Skip to main content
SpringerLink
Log in

An improved method for chloroplast genome sequencing in non-model forest tree species

  • Original Article
  • Published:
Tree Genetics & Genomes Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Alexander LW, Woeste KE (2014) Pyrosequencing of the northern red oak (Quercus rubra L.) chloroplast genome reveals high quality polymorphisms for population management. Tree Genet Genomes 10:803–12

    Article  Google Scholar 

  • Aslan CE, Zavaleta ES, Tershy B, Croll D (2013) Mutualism disruption threatens global plant biodiversity: a systematic review. PLoS One 8:e66993. doi:10.1371/journal.pone.0066993

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Atherton RA, McComish BJ, Shepherd LD, Berry LA, Albert NW, Lockhart PJ (2010) Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform. Plant Methods 6:22

    Article  PubMed Central  PubMed  Google Scholar 

  • Benson G (1999) Tandem repeats finder: a program to analyze DNA sequences. Nucl Acids Res 27:573

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Blanco L, Bernad A, Lázaro JM, Martin G, Garmendia C, Salas M (1989) Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem 264:8935–8940

    CAS  PubMed  Google Scholar 

  • Burger G, Lavrov DV, Forget L, Lang BF (2007) Sequencing complete mitochondrial and plastid genomes. Nat Protoc 2:603–614

    Article  CAS  PubMed  Google Scholar 

  • Chung SM, Gordon VS, Staub JE (2007) Sequencing cucumber (Cucumis sativus L.) chloroplast genomes identifies differences between chilling-tolerant and -susceptible cucumber lines. Genome 50:215–225

    Article  CAS  PubMed  Google Scholar 

  • Cronn R, Liston A, Parks M et al (2008) Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology. Nucleic Acids Res 36:122–122

    Article  Google Scholar 

  • Cronn R, Knaus BJ, Liston A, Maughan PJ, Parks M, Syring JV et al (2012) Targeted enrichment strategies for next-generation plant biology. Am J Bot 99:291–311

    Article  CAS  PubMed  Google Scholar 

  • Dean FB, Hosono S, Fang L, Wu X, Faruqi FA, Bray-Ward P et al (2002) Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci U S A 99:5261–5266

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Denk T, Grimm GW (2010) The oaks of western Eurasia: traditional classifications and evidence from two nuclear markers. Taxon 59:351–366

    Google Scholar 

  • Diekmann K, Hodkinson TR, Fricke E, Barth S (2008) An optimized chloroplast DNA extraction protocol for grasses (Poaceae) proves suitable for whole plastid genome sequencing and SNP detection. PLoS One 3:e2813. doi:10.1371/journal.pone.0002813

    Article  PubMed Central  PubMed  Google Scholar 

  • Esteban JA, SalasM BL (1993) Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization. J Biol Chem 268:2719–2726

    CAS  PubMed  Google Scholar 

  • Goremykin VV, Hirsch-Ernst KI, Wölfl S et al (2003a) Analysis of the Amborella trichopoda chloroplast genome sequence suggests that Amborella is not a basal angiosperm. Mol Biol Evol 20:1499–1505

    Article  CAS  PubMed  Google Scholar 

  • Goremykin VV, Hirsch-Ernst KI, Wolfl S, Hellwig FH (2003b) The chloroplast genome of the “basal” angiosperm Calycanthus fertilis—structural and phylogenetic analyses. Plt Syst Evol 242:119–135

    Article  CAS  Google Scholar 

  • Goremykin VV, Hirsch-Ernst KI, Wolfl S, Hellwig FH (2004) The chloroplast genome of Nymphaea alba: whole-genome analyses and the problem of identifying the most basal angiosperm. Mol Biol Evol 21:1445–1454

    Article  CAS  PubMed  Google Scholar 

  • Goremykin VV, Holland B, Hirsch-Ernst KI, Hellwig FH (2005) Analysis of Acorus calamus chloroplast genome and its phylogenetic implications. Mol Biol Evol 22:1813–1822

    Article  CAS  PubMed  Google Scholar 

  • Hahn C, Bachmann L, Chevreux B (2013) Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads-a baiting and iterative mapping approach. Nucleic Acids Res 41:9

    Article  Google Scholar 

  • Huang DI, Hefer CA, Kolosova N, Douglas CJ, Cronk QCB (2014) Whole plastome sequencing reveals deep plastid divergence and cytonuclear discordance between closely related balsam poplars, Populus balsamifera and P. trichocarpa (Salicaceae). New Phytol 204:693–703

    Article  PubMed  Google Scholar 

  • Hutchison CA, Smith HO, Pfannkoch C, Venter JC (2005) Cell-free cloning using φ29 DNA polymerase. Proc Natl Acad Sci U S A 102:17332–17336

