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Tree Genetics & Genomes

, Volume 9, Issue 2, pp 601–612 | Cite as

The Hypomethylated Partial Restriction (HMPR) method reduces the repetitive content of genomic libraries in Norway spruce (Picea abies)

  • Hanna LarssonEmail author
  • Emanuele De Paoli
  • Michele Morgante
  • Martin Lascoux
  • Niclas Gyllenstrand
Original Paper

Abstract

To evaluate the usefulness of Reduced Representation Libraries (RRL) in species with large and highly repetitive genomes such as conifers, we employed Hypomethylated Partial Restriction (HMPR) on the genome of Norway spruce (Picea abies). The HMPR method preferentially removes the commonly hypermethylated, repetitive fraction of the genome. Hence, RRLs should be enriched for the hypomethylated gene space. For comparison, a standard shotgun library was constructed and samples of the respective libraries were obtained through Sanger sequencing. We obtained a 9-fold gene enrichment, a value which is slightly higher than for other plant species. The amount of repetitive DNA was reduced by 45 % in the RRLs, demonstrating the ability to efficiently remove hypermethylated DNA. Annotating sequences in an uncharacterized genome remains challenging and a large number of sequences could not be classified as either repetitive DNA or as belonging to the gene space. Upon further investigation, we found that some of these uncharacterized fragments were expressed, and in most cases the expression was spatially differentiated, indicating that they might have a function. Full-length transcripts of a subset of expressed fragments also revealed that these could be long non-coding RNAs. In conclusion, our study shows that the HMPR method is effective in constructing libraries enriched for the genic fraction of the genome, while simultaneously reducing the repetitive fraction, in P. abies and may prove a valuable tool for the discovery, validation, and assessment of genetic markers in population studies and breeding efforts when combined with next-generation sequencing technology.

Keywords

HMPR libraries Reduced representation Picea 

Notes

Acknowledgments

This work was supported by the European Community’s Sixth Framework Programme, under the Network of Excellence Evoltree; by the Seventh Framework Programme (FP7/2007–2013), under grant agreement 211868 (Project Noveltree), by the Nilsson-Ehle foundation and the Swedish research council FORMAS. We thank Thomas Källman for the use of P. abies RNAseq data and Jun Chen for assistance with data analysis. We thank two anonymous reviewers for helpful comments and improvements to the manuscript.

Supplementary material

11295_2012_582_MOESM1_ESM.xls (1.5 mb)
ESM 1 (XLS 1571 kb)
11295_2012_582_MOESM2_ESM.doc (236 kb)
ESM 2 (DOC 236 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Hanna Larsson
    • 1
    Email author
  • Emanuele De Paoli
    • 2
  • Michele Morgante
    • 2
  • Martin Lascoux
    • 1
  • Niclas Gyllenstrand
    • 3
  1. 1.Department of Ecology and Genetics, EBCUppsala UniversityUppsalaSweden
  2. 2.Dipartimento di Scienze Agrarie e AmbientaliUniversità di UdineUdineItaly
  3. 3.Department of Plant Biology and Forest GeneticsSwedish University of Agricultural SciencesUppsalaSweden

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