Complete sequence of heterogenous-composition mitochondrial genome (Brassica napus) and its exogenous source
- Juan Wang†1,
- Jinjin Jiang†1,
- Xiaoming Li1,
- Aimin Li2,
- Yongtai Zhang2,
- Rongzhan Guan3 and
- Youping Wang1Email author
© Wang et al.; licensee BioMed Central Ltd. 2012
Received: 24 July 2012
Accepted: 27 November 2012
Published: 28 November 2012
Unlike maternal inheritance of mitochondria in sexual reproduction, somatic hybrids follow no obvious pattern. The introgressed segment orf138 from the mitochondrial genome of radish (Raphanus sativus) to its counterpart in rapeseed (Brassica napus) demonstrates that this inheritance mode derives from the cytoplasm of both parents. Sequencing of the complete mitochondrial genome of five species from Brassica family allowed the prediction of other extraneous sources of the cybrids from the radish parent, and the determination of their mitochondrial rearrangement.
We obtained the complete mitochondrial genome of Ogura-cms-cybrid (oguC) rapeseed. To date, this is the first time that a heterogeneously composed mitochondrial genome was sequenced. The 258,473 bp master circle constituted of 33 protein-coding genes, 3 rRNA sequences, and 23 tRNA sequences. This mitotype noticeably holds two copies of atp9 and is devoid of cox 2-2. Relative to nap mitochondrial genome, 40 point mutations were scattered in the 23 protein-coding genes. atp6 even has an abnormal start locus whereas tatC has an abnormal end locus. The rearrangement of the 22 syntenic regions that comprised 80.11% of the genome was influenced by short repeats. A pair of large repeats (9731 bp) was responsible for the multipartite structure. Nine unique regions were detected when compared with other published Brassica mitochondrial genome sequences. We also found six homologous chloroplast segments (Brassica napus).
The mitochondrial genome of oguC is quite divergent from nap and pol, which are more similar with each other. We analyzed the unique regions of every genome of the Brassica family, and found that very few segments were specific for these six mitotypes, especially cam, jun, and ole, which have no specific segments at all. Therefore, we conclude that the most specific regions of oguC possibly came from radish. Compared with the chloroplast genome, six identical regions were found in the seven mitochondrial genomes, which show that the Brassica family has a stable chloroplast-derived source.
The major function of the mitochondria (mt), as a semiautonomous organelle, in plant growth and development is to provide energy through oxidative phosphorylation . In different to the small mt genome of animals (~16 kb), plants have longer mtDNA ranging from 200kb to 2000kb [2, 3]. To date, several mt genomes from fertile and sterile plant species have been sequenced, including Arabidopsis thaliana, Oryza sativa[5–7], Beta vulgaris[8, 9], Zea mays[10, 11], Nicotiana tabacum, Triticum aestivum[13, 14], and five species from the Brassica genus, i.e., B. napus (pol, nap), B. rapa (cam), B. oleracea (ole), B. juncea (jun), and B. carinata (car) [15–17]. The sequencing results indicated that apart from ribosomal protein genes, protein-coding genes are also relatively conserved both in nucleotide sequence and in number. However, the non-coding sequences are quite inconsistent among species, and even within the same species. The presence of large and short repeats is responsible for the dynamic multipartite structures, reorganization, and recombination .
In higher plants, mitochondrial inheritance usually follows the maternal origin during sexual hybridization. However, much more complicated modes are detected in somatic hybridization, wherein mt genome inheritance is derived from either or both biparents . In the latter pattern, part of the mt genome, including cytoplasmic male sterility (CMS) genes, can be transferred from the donor parent to the receptor parent, and the introgressed segment experiences extensive rearrangement and recombination with the mtDNA of the receptor one. Orf138, originally identified in radish, was transferred successfully to various species, including Arabidopsis, B. napus, and B. oleracea by somatic hybridization [19–25].
CMS genes have a defect in the production of functional pollen. Generally, genes associated with CMS genes are located in the periphery of certain known mitochondrial genes and are cotranscribed with them . T-urf13 (orf115) was the first identified aberrant gene in the Texas (T)-cytoplasm of maize, which encodes a 13 kDa membrane-spanning polypeptide that depolarizes the mitochondria and leads to cell death [27–29]. In the BT (Boro II)-type CMS line of rice, orf79 was cotranscribed with the atp6 gene forming a 2.0 kb transcript . The expressed protein contains a predicted transmembrane domain .
In the Brassica genus, the complete mt genomes of five species are sequenced, coupled with the basic feature of published CMS genes, which allows the detection of the extraneous source from donor parent (radish) of somatic hybrids.
