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

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 separati...

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Autores principales: Islam, Md. Shofiqul, Buttelmann, Gretta L., Chekhovskiy, Konstantin, Kwon, Taegun, Saha, Malay C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963596/
https://www.ncbi.nlm.nih.gov/pubmed/31847311
http://dx.doi.org/10.3390/plants8120606
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author Islam, Md. Shofiqul
Buttelmann, Gretta L.
Chekhovskiy, Konstantin
Kwon, Taegun
Saha, Malay C.
author_facet Islam, Md. Shofiqul
Buttelmann, Gretta L.
Chekhovskiy, Konstantin
Kwon, Taegun
Saha, Malay C.
author_sort Islam, Md. Shofiqul
collection PubMed
description 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.
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spelling pubmed-69635962020-01-27 Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species Islam, Md. Shofiqul Buttelmann, Gretta L. Chekhovskiy, Konstantin Kwon, Taegun Saha, Malay C. Plants (Basel) Protocol 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. MDPI 2019-12-14 /pmc/articles/PMC6963596/ /pubmed/31847311 http://dx.doi.org/10.3390/plants8120606 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Islam, Md. Shofiqul
Buttelmann, Gretta L.
Chekhovskiy, Konstantin
Kwon, Taegun
Saha, Malay C.
Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species
title Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species
title_full Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species
title_fullStr Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species
title_full_unstemmed Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species
title_short Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species
title_sort isolation of intact chloroplast for sequencing plastid genomes of five festuca species
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963596/
https://www.ncbi.nlm.nih.gov/pubmed/31847311
http://dx.doi.org/10.3390/plants8120606
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