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 x 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.