An optimized chloroplast DNA extraction protocol for grasses (Poaceae) proves suitable for whole plastid genome sequencing and SNP detection.
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.


RESULTS
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
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 x 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.
Diekmann
Kerstin
K
Teagasc Crops Research Centre, Carlow, Ireland.
Hodkinson
Trevor R
TR
Fricke
Evelyn
E
Barth
Susanne
S
D001483:Base Sequence; D002736:Chloroplasts / Q000378:metabolism; D002874:Chromosome Mapping; D018742:DNA, Chloroplast / Q000235:genetics*; D018744:DNA, Plant; D017343:Genes, Plant; D005821:Genetic Techniques; D018745:Genome, Plant; D054627:Genome, Plastid; D008969:Molecular Sequence Data; D010802:Phylogeny; D018087:Plastids / Q000378:metabolism*; D006109:Poaceae / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA
D018742:DNA, Chloroplast; D018744:DNA, Plant