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Nucleic Acids Res. 2005; 33(19): 6235–6250.
Published online Oct 31, 2005. doi:  10.1093/nar/gki925
PMCID: PMC1275586

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.

INTRODUCTION

The mitochondrial genome is important in plant development, as well as in productivity (13), and extensive studies have been done on its functions (4). Although the complete nucleotide sequence has been determined for seven land plant species (511), the genomic makeup is not well understood (1113) because of the multipartite structure of the genome (1416). With a new gene-based strategy for sequencing the wheat mitochondrial genome, we obtained a number of recombinant molecules, analyses of which for the first time have provided proof, at the nucleotide sequence level, of the mechanism that produces multipartite molecules in the mitochondrial genome. Moreover, we demonstrate by gene map comparison that thorough gene shuffling occurred during the speciation of three cereals (wheat, rice and maize), leading to remarkable changes in their mitochondrial genome structures, as previously shown by the restriction fragment mapping of maize mitochondrial DNA (mtDNAs) (17) and by MultiPipMaker analysis of several sequenced plant mitochondrial genomes (10). Based on this information, we propose a new method for quantifying genome-wide molecular changes in mitochondrial genomes, which result in ontogenetic as well as phylogenetic variability of the cereal mitochondrial genomes.

In the wheat complex, Triticum (wheat) and Aegilops (goat grass), inter- as well as intra-specific molecular diversity of both the chloroplast and the mitochondrial genomes were studied in order to clarify the phylogenetic relationships of various taxa of the complex, including the origin of wheat (18,19). Diversity among plasmons of their phenotypic effects on various wheat characters also was investigated [for review see (20)]. However, we have not studied the functional relationships between molecular variation and differential phenotypic effects. We determined recently the complete nucleotide sequence and gene content of the wheat chloroplast genome (21). Here we report those of the mitochondrial genome. The information obtained provides a basis for future studies on the linkage of molecular diversity and phenotypic variability in the wheat complex.

MATERIALS AND METHODS

Plant material

The common wheat, Triticum aestivum cv. Chinese Spring, was the source of the mtDNA studied here, that was obtained from mitochondria of 14-day-old etiolated seedlings (22) and was purified before use (23).

MtDNA library construction and clone sequencing

An mtDNA library was constructed by the SuperCos1 in vitro packaging method (Stratagene, LaJolla) from partially digested wheat mtDNA with Sau3AI. From this library, 232 clones were randomly selected and dot-blotted with 32 mitochondrial genes as probes (24). All probe genes, except rps13, hybridized with 7 or more clones, from which 23 clones were selected to cover 31 probe genes and were sequenced by the shotgun method. Sequenced fragments were aligned using BLASTn (25) to determine the entire sequence of each clone.

Sequence assembly and gene analysis

Alignment of the 23 clones gave two linear molecules of ~350 and 76 kb. Two additional clones, #194 and #204, whose ends hybridized to one end each of the two linear molecules, were selected and sequenced. Phrap (http://www.genome.washington.edu/UWGC/analysistools/phrap.htm), BLASTn and blast2sequences programs were used for the primary assembly of all the clones. Manual fine tuning was done to generate the final master circle (MC). Repeat sequences were analyzed by in-house script, window size 8 bp, and represented as a dot-plot image. Open reading frames (ORFs) were identified by a Genome Gambler (Xanagen Co.) and ORFfinder (http://www.ncbi.nlm.nih.gov/projects/gorf/). tRNA genes were searched for by tRNAscan-SE (26). The annotated rice and maize mitochondrial genes, BA000029 and AY506529, respectively, as well as individual wheat mitochondrial genes submitted to the DNA databank, were compared with our sequence data to annotate all the genes. Sequences homologous to known cereal transposable elements were searched for, referring to the TIGR grass transposable elements database after Clifton et al. (10).

Gene nomenclature and nucleotide position

The nomenclature of Clifton et al. (10) for maize mitochondrial genes was adopted, except for the designation of exons, for which ex-1 to ex-5 of a given gene are indicated by a to e, affixed to its gene symbol. Positions of a forward-strand nucleotide in the MC molecule and in a gene or repeat sequence, respectively, are shown as the ‘MC coordinate’ and ‘gene or repeat coordinate’.