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Jansen RK, Raubeson LA, Boore JL et al (2005) Methods for obtaining and analyzing whole chloroplast genome sequences. Methods Enzymol 395:348–384

    Article  CAS  PubMed  Google Scholar 

  • Jansen RK, Saski C, Lee SB, Hansen AK, Daniell H (2011) Complete plastid genome sequences of three rosids (Castanea, Prunus, Theobroma): evidence for at least two independent transfers of rpl22 to the nucleus. Mol Biol Evol 28:835–847

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Katoh K, Misawa K, Kuma KI, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kremer A, Petit RJ (1993) Gene diversity in natural populations of oak species. Ann Sci For 50:186s–203s

    Article  Google Scholar 

  • Kremer A, Sederoff R, Wheeler N (2010) Genomics of forest and ecosystem health in the Fagaceae: meeting report. Tree Genet Genome 6:815–820

    Article  Google Scholar 

  • Kremer A, Abbott AG, Carlson JE et al (2012) Genomics of Fagaceae. Tree Genet Genomes 8:583–610

    Article  Google Scholar 

  • Kunkel TA (2004) DNA replication fidelity. J Biol Chem 279:16895–16898

    Article  CAS  PubMed  Google Scholar 

  • Kurtz S, Schleiermacher C (1999) REPuter: fast computation of maximal repeats in complete genomes. Bioinformatics 15:426–427

    Article  CAS  PubMed  Google Scholar 

  • Leseberg CH, Duvall MR (2009) The complete chloroplast genome of Coix lacryma-jobi and a comparative molecular evolutionary analysis of plastomes in cereals. J Mol Evol 69:311–318

    Article  CAS  PubMed  Google Scholar 

  • Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N et al (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079

    Article  PubMed Central  PubMed  Google Scholar 

  • Liu C, Shi L, Zhu Y, Chen H, Zhang J, Lin X, Guan X (2012) CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences. BMC Genomics 13:715

    Article  PubMed Central  PubMed  Google Scholar 

  • Lu S, Hou M, Du FK, Li J, Yin K (2015) Complete chloroplast genome of the Oriental white oak: Quercus aliena Blume. Mitochondrial DNA (ahead-of-print) 1–3

  • Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J et al (2012) SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 1:18

    Article  PubMed Central  PubMed  Google Scholar 

  • Mardanov AV, Ravin NV, Kuznetsov BB, Samigullin TH, Antonov AS, Kolganova TV et al (2008) Complete sequence of the duckweed (Lemna minor) chloroplast genome: structural organization and phylogenetic relationships to other angiosperms. J Mol Evol 66:555–564

    Article  CAS  PubMed  Google Scholar 

  • Mariac C, Scarcelli N, Pouzadou J, Barnaud A, Billot C, Faye A et al (2014) Cost-effective enrichment hybridization capture of chloroplast genomes at deep multiplexing levels for population genetics and phylogeography studies. Mol Ecol Resour 14:1103–1113

    Article  CAS  PubMed  Google Scholar 

  • Moore MJ, Dhingra A, Soltis PS, Shaw R, Farmerie WG, Folta KM et al (2006) Rapid and accurate pyrosequencing of angiosperm plastid genomes. BMC Plant Biol 6:17

    Article  PubMed Central  PubMed  Google Scholar 

  • Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C et al (2009) Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461:272–276

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Nobel PS (1974) Rapid isolation techniques for chloroplasts. Meth Enzym 31:600–606

    Article  CAS  PubMed  Google Scholar 

  • Nock CJ, Waters DL, Edwards MA, Bowen SG, Rice N, Cordeiro GM et al (2011) Chloroplast genome sequences from total DNA for plant identification. Plant Biotechnol J 9:328–333

    Article  CAS  PubMed  Google Scholar 

  • Parks M, Cronn R, Liston A (2012) Separating the wheat from the chaff: mitigating the effects of noise in a plastome phylogenomic data set from Pinus L. (Pinaceae). BMC Evol Biol 12:100

    Article  PubMed Central  PubMed  Google Scholar 

  • Plomion C, Aury JM, Amselem J et al (2015) Decoding the oak genome: public release of sequence data, assembly, annotation and publication strategies. Mol Ecol Resour. doi:10.1111/1755-0998.12425

    Google Scholar 

  • Richardson PM, Detter C, Schweitzer B et al (2003) Practical applications of rolling circle amplification of DNA templates. Genet Eng 25:51–63

