An improved method for chloroplast genome sequencing in non-model forest tree species
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Chloroplast genomes can provide a large amount of information and resources for use in studies on plant evolution and molecular ecology. However, a rapid and efficient method for obtaining chloroplast genome sequences is still lacking. In this study, we report a modified method for the isolation of intact chloroplasts, which needs less than 0.5 g leaf material. Coupled with rolling circle amplification (RCA), next-generation sequencing, and a pipeline combining de novo assembly and reference-guided assembly (RGA), we successfully obtained a complete chloroplast genome for the non-model forest tree species, evergreen oak Quercus spinosa, with as many as 36 % of the sequence reads mapped to the chloroplast genome. The Q. spinosa cpDNA is 160,825 bp in length and codes for 134 genes (89 protein coding, 8 ribosomal RNAs (rRNAs), and 36 distinct transfer RNAs (tRNAs)). The genome organization and arrangement are similar to those found among most angiosperm chloroplast genomes. Our inexpensive and efficient protocol can be applied to the reconstruction of chloroplast genomes for plant evolutionary studies, especially in non-model tree species.
KeywordsChloroplast Next-generation sequencing Rolling circle amplification (RCA) Oak
The authors thank Dr. Rémy J. Petit, Dr. Antoine Kremer working in INRA Pierroton, France, Dr. Liuyang Wang working in Duke University, USA, and Dr. Saneyoshi Ueno working in Forestry and Forest Products Research Institute, Japan, for revision of, and suggestions about, the preliminary version of this paper. The authors thanks the comments and suggestions from three anonymous reviewers. The research was funded by Beijing Nova Program (grant number: Z151100000315056), National Natural Science Foundation of China (grant number 41201051; 41430749), 111 Project (grant number B13007), and Program for Changjiang Scholars, Innovative Research Team in University (grant number IRT13047) to FKD and the Major projects on control and rectification of water body pollution (2012ZX07105-002-03) to JL
Data Archiving Statement
The Q. spinosa cp genome sequence data has been deposited into GenBank and released to public under the accession number KM841421.1. The sequencing reads were submitted to SRA in NCBI under the accession number SRP061187.
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