Ogura cytoplasmic male sterility (CMS) in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene, Orf138, that prevents the production of functional pollen without affecting female fertility. Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the post‐transcriptional level. The Ogura CMS/Rfo two‐component system is a useful model for investigating nuclear–cytoplasmic interactions, as well as the physiological basis of fertility restoration. Using a combination of positional cloning and microsynteny analysis of Arabidopsis thaliana and radish, we genetically and physically delimited the Rfo locus to a 15‐kb DNA segment. Analysis of this segment shows that Rfo is a member of the pentatricopeptide repeat (PPR) family. In Arabidopsis, this family contains more than 450 members of unknown function, although most of them are predicted to be targeted to mitochondria and chloroplasts and are thought to have roles in organellar gene expression.
Cytoplasmic male sterility (CMS) occurs in higher plants and is a maternally inherited trait that prevents the production of functional pollen, but maintains female fertility. Molecular studies have shown that CMS is determined by mitchondrial genes that are cotranscribed with essential genes in bicistronic messenger RNAs (Schnable & Wise, 1998). This is the case for Ogura CMS in radish (Ogura, 1968), which is controlled by the orf138 mitochondrial locus. This locus comprises two cotranscribed open reading frames, Orf138 and OrfB. orf138 is similar to several mitochondrial genes, and is the sterility‐inducing gene (Bonhomme et al., 1992). orfB encodes subunit eight of the ATP‐synthase complex (Gray et al., 1998).
Nuclear genes that restore male fertility in plants showing Ogura CMS occur naturally in wild radish populations. The effect of these restorer genes on the transcription and translation of orf138 has been investigated. It was shown that the restoration of male fertility correlates with a lower accumulation of Orf138 protein, but has no significant effect on the transcription level of orf138 (Bellaoui et al., 1997).
CMS systems are widely used for the production of commercial F1 hybrid plants. However, when the harvested crop is the seed, CMS systems are exploitable only if a nuclear restorer gene is introduced to suppress male‐sterility in the hybrid plants. Ogura CMS, and the corresponding nuclear restorer locus, Rfo, have been introgressed from radish into rapeseed (Pelletier et al., 1983; Heyn, 1976). However, the introgression of the Rfo locus introduced linked deleterious genetic characteristics and led to a loss of rapeseed genetic information (Delourme et al., 1998). Classical breeding methods have been used to improve the lines, but with limited success, due to reduced homologous recombination between radish and rapeseed DNA (Delourme et al., 1998).
In this context, it would be useful to clone the male‐fertility restorer locus, Rfo, and to use it alone to restore fertility. In addition to simplifying the commercial exploitation of the Ogura CMS/Rfo two‐component system, detailed analysis of Orf138 and the corresponding restorer locus, Rfo, will provide insight into the understanding of nuclear–cytoplasmic interactions, as well as into the physiological basis of fertility restoration.
In this study, we describe our strategy to clone the Rfo locus using a positional cloning approach and making use of the microsynteny between Arabidopsis and radish. From this analysis, we concluded that the protein encoded by Rfo is a member of the pentatricopeptide repeat (PPR) family of proteins.
Rfo‐linked markers match Arabidopsis chromosome I
The fertility restorer gene, Rfo, has been mapped previously in radish, and amplified fragment length polymorphism (AFLP) markers linked to Rfo have been identified (Giancola et al., unpublished data). The localization of Rfo‐linked AFLPs spanning a 3‐cM genetic region of the Arabidopsis genome using BLAST analysis has identified more than one putative Arabidopsis syntenic region. This indicates that, near Rfo, the sequence colinearity between radish and Arabidopsis is limited to a small interval. On the basis of this analysis, we hypothesized that AFLP markers that are tightly linked to Rfo in radish should match a single genomic region in Arabidopsis. Thus, we combined our AFLP analysis with bulked segregant analysis. The DNA that constitutes the bulk was derived from the progeny of a cross between a male‐sterile European radish line, 7ms, and a radish line that is homozygous for Rfo. In this analysis, 800 Pst I–Mse I primer combinations were analysed for polymorphisms, and 3 new AFLP markers that are tightly linked to Rfo (R3, R15 and R5) were identified. R3 and R15 cosegregate with Rfo in a population of more than 900 segregant plants, and R5 maps at 0.1 cM from Rfo (Fig. 1). The markers R3, R5 and R15 that were identified in this AFLP screen, as well as the markers pVR3, R22 and pVR1, which were identified in a previous screen, were cloned, sequenced and mapped in Arabidopsis using BLAST analysis. The markers R3, R15 and pVR3 map within a 40‐kb genomic region on Arabidopsis chromosome I, and R5, R22 and pVR1 match a region 150 kb away from this (Table 1). From these data, we conclude that radish genomic DNA near the Rfo locus is likely to be colinear with Arabidopsis DNA around the region contained in bacterial artificial chromosome (BAC) F24D7 (Table 1).
Arabidopsis chromosome I markers map near Rfo in radish
To further test the sequence colinearity between Arabidopsis chromosome I and the Rfo locus in radish, PCR markers were derived from Arabidopsis chromosome I in the vicinity of BAC F24D7 (Table 2), and were mapped to Rfo in radish. The markers were derived from six Arabidopsis BAC clones that spanned a 1100‐kb region, and were based on AGI‐predicted coding sequences (CDSs). Primers were designed in exons flanking a predicted intron, to increase the chance of identifying polymorphisms between male‐fertile and male‐sterile radishes. In this analysis, primers were designed for 60 CDSs and PCR was carried out on Arabidopsis and on fertile and sterile radishes. All of the primer pairs amplified the CDSs from the corresponding genomic sequence in Arabidopsis. However, only 17 primer pairs efficiently amplified the corresponding CDSs in radish. Out of these 17 amplified CDSs (Table 2), 13 were polymorphic between fertile and sterile radishes, and these were mapped relative to Rfo in a population of 900 F2 plants. The markers M‐T12P18.15, M‐F24D7.4, M‐F24D7.7, M‐F24D7.9, M‐F24D7.13, M‐F24D7.14, M‐F24D7.16, M‐F24D7.17 and M‐F2K11.1 cosegregated genetically with Rfo. On one side of Rfo, the markers M‐F16M19.21 and M‐F2K11.19 were mapped at 1 cM and 0.6 cM from Rfo, respectively. On the other side, M‐F22C12.1 and M‐T12P18.9 were mapped at 0.2 cM and 0.1 cM from Rfo, respectively. From these analyses, we conclude that Rfo is genetically delimited to a region of 0.7 cM between the markers M‐F2K11.19 and M‐T12P18.9.
A high‐resolution genetic map of the radish Rfo locus
To more accurately position the nine markers that cosegregate with Rfo, it was necessary to identify members of the Rfo mapping population that had recombination events close to the Rfo locus. This process involved genotyping DNA samples from 6,907 plants in the mapping populations with the Rfo‐ flanking markers M‐F2K11.19 and M‐T12P18.9. From this screen, 43 plants carrying recombination events in the M‐F2K11.19–M‐T12P18.9 region were identified. These plants were used to map the nine markers relative to each other and to Rfo. In this analysis, Rfo was mapped to a genetic region of 0.042 cM, which is delimited by the markers M‐F24D7.13 and M‐F24D7.9 (Fig. 2; Table 3). In Arabidopsis, this region contains three genes that encode an unknown protein (F24D7.12), a putative protein kinase (F24D7.11) and a putative cytochrome P450 (F24D7.10). Thus, we might predict that Rfo is orthologous to one of these genes. Alternatively, the Rfo locus might be absent from Arabidopsis, and the microsynteny between radish and Arabidopsis in the M‐F24D7.13–M‐F24D7.9 region might not be conserved. The construction of a BAC library from radishes homozygous for the Rfo locus and the identification of radish DNA fragments carrying both of the Rfo flanking markers M‐F24D7.13 and M‐F24D7.9 was the only reliable way to clone Rfo.
A high‐resolution physical map of the radish Rfo locus
To ascertain if there is micro‐colinearity between radish and Arabidopsis within the M‐F2K11.19–M‐T12P18.9 region, a BAC library was constructed from nuclear DNA derived from the line D81, which is homozygous for Rfo. The library consists of 120,000 clones and represents the haploid radish genome at least 23 times over. Using a systematic PCR‐based procedure, the library was screened with markers tightly linked to Rfo (Fig. 2). Positive BAC clones were isolated and aligned in a single predicted contig. The order of the BAC clones in the contig was consistent with the genetic distribution of the markers relative to Rfo. The Rfo locus was physically delimited to a single BAC clone, BAC64, which is positive for both of the Rfo flanking markers, M‐F24D7.13 and M‐F24D7.9 (Fig. 3).
