Open Access Highly Accessed Research article

De novo assembly and characterization of leaf transcriptome for the development of functional molecular markers of the extremophile multipurpose tree species Prosopis alba

Susana L Torales1*, Máximo Rivarola25, María F Pomponio1, Sergio Gonzalez2, Cintia V Acuña2, Paula Fernández25, Diego L Lauenstein3, Aníbal R Verga3, H Esteban Hopp24, Norma B Paniego25 and Susana N Marcucci Poltri2

Author Affiliations

1 Instituto de Recursos Biológicos, 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 Instituto de Fisiología y Recursos Genéticos Vegetales (IFRGV), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria (INTA), Camino 60 Cuadras, km 5.5, X5020ICA, Córdoba, 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 2013, 14:705  doi:10.1186/1471-2164-14-705

Published: 14 October 2013

Abstract

Background

Prosopis alba (Fabaceae) is an important native tree adapted to arid and semiarid regions of north-western Argentina which is of great value as multipurpose species. Despite its importance, the genomic resources currently available for the entire Prosopis genus are still limited. Here we describe the development of a leaf transcriptome and the identification of new molecular markers that could support functional genetic studies in natural and domesticated populations of this genus.

Results

Next generation DNA pyrosequencing technology applied to P. alba transcripts produced a total of 1,103,231 raw reads with an average length of 421 bp. De novo assembling generated a set of 15,814 isotigs and 71,101 non-assembled sequences (singletons) with an average of 991 bp and 288 bp respectively. A total of 39,000 unique singletons were identified after clustering natural and artificial duplicates from pyrosequencing reads.

Regarding the non-redundant sequences or unigenes, 22,095 out of 54,814 were successfully annotated with Gene Ontology terms. Moreover, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 5,992 and 6,236 markers, respectively, throughout the genome. For the validation of the the predicted SSR markers, a subset of 87 SSRs selected through functional annotation evidence was successfully amplified from six DNA samples of seedlings. From this analysis, 11 of these 87 SSRs were identified as polymorphic. Additionally, another set of 123 nuclear polymorphic SSRs were determined in silico, of which 50% have the probability of being effectively polymorphic.

Conclusions

This study generated a successful global analysis of the P. alba leaf transcriptome after bioinformatic and wet laboratory validations of RNA-Seq data.

The limited set of molecular markers currently available will be significantly increased with the thousands of new markers that were identified in this study. This information will strongly contribute to genomics resources for P. alba functional analysis and genetics. Finally, it will also potentially contribute to the development of population-based genome studies in the genera.

Keywords:
Prosopis alba; Fabaceae; Pyrosequencing; Transcriptome assembly; SSRs; SNPs; Functional annotation