Dataset Information

Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species.

ABSTRACT: Isolation of good quality chloroplast DNA (cpDNA) is a challenge in different plant species, although several methods for isolation are known. Attempts were undertaken to isolate cpDNA from Festuca grass species by using available standard protocols; however, they failed due to difficulties separating intact chloroplasts from the polysaccharides, oleoresin, and contaminated nuclear DNA that are present in the crude homogenate. In this study, we present a quick and inexpensive protocol for isolating intact chloroplasts from seven grass varieties/accessions of five Festuca species using a single layer of 30% Percoll solution. This protocol was successful in isolating high quality cpDNA with the least amount of contamination of other DNA. We performed Illumina MiSeq paired-end sequencing (2 × 300 bp) using 200 ng of cpDNA of each variety/accession. Chloroplast genome mapping showed that 0.28%-11.37% were chloroplast reads, which covered 94%-96% of the reference plastid genomes of the closely related grass species. This improved method delivered high quality cpDNA from seven grass varieties/accessions of five Festuca species and could be useful for other grass species with similar genome complexity.

SUBMITTER: Islam MS

PROVIDER: S-EPMC6963596 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five <i>Festuca</i> Species.

Islam Md Shofiqul MS Buttelmann Gretta L GL Chekhovskiy Konstantin K Kwon Taegun T Saha Malay C MC

Plants (Basel, Switzerland) 20191214 12

Isolation of good quality chloroplast DNA (cpDNA) is a challenge in different plant species, although several methods for isolation are known. Attempts were undertaken to isolate cpDNA from <i>Festuca</i> grass species by using available standard protocols; however, they failed due to difficulties separating intact chloroplasts from the polysaccharides, oleoresin, and contaminated nuclear DNA that are present in the crude homogenate. In this study, we present a quick and inexpensive protocol for ...[more]

PMID: 31847311

Similar Datasets

Project description:BackgroundCamellia, comprising more than 200 species, is a valuable economic commodity due to its enormously popular commercial products: tea leaves, flowers, and high-quality edible oils. It is the largest and most important genus in the family Theaceae. However, phylogenetic resolution of the species has proven to be difficult. Consequently, the interspecies relationships of the genus Camellia are still hotly debated. Phylogenomics is an attractive avenue that can be used to reconstruct the tree of life, especially at low taxonomic levels.Methodology/principal findingsSeven complete chloroplast (cp) genomes were sequenced from six species representing different subdivisions of the genus Camellia using Illumina sequencing technology. Four junctions between the single-copy segments and the inverted repeats were confirmed and genome assemblies were validated by PCR-based product sequencing using 123 pairs of primers covering preliminary cp genome assemblies. The length of the Camellia cp genome was found to be about 157kb, which contained 123 unique genes and 23 were duplicated in the IR regions. We determined that the complete Camellia cp genome was relatively well conserved, but contained enough genetic differences to provide useful phylogenetic information. Phylogenetic relationships were analyzed using seven complete cp genomes of six Camellia species. We also identified rapidly evolving regions of the cp genome that have the potential to be used for further species identification and phylogenetic resolution.Conclusions/significanceIn this study, we wanted to determine if analyzing completely sequenced cp genomes could help settle these controversies of interspecies relationships in Camellia. The results demonstrate that cp genome data are beneficial in resolving species definition because they indicate that organelle-based "barcodes", can be established for a species and then used to unmask interspecies phylogenetic relationships. It reveals that phylogenomics based on cp genomes is an effective approach for achieving phylogenetic resolution between Camellia species.

Dataset Information

Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species.

Publications

Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five <i>Festuca</i> Species.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure