Origins and recombination of the bacterial-sized multichromosomal mitochondrial genome of cucumber

Abstract

Members of the flowering plant family Cucurbitaceae harbor the largest known mitochondrial genomes. Here, we report the 1685-kb mitochondrial genome of cucumber (Cucumis sativus). We help solve a 30-year mystery about the origins of its large size by showing that it mainly reflects the proliferation of dispersed repeats, expansions of existing introns, and the acquisition of sequences from diverse sources, including the cucumber nuclear and chloroplast genomes, viruses, and bacteria. The cucumber genome has a novel structure for plant mitochondria, mapping as three entirely or largely autonomous circular chromosomes (lengths 1556, 84, and 45 kb) that vary in relative abundance over a twofold range. These properties suggest that the three chromosomes replicate independently of one another. The two smaller chromosomes are devoid of known functional genes but nonetheless contain diagnostic mitochondrial features. Paired-end sequencing conflicts reveal differences in recombination dynamics among chromosomes, for which an explanatory model is developed, as well as a large pool of low-frequency genome conformations, many of which may result from asymmetric recombination across intermediate-sized and sometimes highly divergent repeats. These findings highlight the promise of genome sequencing for elucidating the recombinational dynamics of plant mitochondrial genomes.

Figure 1.

The 1685-kb Mitochondrial Genome of Cucumber. The genome consists of three circular-mapping chromosomes whose relative abundance varies over a twofold range. For the main 1556-kb chromosome, features on transcriptionally clockwise and counterclockwise strands are drawn on the inside and outside of the circle, respectively. Chloroplast-derived sequences (labeled “chloroplast”) were arbitrarily drawn on the counterclockwise strand. Genes from the same protein complexes are similarly colored, as are rRNAs and tRNAs. The inner circle shows the locations of direct (blue) and inverted (red) repeats (A to I) with the most compelling evidence for recombination activity (see Methods and Supplemental Data Set 2 online). Numbers on the inner circle represent genome coordinates (kb). No features are shown on the two relatively featureless small chromosomes.

Figure 2.

Many Introns in the Cucumber Mitochondrial Genome Are Substantially Larger Than Homologous Introns in Other Seed Plants. Mean (±sd) intron lengths were calculated from the set of fully sequenced mitochondrial genomes (except cucumber) (n = 20) for all but the cox1 group I intron, which among the fully sequenced genomes, is present only in watermelon and cucumber. Mean length of the cox1 intron therefore was based on full-length intron sequences in the alignment used by Sanchez-Puerta et al. (2008). Intron sizes for two other cucurbits (Cucurbita [zucchini] and Citrullus [watermelon]) and their phylogenetic relationships to cucumber (Cucumis) are shown to highlight the recency of intron expansions in cucumber. Introns are sequentially numbered beginning from the 5′ end of the gene, and trans-spliced introns in cucumber are marked with an asterisk. Not included are the two introns in the cox2 gene that are widely present in angiosperms (Qiu et al., 1998) but absent from cucumber. [See online article for color version of this figure.]

Figure 3.

Coverage of the Cucumber Mitochondrial Genome by Identifiable Coding and Noncoding Features. Genes includes all protein exons, rRNAs, and tRNAs. Introns include both cis- and trans-spliced forms. The mitochondrial genome contains DNA with identifiable nuclear features (genes and transposable elements) as well as 514 kb of shared nuclear-mitochondrial DNA whose origin and direction of intercompartmental transfer is ambiguous. A little more than half of the nuclear-like sequences are part of the repetitive fraction of the mitochondrial genome, so the dashed line represents the total sequence complexity of the nuclear-like component of the cucumber mitochondrial genome (i.e., each repeated site is counted only once). [See online article for color version of this figure.]

Figure 4.

Characteristics of Numts and Other Shared Nuclear-Mitochondrial Sequences in Cucumber. Distributions of lengths and percent identities between nuclear-mitochondrial matches corresponding to identifiable numts ([A] and [B]) and to shared sequences whose origin and direction of intercompartmental transfer are ambiguous ([C] and [D]). Results are based on a BLAST e-value cutoff of 1e^–12. The number of matches is shown by gray boxes and plotted on the left ordinate of each panel, whereas nuclear and mitochondrial genome coverage by sequences in each bin is plotted on the right ordinate. [See online article for color version of this figure.]

Figure 5.

Homologous Recombination across a Direct Repeat Strongly Favors the Subdivision of a 129-kb Mitochondrial Chromosome into Two Smaller Chromosomes in Cucumber. Recombination across a 3.6-kb direct repeat (black rectangles) splits a 129-kb (green) mitochondrial chromosome into two subgenomes of lengths 45 kb (blue) and 84 kb (red), and recombination between the latter two reconstitutes the 129-kb chromosome (A). Under the recombination model in (A), the expected (B) and observed (C) patterns from DNA gel blot hybridization of a probe located within the 3.6-kb repeat to purified mitochondrial DNA digested with HindIII, XbaI, and HindIII+XbaI, respectively. The sizes of the bands (kb) are shown to the left of (B). Results show that the 3.6-kb direct repeat mediates homologous recombination. The much stronger hybridization intensity of fragments diagnostic of the 45-kb ([B], blue) and 84-kb ([B], red) chromosomes relative to those of the cointegrated 129-kb chromosome ([B], green) shows that the split conformation predominates (C). Sixty-two (90%) of the 69 informative shotgun clones sequenced for this repeat support the split (45 kb + 84 kb) conformation, whereas just seven of the clones (10%) support the integrated conformation (D). A total of 36 of the 62 clones (58%) supporting the split conformation derived from the 84-kb chromosome (D).

Figure 6.

Variation in the Recombinational Equilibria of Repeats on the Large and Small Mitochondrial Chromosomes in Cucumber. Relative proportion of shotgun clones supporting recombinant genome conformations for 10 repeats with compelling evidence of recombinational activity, based on the support of at least three shotgun clones. In this graph, a repeat with 60 clones supporting the configuration of the reference assembly and 40 clones supporting the recombinant conformation would have a value of 0.4. All repeats shown are on the main chromosome (Figure 1) except for the labeled 3.6-kb repeat on the small chromosomes (Figure 5). As analyzed, direct repeats with points above the dashed line exist predominantly in the split conformation, whereas points below the line favor the integrated conformation. [See online article for color version of this figure.]

Figure 7.

A Recombination-Based Model to Account for the Apparent Recombinational Equimolarity of Large Direct Repeats on the Main Mitochondrial Chromosome in Cucumber. The model may account for the apparent recombinational equimolarity of the largest repeats in the main chromosome versus the nonequimolarity of the large 3.6-kb repeat in the small chromosomes (Figures 5 and 6). Repeats in the master circle are represented by gray and white boxes, with letters identifying their unique flanking sequences. Recombinational equimolarity is the state in which active recombination leads to near-equal concentrations of the four possible configurations for a given direct repeat.