A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.)

Abstract

Background: Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138.

Results: Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura-type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3 kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences.

Conclusions: Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.

Figure 1

The organization of Ogura-type (A) and normal-type (B) mitochondrial genomes represented as a "master circle". Features on forward and reverse strands are drawn on the outside and inside of the circles, respectively. Protein-coding genes are shown in red, rRNAs in blue, tRNAs in orange and orf138 in lime green. The arcs in the same colors indicate syntenic regions between Ogura- and normal-type genomes (refer to Figure 2). Genome maps were made with CGviewer [49].

Figure 2

Comparison of the location and orientation of syntenic regions between Ogura- and normal-type mitochondrial genomes. (A) Ogura-type genome on the X-axis, plotted against normal-type genome on the Y-axis. The numbers for the syntenic regions correspond to those indicated in (B). (B) Schematic illustration of the eleven syntenic regions in radish mitochondrial genomes.

Figure 3

The possible rearrangements that had occurred between two Ogura- and normal-type genomes via short repeated sequences. Large blank arrows indicate each syntenic region. Small arrows in the same color show the position and orientation of repeats. The length and location of the repeats are summarized in Additional file 1 and Additional file 2.

Figure 4

The sequences homologous to Brassicaceae mitochondrial genome plotted on Ogura-type specific region IV. A black line indicates the region specific to the Ogura-type mitochondrial genome (15,255 bp in size) located between syntenic regions 7 and 11. The gene orf138 is located at the edge of this region. Black boxes indicate the sequence homologous to radish and/or other Brassicaceae mitochondrial genomes.

Figure 5

A possible model for integration of the sequence (unique region IV) in Ogura-type mitochondrial genome. Yellow small arrows indicate identical 176-bp inverted repeats at the edges of regions 7 and 11. On the insides of these repeated sequences, another pair of 28-bp inverted repeats is present (purple small arrows). Unique region IV had been integrated into radish mitochondria genome via these repeats when recombination between syntenic regions 7 and 11 occurred.

Figure 6

An atp8 promoter sequence affected by rearrangement among syntenic regions. Alignment of nucleotide sequences shows the comparison of predicted promoters for the gene atp8 among three mitochondrial genomes including Arabidopsis, and Ogura and normal radish. In Ogura-type genome, a promoter core (in a dotted box) is lost. This nucleotide change can affect transcription of the gene atp8.