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.
Highlights
Chloroplasts are cytoplasmic organelles of green plants that are the active sites of photosynthesis [1].They play vital roles in plant physiology and development, including synthesis of chlorophyll, carotenoids, and fatty acids
To be able to take advantage of this information, it is important that the chloroplast DNA be extracted with the least possible amount of contamination from nuclear and mitochondrial DNA or substances such as polysaccharides and resins in preparation for generation sequencing [6,7]
By attempting to adapt chloroplast DNA (cpDNA) extraction protocol to Festuca grasses, we aimed to develop further progress in the study of plastid genomes and to encourage more research in the sequencing of plastid genomes for different plant species
Summary
Chloroplasts are cytoplasmic organelles of green plants that are the active sites of photosynthesis [1]. They play vital roles in plant physiology and development, including synthesis of chlorophyll, carotenoids, and fatty acids. Chloroplasts have their own genome of 120–160 kb in length, with the quadripartite structure found in most of land plants [2]. Being maternally inherited [5], the chloroplast genome is useful for developing cytoplasmic gene pools in plant breeding, and tracking the maternal parent of interspecific hybrids.
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