Comparative analysis of mitochondrial genomes between a wheat K-type cytoplasmic male sterility (CMS) line and its maintainer line

Abstract

Background: Plant mitochondria, semiautonomous organelles that function as manufacturers of cellular ATP, have their own genome that has a slow rate of evolution and rapid rearrangement. Cytoplasmic male sterility (CMS), a common phenotype in higher plants, is closely associated with rearrangements in mitochondrial DNA (mtDNA), and is widely used to produce F1 hybrid seeds in a variety of valuable crop species. Novel chimeric genes deduced from mtDNA rearrangements causing CMS have been identified in several plants, such as rice, sunflower, pepper, and rapeseed, but there are very few reports about mtDNA rearrangements in wheat. In the present work, we describe the mitochondrial genome of a wheat K-type CMS line and compare it with its maintainer line.

Results: The complete mtDNA sequence of a wheat K-type (with cytoplasm of Aegilops kotschyi) CMS line, Ks3, was assembled into a master circle (MC) molecule of 647,559 bp and found to harbor 34 known protein-coding genes, three rRNAs (18 S, 26 S, and 5 S rRNAs), and 16 different tRNAs. Compared to our previously published sequence of a K-type maintainer line, Km3, we detected Ks3-specific mtDNA (> 100 bp, 11.38%) and repeats (> 100 bp, 29 units) as well as genes that are unique to each line: rpl5 was missing in Ks3 and trnH was absent from Km3. We also defined 32 single nucleotide polymorphisms (SNPs) in 13 protein-coding, albeit functionally irrelevant, genes, and predicted 22 unique ORFs in Ks3, representing potential candidates for K-type CMS. All these sequence variations are candidates for involvement in CMS. A comparative analysis of the mtDNA of several angiosperms, including those from Ks3, Km3, rice, maize, Arabidopsis thaliana, and rapeseed, showed that non-coding sequences of higher plants had mostly divergent multiple reorganizations during the mtDNA evolution of higher plants.

Conclusion: The complete mitochondrial genome of the wheat K-type CMS line Ks3 is very different from that of its maintainer line Km3, especially in non-coding sequences. Sequence rearrangement has produced novel chimeric ORFs, which may be candidate genes for CMS. Comparative analysis of several angiosperm mtDNAs indicated that non-coding sequences are the most frequently reorganized during mtDNA evolution in higher plants.

Figure 1

Physical map of the Triticum aestivum cv. Yumai 3 K-type CMS line (Ks3) mitochondrial genome. Circles display (from outside): (1) physical map scaled in kilobase pairs; different colored arcs indicate repeats of more than 20 kb, as in the legend shown in the bottom left corner: red, R1; blue-green, R2; blue, R3; golden yellow, R4; (2) and (3) coding sequences transcribed clockwise and counterclockwise, respectively: different colors represent the different genes, as in the legend shown in the bottom right corner: blue violet, ATP synthases; blue, NADH dehydrogenases; red, cytochrome c biogenesis proteins; purple-red, cytochrome c oxidases; black, cytochrome b oxidase; blue-green, ribosomal proteins; golden yellow, tRNA and rRNA genes; yellow-green, maturase and mttB; (4) GC content variations (in a 1000-bp window and 100-bp increments).

Figure 2

Distribution of unique sequences and sequences showing homology to wheat ctDNA sequences in Ks3 mtDNA. Circles display (from outside): (1) physical map scaled in kilobase pairs; (2) the locations of unique sequences; (3) the locations of chloroplast homologous sequences. Different colors represent different sequence lengths: blue, 100-500 bp; dark green, 500-1000 bp; golden yellow, 1-1.5 kb; blue-green, 1.5-2 kb; purple-red, 2-2.5 kb; and red, 2.5-7 kb.

Figure 3

Distribution of repeats on the physical map of Ks3 mtDNA. Repeats are indicated by blue bars above or below the horizontal lines; repeats marked with bars above and below the line are direct and inverted, respectively. Genes or Ks3-specific ORFs are indicated by red bars above and below the horizontal lines.

Figure 4

The predicted multipartite structures of Ks3 mtDNA. (A) Three pairs of subgenomic molecules produced by recombination of the DR pairs: 65 kb (R2), 33 kb (R3), and 28 kb (R4). (B) Four isomers of the MC molecule produced by recombination of the IR pairs: 99 kb (R1), 33 kb (R3), 33 kb (R3), and 8.8 kb (R5).

Figure 5

Unique ORFs of Ks3 mtDNA. (A) ORFs located in the vicinity of known genes; (B) ORFs overlapping with known genes; (C) partial sequences of ORFs that are homologous to Km3 mtDNA; (D) ORFs that are homologous to two discrete segments of Km3 mtDNA. Red bars indicate the unique sequences of Ks3 mtDNA. Blue bars and blue-green bars indicate the homology of Ks3 mtDNA to Km3 mtDNA. Yellow bars indicated known genes of Ks3 mtDNA. The vertical numbers show the coordinates of the homologous fragments of ORFs in the Km3 mtDNA MC molecule. The numbers in parentheses indicate the coordinates of ORFs in the Ks3 mtDNA MC molecule.

Figure 6

Dot matrix alignment of the Km3 (x-axis, 1-452526) and Ks3 (y-axis, 1-647559) mtDNAs.