Genetic characterization of a pentatricopeptide repeat protein gene,orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish

Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.     

Identification and expression of the kosena radish (Raphanus sativus cv. Kosena) homologue of the ogura radish CMS-associated gene,orf138

A CMS-associated gene, orf125, present in the Japanese radish cultivar Kosena, has a sequence homologous to that of the ogura CMS-associated gene, orf138, except for two amino acid substitutions and a 39 bp deletion in the orf138 coding region. In Kosena radish, orf125 is linked with orfB, whereas the orf125 locus differs in a Brassica napus CMS cybrid derived from protoplast fusion between Kosena radish and B. napus. A novel mtDNA sequence is present in the 3-flanking region of orf125 in the B. napus kosena CMS cybrid. The orf125 is expressed both in the radish and the B. napus kosena CMS cybrid. Its accumulation is strongly associated with the CMS phenotype in B. napus. Fertility restoration was accompanied by a decrease in the amount of ORF125 in B. napus.     

A new fertility restorer locus linked closely to the Rfo locus for cytoplasmic male sterility in radish

In this study, we have investigated a new fertility restorer (Rf) locus for cytoplasmic male sterility (CMS) in radish. We have obtained a CMS-Rf system consisting of sterile line '9802A1', maintainer line '9802B1' and restorer line '9802H'. F(1) plants from cross between sterile line '9802A1' and restorer line '9802H' were all male fertile, self pollination of F(1) plants produced an F(2) segregating population consisting of 600 individuals. The segregating population was found to fit a segregation ratio 3:1 for male fertile and sterile types, indicating that male fertility is restored by a single dominant gene (termed Rfo2) in the CMS-Rf system. Based on the DNA sequence of Rfo/Rfk1 (AJ535623), just one full length gene in the sterile line '9802A1', in the restorer line '9802H' and in the male fertile line '2006H', was cloned, respectively. The three sequences correspond to the same gene with two alleles: Rfob in '9802H' and rfob in '9802A1' and '2006H'. These two alleles differ from Rfo/Rfk1 and rfk1 (AJ535624) alleles by two synonymous base substitutions, respectively. Based on the differences between the Rfob and rfob genes, one PCR-based marker was developed, and designated Marker 1, which is identical to the corresponding region of Rfob by sequence analysis. In the F(2) segregating population described above, the Marker 1 was present in 5 sterile plants and in 453 fertile plants, absent in 4 fertile plants and in 138 sterile plants, and was found to fit a segregation ratio 3:1 indicating that Rfob was single copy in '9802H'. Linkage analysis showed that the Rfo2 locus for our CMS-Rf system was distant from the Rfo locus by about 1.6 cM. The sterile line '9802A1' was pollinated by the male fertile line '2006H' and the resulting F(1) plants were all male fertile. These results indicated that the male fertility of radish CMS can be restored by a new Rf locus, which linked tightly to the Rfo locus.     

Identification of promoter exchange at a male fertility restorer locus for cytoplasmic male sterility in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

The cytoplasmic male sterility (CMS)/nucleus-controlled fertility restoration (Rf) system is a major component that exploits hybrid vigor in crops. Here, we have identified a new pentatricopeptide repeat (PPR)-encoding allele at the RsRf3 locus linked to the fertility restoration to CMS in radish. This allele was named as RsRf3-5 and encodes a putative protein with 18 PPRs and a predicted mitochondrial targeting signal. Another allele RsRf3-6 encoding the same protein with RsRf3-5 possesses a different promoter region and has failed to restore fertility to CMS. The non-restoring allele RsRf3-7 encoding a predicted protein with 15 PPRs has an identical promoter region with RsRf3-6, suggesting that RsRf3-6 could result from a recent crossover between RsRf3-5 and RsRf3-7. Interestingly, the RsRf3-5 allele shows higher RNA expression levels in the CMS cytoplasmic background compared with the RsRf3-6 allele.     

Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice

Cytoplasmic male sterility (CMS) is an important agricultural trait characterized by lack of functional pollen, and caused by ectopic and defective mitochondrial gene expression. The pollen function in CMS plants is restored by the presence of nuclear‐encoded restorer of fertility (Rf) genes. Previously, we cloned Rf2, which restores the fertility of Lead Rice (LD)‐type CMS rice. However, neither the function of Rf2 nor the identity of the mitochondrial gene causing CMS has been determined in LD–CMS rice. Here, we show that the mitochondrial gene orf79 acts as a CMS‐associated gene in LD–CMS rice, similar to its role in BT–CMS rice originating from Chinsurah Boro II, and Rf2 weakly restores fertility in BT–CMS rice. We also show that RF2 promotes degradation of atp6–orf79 RNA in a different manner from that of RF1, which is the Rf gene product in BT–CMS rice. The amount of ORF79 protein in LD–CMS rice was one‐twentieth of the amount in BT–CMS rice. The difference in ORF79 protein levels probably accounts for the mild and severe pollen defects in LD–CMS and BT–CMS rice, respectively. In the presence of Rf2, accumulation of ORF79 was reduced to almost zero and 25% in LD–CMS and BT–CMS rice, respectively, which probably accounts for the complete and weak fertility restoration abilities of Rf2 in LD–CMS and BT–CMS rice, respectively. These observations indicate that the amount of ORF79 influences the pollen fertility in two strains of rice in which CMS is induced by orf79.     

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.     

Marker-assisted identification of restorer gene(s) in iso-cytoplasmic restorer lines of WA cytoplasm in rice and assessment of their fertility restoration potential across environments

Iso-cytoplasmic restorers possess the same male sterile cytoplasm as the cytoplasmic male sterile (CMS) lines, thereby minimizing the potential cyto-nuclear conflict in the hybrids. Restoration of fertility of the wild abortive CMS is governed by two major genes namely, Rf3 and Rf4. Therefore, assessing the allelic status of these restorer genes in the iso-cytoplasmic restorers using molecular markers will not only help in estimating the efficiency of these genes either alone or in combination, in fertility restoration in the hybrids in different environments, but will also be useful in determining the efficacy of these markers. In the present study, the efficiency of molecular markers in identifying genotypes carrying restorer allele of the gene(s) Rf3 and Rf4, restoring male fertility of WA cytoplasm in rice was assessed in a set of 100 iso-cytoplasmic rice restorers using gene linked as well as candidate gene based markers. In order to validate the efficacy of markers in identifying the restorers, a sub-set of selected 25 iso-cytoplasmic rice restorers were crossed with four different cytoplasmic male sterile lines namely, IR 79156A, IR 58025A, Pusa 6A and RTN 12A, and the pollen and spikelet fertility of the F₁s were evaluated at three different locations. Marker analysis showed that Rf4 was the predominant fertility restorer gene in the iso-cytoplasmic restorers and Rf3 had a synergistic effect on fertility restoration. The efficiency of gene based markers, DRCG-RF4-14 and DRRM-RF3-10 for Rf4 (87%) and Rf3 (84%) genes was higher than respective gene-linked SSR markers RM6100 (80%) and RM3873 (82%). It is concluded that the gene based markers can be effectively used in identifying fertility restorer lines obviating the need for making crosses and evaluating the F₁s. Though gene based markers are more efficient, there is a need to identify functional polymorphisms which can provide 100% efficiency. Three iso-cytoplasmic restorers namely, PRR 300, PRR 363 and PRR 396 possessing both Rf4 and Rf3 genes and good fertility restoration have been identified which could be used further in hybrid rice breeding.     

RFLP mapping of the maize gametophytic restorer-of-fertility locus (rf3) and aberrant pollen transmission of the nonrestoring rf3 allele

 Cytoplasmic male sterility (CMS) is the maternally inherited inability to produce functional pollen. The Rf3 allele of the nuclear gene rf3 gametophytically restores male fertility to maize plants with the S-type of CMS. The rf3 locus is on the long arm of maize chromosome two (2L). Using 2L RFLPs and three-point mapping analysis we showed that the rf3 locus is located an estimated 4.3 cM distal to the whp locus and 6.4 cM proximal to the bnl17.14 locus. This information was used in combination with RFLPs on two additional maize chromosomes to show that Rf3/rf3 CMS-S plants may aberrantly transmit the nonrestoring allele, rf3, through the male gametophyte. Received: 30 September 1996/Accepted: 21 March 1997     

