Fertility Restoration Is Associated with Loss of a Portion of the Mitochondrial Genome in Cytoplasmic Male-Sterile Common Bean

Restoration of pollen fertility to cytoplasmic male-sterile common bean by nuclear gene Fr is accompanied by mitochondrial (mt) DNA rearrangements within restored plants. These rearrangements are also observed upon spontaneous cytoplasmic reversion to fertility. An mtDNA fragment of at least 25 kilobases was lost from the genome upon restoration or reversion. This fragment contained DNA segments that were not repeated elsewhere in the genome and, therefore, were not detected within the genome upon fertility restoration. This result suggested that the particular mtDNA configuration absent from restored plants could not be maintained by a constant process of recombination but rather by autonomous replication. No evidence of excision of this region from the mt genome, in the form of a junction fragment associating flanking DNA regions, was detected in fertile restored plants. DNA gel blot hybridization of this mtDNA region, compared with hybridization to related regions of the mitochondrial genome that shared sequence homology, indicated that the mtDNA region associated with sterility was present in lower copy number. These observations, as well as the occurrence of similar or identical rearrangements upon spontaneous cytoplasmic reversion, indicate that the restoration of pollen fertility may be accompanied by loss of an independently replicating subgenomic DNA molecule from the mitochondrial genome.     

Reorganization of mitochondrial genomes of cytoplasmic revertants in cms-S inbred line WF9 in maize

Summary Cytoplasmic reversion to fertility in cms-S maize has been previously correlated with changes in mitochondrial genome organization, specifically with loss of the autonomously replicating linear plasmid-like DNAs, S1 and S2, and with accompanying alterations in the high molecular weight mtDNA (main genome) that specifically involved S1 and S2 sequences. These studies, however, dealt with cytoplasmic revertants occurring in the cms-VG M825 inbred line and in the cms-VG M825/Oh07 F₁ hybrid. This paper deals principally with patterns of mitochondrial DNA reorganization accompanying cytoplasmic reversion to fertility in the WF9 inbred line nuclear background. Here the free S1 and S2 plasmid-like DNAs are retained in the revertants. Mitochondrial DNA analysis by Southern hybridization using cloned fragments of S1 and S2 shows altered organization around S-homologous regions in the main mitochondrial genome of revertants as compared with that of the male-sterile parental controls, but the pattern of main genome changes involving these regions differs from that of the cytoplasmic revertants that occurred in M825 and M825/Oh07 backgrounds. Similar experiments using a clone of the cytochrome oxidase I (COX I) gene of maize as a probe indicate that reorganization in this region is also involved in the changes in mtDNA that accompany cytoplasmic reversion to male fertility in cms-S WF9. The heterogeneity in patterns of reorganization of the main mtDNA genome that accompany cytoplasmic reversion in the same and different nuclear backgrounds are discussed in relation to cytoplasmic male sterility (CMS).     

Mitochondrial DNA variation in pearl millet and related species

Summary Mitochondrial DNA (mtDNA) restriction endonuclease fragment patterns and patterns of mtDNA hybridized by mitochondrial gene probes were used to study phylogenetic relationships of seven Pennisetum species, including five P. americanum (pearl millet) ecotypes and a reference species from the distantly related genus, Panicum. The restriction patterns of the pearl millet ecotypes were uniform with the exception of the ecotype collected in Ethiopia. The probe hybridization method revealed more variability, with both the Rhodesian and Ethiopian ecotypes differing from the others and from each other. Considerable restriction pattern polymorphism was noted among different species of Pennisetum, and Panicum. Significant relationships were noted of Pennisetum polystachyon to P. pedicellatum and of P. purpureum to P. squamulatum using the restriction pattern method. In addition to those relationships, the hybridization method showed relationships of pearl millet to P. purpureum and to P. squamulatum. The relationships noted between species by the hybridization method agreed more closely to the cytological data than those indicated by the restriction pattern method. Therefore, the hybridization method appeared to be the preferred method for studying species relationships. The mitochondrial genome size of pearl millet was calculated to be 407 kb and the mitochondrial genome sizes of other Pennisetum species ranged from 341 to 486 kb.Florida Agricultural Experiment Station Journal Series No. 8485.     