Sequence analysis of BAC64
BAC64 was sequenced using a shotgun sequencing procedure and the sequence was assembled into a single‐sequence contig of 127 kb. The redundancy of the sequence coverage was at least ten times the BAC sequence‐length. Two other tests were carried out to check the quality of the sequence consensus. First, the predicted restriction map was compared to the fingerprint of BAC64, and was found to be consistent. Second, the sequences of the markers linked to Rfo were aligned with the BAC64 sequence, and the genetic order of the markers was shown to match the physical order in the sequence.
In the genetic analysis, Rfo was delimited to a genetic interval of 0.042 cM between the markers F24D7.9 and F24D7.13. These two markers physically delimit Rfo to a 22‐kb region. The M‐F24D7.9– M‐F24D7.13 region encodes three predicted proteins, PPR‐A, PPR‐B and PPR‐C, that belong to the PPR family of proteins (Small & Peeters, 2000) (Fig. 4). We further characterized the three plants, RcE8, RcE14 and RcE12, that carry recombination events in the 22‐kb M‐F24D7.9–M‐F24D7.13 physical region (Table 3). Markers were derived from Ppr‐A, Ppr‐B and Ppr‐C, and were mapped relative to Rfo in the RcE8, RcE14 and RcE12 recombinant plants. The RcE8 plant carries a recombination event between the marker M‐F24D7.13 and Ppr‐A. In this plant, Rfo cosegregates genetically with Ppr‐A, Ppr‐B and Ppr‐C. Thus, this recombination event eliminates the gene encoding UDP‐N‐acetylmuramoylanalyl‐D‐glutamate‐2,6‐diaminoligase (corresponding to the marker M‐F24D7.13) as a candidate for Rfo. The RcE14 plant carries a recombination event between Ppr‐C and M‐F24D7.9. In this plant, Rfo cosegregates genetically with Ppr‐C, Ppr‐B and Ppr‐A. Thus, this recombination event eliminates the gene F24D7.9, which is of unknown function, as a candidate for Rfo. The RcE12 plant carries a recombination event between Ppr‐B and Ppr‐C. In this plant, Rfo cosegregates genetically with Ppr‐B and Ppr‐A. Thus, this recombination event eliminates Ppr‐C as a candidate for Rfo. This statement is reinforced by the sequence analysis of Ppr‐C, which suggests that Ppr‐C is a pseudogene (Fig. 5). In conclusion, Rfo is likely to correspond to Ppr‐A or Ppr‐B or both.
In the Brassicaceae, genome synteny analysis between A. thaliana and other Brassica species has shown that large regions might be colinear, whereas, in some cases, only small sequence islands showed colinearity (reviewed in Schmidt, 2002). One might expect that exploitation of information from the Arabidopsis genome will facilitate the cloning and characterization of economically important genes in crop plants, especially in the Brassicaceae family. In this study, we have used the microsynteny between Arabidopsis and radish, combined with a positional cloning approach, to clone the Rfo locus. It is, to our knowledge, the first study in which exploitation of microcolinearity and map‐based cloning were used together for gene cloning. Moreover, it is the first precise synteny study at the physical level between radish and Arabidopsis genomic regions. The use of the syntenic region from Arabidopsis facilitated the development of closely linked markers for the analysis of the Rfo region, and even if the Rfo locus itself has no counterpart at the equivalent site in Arabidopsis, the comparison accelerated the cloning of Rfo.
The genetic and physical maps around the locus formally delimit Rfo as one of two highly similar PPR proteins, PPR‐A and PPR‐B. PPR‐C, which was excluded genetically, seems to be a pseudogene, as it contains a 17‐bp deletion with respect to the other two genes, that leads to a frameshift and a premature stop codon. PPR‐A and PPR‐C contain a 12‐nucleotide deletion in the third PPR repeat that reduces the similarity of this repeat to the canonical PPR structure, and may prevent these proteins from functioning (Fig. 5). PPR‐B is probably the best candidate for Rfo.
