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An improved protocol for intact chloroplasts and cpDNA isolation in conifers
Leila do Nascimento Vieira1, Helisson Faoro2, Hugo Pacheco de Freitas Fraga1
1Departamento de Fitotecnia, Programa de Pós Graduação em Recursos Genéticos Vegetais, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Plos One
|January 7, 2014
Summary
This study presents an improved protocol for isolating chloroplast DNA (cpDNA) from conifers, overcoming previous challenges. The new method enhances chloroplast isolation and yields high-quality cpDNA suitable for sequencing.
Area of Science:
- Plant Biology
- Molecular Biology
- Genomics
Background:
- Chloroplast DNA (cpDNA) isolation is challenging in conifers using conventional methods.
- Existing plastid genome sequencing protocols for conifers rely on time-consuming long-range PCR.
Purpose of the Study:
- To develop an efficient protocol for high-quality chloroplast DNA isolation from diverse conifer species.
- To enable robust plastid genome sequencing in conifers.
Main Methods:
- Developed a protocol combining a high salt isolation buffer with a saline Percoll gradient.
- Applied the protocol to Araucariaceae, Podocarpaceae, and Pinaceae families.
- Sequenced isolated cpDNA using Illumina MiSeq, starting with 50 ng of DNA.
Main Results:
- Achieved enhanced chloroplast isolation with reduced contamination from polysaccharides, polyphenols, proteins, and nuclear DNA.
- Microscopy confirmed abundant, intact chloroplasts.
- High average coverage was obtained for chloroplast genome mapping across all tested species.
Conclusions:
- The improved protocol provides high quality and yield of chloroplasts and cpDNA from conifers.
- This method is a valuable tool for studies requiring isolated chloroplasts or whole cpDNA sequences in conifers.