Analysis of mitochondrial DNA from somatic hybrid cell lines of Saccharum officinarum (sugarcane) and Pennisetum americanum (pearl millet)

Mitochondrial DNA (mtDNA) from cell suspension cultures of two intergeneric somatic hybrids of Pennisetum americanum (pearl millet) + Saccharum officinarum (sugarcane) was examined by restriction endonuclease digestion and hybridization with sorghum mtDNA cosmids. The mtDNA of one somatic hybrid was indistinguishable from that of pearl millet, while the second exhibited a combination of parental mtDNAs, suggesting mitochondrial fusion. Several novel, possibly recombinant, mtDNA restriction fragments were detected in this hybrid, which may have resulted from intergenmic recombination.Florida Agriculture Experiment Station Journal Series No: 8090.     

Analysis of recombination rate in female and male gametogenesis in pearl millet (Pennisetum glaucum) using RFLP markers

Sex as a factor affecting recovered recombination in plant gametes was investigated in pearl millet, Pennisetum glaucum, by using reciprocal three-way crosses [(AxB)xCvCx(A x B)]. The two populations were mapped at 42 loci pre-selected to cover the majority of the genome. No differences in recombination distances were observed at the whole-genome level and only a few individual linkage intervals were found to differ, all in favour of increased recombination through the male. Distorted segregations found in the three-way crosses provide evidence of post-gametic selection for particular gene(s) or chromosome regions. The significance of these results for the design of pearl millet breeding programmes and inheritance experiments, as well as for other experimental strategies, is discussed.     

Cytogenetics of pearl millet

Summary The somatic karyotype of pearl millet Pennisetum americanum (L.) Leeke. (2n = 14) has been studied in several cultivars, but few cytological markers have been discovered which could help in the easy identification of the chromosomes. Analysis of pachytene bivalents permits such identification but is feasible only in a few cultivars. Recently, several lines having telocentric chromosomes have been produced and classified but their potentialities as cytogenetic tools have yet to be explored. Some African populations of pearl millet carry B-chromosomes in their karyotype. Cytogenetics of B-chromosomes has been reported in great detail. Bs undergo spontaneous changes to produce deficient- and iso-chromosomes. The main effect of B-chromosomes is on chiasma frequency which is exerted by the relative amounts of chiasma promoting euchromatin and the chiasma depressing heterochromatin in the Bs. Haploid plants occur occasionally and sometimes show a low degree of seed set, offering a possibility of establishing homozygous inbred lines. Cytogenetics of several spontaneous and induced autotetraploids have been reported. In general quadrivalent formation between the seven sets of four homologues was random. Seed set of the autotetraploids could be improved by selection; improved seed fertility was found to be associated with increased chiasma frequency, increased quadrivalent frequency and regular distribution of chromosomes at anaphase I. Genes controlling morphological characters of plant phenotype segregate independent of those controlling fertility and in pearl millet polyploidy per se is not limiting to plant vigour. Primary trisomics represent the best studied among the aneuploids of pearl millet. All the seven primary trisomics have been identified and described. Some were used in assigning genes to specific chromosomes but in general trisomies have poor vigour and fertility, and show low frequency of transmission. Apart from B-chromosomes, cytogenetics of interchanges has been the best studied aspect of pearl millet. The frequency of co-orientation of an interchange complex at metaphase I, which determines the fertility or sterility of the interchange heterozygote, is influenced by the genetic background and thus is theoretically amenable for selection leading to improved fertility of the heterozygote. Interchange tester-stocks have been assembled which can be used to identify the chromosomes involved in any newly obtained interchange. A complex interchange line involving all the chromosomes of the complement has also been produced, but the ring-of-fourteen produces total male and female sterility.Genotypic control of mitosis and meiosis has been reported, with reference to chromosome numerical mosaicism, multiploid sporocytes, desynapsis and chromosome fragmentation, and male sterility. Pearl millet being a largely outbreeding species, forced inbreeding was mainly found to result in loss of morphological vigour and reduction in mean chiasma frequency per PMC. Interspecific hybrids between pearl millet and several related species have been cytologically investigated and homology of the seven chromosomes of pearl millet with seven of the fourteen chromosomes of P. purpureum has been demonstrated. Cytogenetic evidence from haploids, autopolyploids and interspecific hybrids has indications to suggest that the haploid number of x = 7 is derived from x = 5, but the evidence is inconclusive and needs critical evaluation.     

Molecular Evolution of the Ac/Ds Transposable-Element Family in Pearl Millet and Other Grasses

We report an Ac-like sequence from pearl millet (Pennisetum glaucum) and deletion derivative Ac-like sequences from pearl millet and another grass species, Bambusa multiplex. Sequence relationships between the pearl millet and maize Ac elements suggest the Ac/Ds transposable-element family is ancient. Further, the sequence identity between the Bambusa Ac-like sequence and maize Ac implies that the Ac/Ds transposable-element family has been in the grass family since its inception. The Ac-like sequences reported from pearl millet and maize Ac are statistically heterogeneous in pair-wise distance comparisons to each other. Yet, we are unable to discriminate between differential selection or ectopic exchange (recombination and conversion) between nonidentical transposable element homologues, as the cause of the heterogeneity. However, the more extreme heterogeneity exhibited between the previously described pearl millet element and maize Ac seems likely to derive from ectopic exchange between elements with different levels of divergence.     