RESULTS

Sequencing of individual clones and their alignment

Twenty-five wheat mtDNA clones were sequenced (Table 1). Their sizes ranged from 27 to 44 kb, except for two (#27 and #39) ~16 kb in size. The average size was 34 898 bp, and the total size was 872 455 bp. Alignment showed a single 452 528 bp MC molecule (Figure 1). Fifteen clones occupied single locations in the genome (‘intact clone’), while the remaining 10 were split into two or three segments located in different parts of the genome, tentatively called the ‘recombinant clone’. Quetier et al. (15) estimated size of the wheat mitochondrial genome to be ~430 kb, based on its SalI restriction map. Their estimate is very close to the size, 452 528 bp, determined by the present sequencing work.

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 ...
Table 1
Wheat mtDNA clones sequenced, showing their size, type, marker genes used and genes other than probe genes identified by sequencing

Intra-molecular recombination and site of recombination

Of the 10 recombinant clones, 8 were split into two segments. The other two (#24 and #224) were cleaved into three segments. Without exception, there was a pair of direct repeats (DRs) or inverted repeats (IRs) at the split site (Figure 1). All the recombinant clones carried a completely or nearly identical copy of the same repeat at the recombination site (details in the next paragraph). DRs connected split fragments head-to-tail, whereas IRs connected them head-to-head or tail-to-tail. These facts indicate that the split clones were produced by intra-molecular recombination between the relevant repeats. In sum, nine repeat pairs, R1 to R9, were responsible for the production of all of the recombinant clones (Table 2). The production of clones #24 and #51 was mediated by the same R7 repeats, whereas #75 (#162 as well) and #96 were reciprocal products of recombination of R8 repeats (Figure 2A). Two clones, #24 and #224, were double recombinants (Figure 2B and C). The former was produced by recombination between two DR pairs, R3 and R7, and the latter recombination between two IR pairs, R2 and R6. Seven additional repeats, R10 to R16, larger than 100 bp were present in the genome (Table 2). Three repeats, R1, R7 and R10, shared a 1634 bp sequence in common, containing a part of rrn26. Similarly, three other repeats, R2, R3 and R4, shared a 4430 bp common sequence that carried trnfM, rrn18 and rrn5. In addition, small repeats of 30–100 bp in size were detected in a dot-matrix image, of which 35 were the direct and 38 were the inverted types. All those repeats are shown in Figure 3, in which R1 to R16 are marked by arrows. We need to search for whether all of them serve as recombination sites or not, although our results showed that a repeat pair as small as 197 bp in size (= R9) mediated 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 ...
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).
Table 2
Repeats involved in intra-molecular recombination, and other repeats larger than 100 bp found in the wheat mitochondrial genome

We tried to identify the recombination site in each repeat pair. Four, R3, R5, R6 and R7, had identical copies. Four others, R1, R2, R4 and R9, had only 1 nt difference between the two copies, located at the extreme end of the repeat (Table 2). Identification of the recombination site therefore was informative only for repeat pair R8, which was involved in the production of three recombinant clones; #75, #96 and #162 (Figure 4). Two copies of this repeat, R8-1 and -2, which carried atp6-1 and atp6-2 at the same R8 coordinates, 91–1251, had 8 nt differences; one at R8 coordinate 6, the others between coordinates 1301 and 1316 (Figure 4A). Nucleotide sequences of the two R8 copies and their 5′- and 3′-flanking segments were compared with those of the three recombinants. As for the 5′-flanking sequence and sixth nucleotide of R8, #75 and #162 were the same as the R8-1 copy, whereas #96 was the same as R8-2. As for the 3′-flanking sequence and seven variable nucleotides at R8 coordinates 1301–1316, #75 and #162 were the same as the R8-2 copy, whereas #96 was identical to the R8-1 copy. These findings indicate that all three recombinant clones were produced by recombination in the same 1294 bp segment of the R8 repeat (Figure 4A). Previously, Bonen and Bird (27) sequenced wheat mtDNA segments flanking atp6, and found that there are two molecular forms at both the 5′ (‘downstream’ in their designation) and 3′ (‘upstream’) borders of the gene. Their nucleotide sequences were in complete agreement with ours, except for a 1 bp deletion in our R8-2 copy between MC coordinates 86 217 and 86 218. Their sequences 3 and 2 correspond to the 5′ and 3′ borders of the R8-1 copy, and the sequences 4 and 1 to the 5′ and 3′ borders of the R8-2 copy (Figure 4B). They located a 6 bp insertion in sequence 3, extending the homologous region between sequences 3 and 4 by 22 bp downstream (toward the 5′ end), which was confirmed by our findings. The sequence comparison (Figure 4B) indicated that the three recombinant clones were produced by recombination in the same 1291 bp segment (3 bp smaller, comparing with the alignment in Figure 4A). Because this segment occupies ~95% of the R8 repeat, it is not surprising that three independent recombination events occurred within this segment.

