An efficient procedure for plant organellar genome assembly, based on whole genome data from the 454 GS FLX sequencing platform
© Zhang et al; licensee BioMed Central Ltd. 2011
Received: 11 September 2011
Accepted: 29 November 2011
Published: 29 November 2011
Complete organellar genome sequences (chloroplasts and mitochondria) provide valuable resources and information for studying plant molecular ecology and evolution. As high-throughput sequencing technology advances, it becomes the norm that a shotgun approach is used to obtain complete genome sequences. Therefore, to assemble organellar sequences from the whole genome, shotgun reads are inevitable. However, associated techniques are often cumbersome, time-consuming, and difficult, because true organellar DNA is difficult to separate efficiently from nuclear copies, which have been transferred to the nucleus through the course of evolution.
We report a new, rapid procedure for plant chloroplast and mitochondrial genome sequencing and assembly using the Roche/454 GS FLX platform. Plant cells can contain multiple copies of the organellar genomes, and there is a significant correlation between the depth of sequence reads in contigs and the number of copies of the genome. Without isolating organellar DNA from the mixture of nuclear and organellar DNA for sequencing, we retrospectively extracted assembled contigs of either chloroplast or mitochondrial sequences from the whole genome shotgun data. Moreover, the contig connection graph property of Newbler (a platform-specific sequence assembler) ensures an efficient final assembly. Using this procedure, we assembled both chloroplast and mitochondrial genomes of a resurrection plant, Boea hygrometrica, with high fidelity. We also present information and a minimal sequence dataset as a reference for the assembly of other plant organellar genomes.
Organellar genomes are widely used in evolutionary and population genetics studies. The plastid genome contains many essential genes, especially those required for photosynthesis. Information from multiple plastid genomes harbors suites of characters that transcend the green plant branch in the tree of life . There are multiple copies of the organellar genomes in plant cells, e.g. plant leaf cells often contain 400 to 1,600 copies of the plastid genome . In angiosperms, most chloroplast (cp) genomes are circular DNA molecules ranging from 120 to 160 kb. They have a quadripartite organization, consisting of two copies of inverted repeats (IRs) of 20-28 kb in size, which divides the rest of the genome into a large-single-copy region (LSC; 80-90 kb) and a small-single-copy (SSC; 16-27 kb) region . Plants have larger and more complex mitochondrial (mt) genomes than other unicellular and multicellular eukaryotes. Mitochondrial genomes, especially those in seed plants, are exceptionally varied in size and structure, and their sequence contents accumulate many repetitive sequences [4, 5].
Recently, there has been a dramatic increase in the number of completely sequenced organellar genomes. To date, sequences from 206 cp genomes and 47 mt genomes have been deposited in the GenBank Organelle Genome Resources. Most of them are sequences generated by the Sanger capillary sequencers . With the emergence of next-generation sequencing technologies, new approaches for cp genome sequencing and assembly have been proposed because of their timesaving, high-throughput, and low-cost advantages [7–9]. As for mt genomes, three main strategies have been used: physical map-based [10–12], shotgun-based [13–15], and gene-based . However, all these strategies for sequencing organellar genomes either require the isolation of cp or mt DNA from nuclear DNA  or are difficult to assemble because of the dynamic structure of multipartite molecules [18–20]. Isolating mitochondria and their DNA is often challenging, so that it is imperative to develop better methods for sequencing and assembling these genomes that do not include experimental sample enrichment.
In this study, we present a rapid procedure for complete cp and mt genome sequence assembly from whole genome shotgun data, without organellar DNA isolation. Using this procedure, we successfully assembled the complete cp and mt genomes of a resurrection plant, Boea hygrometrica (Bunge) R Br of the Gesneriaceae family. This is the first mitochondrial genome to be sequenced from a resurrection plant. Boea hygrometrica is an unusual, desiccation-tolerant angiosperm native to China [21, 22]. Comprehensive analyses of the organellar genomes of this particular plant, and comparison with those of other plants, will help us to understand the evolution of Boea hygrometrica.