Molecular mapping and candidate gene identification of the Rf2 gene for pollen fertility restoration in sorghum [Sorghum bicolor (L.) Moench]

The A1 cytoplasmic–nuclear male sterility system in sorghum is used almost exclusively for the production of commercial hybrid seed and thus, the dominant genes that restore male fertility in F₁ hybrids are of critical importance to commercial seed production. The genetics of fertility restoration in sorghum can appear complex, being controlled by at least two major genes with additional modifiers and additional gene–environment interaction. To elucidate the molecular processes controlling fertility restoration and to develop a marker screening system for this important trait, two sorghum recombinant inbred line populations were created by crossing a restorer and a non-restoring inbred line, with fertility phenotypes evaluated in hybrid combination with three unique cytoplasmic male sterile lines. In both populations, a single major gene segregated for restoration which was localized to chromosome SBI-02 at approximately 0.5 cM from microsatellite marker, Xtxp304. In the two populations we observed that approximately 85 and 87% of the phenotypic variation in seed set was associated with the major Rf gene on SBI-02. Some evidence for modifier genes was also observed since a continuum of partial restored fertility was exhibited by lines in both RIL populations. With the prior report (Klein et al. in Theor Appl Genet 111:994–1012, 2005) of the cloning of the major fertility restoration gene Rf1 in sorghum, the major fertility restorer locus identified in this study was designated Rf2. A fine-mapping population was used to resolve the Rf2 locus to a 236,219-bp region of chromosome SBI-02, which spanned ~31 predicted open reading frames including a pentatricopeptide repeat (PPR) gene family member. The PPR gene displayed high homology with rice Rf1. Progress towards the development of a marker-assisted screen for fertility restoration is discussed.     

Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.     

小麦T型细胞质雄性不育系与可育系叶片蛋白质双向电泳分析

利用育性恢复基因(Rf3)的近等基因系1031-1、S-165和1031-1与S-165之间的正交与反交,创建了四个实验品系(1031-1、S-165、不育品系、反交品系);采用改进的蛋白质聚丙烯酰胺凝胶双向电泳技术,从发育遗传学的角度,对小麦T型细胞质雄性不育和可育株旗叶表达的相关蛋白产物进行差异分析。通过对旗叶蛋白的双向电泳分析,发现4个品系有相近的蛋白质双向电泳图谱。没有相应穗中差异蛋白质的出现。从蛋白质水平上证实了不育基因与恢复基因表达具有器官特异性特征。     

Comparative analysis of fertility restoration genes for WA,Y, and DA cytoplasmic male sterility in rice

Rice chromosome single segment substitution line (SSSL) W23-19-06-06-11 with the genotype Rf3Rf3/Rf4Rf4, a strong restorer line for wild-abortive (WA) cytoplasmic male sterility (CMS), was recently identified from the SSSL library. To investigate the genetic mode of Rf genes and the genetic relationship among WA, yegong (Y), and dwarf-wildabortive (DA) CMS systems, the plants derived from three BC3F2 populations involving W23-19-06-06-11 and the three CMS lines, that carried the Rf3Rf3/Rf4Rf4, Rf3Rf3/rf4rf4, and rf3rf3/Rf4Rf4 genotypes and WA-, Y-, and DA-CMS cytoplasm, were selected and their pollen and spikelet fertility were evaluated. The results show that the genetic effect displayed a trend of Y-CMS > WA-CMS > DA-CMS in the genetic background of W23-19-06-06-11, the effect of Rf4 appeared to be slightly larger than that of Rf3, and their effects were additive for the three CMS systems. Two pairs of dominant genes governed the fertility restoration in pollen and spikelet in the W23-19-06-06-11 which indicates that the genetic mode of the Rf genes was a qualitative character for the three CMS systems.     

Introgression and molecular tagging of <Emphasis Type="Italic">Rf</Emphasis><Subscript>4</Subscript>, a new male fertility restoration gene from wild sunflower <Emphasis Type="Italic">Helianthus maximiliani</Emphasis> L.

