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Development and Characterization of Three Highly Informative EST-SSR Multiplexes for Pinus halepensis mill. and their Transferability to Other Mediterranean Pines

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Abstract

Aleppo pine (Pinus halepensis Mill.) is an important component of Mediterranean forests. However, the scarcity of molecular resources limits population genetics studies shedding light on biogeographical patterns and/or within-population dynamics in this species. This is due to the complexity of conifers’ genome, which includes many repetitive elements, and to the low genetic variation found in large parts of Aleppo pine distribution. The use of Next-Generation Sequencing (NGS) allows the development of molecular resources even for non-model species with complex genomes (i.e., complete genome and transcriptome sequencing, high numbers of neutral and putative functional markers, EST-SSRs, and SNPs). In this study, we developed a set of 14 polymorphic microsatellites (EST-SSRs) derived from transcriptome data, and combined them in three highly informative multiplexes for cost-effective genetic studies in P. halepensis. The three multiplexes were designed to allow addition of previously available SSRs of adequate size. Loci included in the EST-SSR multiplexes generally showed no null alleles and replicable, easily scorable amplification patterns. Furthermore, the cross-species transferability to two other Mediterranean pines (Pinus pinaster Aiton and Pinus heldreichii H. Christ 1863) was tested, revealing that these EST-SSRs are highly transferable and polymorphic across Mediterranean Pinus species. Finally, preliminary genetic analyses on multiple populations showed some potential applications of the newly developed EST-SSRs to population genetics studies within and among Mediterranean pine populations.

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Acknowledgements

This study was carried out with the financial support of the European Union Seventh Framework Programme project Forger (no. KBBE-289119), and the Italian Ministry of Education, University and Scientific Research project Biodiversitalia (RBAP10A2T4).

We thank Francesca Bagnoli and Sara Pinosio for valuable help with data analysis, Sara Chiussi for producing GIS images, and Enrique Hernández Tecles, Ricardo Alía, Dalibor Ballian, Dragos Postolache, Nicola Moretti, Tiziana Gentilesca, Sebastiano Pini, Marco Borghetti, and the staff of the “Riserva Naturale Statale Montefalcone” and of Pollino National Park for help in collecting plant material.

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    1. Institute of Biosciences and BioResources, National Research Council (CNR), Via Madonna del Piano 10, 50019, Sesto Fiorentino (FI), Italy

      C. Leonarduzzi, I. Spanu, M. Labriola, A. Piotti & G. G. Vendramin

    2. Institute of Biosciences and BioResources, National Research Council (CNR), Division of Palermo, Corso Calatafimi 414, 90129, Palermo, Italy

      C. Leonarduzzi

    3. INRA, UMR1202 BIOGECO, 33610, Cestas, France

      S. C. González-Martínez

    4. University of Bordeaux, BIOGECO, UMR 1202, 33400, Talence, France

      S. C. González-Martínez

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    1. C. Leonarduzzi
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    C. Leonarduzzi and I. Spanu contributed equally to this work.

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    Leonarduzzi, C., Spanu, I., Labriola, M. et al. Development and Characterization of Three Highly Informative EST-SSR Multiplexes for Pinus halepensis mill. and their Transferability to Other Mediterranean Pines. Plant Mol Biol Rep 34, 993–1002 (2016). https://doi.org/10.1007/s11105-016-0980-4

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