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The HypoMethylated Partial Restriction (HMPR) method reduces the repetitive content of genomic libraries in Norway spruce (Picea abies)
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Växtekologi och evolution.
Dipartimento di Scienze Agrarie e Ambientali, Universita di Udine, Italy.
Dipartimento di Scienze Agrarie e Ambientali, Universita di Udine, Italy.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Växtekologi och evolution.
Vise andre og tillknytning
2013 (engelsk)Inngår i: Tree Genetics & Genomes, ISSN 1614-2942, E-ISSN 1614-2950, Vol. 9, nr 2, s. 601-612Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

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.

sted, utgiver, år, opplag, sider
2013. Vol. 9, nr 2, s. 601-612
Emneord [en]
HMPR libraries, Reduced representation, Picea
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-180368DOI: 10.1007/s11295-012-0582-8ISI: 000316366200023OAI: oai:DiVA.org:uu-180368DiVA, id: diva2:549706
Tilgjengelig fra: 2012-09-05 Laget: 2012-09-05 Sist oppdatert: 2017-12-07bibliografisk kontrollert
Inngår i avhandling
1. Population Genetics and Genome Organization of Norway Spruce
Åpne denne publikasjonen i ny fane eller vindu >>Population Genetics and Genome Organization of Norway Spruce
2012 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Understanding the underlying genetic causes of adaptation to local conditions is one of the main goals of population genetics. A strong latitudinal cline in the phenotypic trait of bud set is observed in present day populations of Norway spruce (Picea abies (L.) Karst). The first step towards determining how this strong selection on adaptive traits translates at the loci underlying the trait was to use multilocus sequence data to gain information on the fundamental population genetic properties of Norway spruce. We determined that the level of LD was low and genetic diversity was in the low range. Coalescent simulations revealed a demographic scenario of a fairly old and severe bottleneck as consistent with the observed data. To examine the role of selection at genes putatively involved in the control of bud set we, again, used a multilocus data set to test for deviations from neutrality and demographic scenarios inferred from background loci. Different candidate genes were identified by using different approaches, highlighting the difficulty in predicting how local adaptation will manifest itself on different time scales and in rangewide samples. When examining properties important in the design of association studies, the inevitable next step in identifying genes involved in local adaptation, we found that previous estimates of a low level of LD were highly influenced by the joint analysis of several loci over a large distribution range and that estimates of LD was in fact heterogeneous across loci and increased within populations. In addition, we found that within species tests for deviations from neutral expectations were highly sensitive to sample size. Additional genomic sequence characterization in Norway spruce is necessary to provide more comprehensive sets of markers for association studies, also including gene promoters and non-genic regions of the genome. In the final paper we show that the HMPR method is effective in constructing libraries enriched for the single copy fraction of the genome when applied to the large and dominantly repetitive genome of Norway spruce.

In summary, the studies presented in this thesis together constitute a foundation for future studies on adaptive evolution in Norway spruce.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2012. s. 44
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 969
Emneord
Picea abies, demographic history, linkage disequilibrium, HMPR
HSV kategori
Forskningsprogram
Biologi med inriktning mot evolutionär funktionsgenomik
Identifikatorer
urn:nbn:se:uu:diva-180370 (URN)978-91-554-8465-1 (ISBN)
Disputas
2012-10-19, Zootissalen, Evolutionsbiologiskt Centrum (EBC), Villavägen 9, Uppsala, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2012-09-28 Laget: 2012-09-05 Sist oppdatert: 2013-01-23bibliografisk kontrollert

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