Genome size and nucleotide sequence in the genic region
SNP in protein-coding genes of mtDNA between oguC and nap
Reconstruction of the nap-CMS cybrid mitochondrial genome
Reorganization of the mitochondrial genome
Unique region of the genome
Unique region found in oguC
included in the large repeats
part of orf101-4
Homology with rapeseed chloroplast genome
Homologous segments to chloroplast of rapeseed found in oguC mitotype
ORFs and predicted CMS-related chimeric ORF in this genome
We detected 39 ORFs in this genome, with the shortest size equal to 303 bp, which summed to 7.41% of the mitotype. Of the 39 ORFs, 23 (similarity ≥ 99%) were shared in one or more Brassica genomes, which were remotely related to CMS. However, they are likely functional genes as these later-discovered genes ccm, orf25 (atp4), and orfB (atp8) [37, 38]. Of the remaining 16 unique ORFs in the oguC mitochondrial genome, 8 ORFs were totally not matched and 8 were partly identical to those present in the other six mitotypes. Five, including orf138, which is the oguC-related CMS gene, out of eight non-matched ORFs were completely or partially situated in the unique regions (Table 2). Three common ORFs and three unique ORFs were located in the cp-derived domains (Table 3). Among them, orf210 and orf344-1 were highly similar to 2 segments of the beta subunit of RNA polymerase, which were wholly situated in the chloroplast genome with a length of 1072 amino acids. When the intact nucleotide sequence of beta subunit of RNA polymerase was aligned with oguC mtDNA, H1 was found to be a truncated RNA polymerase beta subunit with 97% similarity. Some point mutations and indels resulted in the production of these two ORFs. Similar to orf344-1, a truncated ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene from cp genome evolved into orf313. Based on the three features of CMS related genes, namely, unique to the given mitotype, membrane-spanning domains and near to the functional genes [26, 39], orf138, which encodes a 19 kDa transmembrane protein that showed toxicity to bacterial growth, can be suppressed by the nuclear Rfo locus [40–43].
When nap mtDNA was taken as the control, 22 syntenic regions were detected in total. Estimating the number of recombination events was difficult because of the many syntenic regions. However, relative to two more similar mitotypes (pol and nap) that have 13 syntenic region with the same analysis criterion (length ≥ 1000 bp, similarity ≥ 95%) , it showed complex reconstruction. oguC mitotype must have undergone complicated changes and evolutionary events when the cytoplasm of two cells contacted each other.
The composition of mitochondrial genomes of 7 mitotypes from Brassica family*
Genome size (bp)
Unique region (%)
cp-derived sequences (%)
Large repeat (bp)
Short repeat (%)
Using one genome of the seven mitotypes as the control to find the unique regions for every mitotype, the percentage ranged from 0% in three mitotypes (cam, jun, and ole) to 8.60% in oguC. Both nap and car contained three shorter specific segments constituting 0.74% and 1.57% of these two genomes, respectively. A 620 bp unique segment located in pol contributed 0.28% to its total genome. In terms of the percentage, at least 7% of the exogenous sequences from radish mtDNA coexist with the oguC mtDNA (Table 4). When searched against the NCBI databases using those specific segments, similar alignments to that of oguC were obtained, some of which resembled those in Arabidopsis thaliana.
We also predicted the cp-derived sequence, which was intriguing because the seven mitotypes were blasted for the identical six segments with identities more than 95% (Table 4). However, because of the large copy of R1, five cp-derived segments had two copies in ole. From the cp-derived data, we found that Brassica species have stable sources of chloroplast sequence.
This study finished mtDNA sequencing of a Ogura-cms-cybrid (oguC), which derived from somatic fusion between Brassica napus and sterile radish. By contrast to one or more of six other Brassica lines, we reasonably speculated that tatC gene and 2 unique regions, U3 and U7, must be introgressed from radish. In addition, the rearrangement mediated by large and short repeats between these two parental mtDNAs extensively existed. With regard to the evolution of this integrated CMS mtDNA, more data need to be known.
Plant material and mitochondrial genome extraction
Seed of Brassica napus (ogu-CMS cybrid, oguC) was kindly provided by Norddeutsche Pflanzenzucht, Hans-Georg Lembke KG (Germany). The etiolated one-week-old Brassica napus seedlings were prepared; the mitochondria and mtDNA extraction were performed following previously published methods . To satisfy the requirements for 454 sequences, the minimum criterion for sample concentration was 50 ng/μl and total amount was equal to at least 20 μg.
A shotgun library that includes short and long paired end libraries were constructed simultaneously, which was followed by emulsion-based clonal amplification (emPCR) for DNA library bead enrichment. Finally, a genome sequencer FLX operation was conducted and the system output was derived. The contigs were joined by PCR sequencing. For the oguC genome, high quality read number, high quality bases, average read length, and sequencing depth were 8387, 3,913,351 bp, 469.3 bp, 15.2X, respectively. For SNP analysis and unique regions in oguC resequence was done.
The genes scattered in this genome were annotated using the Blast service of NCBI. tRNAscan  and ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) were used to identify the tRNA sequences and potential ORFs, respectively. The unique regions of seven genomes were dug out with MultiPipMaker . BlastN was used to discover large repeats. Short repeats were detected using commercial software developed by Shanghai Majorbio Bio-pharm Biotechnology Company (China). The accession numbers of the mtDNA are listed in Table 4.
This work was supported by the NSFC project (30971812, 31171581), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Program of International S &T Cooperation of China (1021) and the project of Jiangsu Province (BRA2010141, CXLX11_0998).
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