Transgenic rice hybrids that carry the Rf-1 gene at multiple loci show improved fertility at low temperature

By using a genomic fragment that carries the rice (Oryza sativa L.) fertility restorer gene, Rf-1, rice restorer lines harbouring multiple Rf-1 genes on different chromosomes were developed by genetic engineering and crossing. Hybrid lines that were obtained by crossing the restorer lines having two and three Rf-1 genes with a cytoplasmic male sterile (CMS) line had nearly 75 and 87.5% pollen fertility rates under a normal condition, respectively, whereas a conventional hybrid line showed a 50% pollen fertility rate. Furthermore, the seed set percentage under low temperature conditions was much higher in the hybrid lines with multiple Rf-1 genes than the conventional hybrid line. These results indicate that multiplication of the Rf-1 gene conferred cold tolerance at the booting stage to hybrid rice through increasing the potentially fertile pollen grains. This strategy to improve fertility at low temperature of hybrids could be applied to any grain crops that are developed based on CMS and its gametophytic restorer gene, let alone rice.     

Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryza sativa L.)

A rice nuclear gene, Rf-1, restores the pollen fertility disturbed by the BT-type male sterile cytoplasm, and is widely used for commercial seed production of japonica hybrid varieties. Genomic fragments carrying Rf-1 were identified by conducting chromosome walking and a series of complementation tests. Isolation and analysis of cDNA clones corresponding to the fragments demonstrated that Rf-1 encodes a mitochondrially targeted protein containing 16 repeats of the 35-aa pentatricopeptide repeat (PPR) motif. Sequence analysis revealed that the recessive allele, rf-1, lacks one nucleotide in the putative coding region, presumably resulting in encoding a truncated protein because of a frame shift. Rice Rf-1 is the first restorer gene isolated from cereal crops that has the property of reducing the expression of the cytoplasmic male sterility (CMS)-associated mitochondrial gene like many other restorer genes. The present findings may facilitate not only elucidating the mechanisms of male sterility by the BT cytoplasm and its restoration by Rf-1 but also isolating other restorer genes from cereal crops, especially rice.     

Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.     

甘蓝型油菜pol CMS育性恢复基因对orf224/atp6的转录调控

用 10个线粒体基因探针对波里马细胞质雄性不育 (polimaCMS)三系 1141A(pol) ,1141B(nap)和 1141R(pol)的花蕾线粒体RNA进行了Northern检测。结果表明 ,只有 3个探针atp6、orf2 2 4和orf2 2 2检测到转录本的差异。atp6在可育的 1141B中只转录产生一个丰度很高的 1 1kb转录本 ,在雄性不育的 1141A和pol胞质恢复系 1141R中 ,这个转录本的丰度明显减少并出现了分子量较大的 2个转录本 2 2kb、1 9kb转录本。与 1141A相比 ,恢复系1141R的 2 2kb和 1 9kb转录本丰度明显减少 ,并伴随着两个新的转录本 1 4kb和 1 3kb。表明orf2 2 4 atp6的表达与polCMS有关 ,并且其转录受到恢复基因Rfp的调控。同时通过对杂种F1 ( 1141A× 1141R)与另一个恢复系RS35 (pol)的比较证实 ,Rfp对orf2 2 4 atp6的调控与Rfp纯合与否无关。orf2 2 4 atp6在 1141A的苗期叶片中还转录产生育性恢复特异的 1 4kb转录本 ,这可能与细胞核基因型和相对低温条件有关。     

红莲型细胞质雄性不育水稻线粒体atp6基因转录本的编辑位点研究

以红莲（HL）型水稻细胞质雄性不育系A、保持系B及杂种一代F₁为材料,首次比较研究了红莲型水稻线粒体atp6基因转录本的编辑位点及各位点的编辑频率.结果表明atp6基因的转录本有18个编辑位点,其中有15个发生在密码子的第一和第二位点上,这些位点的编辑最终会导致氨基酸种类的变化.18个编辑位点在A、B和F₁中没有差异,但各位点的编辑频率在引入了核恢复基因的条件下发生了较大的变化,完全编辑的比例增加.这些结果首次证明HL型细胞质雄性不育与线粒体atp6转录本的编辑有一定相关性,编辑不充分的转录产物最终会干扰线粒体功能的正常发挥.     

