Laboratory of Plant Breeding and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan E-mail: email@example.com
An ms-bo type cytoplasmic male sterile (CMS) line and its fertility restorer line are widely used for F1 hybrid seed production of japonica rice cultivars. We have carried out map-based cloning of the Rf1 gene and found that a 4.7-kb genomic fragment of a restorer line restored the fertility when introduced into a CMS line. The genomic fragment contained a single ORF encoding 791 amino acids protein, and the corresponding cDNA was isolated. The Rf1 gene encodes a protein with a mitochondrial targeting presequence of 26 amino acids and 18 repeats of the 35-amino-acid pentatricopeptide repeat (PPR) motif. A nonrestoring genotype (rf1) was identified to have deletions within the coding region. Introduction of the Rf1 gene is shown to promote the processing of an aberrant B-atp6 RNA in transgenic CMS lines with the Rf1 gene.
A fertility restorer gene, Rf1, which is important for commercial F1 hybrid seed production of rice has been cloned and characterised.
cytoplasmic male sterility; restorer fertility gene; pentatricopeptide repeat (PPR); Oryza sativa L.
Cytoplasmic male sterility (CMS) is a maternally inherited trait that results in the inability to produce fertile pollen. In some cases, pollen fertility is recovered by a nuclear-encoded gene called a fertility restorer gene (Rf). CMS/Rf systems greatly facilitate hybrid seed production (Schnable and Wise 1998).
Recently, Rf genes have been cloned in Petunia (Bentolila et al. 2002) and radish (Brown et al. 2003; Koizuka et al. 2003) by using a positional cloning strategy. Both of the Rf genes have been shown to encode a protein with mitochondrial transit peptide and pentatricopeptide repeat (PPR) motif. PPR protein contains a characteristic tandem array of a 35-amino-acid motif, and therefore is termed a pentatricopeptide repeat. There are more than 450 members of the PPR family in Arabidopsis thaliana (Aubourg et al. 2000). The majority of these proteins are predicted to be targeted to mitochondria or chloroplasts. The PPR proteins are considered to react with specific RNA in organelles and play a role in RNA processing or translation (Small and Peeters 2000). The identification of Rf genes in Petunia and radish as PPR-containing genes suggests that searching for PPR motif genes near known restorer loci should be a useful strategy to identify Rf genes in other species.
In rice (Oryza sativa L.), one particular CMS system has been obtained by combining the cytoplasm of Chinsurah Boro II (indica rice) with the nuclear genome of Taichung 65 (japonica rice). It is called the ms-bo type or BT type. In this ms-bo type CMS line, pollen abortion is initiated after pollen mitosis. However, pollen fertility can be restored gametophytically by the gene product of a single dominant nuclear gene, Rf1 (Shinjyo 1975). It has been reported that a unique sequence (orf79) located downstream from mitochondrial atp6 causes male sterility. In CMS lines, the abnormal atp6 (B-atp6) is transcribed as a 2.0-kb RNA consisting of normal atp6 and a unique sequence containing orf79, whereas two discontinuous RNA of 1.5 and 0.45-kb are generated from the 2.0-kb RNA by RNA processing in the presence of the Rf1 gene (Kadowaki et al. 1990; Iwabuchi et al. 1993; Akagi et al. 1994).
We followed a positional cloning strategy to identify the Rf1 gene. After mapping of the Rf1 locus within 1 cM, we searched for ORFs containing a mitochondrial targeting presequence and PPR-motif in the genome of Nipponbare, even though the genotype of Nipponbare is rf1/rf1, using a rice genome automated annotation system RiceGAAS and found four such genes. Three genes existed in a tandem array. Each gene was named PPR8-1, PPR8-2 and PPR8-3, respectively. A BAC clone containing the PPR8-1, PPR8-2 and PPR8-3 genes was screened from BAC libraries of cultivar Milyang 23, whose genotype is Rf1/Rf1. A 4.7 -kb Xba I fragment containing the PPR8-1 gene, a 9.5-kb Sac I fragment containing the PPR8-2 gene, and a 8.7-kb Sac I fragment containing the PPR8-3 gene were then subcloned. Each of the genomic fragments was introduced into a CMS line, BTA (rf1/rf1), by Agrobacterium-mediated gene transfer (Kazama and Toriyama 2003).
Here we report the pollen and seed fertility in the complementation test. Restoration of fertility by the PPR8-1 gene demonstrated that the PPR8-1 was the Rf1 gene. Processing of the B-atp6 RNA was examined in the transgenic plants.
