The 5′-leader sequence of sugar beet mitochondrial <Emphasis Type="Italic">atp6</Emphasis> encodes a novel polypeptide that is characteristic of Owen cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is a mitochondrially encoded trait, which is characterized by a failure of plants to produce viable pollen. We have investigated the protein profile of mitochondria from sugar beet plants with normal (fertile) or CMS cytoplasm, and observed that a 35-kDa polypeptide is expressed in Owen CMS plants but not in normal plants. The variant 35-kDa polypeptide was found in CMS mitochondria placed in five different nuclear backgrounds. Interestingly, this polypeptide proved to be antigenically related to a 387-codon ORF (preSatp6) that is fused in-frame with the downstream atp6. The presequence extension of the atp6 ORF is commonly found in higher plants, but whether or not it is normally expressed has hitherto remained unclear. Our study is thus the first to demonstrate that the atp6 presequence is actually translated in mitochondria. We also observed that preSATP6 is a mitochondrial membrane protein that assembles into a homogeneous 200-kDa protein complex. In organello translation experiments in the presence of protease inhibitors showed a reduction in the abundance of mature preSATP6 with time, suggesting that the mature preSATP6 may be derived by proteolytic processing of a translation product of the preSatp6/Satp6 ORF.     

High intraspecific diversity of Restorer‐of‐fertility‐like genes in barley

Nuclear restorer of fertility (Rf) genes suppress the effects of mitochondrial genes causing cytoplasmic male sterility (CMS), a condition in which plants fail to produce viable pollen. Rf genes, many of which encode RNA‐binding pentatricopeptide repeat (PPR) proteins, are applied in hybrid breeding to overcome CMS used to block self‐pollination of the seed parent. Here, we characterise the repertoire of restorer‐of‐fertility‐like (RFL) PPR genes in barley (Hordeum vulgare). We found 26 RFL genes in the reference genome (‘Morex’) and an additional 51 putative orthogroups (POGs) in a re‐sequencing data set from 262 barley genotypes and landraces. Whereas the sequences of some POGs are highly conserved across hundreds of barley accessions, the sequences of others are much more variable. High sequence variation strongly correlates with genomic location – the most variable genes are found in a cluster on chromosome 1H. A much higher likelihood of diversifying selection was found for genes within this cluster than for genes present as singlets. This work includes a comprehensive analysis of the patterns of intraspecific variation of RFL genes. The RFL sequences characterised in this study will be useful for the development of new markers for fertility restoration loci.     

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.     

Mapping of a chloroplast RFLP marker associated with the CMS cytoplasm of sugar beet (Beta vulgaris)

The Owen cytoplasm of male-sterile sugar beet is associated with several alterations of mitochondrial DNA and one additional HindIII site of chloroplast DNA. The region of this HindIII site has been cloned and sequenced. The site maps in a small reading frame (orf32) close to the ycf7 (orf31) gene in the petG-psbE region of chloroplast DNA. Possible functional implications of the results are discussed. The chloroplast RFLP marker described could be useful for studies on chloroplast-mitochondrial interactions, CMS of sugar beet, and the origin of the Owen cytoplasm.     

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orf H522 induces male sterility in transgenic tobacco plants

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic analysis of fertility-restorer genes in rice

Wild-abortive (WA), Honglian (HL) and Baro-II (BT) are three important cytoplasmic male sterility (CMS) types in rice. It is essential to investigate genetic mode and allelism of fertility restorer (Rf) genes and the relationship between Rf and CMS. Fertility of the all test-cross F₁ plants shows that the restorer-maintainer relationship is similar for HL-CMS and BT-CMS, while that is variance for WA-CMS and HL-CMS (or BT-CMS), respectively. Genetic analysis of Rf genes indicates that HL-or BT-CMS are controlled by single dominant Rf gene and WA-CMS is controlled by one or two pairs of dominant Rf genes, which reflects the characters of the gametophytic and sporophytic restoration CMS type. It is concluded that there are at least three Rf loci in different accessions with Rf genes for each CMS type.     

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.     

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs

The complete nucleotide sequence (501,020 bp) of the mitochondrial genome from cytoplasmic male-sterile (CMS) sugar beet was determined. This enabled us to compare the sequence with that previously published for the mitochondrial genome of normal, male-fertile sugar beet. The comparison revealed that the two genomes have the same complement of genes of known function. The rRNA and tRNA genes encoded in the CMS mitochondrial genome share 100% sequence identity with their respective counterparts in the normal genome. We found a total of 24 single nucleotide substitutions in 11 protein genes encoded by the CMS mitochondrial genome. However, none of these seems to be responsible for male sterility. In addition, several other ORFs were found to be actively transcribed in sugar beet mitochondria. Among these, Norf246 was observed to be present in the normal mitochondrial genome but absent from the CMS genome. However, it seems unlikely that the loss of Norf246 is causally related to the expression of CMS, because previous studies on mitochondrial translation products failed to detect the product of this ORF. Conversely, the CMS genome contains four transcribed ORFs (Satp6presequence, Scox2-2 , Sorf324 and Sorf119) which are missing from the normal genome. These ORFs, which are potential candidates for CMS genes, were shown to be generated by mitochondrial genome rearrangements.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann     

Genetical and molecular analysis reveals a cooperating relationship between cytoplasmic male sterility- and fertility restoration-related genes in <Emphasis Type="Italic">Oryza</Emphasis> species

Although the characterization of genes associated with cytoplasmic male sterility (CMS) and fertility restoration (Rf) has been well documented, the evolutionary relationship between nuclear Rf and CMS factors in mitochondria in Oryza species is still less understood. Here, 41 accessions from 7 Oryza species with AA genome were employed for analyzing the evolutionary relationships between the CMS factors and Rf candidates on chromosome 10. The phylogenetic tree based on restriction fragment length polymorphism patterns of CMS-associated mitochondrial genes showed that these 41 Oryza accessions fell into 3 distinct groups. Another phylogenetic tree based on PCR profiles of the nuclear Rf candidates on chromosome 10 was also established, and three groups were distinctively grouped. The accessions in each subgroup/group of the two phylogenetic trees are well parallel to each other. Furthermore, the 41 investigated accessions were test-crossed with Honglian (gametophytic type) and Wild-abortive (sporophytic type) CMS, and 5 groups were classified according to their restoring ability. The accessions in the same subgroup of the two phylogenetic trees shared similar fertility restoring pattern. Therefore, we conclude that the CMS-associated mitotypes are compatible to the Rf candidate-related nucleotypes, CMS and Rf have a parallel evolutionary relation in the Oryza species.     