The PPR gene family is a large family in plants, consisting of more than 450 genes in Arabidopsis (Aubourg et al., 2000; Small & Peeters, 2000). The functions of these genes are mainly unknown, although most of them are predicted to be targeted to mitochondria and chloroplasts and may have roles in organellar gene expression (Small & Peeters 2000; Barkan & Goldschmidt‐Clermont, 2000). There is some evidence that members of this family are sequence‐specific RNA‐binding proteins (Lahmy et al., 2000; Mancebo et al., 2001). The few PPR mutants that have been described have defects that are limited to a failure to express specific organellar transcripts (Manthey & McEwen, 1995; Fisk et al., 1999). As most CMS restorer genes seem to function by preventing the expression of mitochondrial CMS‐inducing transcripts or proteins (Budar & Pelletier, 2001), PPR proteins are logical candidates for the products of nuclear restorer genes. An important breakthrough in the understanding of CMS restoration was made last year when the Rf restorer locus in Petunia hybrida was shown to encode a mitochondrial PPR protein (Bentolila et al., 2002), which was the first demonstration of the involvement of a member of this family in fertility restoration. With the subsequent demonstration that the Ogura restorer locus also encodes PPR proteins, it became even more tempting to speculate that many restorer genes, in a wide range of plant species, are also PPR genes. The only exception described so far relates to Texas maize (Zea mays) CMS, in which the nuclear restorer gene Rf2 encodes an aldehyde dehydrogenase (Cui et al., 1996). Rf2 has no effect on the RNA or protein levels of the CMS protein URF13. The authors proposed that RF2 protein compensates for a metabolic defect caused by the CMS protein.
A surprising observation is that both petunia Rf and radish Rfo (whichever of the 2 PPR genes is taken to be Rfo in radish) are most similar to the same group of 20 or so Arabidopsis PPR proteins, out of the more than 450 possible, when compared using BLASTP searches (data not shown). A distance tree (Fig. 6) shows the relationship of the RFO proteins (and, to a lesser extent, petunia RF) to this group of Arabidopsis PPR proteins. However, it is impossible to identify obvious single Arabidopsis orthologues of the CMS restorer proteins, whereas putative orthologues of two other characterized PPR proteins (maize CRP1 and radish p67) are easy to identify by sequence similarity. The Arabidopsis Rfo‐like genes closely resemble each other, and are predominantly arranged in two loose clusters on chromosome I, one of which is close to the zone that is syntenic to the Rfo locus. It is intriguing to speculate about the function of these genes in a highly autogamous species where, to our knowledge, CMS has never been described.
Mapping of Rfo.
The Rfo segregating population was obtained by selfing F1 hybrids that carried Ogura CMS, which were derived from the cross between a European male‐sterile radish line, 7ms, and the radish line D81.8, which is homozygous for Rfo. AFLP analysis was performed on bulked DNA samples of male‐fertile and male‐sterile plants, as described previously (Bendahmane et al., 1997). To identify plants carrying recombination events linked to Rfo in radish, DNA samples were extracted from 6,907 F2 plants and analysed using the Rfo‐flanking markers M‐T12P18.9 and M‐F2K11.19. Map distances are given in centimorgans, and represent the percentage of recombinant plants in the total number of plants analysed.
Construction and screening of a radish bacterial artificial chromosome library.
The BAC library was prepared from nuclei extracted from radish line D81.8, which is homozygous for Rfo, based on the method described in Peterson et al. (2000). The library consists of 120,000 BAC clones. To assess the insert size of the BAC clones, plasmid DNA was isolated from more than 100 randomly chosen clones, digested with Not I and analysed by pulsed‐field gel electrophoresis, as described previously (Kanyuka et al., 1999). The insert‐size distribution of the library is as follows: 13.2% of the colonies have an insert size in the range of 150–200 kb, 51.5% in the range of 100–150 kb, 33.8% in the range of 50–100 kb, and 1.5% have inserts of less than 50 kb. The screening of the BAC library was performed as described previously (Kanyuka et al., 1999).
DNA sequencing and analysis.
A shotgun cloning strategy was used for sequencing BAC64. Sequence contigs were assembled using UNIX versions of the Staden programmes package (Staden et al., 1998). Gene prediction was performed using GENSCAN software (Burge & Karlin, 1997). BLAST analysis was used for the prediction of gene function and for mapping radish AFLP sequences in the Arabidopsis genome. Multiple sequence alignment was carried out using ClustalW software (Thompson et al., 1994). The genomic DNA sequence containing Rfo is deposited in the EMBL nucleotide sequence database under the accession number AJ550021.
This work was supported by Génoplante, the French consortium for plant genomics. The work was carried out in compliance with the current laws governing research and development programmes in France.
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