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An Optimized Chloroplast DNA Extraction Protocol for Grasses (Poaceae) Proves Suitable for Whole Plastid Genome Sequencing and SNP Detection
Diekmann, Kerstin Hodkinson, Trevor R Fricke, Evelyn Barth, Susanne
Diekmann, Kerstin
Hodkinson, Trevor R
Fricke, Evelyn
Barth, Susanne
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30/07/2008
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PloSone e2813.pdf
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Diekmann K, Hodkinson TR, Fricke E, Barth S (2008) An Optimized Chloroplast DNA Extraction Protocol for Grasses (Poaceae) Proves Suitable for Whole Plastid Genome Sequencing and SNP Detection. PLoS ONE 3(7): e2813. doi:10.1371/journal.pone.0002813
Abstract
Background
Obtaining chloroplast genome sequences is important to increase the knowledge about the fundamental biology of plastids, to understand evolutionary and ecological processes in the evolution of plants, to develop biotechnological applications (e.g. plastid engineering) and to improve the efficiency of breeding schemes. Extraction of pure chloroplast DNA is required for efficient sequencing of chloroplast genomes. Unfortunately, most protocols for extracting chloroplast DNA were developed for eudicots and do not produce sufficiently pure yields for a shotgun sequencing approach of whole plastid genomes from the monocot grasses.
Methodology/Principal Findings
We have developed a simple and inexpensive method to obtain chloroplast DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Many protocols for extracting chloroplast DNA require an ultracentrifugation step to efficiently separate chloroplast DNA from nuclear DNA. The developed method uses two more centrifugation steps than previously reported protocols and does not require an ultracentrifuge.
Conclusions/Significance
The described method delivered chloroplast DNA of very high quality from two grass species belonging to highly different taxonomic subfamilies within the grass family (Lolium perenne, Pooideae; Miscanthus×giganteus, Panicoideae). The DNA from Lolium perenne was used for whole chloroplast genome sequencing and detection of SNPs. The sequence is publicly available on EMBL/GenBank.