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

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 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.

SUBMITTER: Diekmann K

PROVIDER: S-EPMC2474675 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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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 K Hodkinson Trevor R TR Fricke Evelyn E Barth Susanne S

PloS one 20080730 7

<h4>Background</h4>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 ...[more]

PMID: 18665252

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Project description:Date palm is a very important crop in western Asia and northern Africa, and it is the oldest domesticated fruit tree with archaeological records dating back 5000 years. The huge economic value of this crop has generated considerable interest in breeding programs to enhance production of dates. One of the major limitations of these efforts is the uncertainty regarding the number of date palm cultivars, which are currently based on fruit shape, size, color, and taste. Whole mitochondrial and plastid genome sequences were utilized to examine single nucleotide polymorphisms (SNPs) of date palms to evaluate the efficacy of this approach for molecular characterization of cultivars. Mitochondrial and plastid genomes of nine Saudi Arabian cultivars were sequenced. For each species about 60 million 100 bp paired-end reads were generated from total genomic DNA using the Illumina HiSeq 2000 platform. For each cultivar, sequences were aligned separately to the published date palm plastid and mitochondrial reference genomes, and SNPs were identified. The results identified cultivar-specific SNPs for eight of the nine cultivars. Two previous SNP analyses of mitochondrial and plastid genomes identified substantial intra-cultivar ( = intra-varietal) polymorphisms in organellar genomes but these studies did not properly take into account the fact that nearly half of the plastid genome has been integrated into the mitochondrial genome. Filtering all sequencing reads that mapped to both organellar genomes nearly eliminated mitochondrial heteroplasmy but all plastid SNPs remained heteroplasmic. This investigation provides valuable insights into how to deal with interorganellar DNA transfer in performing SNP analyses from total genomic DNA. The results confirm recent suggestions that plastid heteroplasmy is much more common than previously thought. Finally, low levels of sequence variation in plastid and mitochondrial genomes argue for using nuclear SNPs for molecular characterization of date palm cultivars.

Project description:Chloridoideae is one of the largest subfamilies of Poaceae, containing many species of great economic and ecological value; however, phylogenetic relationships among the subtribes and genera of Cynodonteae are controversial. In the present study, we combined 111 plastomes representing all five tribes, including 25 newly sequenced plastomes that are mostly from Cynodonteae. Phylogenetic analyses supported the five monophyletic tribes of Chloridoideae, including Centropodieae, Triraphideae, Eragrostideae, Zoysieae and Cynodonteae. Simultaneously, nine monophyletic lineages were revealed in Cynodonteae: supersubtribe Boutelouodinae, subtribes Tripogoninae, Aeluropodinae, Eleusininae, Dactylocteniinae, supersubtribe Gouiniodinae, Cleistogenes and Orinus, and subtribe Triodiinae. Within the tribe of Cynodonteae, the basal lineage is supersubtribe Boutelouodinae and Tripogoninae is sister to the remaining lineages. The clade formed of Aeluropodinae and Eleusininae is sister to the clade composed of Dactylocteniinae, supersubtribe Gouiniodinae, Cleistogenes and Orinus, and subtribe Triodiinae. The clade comprising Dactylocteniinae and supersubtribe Gouiniodinae is sister to the clade comprising Cleistogenes, Orinus, and Triodiinae. Acrachne is a genus within Eleusininae but not within Dactylocteniinae. Molecular evidence determined that Diplachne is not clustered with Leptochloa, which indicated that Diplachne should not be combined into Leptochloa. Cleistogenes is sister to a clade composed of Orinus and Triodia, whereas the recently proposed subtribe Orininae was not supported. Cynodonteae was estimated to have experienced rapid divergence within a short period, which could be a major obstacle in resolving its phylogenetic relationships. Ancestral state reconstructions of morphological characters showed that the most recent common ancestor (MRCA) of Chloridoideae has a panicle, multiple florets in each spikelet, the peaked type of stomatal subsidiary cells, and a saddle-shaped phytoliths, while the ancestral morphological characters of Cynodonteae are the panicle, peaked type of stomatal subsidiary cells, sharp-cap cell typed and equal-base-cell microhair, and square-shaped phytoliths. Overall, plastome phylogenomics provides new insights into the phylogenetic relationships and morphological character evolution of Chloridoideae.

Dataset Information

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

Publications

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

Similar Datasets

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