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.     

A fertile revertant from petaloid cytoplasmic male-sterile carrot has a rearranged mitochondrial genome

 A spontaneously derived fertile plant was recovered from a petaloid cytoplasmic male-sterile (CMS) carrot inbred line. Genetic analysis indicated a single nuclear gene was responsible for the restoration to fertility. Within a family segregating for the nuclear restorer in combination with the sterility-inducing cytoplasm, fertile plants were recovered that could not restore fertility when crossed to sterile genotypes. Genetic analysis indicated cytoplasmic reversion for fertility, and Southern analysis, comparing mtDNA organization of the fertile revertant and its CMS progenitor, identified mitochondrial genome rearrangements. Hybridization of cosmids representing a 108-kb subgenomic circle of the sterile line to DNA of a fertile maintainer and fertile revertant lines indicated a similar mtDNA organization for these genotypes that was distinct from that of the sterile line. Six restriction fragments totalling 43.2 kb were common to the fertile maintainer and revertant and absent in the sterile; other restriction fragments totalling 38.2 kb were present only for the sterile line. Unique fragments of low stoichiometry, two for the fertile maintainer and three for the revertant, distinguished these lines. The reversion to fertility in the sterile line could have resulted from the amplification of a mitochondrial submolar genome highly homologous to that found in the fertile maintainer line. Received: 4 October 1997/Accepted: 12 December 1997     

Deletion of the last two exons of the mitochondrialnad7 gene results in lack of the NAD7 polypeptide in aNicotiana sylvestris CMS mutant

InNicotiana sylvestris, two cytoplasmic male sterile (CMS) mutants obtained by protoplast culture show abnormal developmental features of both vegetative and reproductive organs, and mitochondrial gene reorganization following homologous recombination between 65 bp repeated sequences. A mitochondrial region of 16.2 kb deleted from both CMS mutants was found to contain the last two exons of thenad7 gene coding for a subunit of the mitochondrial respiratory chain complex I, which is encoded in the nucleus in fungi and animals but was recently found to be encoded by the mitochondrial genome in wheat. Although theN. sylvestris nad7 gene shows strong homology with its wheat counterpart, it contains only three introns instead of four. Polymerase chain reaction (PCR) experiments indicated that the parental gene organization, including the completenad7 gene, is probably maintained at a substoichiometric level in the CMS mutants, but this proportion is too low to have a significant physiological role, as confirmed by expression studies showing the lack of detectable amounts of the NAD7 polypeptide. Consequently, absence of NAD7 is not lethal to plant cells but a deficiency of complex I could be involved in the abnormal CMS phenotype.     

Synergistic effects of Trichoshield on enhancement of growth and resistance to downy mildew in pearl millet

Trichoshield, a talc formulation consisting of spores of Trichoderma harzianum, Trichoderma lignorum, Gliocladium virens and Bacillus subtilis was tested, following different application methods, for its ability to promote growth of pearl millet plants and to induce resistance to downy mildew of pearl millet. Under laboratory conditions, trichoshield seed treatment enhanced seed germination and seedling vigor of pearl millet significantly over the control; under greenhouse conditions vegetative growth parameters like height, fresh and dry weight, leaf area and number of tillers were significantly enhanced over the control: Trichoshield formulation offered greater protection against downy mildew in comparison with individual strains of T. harzianum, T. lignorum, G. virensand B. subtilis. Among the methods of application, foliar spray was found to be a more efficient delivery method than seed treatment or slurry treatment. Combinations of foliar spray with seed treatment and slurry treatment produced the same effect as foliar spray alone. Under field conditions, trichoshield treatment enhanced reproductive parameters like number of earheads, length and girth of earheads, 1000 seed weight and yield significantly over the control. Days required for 50% flowering was reduced by 4 days compared to the control. Yield enhancement of 28% over the control was highly significant. Trichoshield treatment offered protection ranging from 52 to 71% under field conditions, depending on the application method. However, the chemical fungicide metalaxyl Apron provided the highest protection against downy mildew, both under greenhouse and field conditions.     