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, ...

Bonen and Bird (27) also reported the presence of short DRs in three of the above four sequences, corresponding to the present 5′ and 3′ borders of the R8-1 copy and the 5′ border of the R8-2 copy (Figure 4B), where ‘border’ means the boundary between a repeat end and its flanking sequence. We examined 60 bp sequences around the 5′ and 3′ borders (30 bp on both sides of each border) of all repeats shown in Table 2. The complete border sequences are given in Supplementary Table 1. Of 70 border sequences of the 35 repeat copies, 22 contained straight, DRs (no gap, no mismatch) of 3–7 bp while additional 24 possessed aberrant 4–10 bp DRs, having a mismatched nucleotide or a few nucleotides intervening between the repeats, and the remaining 24 did not have short DRs (Table 3). Fourteen repeats had short DRs at both ends, which did not show any sequence similarity, homologous or complementary, to each other. Thus, we conclude that the majority of the repeat ends are associated with short DRs, although their functional role is unknown.

Table 3
Short DRs found in the 5′- and 3′-borders of 16 repeats, R1–R16, in the wheat mitochondrial genome

Stern and Palmer (28) indicated that rrn18 and rrn26 often are contained in recombination sites of the wheat mitochondrial genome. Our results confirmed this because 6 of the 12 recombination events detected are mediated by repeat pairs containing those genes (Table 2).

Genes and the genetic map of the wheat mitochondrial genome

In all, 55 genes and their exons were identified (Table 4) and mapped on the MC molecule (Figure 5). All the protein-, rRNA- and tRNA-coding genes known for wheat (24), rice (8) and maize (10) were present, i.e. 9 Complex I genes, 1 Complex III gene, 3 Complex IV genes, 5 Complex V genes, 4 cytochrome c biogenesis genes, 11 ribosomal protein genes, 2 other protein-coding genes, 3 rRNA genes and 17 tRNA genes. Nucleotide sequences of seven wheat genes, rpl16, rps3, rps4, mttB, trnA, trnI and trnM, were determined here for the first time. Three genes, rpl2-p, rps19-p, and rrn26-p (the third rrn26 copy), were truncated. The first two are functional in rice but missing in maize (10). Nine genes, nad1, nad2, nad4, nad5, nad7, cox2, ccmFC, rps3 and trnA (chloroplast origin), had the exon–intron structure. The chloroplast counterpart of trnA also has an intron (21). All exons of nad4 (exons a-d), nad7 (a-e), cox2 (a,b), ccmC (a,b), rps3 (a,b) and trnA (5′-,3′-ex) were cis-spliced, whereas some exons of nad1, nad2 and nad5, were trans-spliced (the slash indicating trans-spliced exons) as follows: nad1a/nad1b,c/nad1d/nad1e; nad2a,b/nad2c-e; and nad5a,b/nad5c/nad5d,e.

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, ...
Table 4
Genes in the wheat mitochondrial genome

Ten genes were present in multi-copy: atp6, atp8, rrn26, trnD and trnP were duplicated and rrn5, rrn18, trnfM, trnK and trnQ triplicated. In addition, three trnS genes were found, but they greatly differed each other in nucleotide sequence and therefore were considered different genes, confirming the results of two previous works (29,30).