Data summary of Roche/454 GS FLX sequencing
Mean read Length (bp)
aRead peak quality
Data summary of SOLiD4.0 sequencing
Read length (bp)
Total data (Mbp)
aInsert size1 (bp)
bInsert size 2 (bp)
Assembly of a Cp Genome
Assembly of an Mt Genome
In comparison to non-plant unicellular and multicellular eukaryotes, plants have larger and more complex mitochondrial genomes . All the features of plant mt genomes, including RNA editing, genomic recombination, trans-splicing, and insertions of "foreign" DNA from other genomes  make assembling mt genomes difficult. As recent studies have shown, genome sequences vary exceptionally in size, structure, and sequence content, especially among seed plants [4, 5]. However, there are essential genes that are highly conserved in almost all plant mt genomes, such as NADH dehydrogenase, succinate dehydrogenase, ubichinol cytochrome c reductase, cytochrome c oxidase, and ATP synthase. Using these genes, we could identify assembled contigs originated from the mt genome. Such gene-based procedures have been used to enrich plant mtDNA for mt genomic sequencing .
SOLiD mate-pair read links of spanning repetitive contigs
Minimal Sequencing Data for Organellar Genome Assembly
After finishing the organellar genome assembly for B. hygrometrica, we carried out a simulation study to determine a minimal sequencing dataset for our procedure. We randomly sampled 50-1,400 Mbp sequences from the raw Roche/454 data, and assembled the organellar genomes with our procedure. Flow-cytometry study showed that the genome size of B. hygrometrica is about 300 Mbp, which is twice large as that of Arabidopsis thaliana (our unpublished data). The sequencing coverage of B. hygrometrica is about 4.68×.
Analysis of minimal sequencing coverage for the complete organellar genome assembly of B. hygrometrica
Sample data (Mbp)
Sequencing coverage (X)*
We have successfully applied a new, efficient procedure to determine the complete chloroplast and mitochondrial genome sequences of the resurrection plant, Boea hygrometrica. Subsequently, we have also applied this approach to completely assemble the mt genome of Phoenix dactylifera L with only one run of Roche/454 data, and two Hassawi rice (Oryza sativa L. in Saudi Arabia) organellar genomes (both cp and mt genomes) (data not shown). Therefore, we are confident that our efficient and straightforward procedure will prove useful for further organellar genome sequencing and assembly.
Materials and methods
Materials and datasets
Boea hygrometrica plants were collected from their natural habitat in Beijing, and maintained in a greenhouse (approximately 25°C, 16 h/8 h light period) with regular irrigation. After 2 weeks of growth, fresh green leaves were collected. We extracted genomic DNA from 50 g of leaves according to a CTAB-based protocol . According to the manufacturer's manual for the 454 GS FLX Titanium, we used 5 μg of purified DNA to construct the libraries. In addition, two mate-pair libraries were constructed for the SOLiD 4.0 (Applied Biosystems, Foster City, CA) sequencing platform. We downloaded 206 sequenced plant chloroplast genome sequences from the NCBI (National Center for Biotechnology Information) ftp site http://ftp.ncbi.nih.gov/genomes/Chloroplasts/plastids and 47 sequenced plant mitochondrial genome sequences from NCBI Organelle Genome Resources http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=33090&opt=organelle.
The genome data have been submitted to the National Center for Biotechnology Information (NCBI) database. The accession numbers are [GenBank: JN107811] and [GenBank: JN107812] for Boea hygrometrica chloroplast and mitochondrial genomes, respectively.
We wish to thank Xing Deng and Xuming Wang for their preparation of the B. hygrometrica materials for this project. We also thank Douglas Senalik and Simon Gladman for sharing two important perl scripts for this procedure.
This work was supported by grants from the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-R-01-04), the Natural Science Foundation of China (90919024), the Natural Science Foundation of China (30900831) and the National Basic Research Program (973 Program) from the Ministry of Science and Technology of the People's Republic of China (2011CB944100).
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