Intraspecific variations of the cytoplasmic male sterility genes orf108 and orf117 in Brassica maurorum and Moricandia arvensis, and the specificity of the mRNA processing.

The mitochondrial gene orf108, which is co-transcribed with atp1 and causes cytoplasmic male sterility in Brassica crops, is widely distributed across wild species and genera of Brassicaceae. However, to date, intraspecific variations in the presence of orf108 have not yet been studied, and the mechanisms underlying the wide distribution of the gene remain unclear. We analyzed the presence and sequence variations of orf108 in two wild species, Brassica maurorum and Moricandia arvensis. After polymerase chain reaction amplification of the 5' region of atp1 and the coding sequence of orf108, we determined the DNA sequences. Brassica maurorum and M. arvensis showed variations in the presence of orf108 or orf117 (orf108V117) both between and within accessions and were not fixed to the mitochondrial type with the male sterile genes. Sequencing of the amplicons showed that B. maurorum had orf108V117 instead of orf108. Sequencing also indicated mitochondrial heteroplasmy in the two species; in particular, in B. maurorum, one plant possessed both orf108 and orf108V117 sequences. These results suggest that substoichiometric shifting of mitochondrial genomes leads to the acquisition or loss of orf108. Furthermore, fertility restorer genes of the two species were involved in the processing of the mRNA of male sterility genes at different sites.

A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish.

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5' untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.

Appearance of male sterile and black radishes in the progeny of cross between Raphanus raphanistrum and Raphanus sativus.

In addition to Ogura cytoplasmic male sterility (CMS), which is used extensively for F1 hybrid seed production in Brassicaceae crops, two other CMS systems, NWB CMS and DCGMS, have also been identified. The causal gene for the latter two CMS systems has been identified as a novel chimeric gene, orf463. We previously reported that orf463 is specific to black radish cultivars and that it is present in line 'RS-5' of Raphanus raphanistrum; however, the orf463 sequence in 'RS-5' differed from that of black radish cultivars. Though, R. raphanistrum with an orf463 sequence identical to that found in black radish cultivars was recently identified. We therefore sought to determine whether the orf463 gene in line 'RS-5' induces CMS in radishes. We crossed 'RS-5' as a female parent with a cultivated radish, 'Uchiki-Gensuke', as a male parent, and examined the gross plant morphology and pollen fertility of the resulting progeny. The F2 population contained both male sterile plants and plants with black roots. The findings showed that R. raphanistrum contains two types of orf463 genes that induce CMS, and that the origin of black radishes could be attributed to R. raphanistrum having orf463 gene.