Restriction fragment analyses of wheat mtDNA revealed the presence of seven molecular forms of the rrn18-rrn5 cluster (31,32). We identified three copies, Copy-1, -2 and -3, of a three-gene cluster, trnfM-rrn18-rrn5, in the MC molecule, all of which were included in three repeats, R2, R3 and R4 (Table 2). Figure 6 illustrates the production of two recombinant forms of this gene cluster from recombination between Copy-1 and -2 (pathway [A]) and Copy-2 and -3 (pathway [B]). Because recombination also occurs between Copy-1 and -3, six recombinants are expected altogether. We obtained three of them, which were produced by recombination between Copy-1 and -2 (#1L/R), Copy-1 and -3 (#224C/R) and Copy-2 and -3 (#24C/L) (Table 2). None of their reciprocal products was obtained, probably as a matter of chance owing to the small number of the clones examined, because the fourth recombinant molecule is reported by Lejeune et al. (32). As for rrn26, two molecular forms of its 5′ end, and three forms of the 3′ end had been predicted previously (15,32). This prediction was verified by the present findings confirming two complete and one partial copy (422 bp 3′ end) of rrn26.

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 ...

Two copies of atp8 had five mismatched nucleotide pairs scattered within the 471 bp gene region. Sequence analyses of recombinant molecules supposedly produced by recombination between the R11 repeats containing this gene (Table 2) might be useful in specifying recombination site(s) within the repeat.

In addition to those genes, 179 ORFs larger than 300 bp were found (Supplementary Table 2). Their total size amounted to 75 465 bp, occupying ~16.7% of the entire genome. This number greatly exceeds the 121 ORFs of comparable size reported for maize (10), in spite of the fact that the wheat mitochondrial genome is much smaller than the maize genome. Functional analysis of those ORFs will be an important problem in the future mitochondrial genomics.

MtDNA sequences homologous to ctDNA

Homology search using the blast2sequence program revealed that the wheat mitochondrial genome has 55 sequences homologous (mostly with 80% or higher homology on the nucleotide basis) to the corresponding sequences of the wheat chloroplast genome (Table 5). Exceptions were nine sequences question-marked in the last column of Table 5, which were mosaic of highly conserved and variable sequences, showing segmental differentiation of the sequences. Sizes of individual sequences vary between 27 bp for the smallest and 4239 bp for the largest. The total size, 26 264 bp, corresponds to 5.80% of the entire genome.

Table 5
Wheat mtDNA sequences showing homology to ctDNA sequences

Of the above 55 wheat mtDNA sequences, 8 carried native (not chloroplast-derived) mitochondrial genes, atp1, rrn18-1, -2, -3, rrn26-1, -2, -p and trnM, whose total size amounted to 12 809 bp. They showed homology to the ctDNA sequences carrying the corresponding chloroplast genes, atpA, rrn16, rrn23 and trnM (marked by circles in Table 5). Each of the gene pairs, atp1/atpA, rrn18/rrn16, rrn26/rrn23 and mt-trnM/ct-trnM, is assumed to have originated from a common prokaryotic gene, being homoeologous to each other (evidence will be reported elsewhere). The total size of the mtDNA sequences of real chloroplast origin therefore was estimated as 13 455 bp; 2.97% of the wheat mitochondrial genome, compared with 22 593 bp (6.3%) and 25 281 bp (4.4%) reported, respectively, for rice and maize (8,10). Thus, both the total size and proportion of the chloroplast-derived sequences relative to the entire genome were smallest in wheat, comparing with rice and maize.