    Article  CAS  Google Scholar 

  • Schmitz-Linneweber C, Maier RM, Alcaraz JP, Cottet A, Herrmann RG, Mache R (2001) The plastid chromosome of spinach (Spinacia oleracea): complete nucleotide sequence and gene organization. Plt Mol Biol 45:307–315

    Article  CAS  Google Scholar 

  • Schuster SC (2008) Next-generation sequencing transforms today’s biology. Nat Methods 5:16–18

    Article  CAS  PubMed  Google Scholar 

  • Shi C, Hu N, Huang H, Gao J, Zhao YJ, Gao LZ (2012) An improved chloroplast DNA extraction procedure for whole plastid genome sequencing. PLoS One 7:e31468. doi:10.1371/journal.pone.0031468

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T et al (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5:2043–2049

    PubMed Central  CAS  PubMed  Google Scholar 

  • Steane DA (2005) Complete nucleotide sequence of the chloroplast genome from the Tasmanian blue gum, Eucalyptus globulus (Myrtaceae). DNA Res 12:215–220

    Article  CAS  PubMed  Google Scholar 

  • Straub SC, Parks M, Weitemier K, Fishbein M, Cronn RC, Liston A (2012) Navigating the tip of the genomic iceberg: next-generation sequencing for plant systematics. Am J Bot 99:349–364

    Article  CAS  PubMed  Google Scholar 

  • Stull GW, Moore MJ, Mandala VS et al. (2013) A targeted enrichment strategy for massively parallel sequencing of angiosperm plastid genomes. Appl Plant Sci 1:apps.1200497. doi:10.3732/apps.1200497

  • Vieira Ldo N, Faoro H, Fraga HP, Rogalski M, de Souza EM, de Oliveira Pedrosa F et al (2014) An improved protocol for intact chloroplasts and cpDNA isolation in conifers. PLoS ONE 9:e84792. doi:10.1371/journal.pone.0084792

    Article  PubMed  Google Scholar 

  • Wu Z, Raven P (1999) Flora of China. Vol. 4 (Cycadaceae through Fagaceae). Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis

    Google Scholar 

  • Wu FH, Kan DP, Lee SB, Daniell H, Lee YW, Lin CC et al (2009) Complete nucleotide sequence of Dendrocalamus latiflorus and Bambusa oldhamii chloroplast genomes. Tree Physiol 29:847–856

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wu FH, Chan MT, Liao DC, Hsu CT, Lee YW, Daniell H et al (2010) Complete chloroplast genome of Oncidium Gower Ramsey and evaluation of molecular markers for identification and breeding in Oncidiinae. BMC Plant Biol 10:68

    Article  PubMed Central  PubMed  Google Scholar 

  • Wu J, Liu B, Cheng F, Ramchiary N, Choi SR, Lim YP, Wang XW (2012) Sequencing of chloroplast genome using whole cellular DNA and solexa sequencing technology. Front Plant Sci 3:243

    PubMed Central  CAS  PubMed  Google Scholar 

  • Xu B, Li T, Luo Y, Xu R, Cai H (2014) An Empirical Algorithm for Bias Correction Based on GC Estimation for Single Cell Sequencing. Trends and Applications in Knowledge Discovery and Data Mining. Springer International Publishing: 15–21

  • Zhou YH, Zhang XP, Ebright RH (1991) Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase. Nucleic Acids Res 19:6052

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. College of Forestry, Beijing Forestry University, Beijing, 100083, People’s Republic of China

    Fang K. Du, Sihai Lu, Yuyao Wang, Junqing Li & Kangquan Yin

  2. Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, 666303, Yunnan, People’s Republic of China

    Tiange Lang

  3. College of life science, Lanzhou University, Lanzhou, 730000, Gansu, People’s Republic of China

    Sihai Lu

  4. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China

    Kangquan Yin

Authors
  1. Fang K. Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tiange Lang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sihai Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuyao Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Junqing Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kangquan Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Fang K. Du or Kangquan Yin.

Additional information

Communicated by Y. Tsumura

This article is part of the Topical Collection on Genome Biology

Electronic supplementary material

Below is the link to the electronic supplementary material.

Figure S1

Alignment of cp genome assembly results that produced by the pipeline of this study and MITObim. (DOCX 132 kb)

Table S1

Published angiosperm cp genome sequences deposited in the NCBI database. (XLSX 35 kb)

Table S2

Repeats identified in the Quercus spinosa cp genome. (XLSX 16 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Du, F.K., Lang, T., Lu, S. et al. An improved method for chloroplast genome sequencing in non-model forest tree species. Tree Genetics & Genomes 11, 114 (2015). https://doi.org/10.1007/s11295-015-0942-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11295-015-0942-2

Keywords

Search

Navigation