苎麻atp6和atp9基因的克隆表达及与细胞质雄性不育的相关性

摘要: 根据GenBank报道的双子叶植物线粒体atp6和atp9基因编码区保守序列设计简并引物, 通过PCR技术从苎麻细胞质雄性不育系、保持系和恢复系(简称“三系”) mtDNA中扩增目的基因片段, 发现所得序列开放阅读框虽不完整, 但与GenBank报道的其他植物线粒体atp6和atp9基因同源性分别高于94%和85%。采用DNA Walking步移法分别从3′端和5′端扩增两个基因片段的未知侧翼序列, 分离出完整的苎麻线粒体atp6和atp9基因, 包含了完整的开放阅读框。其中“三系”的atp6基因在mtDNA水平、转录和翻译调控水平、蛋白质水平上均无差异。不育系atp9基因在编码区3′端与保持系和恢复系相比存在若干个碱基的差异和缺失; RT-PCR分析还表明, 不育系atp9基因在现蕾期和盛花期的表达量很高。推测不育系atp9基因的结构变异和/或异常表达与苎麻细胞质雄性不育(CMS)的关系密切。     

Genic markers for wild abortive (WA) cytoplasm based male sterility and its fertility restoration in rice

Commercial exploitation of heterosis is essential for enhancing productivity of rice. The use of cytoplasmic male sterility (CMS) and fertility restoration system greatly facilitates large scale production of hybrid seed. The wild abortive (WA) cytoplasm is most widely used for hybrid seed production in rice. The present study was undertaken to develop molecular markers for both WA cytoplasm based male sterility and its fertility restoration for use in efficient hybrid breeding. High degree of genetic differentiation of WA-cytoplasm from its normal fertile counterpart was observed due to DNA rearrangements involving five (coxI, coxIII, cob, atp6 and rps3) mitochondrial genes. Cleaved amplified polymorphic sequence (CAPS) markers based on five mitochondrial genes namely, coxIII, cob, atp9, rps3 and 18SrRNA polymorphic between CMS and maintainer line were developed. The utility of these informative markers was demonstrated in purity testing of the CMS line Pusa6A being used in commercial hybrid seed production. Fertility restoration was found to be controlled by a major locus in the Basmati restorer line PRR78, which was mapped to a short marker interval of 0.8 cM and a physical interval of 163.6 kb on rice chromosome 10. A total of 13 pentatricopeptide repeat (PPR) motif containing genes were predicted in a 1.66 Mb region on the long-arm of this chromosome of which, four were present in the marker interval containing the fertility restorer gene. High degree of conservation of gene order was observed between japonica and indica for the predicted PPR genes. A sequence tagged site (STS) and a genic non-coding microsatellite (GNMS) marker were designed based on one of the candidate PPR motif containing genes present in the marker interval, which were validated using F₂ population and other known restorer lines. The candidate gene based marker identified in the present study would be useful in marker assisted selection (MAS) for fertility restorer gene in hybrid breeding programme based on WA-CMS of rice.     

Mapping of the Rf-3 nuclear fertility-restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers

The cytoplasmic male sterility (CMS) of wild-abortive (WA) cytoplasm has been widely used for breeding hybrid rice. Two restorer genes for the CMS have been found by traditional genetic analysis. To tag the restorer genes we used a set of near-isogenic lines (NILs) of Zhenshan 97 carrying different genotypes for fertility restoration from IR24, to perform RAPD analysis. From the survey of 720 random primers, six RAPD markers were identified to be associated with Rf-3. Three of these OPK05-800, OPU10-1100 and OPW01-350, were mapped on chromosome 1. Two populations from the crosses between Zhenshan 97 A and a near-isogenic restorer line ZSR21 and between Zhenshan 97 A and IR24 were used for mapping Rf-3. The three RAPD markers and three RFLP markers, RG532, RG140 and RG458, were found to be closely linked to Rf-3 in the two populations. The same location of Rf-3 was also found in a population from the cross of IR58025 A//IR36/IR58025 B. At the RG532 locus, different alleles were found between two CMS lines, Zhenshan 97 A and IR58025 A, and between two restorer lines, IR24 and IR36. The use of these molecular markers closely linked to Rf-3 in facilitating the development of hybrid rice is discussed. Received: 3 January 1996 / Accepted: 17 May 1996     

The petunia restorer of fertility protein is part of a large mitochondrial complex that interacts with transcripts of the CMS-associated locus