Gene shuffling in the cereal mitochondrial genome

We compared mitochondrial gene maps of wheat, rice (8) and maize (10), excluding tRNA genes, pseudogenes and ORFs (Figure 7). Five exons of nad7, nad7a to e, showed a common arrangement in the three cereals. This gene was used to mark the common map origin, and the arrangement of nad7a to nad7e to mark the common map direction. A syntenic gene/exon arrangement, then, should appear as a row of genes/exons parallel to either diagonal line. Only a few gene/exon clusters of the three cereals showed synteny. One 5-gene cluster, ccmFN-rps1-matR-nad1e-nad5c, and five 2-gene clusters, rps13-nad1bc, rrn18-rrn5, rps3-rpl16, nad9-nad2cde and nad3-rps12, showed synteny. The third and fourth ones are shown as 3-gene clusters in Figure 7, because maize has an extra copy of both rps3a and nad2de and, for this, rps3a and rps3bcd, and nad2c and nad2de were shown separately. Similarly, nad4abc and nad4d were shown as a 2-gene cluster because rice has two extra copies of nad4d. In addition, three 2-gene clusters, rps19(p)-nad4L, ccmB-nad2ab and nad5ab-rpl2(p), of wheat and rice conserved synteny, and two 2-gene clusters (cox1-rrn26 and nad6-rps4) of wheat and maize preserved synteny. No synteny was detected for any other gene combinations, indicative that frequent gene shuffling occurred during cereal speciation, resulting in remarkable structural differences in the cereal's mitochondrial genomes. Fauron et al. (17) showed by the physical map comparison that mitochondrial genome restructuring has taken place between three maize cytotypes, and Clifton et al. (10) demonstrated by MultiPipMaker analysis that little sequence similarity exists between mitochondrial genomes of six plant species. Those results agree with ours of the above cereal gene map comparison.

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 ...

DISCUSSION

Features of the gene-based strategy for sequencing plant mitochondrial genomes

Two principal strategies have been used to sequence plant mitochondrial genomes; the physical map-based (5,79), and the genome shotgun strategies (6,10,11). We used a new gene-based strategy for wheat, facilitated by the fact that many wheat mitochondrial genes are available as probes (24) for selecting wheat mtDNA clones for sequencing. Use of this strategy gave a complete picture of the wheat mitochondrial genome by sequencing the 872.5 kb mtDNA, less than twice the genome size, 452 528 bp. Comparative values for the genome shotgun strategy are ~4, 8 and >20 times for Arabidopsis, tobacco and maize (6,11,10), indicating apparent high genome sequencing efficiency of the gene-based strategy. However, application of this strategy requires construction of a cosmid mtDNA library and selection of mtDNA clones covering known mitochondrial genes by dot hybridization. The overall efficiency of the gene-based strategy, compared with that of the genome shotgun strategy, is not clear.

The advantage of the gene-based, compared with the physical map-based strategy, is that no physical map construction is required. This is difficult with some plants because of the multipartite structure of the mitochondrial genome. Based on the physical map of the mitochondrial genome of a common wheat cultivar, Capitole (15), Lejeune and Quetier [cited from (24)] constructed the first gene map of the wheat mitochondrial genome, to which 36 genes were allocated. Their map completely matches ours for five local gene maps: (i) rrn18/rrn5cobatp6nad5denad4abcdnad2aborf25 (= atp4)–nad2cdenad9cox2abcox1rrn26, (ii) rps7rrn18/rrn5nad7abcdeatp1atp9nad1bcrps13atp6cox3, (iii) nad1arrn18/rrn5nad5abnad1dnad6rrn26, (iv) nad3rps12orf156 (= atp8) and (v) matRnad1enad5c. The arrangement of these five gene groups within the genome, however, differ both in order and direction. Their order in Lejeune and Quetier's map is that shown above, whereas in our map it is (i)–(iii, reverted)–(iv, reverted)–(ii)–(v, reverted). Whether this discrepancy is due to the different mtDNA sources, or to problems in the physical map they relied upon, needs to be clarified.

The gene-based strategy for complete mitochondrial genome sequencing can not achieve its goal by itself if the genome contains large gene-free region(s) of >35 kb (average insert size of the vector) when Cosmid mtDNA clones are used in sequencing. To cover such regions of the genome, we need to perform sequencing of some additional clones which do not carry any probe genes. In fact, it was necessary for us to sequence a probe gene-free clone, #194, to complete sequencing the wheat mitochondrial genome. The MC molecule obtained successfully integrated all sequenced mtDNAs in it without leaving any pieces out.