Identification of the predominant nonrestoring allele for Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.): development of molecular markers for the maintainer genotype

Hybrid seed production in sugar beet relies on cytoplasmic male sterility (CMS). As time-consuming and laborious test crosses with a CMS tester are necessary to identify maintainer lines, development of a marker-assisted selection method for the rf gene (the nonrestoring allele of restorer-of-fertility locus) is highly desirable for sugar-beet breeding. To develop such a method, we investigated genetic variation at the Rf1 locus, one of two Rf loci known in sugar beet. After HindIII-digestion, genomic DNAs from beet plants known to have a restoring Rf1 allele yielded a range of hybridization patterns on agarose gels, indicating that Rf1 is a multi-allelic locus. However, the hybridization patterns of 22 of 23 maintainer lines were indistinguishable. The nucleotide sequences of the rf1 coding regions of these 22 maintainer lines were found to be identical, confirming that the lines had the same rf1 allele. Two PCR markers were developed that targeted a downstream intergenic sequence and an intron of Rf1. The electrophoretic patterns of both markers indicated multiple Rf1 alleles, one of which, named the dd(L) type, was associated with the maintainer genotype. To test the validity of marker-assisted selection, 147 sugar beet plants were genotyped using these markers. Additionally, the 147 sugar beet plants were crossed with CMS plants to determine whether they possessed the maintainer genotype. Analysis of 5038 F1 offspring showed that 53 % of the dd(L) plants, but none of the plants with other alleles, had the maintainer genotype. Thus, selection for the dd(L) type considerably enriched the proportion of plants with the maintainer genotype.     

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.     

Effects of fine‐scale genetic structure on male mating success in gynodioecious Beta vulgaris ssp. maritima

Plant mating systems are known to influence population genetic structure because pollen and seed dispersal are often spatially restricted. However, the reciprocal outcomes of population structure on the dynamics of polymorphic mating systems have received little attention. In gynodioecious sea beet (Beta vulgaris ssp. maritima), three sexual types co‐occur: females carrying a cytoplasmic male sterility (CMS) gene, hermaphrodites carrying a non‐CMS cytoplasm and restored hermaphrodites that carry CMS genes and nuclear restorer alleles. This study investigated the effects of fine‐scale genetic structure on male reproductive success of the two hermaphroditic forms. Our study population was strongly structured and characterized by contrasting local sex‐ratios. Pollen flow was constrained over short distances and depended on local plant density. Interestingly, restored hermaphrodites sired significantly more seedlings than non‐CMS hermaphrodites, despite the previous observation that the former produce pollen of lower quality than the latter. This result was explained by the higher frequency of females in the local vicinity of restored (CMS) hermaphrodites as compared to non‐CMS hermaphrodites. Population structure thus strongly influences individual fitness and may locally counteract the expected effects of selection, suggesting that understanding fine scale population processes is central to predicting the evolution of gender polymorphism in angiosperms.     

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.     

Mitochondrially-targeted expression of a cytoplasmic male sterility-associated <Emphasis Type="Italic">orf220</Emphasis> gene causes male sterility in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Background  

The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility.     

A Cytoplasmic Male Sterility-Associated Rearrangement of the Mitochondrial cobGene Region in Sugar Beet Beta vulgarisL.

Physical maps of the cobmtDNA region were constructed and compared between sugar beet Beta vulgarisL. plants with normal fertility and with cytoplasmic male sterility (CMS). A CMS-associated rearrangement did not affect the coding region of coband combined two mtDNA regions which are normally about 150 kb apart. Two point substitutions were found in the 3"-untranslated region of cob.     

Allelic discrimination of the <Emphasis Type="Italic">Restorer-of-fertility</Emphasis> gene and its inheritance in peppers (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS), one of the most important traits in crop breeding, is used for commercial F₁-hybrid seed production in peppers (Capsicum annuum L.). A nuclear gene, Restorer-of-fertility (Rf), can induce normal pollen production in CMS plants resulting in fertility. Since the first report of fertility restoration in peppers, various inheritance modes have been suggested, including the presence of a third haplotype of the locus. The pepper Rf gene has not been cloned, and calculated genetic distances of linked markers have varied between research groups. A more precise allelic test and additional genetic mapping are needed to accurately select recombinants for use in marker-assisted backcrossing (MAB). Therefore, the reliability and application of these markers for allelic selection of the Rf gene was tested. Two different F₂ populations, Buja and Tamna, were used for the construction of a linkage map. From these linkage groups, a new closely linked flanking marker of the Rf gene were identified. Previous allelic testing revealed the existence of a third haplotype, Rfls ⁷⁷⁰¹ , which can function as dominant (Rf) or recessive (rf). In a previous report, Rfls ⁷⁷⁰¹ was considered to be linked to unstable male sterility (MS). However, our results suggest that unstable MS was induced by a gene residing at another locus rather than by Rfls ⁷⁷⁰¹ haplotype-linked allele.