Extensive structural variations between mitochondrial genomes of CMS and normal peppers (Capsicum annuum L.) revealed by complete nucleotide sequencing

Abstract

Background: Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp.

Results: We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (ψatp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and ψatp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes.

Conclusions: Although large portion of sequence context was shared by mitochondrial genomes of CMS and male-fertile pepper lines, extensive genome rearrangements were detected. CMS candidate genes located on the edges of highly-rearranged CMS-specific DNA regions and near to repeat sequences. These characteristics were detected among CMS-associated genes in other species, implying a common mechanism might be involved in the evolution of CMS-associated genes.

Figure 1

Gene maps of the mitochondrial genomes of CMS and male-fertile pepper lines. (a) Gene map of FS4401 (CMS) (b) Gene map of Jeju (male-fertile). The genes drawn outside of the circle are transcribed clockwise and those inside, counterclockwise. The colors of the genes denote the functions of the gene products. Large repeat sequences (>1 kb) are shown as colored arrows on the outer circle. Sequence blocks that were syntenic between genomes (>2 kb; > 95% similarity) are depicted on the inner circles. They were drawn in two lines of inner circles to separate blocks in different directions.

Figure 2

Structure of the atp6 gene copies in Jeju and FS4401. The sequences correspond to gene-coding region are drawn as the wider rectangles and the upstream or downstream regions as the narrower bars. The sequence units that show high similarity (>99%) to each other and included in the same category of sequence characteristics (non-coding region/coding region, atp6 region showing high conservation/poor conservation among plant taxa) are depicted as the same color. The overall scheme of figure was adopted from Kim et al. [26].

Figure 3

Distribution of specific ORF s, sequences showing similarity with the other pepper mtDNA, tobacco mtDNA and FS4401 plastid genome, repeated sequences in FS4401 and Jeju mtDNA. Locations of ORFs (longer than 300 bp) that are specific to FS4401 (above) or Jeju (below) are shown on FS4401 or Jeju mtDNA, respectively. Red-colored ORFs are specifically present only in one of genomes or carry structural rearrangements. Blue-colored ORFs show polymorphism in length or sequence compared to its counterpart. Known genes are depicted in grey. The sequences showing similarity between genomes were determined based on alignment generated using default parameters of the BLASTN algorithm and is depicted by black rectangles or bars. The distribution of repeated sequences in each genome (>100 bp; > 95%) is depicted with black bars and rectangles. The name of ORFs indicates the number of amino acids in encoded proteins except for the case of ‘orf507’ for which the number of nucleotides in the ORF was adopted to name the ORF in consistent with the previous research [27].

Figure 4

Comparison of sequence structure around orf507 and ψatp6-2 between FS4401, Jeju, and CM334. The sequence blocks conserved between two lines are depicted in the same colors.

Figure 5

Localization of syntenic sequence blocks of mitochondrial genomes in other crops. Sequence blocks showing synteny (>2 kb, > 95%) between a CMS line and a different line were depicted as blue-green color. mtDNAs of CMS lines were used as the reference genome in each comparison. Distribution of repeated sequences (>100 bp, > 95%) in CMS lines is shown with brown bars and boxes. The CMS-associated genes in each crop are indicated above the alignments. Sequence blocks and repeated sequences are depicted in two layers to show the direction and length.