Blast search on the sequence homology between the present wheat and previously reported rice and maize mitochondrial genomes (8,10) provided supporting evidence that the present MC molecule represents the wheat mitochondrial genome. The rice and maize mitochondrial genomes were divided into successive 30 kb sections (sizes of the end sections were somewhat different), and sequences homologous to wheat mtDNA were investigated for each section (Table 6). All the sections contained homologous sequences of ~3 kb or larger (up to 15 kb) to wheat mtDNA. MtDNA sequences conserved between wheat and rice, and between wheat and maize were distributed all over the rice and maize genomes, with no large conserved sequence-free regions (larger than 10 kb; detailed data omitted) in the genomes. This fact indicates that rice and maize mitochondrial genomic sequences are well represented in the wheat MC molecule.

Table 6
Rice and maize mtDNA sequences homologous to wheat mtDNA in different sections of the genome

The most essential feature of the present gene-based strategy is that it facilitated the recovery of recombinant molecules. Restriction fragment mapping of plant mtDNA shows a multipartite structure of the mitochondrial genome, consisting of isomeric as well as subgenomic molecules produced by intra-molecular recombination (12,1417,33). None of the previous works on complete sequencing of flowering plant mitochondrial genomes, by use of either the genome shotgun or physical map-based strategies, has recovered recombinant molecules. This is why recombination events have not been analyzed at the nucleotide sequence level. By virtue of the gene-based strategy, we obtained 10 recombinant clones among 25 examined, determined their nucleotide sequences, and identified repeat sequences responsible for their formation.

Structural features of the wheat mitochondrial genome

The wheat mitochondrial genome was assumed to be a 452 528 bp MC molecule (Figure 1), that was ~92 and 79% the size of the rice and maize mitochondrial genomes, and possessed all the protein-, rRNA- and tRNA-coding genes known to be present in rice and maize (8,10). These facts indicate that wheat has the most compact mitochondrial genome among the three cereals.

Multicopy mitochondrial genes were compared between wheat, rice and maize (Table 7). Gene amplification in general was species-specific. All of the multicopy wheat genes were located in the repeated sequences (Table 2). With the exceptions of atp8 and trnQ, multicopies of all the wheat genes had identical nucleotide sequences. As for trnQ, two copies were identical, whereas the third copy differed from them by a single nucleotide. These facts suggest their recent amplification, comparing with the divergence time of three cereals. One alternative possibility is copy correction through homologous recombination, which is known to occur in the case of chloroplast IRs (34).

Table 7
Copy numbers of mitochondrial genes that differ in number in wheat, rice and maize: gene fragments, pseudogenes and chloroplast-derived genes are excluded

To account for the observed species-specific gene amplification, a mechanistic model can be proposed. Recombination between the same repeat sequences in two subgenomic molecules produced by recombination between different repeat pairs will give rise to an aberrant MC molecule having a duplicate segment. Figure 6 illustrates an example, using a simplified MC molecule, in which only three copies (Copy-1, -2 and -3) of the trnfM-rrn18-rrn5 cluster and two repeat pairs, R5 and R6, are shown. Recombination between the R6 sequences in two subgenomic molecules, II and III, which are produced in pathways [B] and [C], gives a new MC molecule with an extra copy of the trnfM-rrn18-rrn5 cluster and R5 repeat together with their flanking regions. The size of the duplication corresponds to the sum of two segments, one between the recombination breakpoints in Copy-2 and one R5 copy, and the other between those in Copy-3 and the other R5 copy.

Search for transposable element sequences in the wheat mitochondrial genome revealed presence of five sequences, three of which were different partial sequences of the wheat Sabrina retrotransposon, and two others were a part of a rice Tos-14 retrotransposon and wheat Tar1 retrotransposon. Total size of the five sequences was 805 bp, being ~0.2% of the mitochondrial genome. Comparable figures for rice and maize were 20 sequences (total size 7003 bp, 14.3% of the genome) and 4 sequences (total size 641 bp, 0.1% of the genome), respectively (8,10). In this respect, wheat mitochondrial genome is similar to maize than to rice mitochondrial genome.

