Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome

Abstract

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon-intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Figure 1

Alignment of 25 mtDNA clones in the 452 528 bp MC molecule of the wheat mitochondrial genome. Broad, light-green bar shows the MC molecule cleaved between MC coordinates 452 528 and 1. Numbers on the MC molecule show the MC coordinates of the ends of all the clones, their segments and repeat sequences. Rectangle with projection in the broad bar: R1–R9 repeat pairs involved in recombinant clone formation. DRs are dark green, IRs dark brown. The projection shows the direction of each repeat copy. Slender bar: individual clones; light blue, yellow and orange represent intact, single recombinant and double recombinant clones, respectively. L, C or R affixed to clone numbers: Left, central and right segments of a recombinant clone. Note that L and R segments of a single-recombinant clone have the same repeat copy at the end connecting two segments; head-to-tail for DRs, and head-to-head or tail-to-tail for IRs. The double-recombinant clone has a copy of one repeat pair at one end each of its L and C segments and a copy of another repeat pair at the other end of C and at one end of R that, respectively, connect the L and C segments and the C and R segments by recombination.

Figure 2

Origins of four recombinant clones obtained by recombination mediated by different repeat pairs. Rectangle: MC molecule. Arrows: DR or IR pairs. Broken line: fusion of separate segments by recombination. Thick and thin lines: cloned DNA segment and remaining part of the recombinant molecule not included in the clone. Numbers on MC molecules: MC coordinates at the ends of repeats and the cloned molecule. Note: DRs should be drawn in the same direction by folding the MC molecule with a 180° twist. This was omitted to simplify the figure. (A) Clones #75 and #96 as reciprocal products of R8-mediated recombination. They are part of two subgenomic molecules; (B) clone #24 is the product of double recombination at two DR pairs, R3 and R7; (C) clone #224 is the product of double recombination at two IR pairs, R2 and R6.

Figure 3

Dot matrix of the MC molecule of wheat mitochondrial genome, showing direct (blue) and inverted (orange) repeat pairs of larger than 30 bp. Sixteen repeat pairs, R1–R16, of larger than 100 bp are marked by arrows (Table 2).

Figure 4

(A and B) Recombination site in R8 repeats which produced the three recombinant clones, #75, #96 and #162. Nucleotide sequences in pink, light green and yellow backgrounds, respectively, are sequences homologous to an R8 copy (R8-1) and its flanking regions, sequences homologous to the other R8 copy (R8-2) and its flanking regions, and the recombination site sequence. In this figure, the forward strands are shown, which are antisense relative to the apt6-coding sequence. Numbers outside and inside the R8 or R8′ repeat: MC coordinates of the nucleotides flanked respectively by two R8 copies and the R8 or R8′ coordinates of the variable nucleotides between them. Capital and lower case letter: Consensus and unique nucleotide between two R8 copies and their flanking regions. Asterisk: deficient nucleotide.

Figure 5

Genetic map of the wheat mitochondrial genome showing the location of all the genes and their exons in the outer-most circle, of ORFs larger than 300 bp in the central circle, and of chloroplast-derived DNA segments in the inner-most circle. The broad, outer-most circle represents the MC molecule, in which the nine repeat pairs, R1–R9, that mediate production of all the recombinant clones and an additional 7035 bp repeat pair, R10, are shown. Genes and exons coded by the forward and reverse DNA strands are shown outside and inside the MC molecule, respectively.

Figure 6

Production of various molecular forms from the MC molecule by intra-molecular recombination between different repeat pairs. Copy-1, -2 and -3 are three copies of the trnfM-rrn18-rrn5 gene cluster. Copy-2 and -3 are inverted relative to Copy-1. R5 and R6 represent a DR and an IR pair, respectively. A/B, C/D and E/F are PCR primer pairs to mark the 5′- and 3′-flanking regions of Copy-1, -2 and -3, respectively. [A]: production of an isomer (flop form) of the MC molecule (flip form) by recombination between an IR pair, Copy-1 and -2. [B] and [C]: production of two complementary subgenomic molecules by recombination between a DR pair, Copy-2 and -3, and two R5 copies, respectively. [D]: production of an aberrant MC molecule having extra copies of the three-gene cluster (Copy-3/2) and R5 repeat by recombination between R6 repeats in two subgenomic molecules, II and III.

Figure 7

Correlation of gene order between the mitochondrial gene maps of wheat and rice (A) and wheat and maize (B). All the protein- and rRNA-coding genes and the former's trans-spliced exons are arranged from top to bottom for wheat, and from left to right for rice and maize, based on their order in the respective gene maps. Genes of rice and maize are indicated by code numbers given to the corresponding wheat genes in the left margin of figures. Duplicate genes carry the same number.