Whole Mitochondrial Genome Sequencing and Re-Examination of a Cytoplasmic Male Sterility-Associated Gene in Boro-Taichung-Type Cytoplasmic Male Sterile Rice

Abstract

Nuclear genome substitutions between subspecies can lead to cytoplasmic male sterility (CMS) through incompatibility between nuclear and mitochondrial genomes. Boro-Taichung (BT)-type CMS rice was obtained by substituting the nuclear genome of Oryza sativa subsp. indica cultivar Chinsurah Boro II with that of Oryza sativa subsp. japonica cultivar Taichung 65. In BT-type CMS rice, the mitochondrial gene orf79 is associated with male sterility. A complete sequence of the Boro-type mitochondrial genome responsible for BT-type CMS has not been determined to date. Here, we used pyrosequencing to construct the Boro-type mitochondrial genome. The contiguous sequences were assembled into five circular DNA molecules, four of which could be connected into a single circle. The two resulting subgenomic circles were unable to form a reliable master circle, as recombination between them was scarcely detected. We also found an unequal abundance of DNA molecules for the two loci of atp6. These results indicate the presence of multi-partite DNA molecules in the Boro-type mitochondrial genome. Expression patterns were investigated for Boro-type mitochondria-specific orfs, which were not found in the mitochondria from the standard japonica cultivar Nipponbare. Restorer of fertility 1 (RF1)-dependent RNA processing has been observed in orf79-containing RNA but was not detected in other Boro-type mitochondria-specific orfs, supporting the conclusion that orf79 is a unique CMS-associated gene in Boro-type mitochondria.

Fig 1. Boro-type mitochondria sequences are assembled into five circular chromosomes.

(a) Compositions of contigs in five assembled mitochondrial chromosomes (MC). The arrow box indicates a contig and its direction. Arrows represent primers used for confirmation of contig connections. The same contigs are filled with the same colors. (b) Each of the five circular chromosomes was validated by PCR analysis using the primer sets indicated. (c) The inverse order of the contigs in MC-3 was detected by PCR analysis. (d) Integration of MC-5 into MC-2 was validated by PCR analysis.

Fig 2. Graphical genome maps of subgenome-1 and subgenome-2 with all of the known genes and Boro-type mitochondria-specific orfs.

The outer circle indicates the graphical genome maps generated by using OGdraw. Colors in the inner circle correspond to those of the contigs listed in Fig 1.

Fig 3. Recombination between subgenome-1 and subgenome-2 via the 450-bp identical sequence is possible but rare.

(a) Schematic model of recombination between subgenome-1 and subgenome-2. Arrows indicate primers used for detection of recombination. EcoRI sites are indicated as E. (b) Recombinant fragments were detected by PCR. (c) Southern blot analysis detected the recombination molecule between subgenome-1 and subgenome-2; however, the frequency of recombination was lower than that of the non-recombinant subgenome-1 molecule.

Fig 4. Expression patterns of orfs specific to Boro-type mitochondria were not altered in the presence or absence of the restorer of fertility gene, Rf1.

Northern blot analysis was performed using a BT-type CMS rice (BTA), a fertility restorer with Rf1 (BTR), a transgenic BTA complemented with Rf1 (Compl), and a maintainer line (T65).