Substoichiometrically different mitotypes coexist in mitochondrial genomes of Brassica napus L

Abstract

Cytoplasmic male sterility (CMS) has been identified in numerous plant species. Brassica napus CMS plants, such as Polima (pol), MI, and Shaan 2A, have been identified independently by different researchers with different materials in conventional breeding processes. How this kind of CMS emerges is unclear. Here, we report the mitochondrial genome sequence of the prevalent mitotype in the most widely used pol-CMS line, which has a length of 223,412 bp and encodes 34 proteins, 3 ribosomal RNAs, and 18 tRNAs, including two near identical copies of trnH. Of these 55 genes, 48 were found to be identical to their equivalents in the "nap" cytoplasm. The nap mitotype carries only one copy of trnH, and the sequences of five of the six remaining genes are highly similar to their equivalents in the pol mitotype. Forty-four open reading frames (ORFs) with unknown function were detected, including two unique to the pol mitotype (orf122 and orf132). At least five rearrangement events are required to account for the structural differences between the pol and nap sequences. The CMS-related orf224 neighboring region (∼5 kb) rearranged twice. PCR profiling based on mitotype-specific primer pairs showed that both mitotypes are present in B. napus cultivars. Quantitative PCR showed that the pol cytoplasm consists mainly of the pol mitotype, and the nap mitotype is the main genome of nap cytoplasm. Large variation in the copy number ratio of mitotypes was found, even among cultivars sharing the same cytoplasm. The coexistence of mitochondrial mitotypes and substoichiometric shifting can explain the emergence of CMS in B. napus.

Figure 1. Organization of the pol mitotype in B. napus.

A total of 55 genes with known function were identified, including 34 protein-coding genes (red), 3 ribosomal RNA genes (yellow), and 18 tRNA genes (green). Nine (out of 44) putative ORFs encoding at least 150 amino acids are marked in blue.

Figure 2. Alignment of the nap and pol mitotype genomes.

The numbers refer to the syntenic regions with the pol mitotype sequence as a reference. Comparisons between nap (horizontal axis) and pol (vertical axis) indicated that the nucleotide sequences of the syntenic region are highly conserved, but the syntenic orders and directions were evolutionally rearranged.

Figure 3. Syntenic reordering and structural genomic changes caused by short repetitive sequences.

The numbers refer to the syntenic regions as in Figure 2. The orientation of the sequence is shown by an arrow. (A) A pair of inverted repeats (in red, F) may have induced the re-orientation of region 10. (B) In pol, a pair of inverted repeats (in green, H) is located at the end of linked regions 8 and 7, but in nap, this region is re-orientated and contains a small deletion of a sequence of unknown origin and fewer repeats due to an overlap of H at the terminus of region 6. (C) A 50 bp sequence (in blue, K) is located between syntenic regions 4 and 5 in pol, whereas two copies of K are present at this location in nap, accompanied by a 44 nucleotide insertion of unknown origin.

Figure 4. Structural polymorphism in the region surrounding orf224.

The numbers refer to the syntenic regions as in Figure 2. At least two recombination events are inferred in this region. One of these, located within orf265 in pol, splits this sequence into three segments, and in nap the terminus of orf265 generates orf261 by fusing to a neighboring sequence; the second event occurs between regions 2 and 3 resulting in the formation of orf188, a combination of the terminal 90 bp of region 2 and a new neighboring sequence in the nap mitotype.

Figure 5. PCR amplification of the pol-CMS line and its maintainer.

The pol and nap mitotypes are both present in pol-CMS line NH3A (A) and its maintainer NH3B (B), but are substoichiometrically differentiated in these cytotypes. Primer pairs P1 and P2 targeted the nap mitotype-specific sequences orf117b and orf222, respectively, whereas P3-9 and P11 targeted pol mitotype-specific sequences. P10 amplified two distinct fragments 841 bp (pol-specific) and 226 bp (nap-specific) in size. All PCR reactions used the 10 ng of mtDNA template. All primer pairs were able to amplify target fragments in both lines, but amplification differed in the two lines. The PCR assay indicated that the nap and pol mitotypes coexisted in rapeseed, but the content was substoichiometrically different in the two cytoplasm types. M: DNA ladder.

Figure 6. Alignment of the B. rapa mitochondrial segment with the two B. napus mitotype sequences.

The 124 kb B. rapa mitochondrial segment was plotted on the horizontal axis against the pol subgenome (A) or nap subgenome (B) on the vertical axis.

Figure 7. The tripartite mitochondrial genomic structure of the mitotypes.

The numbers refer to the syntenic regions as in Figure 2. Highly or completely homologous regions are indicated by color. (A) nap; (B) pol. R: 2.4 kb repetitive sequence.