the reconciliation of sequence with gene expression data is underway.

INTEGRATION OF BIOINFORMATICS AND EXPERIMENTAL APPROACHES TO INVESTIGATE USEFUL GENE FUNCTIONALITY IN PETUNIA SPECIES

ZENONI, Sara;TORNIELLI, Giovanni Battista;PEZZOTTI, Mario
2010

Abstract

Even if Petunia has been used as a model plant to enrich our knowledge on several physiological processes, the magnitude of unexplored sequence space is still large. Here, we report the use of RNA-Seq to characterize the transcriptome of both Petunia inflata and Petunia axillaris and to develop affordable tools for functional genomics. RNA samples from five different plant organs were extracted, pooled and used to generate normalized cDNA libraries. Then, 3’ cDNA fragments were sequenced using the Roche GS-FLX platform. The 454 reads were pre-processed, clustered, assembled and annotated in order to obtain a catalogue of unique transcripts (tentative consensus sequences plus singletons) for each species. The subsequent analyses were restricted to tentative consensus sequences (TCs) since they represent a more reliable and reduced set of sequences with respect to singletons. Firstly, we combined P. inflata and P. axillaris TC sequences into a unique collection and organized them into ‘mixed super-assemblies’ in order to compare the transcriptomes and investigate the extent to which they overlap. Secondly, we selected a well-annotated reference proteome and evaluated if proteins encoded by both the transcriptomes were equally represented with respect to the reference proteome. Lastly, oligonucleotide probes were designed based on TCs and two CombiMatrix custom array chips were synthesized per species. These arrays were used for large scale analysis of organ-specific gene expression in both the species. In addition, RNA from seeds of the partially-characterized an11 mutant from Petunia x hybrida was hybridized on both the arrays in order to obtain information about the molecular mechanisms responsible of the observed seed coat morphological alteration. At the moment, the reconciliation of sequence with gene expression data is underway.
petunia; Transcriptome comparison; microarray
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/626352
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