Original researchThe Mitochondrial Genome of Raphanus sativus and Gene Evolution of Cruciferous Mitochondrial Types
Introduction
Mitochondrial genomes are important for supplying ATP in the development and reproduction of plants. Plant mitochondrial genomes vary greatly in size, ranging from 187 kb (Oda et al., 1992) to over 11.3 Mb (Sloan et al., 2012). The organization of plant mitochondrial genomes is known to be reversibly dynamic compared with their counterparts in animals or fungi, which affects the molecular pool of the peculiar mitochondrial genomes of seed plants due to mediation via various large repeated sequences (> 1 kb). Irreversible restructuring of plant mitochondrial genomes has certainly occurred to form genomes corresponding to individual energetic systems, mediated by short repeats (Andre et al., 1992; Newton et al., 2004). The mitochondrial genomes of plant species have lost numerous genes and acquired many genes during their evolution. Based on these properties, conducting sequence analysis of these genomes is important for identifying vital activities in seed plants. Analysis of the evolution of mitochondrial genes or ORFs over a long time-span is necessary to reveal evolutionary sequelae indicative of plant speciation. In this study, the mitochondrial genome of Raphanus sativus (sat), of the Cruciferae family, was sequenced and annotated. How evolutionary factors such as mutation, insertion/deletion and rearrangement resulted in the mitochondrial types within this plant family was also explored.
R. sativus (radish), a member of the Cruciferae family, is widely cultivated in Asia, especially in China, Japan and South Korea (Curtis, 2011). The enlarged root of the radish is commonly consumed as a fresh food or can be utilized as fodder as well as green manure (Noreen and Ashraf, 2009). Classifications of radish mitotypes and certain genes in radish mitochondria have been reported (Sakai and Imamura, 1992; Albaum et al., 1995; Kim et al., 2007). The involvement of short repeat sequences in dynamic mtDNA rearrangements has also been studied in the radish (Lee et al., 2009).
The Cruciferae family was chosen to analyze the evolution of mitochondrial genes because it holds an important position among angiosperm families. The family contains key vegetable and oilseed crops as well as several model organisms, including Arabidopsis thaliana and certain Brassica species (Bailey et al., 2006). As a model for plant biology, the Cruciferae have been extensively studied. Among this family, the first reported mitochondrial genome was that of A. thaliana (termed as the tha mitotype) (Unseld et al., 1997). The mitotype sequence of Brassica napus (nap) was subsequently published (Handa, 2003). In a previous study, we sequenced the mitochondrial genomes of five Brassica mitotypes: cam (B. rapa), ole (B. oleracea), pol (B. napus cultivar Polima), jun (B. juncea) and car (B. carinata) (Chang et al., 2011; Chen et al., 2011).
In this study, we present the complete mitochondrial genome sequence for the radish (referred to as sat). We provide the annotated mitotype of the common radish that is most commonly cultivated in Northern China and analyzed the properties of sat. The sat mitotype and six previously reported cruciferous mitotypes were used as a representative sample of Cruciferae family mitochondrial genomes to analyze the evolution of genes with known functions and the variation among ORFs with unknown functions within this family. The reasons for this variation were also explored.
Section snippets
The mitochondrial genome of R. sativus
For mitochondrial genome sequencing of R. sativus, 40,774 sequencing reads was applied, and the total sequence of 18,393,340 bp was obtained. The initial assembly of the sequences was unsuccessful because of the limited coverage ratio for the mitochondrial genome. Further sequencing to increase the coverage resulted in a coverage ratio of 76×. Assembly of these sequences using Newbler resulted in four large contigs (mean length of 58,414 bp). These contigs were joined through Sanger sequencing
Discussion
In the present study, the genes of the sat mitochondrial genome were identified (Table S1), and a tripartite genome structure and large structural differences from other cruciferous mitochondrial genomes were demonstrated at the sequence level. These data will undoubtedly benefit research on the mechanisms of energetic metabolism, development and reproduction of R. sativus. The examination of mitochondrial genome and gene evolution in the Cruciferae revealed that cruciferous mitotypes have lost
Mitochondrial DNA isolation and sequencing
Mitochondrial DNA was isolated from the seeds of the local Nanjing variety of radish (chuanxinhong) according to the methods described by Chen et al. (2011) and stored in our laboratory. Genome sequencing was performed using the GS-FLX platform (Roche, CT, USA). Newbler v.2.6 (454 Life Science Corp, CT, USA) was used to assemble the reads into contigs. Sanger sequencing of PCR products was conducted to join the contigs to form the complete genome.
Sequence data analysis
The mitochondrial sequences were annotated with
Acknowledgements
This work was supported by the National Basic Research Program of China (973 Program) (No. 2011CB109300), the National Natural Science Foundation of China (No. 30970289), the National Key Technology R&D Program (Nos. 2010BAD01B02 and 2011BAD13B09) in China and the Special Fund for Independent innovation of Agricultural Science and Technology in Jiangsu province (Nos. CX (10) 1030 and CX (11) 1026). The authors thank Shanghai Majorbio Bio-pharm Biotechnology Company (China) for their help on the
References (31)
- et al.
Small repeated sequences and the structure of plant mitochondrial genomes
Trends Genet.
(1992) - et al.
Changes in antioxidant enzymes and some key metabolites in some genetically diverse cultivars of radish (Raphanus sativus L.)
Environ. Exp. Bot.
(2009) - et al.
Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome
J. Mol. Biol.
(1992) - et al.
The Tokumasu radish mitochondrial genome contains two complete atp9 reading frames
Plant Mol. Biol.
(1995) - et al.
Diversity of the Arabidopsis mitochondrial genome occurs via nuclear-controlled recombination activity
Genetics
(2009) - et al.
Toward a global phylogeny of the Brassicaceae
Mol. Biol. Evol.
(2006) - et al.
Mitochondrial genome sequencing helps show the evolutionary mechanism of mitochondrial genome formation in Brassica
BMC Genomics
(2011) The mitochondrial genome of the gymnosperm Cycas taitungensis contains a novel family of short interspersed elements, Bpu sequences, and abundant RNA editing sites
Mol. Biol. Evol.
(2008)- et al.
Substoichiometrically different mitotypes coexist in mitochondrial genomes of Brassica napus L
PLoS ONE
(2011) - et al.
Sequence and comparative analysis of the maize NB mitochondrial genome
Plant Physiol.
(2004)
Genetic engineering of radish: current achievements and future goals
Plant Cell Rep.
progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement
PLoS ONE
The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana
Nucleic Acids Res.
Identification of a novel mitochondrial genome type and development of molecular markers for cytoplasm classification in radish (Raphanus sativus L.)
Theor. Appl. Genet.
The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel gene for tRNACys(GCA)
Nucleic Acids Res.
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