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 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.
© 2019 by the authors. Licensee MDPI, Basel, Switzerland.
Chloroplast, CpDNA, Festuca spp, Forage grass, Sequencing
Islam, Md, Gretta L. Buttelmann, Konstantin Chekhovskiy, Taegun Kwon, and Malay C. Saha. "Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species." Plants 8, no. 12 (2019): 606.