The Hypomethylated Partial Restriction (HMPR) method reduces the repetitive content of genomic libraries in Norway spruce (Picea abies)
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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.
KeywordsHMPR libraries Reduced representation Picea
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.
- Ahuja MR, Neale DB (2005) Evolution of genome size in conifers. Silvae Genetica 54:126–137Google Scholar
- De Paoli E (2006) Diversità genetica, linkage disequilibrium e componente ripetitiva del genoma in Abete Rosso (Picea abies (L.) Karst.). Dissertation, Università Degli Studi di UdineGoogle Scholar
- Hyten DL, Cannon SB, Song Q, Weeks N, Fickus EW, Shoemaker RC, Specht JE, Farmer AD, May GD, Cregan PB (2010) High-throughput SNP discovery through deep resequencing of a reduced representation library to anchor and orient scaffolds in the soybean whole genome sequence. BMC Genomics 11:38–45PubMedCrossRefGoogle Scholar
- Kerstens HHD, Crooijmans RPMA, Veenendaal A, Dibbits BW, Chin-A-Woeng TFCC, den Dunnen JT, Groenen MAM (2009) Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey. BMC Genomics 10:479–489PubMedCrossRefGoogle Scholar
- Morgante M, De Paoli E (2011) Toward the conifer genome sequence. In: C. Plomion & J. Bousquet (eds) Genetics, genomics and breeding of conifers. Series on "Genomics of Industrial Crops" (Ed. C. Kole), pp. 389–403, Science Publishers, New Hampshire.Google Scholar
- Nelson W, Luo M, Ma J, Estep M, Estill J, He R, Talag J, Sisneros N, Kudrna D, Kim HR, Ammiraju JSS, Collura K, Bharti AK, Messing J, Wing RA, SanMiguel P, Bennetzen JL, Soderlund C (2008) Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains. BMC Genomics 9:621–636PubMedCrossRefGoogle Scholar
- Panzitt K, Tschernatsch MM, Guelly C, Moustafa T, Stradner M, Strohmaier HM, Buck CR, Denk H, Schroeder R, Trauner M et al (2007) Characterization of HULC, a novel gene with striking up-regulation in hepatocellular carcinoma, as noncoding RNA. Gastroenterology 132(1):330–342PubMedCrossRefGoogle Scholar
- Rake AV, Miksche JP, Hall RB, Hansen KM (1980) DNA reassociation kinetics of four conifers. Genome 22:69–79Google Scholar
- Scotti I, Burelli A, Cattonaro F, Chagné D, Fuller J, Hedley PE, Jansson G, Lalanne C, Madur D, Neale D, Plomion C, Powell W, Troggio M, Morgante M (2005) Analysis of the distribution of marker classes in a genetic linkage map: a case study in Norway spruce (Picea abies Karst). Tree Genet Gen 1:93–102CrossRefGoogle Scholar
- Smit AFA, Hubley R, Green P (2010) RepeatMasker Open-3.0. 1996–2010 <http://www.repeatmasker.org>
- Whitelaw CA, Barbazuk WB, Pertea G, Chan AP, Cheung F, Lee Y, Zheng L, van Heeringen S, Karamycheva S, Bennetzen JL, SanMiguel P, Lakey N, Bedell J, Yuan Y, Budiman MA, Resnick A, Van Aken S, Utterback T, Riedmuller S, Williams M, Feldblyum T, Schubert K, Beachy R, Fraser CM, Quackenbush J (2003) Enrichment of gene-coding sequences in maize by genome filtration. Enrichment of gene-coding sequences in maize by genome filtration. Science 302(5653):2118–2120PubMedCrossRefGoogle Scholar