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

Abstract

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.

Keywords: Fertility restorer gene; ORF687; Ogura cytoplasmic male sterility; Orf138; PPR-code; Radish.

Fig. 1

Presence of orf138 and Rft, and genotyping of Rfo locus. a Detection of orf138 DNA. b PCR–RFLP of Rfo locus to determine the genotype. c Detection of Rft by the STS marker. 1: ‘Uchiki-Gensuke’ (a maintainer of Ogura CMS). 2: ‘MS-G’. 3: ‘MR’. 4: ‘BK’. 5: ‘SK’. C1: A Japanese wild radish with RfoRfo genotype. C2: A Japanese wild radish having Rft

Fig. 2

Transcript patterns of orf138 and pollen fertility in the progenies of ‘MR’ × (‘BK’ × ‘SK’) and ‘MS-G’ × (‘BK’ × ‘SK’). ‘UG’; the maintainer of ‘MS-G’ (‘Uchiki-Gensuke’) used as a negative control

Fig. 3

5′ and 3′ ends mapping of the orf138 mRNA. Three arrow heads indicate the previously reported 5′ end in different radish varieties. The black arrow indicates the 3′ end of orf138-atp8 co-transcript. Position of 5′ or 3′ ends and their frequency are indicated by bars and numbers, respectively. Red numbers (‘MR’ × (‘BK’ × ‘SK’), Blue numbers: ‘MS-G’ × (‘BK’ × ‘SK’). Black numbers: European wild radish (Giancola et al. 2007). The length of the bars reflects the frequency of each end

Fig. 4

Putative binding sites of ORF687 in the orf138 mRNA were predicted based on the PPR-code. Matching probability to each putative binding site (P value) is blotted. ORF687 of ‘BK’ (Rfo) restores fertility to the Type A orf138 (gray circles), but not to the Type H (open circle). ORF687 of ‘MR’ (rfo) cannot restore fertility to either Type A (gray square) or Type H (white square)