It is important to know what kinds of sequences were involved in the observed mitochondrial genome differentiation. For this purpose, the MC coordinates of all unique wheat sequences larger than 100 bp, comparing with both the rice and maize mtDNA sequences were enumerated (Supplementary Table 3). In total, 227 unique sequences distributed throughout the genome were identified. Comparison between their positions and those of all mitochondrial genes in the genome indicated that almost all unique sequences corresponded to intergenic spaces. The exceptions were nine sequences carrying partial sequence of a gene. Of those, six sequences carried 3–97 bp of the highly variable 3′ end of the sense strand of cob, nad6, rpl2-p, rrn5-1, rrn5-2 and rrn5-3. Two sequences contained a 324 bp segment of atp6-1 and -2, that is located in the 3′-terminal region of these genes. The last sequence carried a 28 bp 5′ end of nad9, that is variable among the three cereals. These facts taken together demonstrate that the mtDNA sequences diversified in the three cereals are mostly redundant DNAs.

In a summary, the wheat mtDNA sequences were partitioned into six categories, genic (including introns), ORF, repetitive, chloroplast-derived, retro-element and unique sequences (Table 8). This partition was not orthogonal, because some sequences were enumerated in more than one category. Sizes of the genic, ORF, repetitive, chloroplast-derived and unique sequences were obtained from the data presented in Table 4, Supplementary Table 2, Table 2, Table 5 and Supplementary Table 3, respectively.

Table 8
Classification of wheat mtDNA sequences into different categories

Structural dynamics of the mitochondrial genome in ontogeny

Arrieta-Montiel et al. (35) reported on the structural dynamics of the common bean mitochondrial genome, which was revealed by studying a single mtDNA segment carrying the cms-associated pvs-orf239 sequence. Using the gene-based strategy, we isolated 10 recombinant mtDNA molecules, and determined the repeat sequences responsible for their production. Many other repeat pairs also were characterized (Table 2 and Figure 3), which are potential sites for additional recombination. Based on the entire wheat mitochondrial genome sequence (DNA Database accession no. AP008982) and the map positions of all repeat pairs larger than 100 bp (Table 2), we may prepare DNA primers for the sequences flanking both ends of those repeats. Their use in long-range PCR will allow efficient screening of recombinant molecules produced by recombination between the marked repeat pairs and quantification of isomeric as well as subgenomic molecules, as proved by Sugiyama et al. (11) in tobacco. They also demonstrated that long-range PCR works for a distance as long as 23 kb between two primers, which is sufficient to cover all repeats present in the wheat mitochondrial genome (Table 2). The same method may also facilitate finding the difference in recombinational activity among various repeat pairs as well as the equality or inequality of the reciprocal recombination products.

The methodological details for such studies are as follows: recombination between an IR pair will produce an isomer (flop form) of the MC molecule (flip form; Figure 6, pathway [A]). This event is detected by long-range PCR using four primer pairs, A/B, C/D, A/D and C/B. If either the A/B or C/D primer pair gives an amplified product in PCR, the template clone is regarded as the original MC molecule, whereas if either the A/D or C/B pair gives an amplified product, the template clone is regarded as the flop configuration of MC, so far as the marked IR is concerned. The ratio of the latter to the former clones in number gives the molar ratio of the recombinant to the non-recombinant clones. Similarly, recombination between a DR pair will produce two subgenomic molecules (Figure 6, pathway [B]), whose production is detected by successful DNA amplification by use of the C/F or E/D primer pair. Quantification of the subgenomic molecules over the non-recombinants is achieved in the same way as described above. Such studies targeted to different repeat pairs enumerated in Table 2, using Cosmid clones of wheat mtDNA extracted from different organs or different ages of the plant as the template, will disclose structural dynamics of the mitochondrial genome in plant development.

Evolutionary change in the mitochondrial genome structures of cereals

The chloroplast genomes of rice, maize and wheat have identical gene arrangements (36,37,21), evidence of the structure's evolutionary stability. In contrast, the mitochondrial genome structure differs markedly in the three cereals (Figure 7) although the kinds of genes present essentially are the same [(8,10), present findings]. We showed that a variety of mtDNA molecules are produced in somatic tissues by intra-molecular recombination mediated by different repeat pairs. The structural differences of several mitochondrial genes in wheat and rice are suspected to be caused by short repeat pairs (data to be published elsewhere). We postulate that the same mechanism operates in germ cell lines, creating structural diversity in the mitochondrial genomes of different plant phylogenies.

