Transcriptome survey of Patagonian southern beech Nothofagus nervosa (= N. alpina): assembly, annotation and molecular marker discovery

Nothofagus nervosa is one of the most emblematic native tree species of Patagoniantemperate forests. Here, the shotgun RNA-sequencing (RNA-Seq) of the transcriptome of N.nervosa, including de novo assembly, functional annotation, and in silico discovery ofpotential molecular markers to support population and associations genetic studies, aredescribed.

Results: Pyrosequencing of a young leaf cDNA library generated a total of 111,814 high quality reads,with an average length of 447 bp.

De novo assembly using Newbler resulted into 3,005tentative isotigs (including alternative transcripts). The non-assembled sequences (singletons)were clustered with CD-HIT-454 to identify natural and artificial duplicates frompyrosequencing reads, leading to 21,881 unique singletons.

15,497 out of 24,886 nonredundantsequences or unigenes, were successfully annotated against a plant proteindatabase. A substantial number of simple sequence repeat markers (SSRs) were discovered inthe assembled and annotated sequences.

More than 40% of the SSR sequences were insideORF sequences. To confirm the validity of these predicted markers, a subset of 73 SSRsselected through functional annotation evidences were successfully amplified from sixseedlings DNA samples, being 14 polymorphic.

Conclusions: This paper is the first report that shows a highly precise representation of the mRNAsdiversity present in young leaves of a native South American tree, N.

nervosa, as well as its insilico deduced putative functionality. The reported Nothofagus transcriptome sequencesrepresent a unique resource for genetic studies and provide a tool to discover genes of interestand genetic markers that will greatly aid questions involving evolution, ecology, andconservation using genetic and genomic approaches in the genus.

Published on: 2012-07-02

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