A class of nuclear genes termed "restorers of fertility" (Rf) acts to suppress the expression of abnormal mitochondrial genes associated with cytoplasmic male sterility (CMS). In petunia, both the nuclear Rf gene and mitochondrial CMS-associated gene have previously been identified. The CMS-associated gene is an aberrant chimera in which portions of several mitochondrially encoded genes are fused to an unknown reading frame. The dominant Rf allele reduces the CMS-associated protein to nearly undetectable levels and alters the RNA population derived from the CMS locus, but its mechanism of action has not been determined. The petuniaRf gene is a member of the pentatricopeptide repeat gene family (PPR), an unusually large gene family in Arabidopsis (approximately 450 genes) compared with yeast (five genes) and mammalian genomes (six genes). The PPR gene family has been implicated in the control of organelle gene expression. To gain insight into the mode of action of PPR genes, we generated transgenic petunia plants expressing a functional tagged version of Rf. Analysis of the restorer protein revealed that it is part of a soluble mitochondrial inner-membrane-associated, RNase-sensitive high-molecular-weight protein complex. The complex is associated with mRNA derived from the CMS locus.     

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

A chimeric <Emphasis Type="Italic">Rfo</Emphasis> gene generated by intergenic recombination cosegregates with the fertility restorer phenotype for cytoplasmic male sterility in radish

In this work, we have identified a chimeric pentatricopeptide repeat (PPR)-encoding gene cosegregating with the fertility restorer phenotype for cytoplasmic male sterility (CMS) in radish. We have constructed a CMS-Rf system consisting of sterile line ‘9802A2’, maintainer line ‘9802B2’ and restorer line ‘2007H’. F₂ segregating population analysis indicated that male fertility is restored by a single dominant gene in the CMS-Rf system described above. A PPR gene named Rfoc was found in the restorer line ‘2007H’. It cosegregated with the fertility restorer in the F₂ segregating population which is composed of 613 fertile plants and 187 sterile plants. The Rfoc gene encodes a predicted protein 687 amino acids in length, comprising 16 PPR domains and with a putative mitochondrial targeting signal. Sequence alignment showed that recombination between the 5′ region of Rfob (EU163282) and the 3′ region of PPR24 (AY285675) resulted in Rfoc, indicating a recent unequal crossing-over event between Rfo and PPR24 loci at a distance of 5.5 kb. The sterile line ‘9802A2’ contains the rfob gene. In the F₂ population, Rfoc and rfob were observed to fit a segregation ratio 1:2:1 showing that Rfoc was allelic to Rfo. Previously we have reported that a fertile line ‘2006H’, which carries the recessive rfob gene, is able to restore the male fertility of CMS line ‘9802A1’ (Wang et al. in Theor Appl Genet 117:313–320, 2008). However, here when conducting a cross between the fertile line ‘2006H’ and CMS line ‘9802A2, the resulting plants were male sterile, which shows that sterile line ‘9802A2’ possesses a different nuclear background compared to ‘9802A1’. Based on these results, the genetic model of fertility restoration for radish CMS is also discussed.     

Structural diversity and evolution of the Rf-1 locus in the genus Oryza

The Rf-1 locus in rice is agriculturally important as it restores fertility in plants with BT-type cytoplasmic male sterility (CMS). The Rf-1 locus contains several duplicated copies of the gene responsible for restoration of fertility. We analyzed the genomic structure of the Rf-1 locus in the genus Oryza to clarify the structural diversity and evolution of the locus. We identified six genes (Rf-1A to Rf-1F) with homology to Rf-1 at this locus in Oryza species with an AA genome. The Rf-1 locus structures in the rice accessions examined were very complex and fell into at least six classification types. The nucleotide sequences of the duplicated genes and their flanking regions were highly conserved suggesting that the complex Rf-1 locus structures were produced by homologous recombination between the duplicated genes. The fact that complex Rf-1 locus structures were common to Oryza species that have evolved independently indicates that a duplication of the ancestral Rf-1 gene occurred early in rice evolution and that homologous recombination resulted in the diversification of Rf-1 locus structures. Additionally, the amino acid sequences of each duplicated gene were conserved between species. This suggests that the duplicated genes in the Rf-1 locus may have divergent functions and may act by controlling mitochondrial gene expression in rice as occurs in the restoration of CMS.