Cytoplasmic male sterility (CMS) and its fertility restoration (Rf) genes are critical tools for hybrid seed production to utilize heterosis. In sunflower, CMS PET1 and the associated Rf gene Rf (1) is the only source extensively used in commercial hybrid production. The objective of this research was to develop new sources of CMS and fertility restorers to broaden the genetic diversity of hybrid seed production. We identified a new type of CMS, named as CMS GIG2, from an interspecific cross between Helianthus giganteus accession1934 and H. annuus cv. HA 89. Based on reactions to a set of standard Rf testers, CMS GIG2 is different from all previously reported CMS types, including the CMS GIG1 from another H. giganteus accession. We also identified an Rf gene for CMS GIG2 from wild species H. maximiliani accession 1631. The CMS GIG2 and its restoration gene were introduced into HA 89 background through recurrent backcross and single plant selection techniques. Genetic analysis revealed that the CMS GIG2-Rf system is controlled by a completely dominant gene, named as Rf (4), and the gene additive and dominance effects were estimated as 39.9 and 42.2%, respectively, in the HA 89 background. The gene Rf (4) was mapped onto linkage group 3 with simple sequence repeat (SSR) markers and RFLP-derived STS-marker, and is about 0.9 cM away from the SSR marker ORS1114 based on a segregation population of 933 individuals. The CMS GIG2-Rf (4) system tagged by molecular markers provides an alternative genetic source for hybrid breeding in the sunflower crop.     

Inheritance and molecular mapping of two fertility-restoring loci for Honglian gametophytic cytoplasmic male sterility in rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

The Honglian cytoplasmic male sterility (cms-HL) system, a novel type of gametophytic CMS in indica rice, is being used for the large-scale commercial production of hybrid rice in China. However, the genetic basis of fertility restoration (Rf) in cms-HL remains unknown. Previous studies have shown that fertility restoration is controlled by a single locus located on chromosome 10, close to the loci Rf1 and Rf4, which respond to cms-BT and cms-WA, respectively. To determine if the Rf locus for cms-HL is different from these Rf loci and to establish fine-scale genetic and physical maps for map-based cloning of the Rf gene, high-resolution mapping of the Rf gene was carried out using RAPD and microsatellite markers in three BCF₁ populations. The results of the genetic linkage analysis indicated that two Rf loci respond to cms-HL, and that these are located in different regions of chromosome 10. One of these loci, Rf5 , co-segregates with the SSR marker RM3150, and is flanked by RM1108 and RM5373, which are 0.9 cM and 1.3 cM away, respectively. Another Rf locus, designated as Rf6(t), co-segregates with RM5373, and is flanked by RM6737 and SBD07 at genetic distances of 0.4 cM. The results also demonstrated these loci are distinct from Rf1 and Rf4. A 105-kb BAC clone covering the Rf6(t) locus was obtained from a rice BAC library. The sequence of a 66-kb segment spanning the Rf6(t) locus was determined by a BLASTX search in the genomic sequence database established for the cultivar 93-11.Communicated by R. Hagemann     

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.     

Fine mapping of a fertility restoring gene for a new CMS hybrid rice system

The Fujian Abortion cytoplasmic male sterility (CMS-FA) system, a new type of sporophytic CMS system in indica rice (Oryza sativa L.), was developed using the cytoplasm and the corresponding fertility-restoring gene from a wild rice (O. rufipogon L.), which originated from Fujian Province, China. Previous studies in combination with several years of production practice demonstrated that CMS-FA hybrid rice was superior to CMS-WA hybrid rice, a prevailing hybrid rice worldwide, and that the male fertility restoration was controlled by a pair of dominant alleles. We tentatively designated the fertility restoration gene as Rf(fa). The analysis of the polymorphism between the fertile and sterile pool DNAs from a mapping segregation population (BC1F1) indicated that Rf(fa) was located on rice chromosome 10. We further delimited the Rf(fa) locus to a 121.1-kb region flanked by RM6100 and MM2023, which were approximately 0.26 cM and 0.18 cM away from Rf(fa), respectively, by simple sequence repeat molecular marker linkage genetic analysis. These results would facilitate the map-based cloning of Rf(fa), the elucidation of a novel molecular mechanism underlying cytoplasm–nucleus interaction in the CMS-FA system, and the production application of this hybrid rice.