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Discovery, validation, and in silico functional characterization of EST-SSR markers in Eucalyptus globulus

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Abstract

Eucalyptus globulus is the most commonly planted hardwood species for pulpwood in temperate regions. We aimed to develop and characterize functional molecular markers for population genetic analyses and molecular breeding in this model tree species. Public expressed sequence tag (EST) databases were screened for nonredundant sequences to predict putative gene functions and to discover simple sequence repeats (EST-SSRs), which were then validated in E. globulus and six other Eucalyptus species. A total of 4,924 nonredundant sequences were identified from 12,690 updated E. globulus ESTs. Approximately 19.3% (952) were unigenes and contained 1,140 EST-SSR markers, which were mainly trimeric (58.6%). A set of 979 primers for putative SSR markers was designed after bioinformatic analysis. The predicted functions of these ESTs containing SSR were classified according to their gene ontology (GO) categories (biological process, molecular function, and cellular component). GO categories were assigned to 226 ESTs (30.2%). Most ESTs containing SSR (78.7%) had significant matches (E ≤ 10−5) with the nonredundant protein database using BLASTX. From a set of 56 random primer pairs, 37 could be validated in eight E. globulus genotypes and were also tested for cross-transferability to other six Eucalyptus species (Eucalyptus grandis, Eucalyptus saligna, Eucalyptus dunnii, Eucalyptus viminalis, Eucalyptus camaldulensis, Eucalyptus tereticornis). Seventeen polymorphic EST-SSR markers for E. globulus were evaluated in 60 unrelated trees, being representative of the species’ natural distribution. As a result, six highly informative markers were proposed for genetic diversity analyses, fingerprinting, and comparative population studies, between different species of E. globulus.

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Acknowledgments

Cintia Acuña thanks Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina for a fellowship to support her PhD studies. Special thanks to Pablo Pathauer for collecting and selecting the plant material. We are grateful to Dr. Norma Paniego for their scientific assistance during the first part of this work. Special thanks go to Eleonora Campos and Verónica Villalba for the critical reading of the manuscript. We gratefully acknowledge the excellent assistance of the Bioinformatics Unit at the Biotechnology Institute, INTA Castelar, especially Dr. Marcelo Soria. We thank the anonymous reviewers for their comments and suggestions on the manuscript. This research was supported by the ANPCyT/FONCYT, BID 1728 OC/AR, PICT-2008-00118, INTA-PE 041120, and BiotecSur UE 127118.

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

    Cintia V. Acuña, Paula Fernandez, Pamela V. Villalba, Martín N. García, H. Esteban Hopp & Susana N. Marcucci Poltri

  2. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

    H. Esteban Hopp

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  1. Cintia V. Acuña
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  2. Paula Fernandez
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  3. Pamela V. Villalba
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  4. Martín N. García
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  5. H. Esteban Hopp
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  6. Susana N. Marcucci Poltri
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Correspondence to Susana N. Marcucci Poltri.

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Communicated by G. G. Vendramin

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CVA is a PhD student supported by a fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). Dr. PF is a career member of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). Dr. HEH is also a career member of the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC) and Professor at the Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (UBA).

Electronic supplementary material

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Online Resource 1

In silico EST-SSRs derived from curated public E. globulus libraries. The data describe the 1,140 EST-SSRs: unigenes names, similarity matches, E value, similarity mean, GO term, sequence length, SSR description, primers description (sequence of forward and reverse primers, annealing temperature (°C)), expected product size (bp), and unigene sequences (XLS 1295 kb)

Online Resource 2

Gene ontology annotation. This table provides the detailed information of sequence distribution in graph levels, GO term, number of sequences and parents for biological process, molecular function, and component categories (XLS 112 kb)

Online Resource 3

Pathways maps. This table provides information on the enzymes putatively encoded by the ESTs containing SSR, based on homology prediction and their associated pathways. This includes KEGG maps, enzyme names, and sequences (XLS 54 kb)

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Acuña, C.V., Fernandez, P., Villalba, P.V. et al. Discovery, validation, and in silico functional characterization of EST-SSR markers in Eucalyptus globulus . Tree Genetics & Genomes 8, 289–301 (2012). https://doi.org/10.1007/s11295-011-0440-0

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