Another possible factor for high phylogenetic variability of the mitochondrial genome, compared with the chloroplast genome, is high DNA redundancy in the former than in the latter genome. The ratio of the genic sequences, including all exons and cis-introns, and excluding the sequences of chloroplast origin and pseudogenes, to the total mitochondrial genome size is 18.0% for rice (8), 11.7% for maize (10) and 15.9% for wheat (Table 8). Comparable values for the chloroplast genome are 58.8% for rice (36) and 60.4% for wheat (21), indicative of the presence of a much larger amount of redundant DNAs in the mitochondrial than in the chloroplast genome.

The MC molecule may represent the intact wheat mitochondrial genome

All previous works on complete sequencing of flowering plant mitochondrial genomes are based upon the MC molecule hypothesis (611). Because of the multipartite structure of the genome and the lack of direct electron-microscopic evidence, however, the existence of the MC molecule is still a matter of debate (1113). After Andre et al. (12), we suspected reality of the MC molecule in wheat and upon this suspicion we adopted the gene-based sequencing strategy. It turns out, however, that analysis of the 10 recombinant clones obtained has given support to the existence of the MC molecule.

If we consider the MC molecule to be a flip configuration of the genome, then recombination between either of the three IR pairs (Table 2) will produce its flop (= isomeric) molecule, as shown in the pathway [A] of Figure 6, whereas recombination between either of the DR pairs produces two complementary, subgenomic molecules (pathways [B] and [C] in Figure 6), where ‘complementary’ means that a complete gene set is shared by two or more molecules (15). The origin of eight recombinant clones can be explained by a single recombination event, while the remaining two double-recombination events occurred in the MC molecule. However, if the genome were in any other configuration, most of the recombinant clones obtained could not have been produced by simple recombination events (Table 9). Consider the following: if the genome existed in the flop configuration of MC (Table 9, Case 1–4), then recombination between any pair of the present DRs should produce double-flop configurations of the genome (Case 1 and 2), and recombination between IRs should produce two subgenomic molecules (Case 3 and 4). Similarly, if the genome consisted of two subgenomic molecules (Case 5–8), recombination between the repeat sequences in two separated molecules should produce the MC molecule (Case 5 and 6), or its double-flop configuration (Case 7 and 8). In all eight postulated cases, the expected recombination products do not match the ones we actually obtained. This fact supports the hypothesis that the MC molecule serves as the basic wheat mitochondrial genome structure.

Table 9
Expected and actual products of recombination when the mitochondrial genome has alternative configurations

A possible alternative is that the wheat mitochondrial genome contains all kinds of isomeric as well as subgenomic molecules (13). Lonsdale et al. (16) and Fauron et al. (17) showed that 5–14 subgenomic molecules are produced from the MC molecule of sugar beet and maize by intra-molecular recombination. In our study we prepared wheat mtDNA from 2-week-old seedlings. Now, if a seedling consists of ~106 cells, it means that 19 successive cell divisions, on the average, occurred before DNA extraction. We do not know how many replication origins exist in the wheat mitochondrial MC molecule. An electron-microscopic study of mtDNA replication in Chenopodium indicates only a few, if not just one, origins in its mtDNA (38). Considering this fact, together with information on the single replication origin of bacterial chromosomes, it is hard to believe that all kinds of subgenomic molecules have replication origins necessary for their maintenance through many cell cycles. This is further support for the presence of the MC molecule.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

Supplementary Material

[Supplementary Material]

Acknowledgments

We thank L. Bonen for her generous gift of the mitochondrial gene probes used in our clone selection, C.-H. Guo and T. Ochiai for technical help, Jeffrey D. Palmer for critical comments on our manuscript, and Val Woodward for valuable advice on improving the manuscript regarding its context and language. This work was supported by a Grant-in-Aid (No. 12309008, 2000–2002) from the Japan Society for the Promotion of Sciences, and research grants from the Iijima Memorial Foundation for the Promotion of Food Science and Technology (2003 and 2004) and the Nisshin Seifun Foundation (2001 and 2004). Funding to pay the Open Access publication charges for this article was provided by the Graduate School of Agriculture, Kyoto University, Japan.

Conflict of interest statement. None declared.

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