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Open Access Research article

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

Susana L Torales1*, Máximo Rivarola25, María F Pomponio1, Paula Fernández25, Cintia V Acuña2, Paula Marchelli35, Sergio Gonzalez2, María M Azpilicueta3, Horacio Esteban Hopp24, Leonardo A Gallo3, Norma B Paniego25 and Susana N Marcucci Poltri2*

Author Affiliations

1 Instituto de RecursosBiológicos, IRB, Instituto Nacional de Tecnología Agropecuaria (INTA Castelar), CC 25, Castelar, B1712WAA, Argentina

2 Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA Castelar), CC 25, Castelar, B1712WAA, Argentina

3 EEA Bariloche, Genética Ecológica y Mejoramiento Forestal, Instituto Nacional de Tecnología Agropecuaria (INTA, Bariloche), CC 277, 8400, Bariloche, Argentina

4 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

5 CONICET, Buenos Aires, Argentina

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BMC Genomics 2012, 13:291  doi:10.1186/1471-2164-13-291

Published: 2 July 2012

Abstract

Background

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

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 3,005 tentative isotigs (including alternative transcripts). The non-assembled sequences (singletons) were clustered with CD-HIT-454 to identify natural and artificial duplicates from pyrosequencing reads, leading to 21,881 unique singletons. 15,497 out of 24,886 non-redundant sequences or unigenes, were successfully annotated against a plant protein database. A substantial number of simple sequence repeat markers (SSRs) were discovered in the assembled and annotated sequences. More than 40% of the SSR sequences were inside ORF sequences. To confirm the validity of these predicted markers, a subset of 73 SSRs selected through functional annotation evidences were successfully amplified from six seedlings DNA samples, being 14 polymorphic.

Conclusions

This paper is the first report that shows a highly precise representation of the mRNAs diversity present in young leaves of a native South American tree, N. nervosa, as well as its in silico deduced putative functionality. The reported Nothofagus transcriptome sequences represent a unique resource for genetic studies and provide a tool to discover genes of interest and genetic markers that will greatly aid questions involving evolution, ecology, and conservation using genetic and genomic approaches in the genus.

Keywords:
Nothofagaceae; Forest genomics; Pyrosequencing; de novo transcriptome assembly; SSRs; Functional annotation