We used near isogenic lines that differ at the Rf1 gene locus. These lines have been derived from a backcross of Chinsurah Boro II x Taichung 65 (Shinjyo 1975). They are [normal]rf1/rf1 Taichung 65 as a maintainer line (BTB), B21F1 [ms-bo]rf1/rf1 as a CMS line (BTA), and B12F8 [ms-bo]Rf1/Rf1 as a restorer line (BTR). The genotype of Nipponbare is rf1/r1, and that of Milyang 23 is Rf1/Rf1 (Ichikawa et al. 1997).
Total RNA was isolated from 100 mg of mature anthers of BTA, BTR and F1(BTA X BTR), and transformants using the RNeasy Plant Mini Kit (Qiagen, Hiden, Germany), and 10 micrograms of each RNA was subjected to Northern blot analysis. A part of the B-atp6 gene of BTA was amplified using primer j (Iwabuchi et al. 1993) and primer GSP (5'-AGGGGTGGGATATTTGCCTGGTCCACC –3') and labelled with digoxigenin using the PCR DIG probe Synthesis Kit.
A 4.7-kb Xba I fragment containing the PPR8-1 gene, a 9.5-kb Sac I fragment containing the PPR8-2 gene, and a 8.7-kb Sac I fragment containing the PPR8-3 gene were introduced into a CMS line, BTA (rf1/rf1), by Agrobacterium-mediated gene transfer. The pollen fertility was examined in 35 transgenic plants with the PPR8-1 gene. Twenty plants showed near 50% of fertility, indicating that the transgene was segregated in the pollen, and pollen with the PPR8-1 restored the fertility (Fig. 1). These transgenic plants with the PPR8-1 showed normal seed set (Fig.1), demonstrating that the PPR8-1 gene restored the fertility. The fertility was not recovered by the PPR8-2 or PPR8-3 genes. These results demonstrated that the PPR8-1 was the Rf1 gene.
We examined the transcripts of 0.45-kb that had been reported to be specific for Rf1 genotype and derived from the processing of the B-atp6 RNA by the action of Rf1 (Iwabuchi et al. 1993; Akagi et al. 1994). Transcripts of B-atp6 were investigated in mature anthers in transgenic plants by Northern blot analysis. A portion of the 3' end of B-atp6 containing orf79 was used as a probe. In anthers of the fertile transgenic plants with the PPR8-1 gene, the 0.45-kb band was detected, as previously observed in the transgenic callus (Kazama and Toriyama 2003). In contrast, the band was not detected in the transgenic lines with the 9.5-kb Sac I fragment containing the PPR8-2 gene or in those with the 8.7-kb Sac I fragment containing the PPR8-3 gene. This result indicates that protein encoded by the Rf1 gene promotes the production of the 0.45-kb RNA of B-atp6 in anthers.
We have previously reported the nucleotide sequence of the 4.7-kb Xba I fragment of Milyang 23, which contained the PPR8-1 gene (Kazama and Toriyama 2003). The fragment contained a single ORF consisting of 2376 nucleotides (accession number AB106867). The ORF predicted a protein of 791 amino acids, a mitochondrial targeting presequence of 26 amino acids and 18 repeats of pentatricopeptide. A cDNA corresponding to this ORF was isolated from BTR using RT-PCR (accession number AB110016), and its sequence was identified to be the same as the genomic sequence of Milyang 23. Then we confirmed that this gene was the Rf1 gene. Comparison of the nucleotide sequences with those of Nipponbare (rf1/rf1) revealed that there is a 1-nucleotide deletion and a 574-nucleotide deletion in the middle of the PPR8-1 gene, which cause the creation of a stop codon in the 5th PPR. There are 22 nucleotide substitutions other than the deletions.
Searching for PPR motif genes with a mitochondrial targeting presequence near known restorer loci was proved to be a useful strategy to identify Rf genes. The nucleotide sequence of the Rf1 gene will greatly facilitate the breeding of restorer lines for ms-bo type CMS and F1 hybrids in rice.
Fig. 1 Restoration of pollen and seed fertility by the complementation test.
(A) Sterile pollen of a CMS line (BTA [ms-bo]rf1/rf1). (B) Pollen of the transgenic BTA line with the PPR8-1 gene. A half of pollen grains was stained with I2-KI solution, indicating that the fertility of pollen grains with the PPR8-1 gene was restored. (C) Normal seed set of the transgenic BTA line with the PPR8-1 gene.
This study was supported by a Grant-in-Aid from the Ministry of Education, Science and culture, Japan, and by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project IP-1008).
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