title: An improved chloroplast DNA extraction procedure for whole plastid genome sequencing.
abstract: BACKGROUND
  Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.
  
  
  RESULTS
  We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40-50% cpDNA purity is achieved with our method.
  
  
  CONCLUSIONS
  Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.
authors.affiliation: Plant Germplasm and Genomics Center, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Shi
authors.forename: Na
authors.initials: N
authors.lastname: Hu
authors.forename: Hui
authors.initials: H
authors.lastname: Huang
authors.forename: Ju
authors.initials: J
authors.lastname: Gao
authors.forename: You-Jie
authors.initials: YJ
authors.lastname: Zhao
authors.forename: Li-Zhi
authors.initials: LZ
authors.lastname: Gao
mesh_terms: D002736:Chloroplasts / Q000378:metabolism*; D002874:Chromosome Mapping; D003362:Cost-Benefit Analysis; D018742:DNA, Chloroplast / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D016678:Genome; D054628:Genome, Chloroplast; D018745:Genome, Plant; D054627:Genome, Plastid; D008957:Models, Genetic; D012275:Oryza / Q000235:genetics; D010944:Plants / Q000378:metabolism; D018087:Plastids / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D018742:DNA, Chloroplast; D018744:DNA, Plant; D003850:Deoxyribonuclease I