Molecular basis of cytoplasmic male sterility and fertility restoration in rice

Cytoplasmic male sterility (CMS) is a maternally inherited trait that causes dysfunctions in pollen and anther development. CMS is caused by the interaction between nuclear and mitochondrial genomes. A product of a CMS-causing gene encoded by the mitochondrial genome affects mitochondrial function and the regulation of nuclear genes, leading to male sterility. In contrast, the RESTORER OF FERTILITY gene (Rf gene) in the nuclear genome suppresses the expression of the CMS-causing gene and restores male fertility. An alloplasmic CMS line is often bred as a result of nuclear substitution, which causes the removal of functional Rf genes and allows the expression of a CMS-causing gene in mitochondria. The CMS/Rf system is an excellent model for understanding the genetic interactions and cooperative functions of mitochondrial and nuclear genomes in plants, and is also an agronomically important trait for hybrid seed production. In this review article, pollen and anther phenotypes of CMS, CMS-associated mitochondrial genes, Rf genes, and the mechanism that causes pollen abortion and its agronomical application for rice are described.     

An apomictic polyhaploid obtained from a pearl millet x Pennisetum squamulatum apomictic interspecific hybrid

Summary In a research program to transfer apomixis from Pennisetum squamulatum Fresen to pearl millet, P. americanum L. Leeke, a polyhaploid plant (2n=21) was discovered in the uniform open-pollinated progeny of an apomictic interspecific hybrid (2n = 41) between pearl millet and P. squamulatum. The polyhaploid was shorter, less vigorous and was smaller morphologically than its maternal parent. It probably originated by parthenogenetic development of a reduced gametophyte in the apomictic interspecific hybrid. The most common metaphase I chromosome association in the polyhaploid was 4 bivalents plus 13 univalents. Irregular chromosome distribution, tripolar spindles, bridges and fragments were observed at anaphase I and telophase I. The polyhaploid was male-sterile and partially female- fertile having multiple aposporous embryo sacs in 95% of the ovules. Seed set was 3% when open-pollinated and 33% when pollinated with pearl millet pollen. Low seed set was due to competition among multiple embryos developing in the same ovule. Seventeen progeny from seed produced under open-pollination on the polyhaploid each had 2n=21 chromosomes and were morphologically uniform and identical to the female parent. The expression of obligate apomixis in the polyhaploid conditioned by the P. squamulatum genome between the simplex and duplex condition indicates that apomictic reproduction is possible in nonpolyploid plants.     

Mitochondrial Respiration Associated with Cytoplasmic Male Sterility in Pearl Millet

Respiratory activities in vegetative tissues and the isolated mitochondria of etiolated seedlings and reproductive tissues were studied in two cytoplasmic male sterile (CMS) lines having the same A, cytoplasm with different nuclear backgrounds of pearl millet along with their maintainer, restorer and restored lines. In addition, the assays of cytochrome c oxidase and succinate dehydrogenase were performed in isolated mitochondria. Cyanide-sensitive, cyanide-insensitive and total respirations in vegetative tissues and isolated mitochondria were lower in both the CMS lines than their respective maintainers, restorers and restored hybrids. Except in CMS lines, uptake of 0₂ during anther development increased from premeiotic to postmeiotic stage in all the lines. Consumption of 0₂,however, declined from meiotic to postmeiotic stage in the anthers of CMS lines. Pure mitochondrial preparations were obtained which were 92-94% intact. Enzymes, cytochrome c oxidase (complex IV) and succinate dehydrogenase (complex II) showed lower activities in both the CMS lines than their respective maintainer, restorer and restored hybrids. The CMS lines also displayed lower levels of nuclear encoded enzymes, viz., alternative oxidase and succinate dehydrogenase.     

Mitochondrial DNA Polymorphisms Revealed by RAPD Assays Distinguish the Male-sterile and Male-fertile Cytoplasms in Pearl Millet

Diversification of cytoplamic male sterile (CMS) sources is of considerable significance in pearl millet, considering that almost all the commercially cultivated hybrids, particularly in India, are based on a single CMS source, A₁. We analyzed the mitochondrial DNA (mtDNA) polymorphisms among five pearl millet CMS sources (A₁ to A₅) and a male-fertile maintainer (B) line, all in an isonuclear background. Analysis using 21 random primers led to identification of polymorphic bands specific to the A₁, A₂, A3 and A₅ CMS lines. Two RAPD primers, OP-G12 and OP-G19, in combination were able to distinguish all the male-sterile and male-fertile cytoplasms. Highly effective for this purpose also were four RAPD markers, OP-B7, OP-D8, OP-F10 and OP-G12. Cluster analysis, followed by bootstrap analysis, of the mtDNA dataset revealed two distinct clusters: cluster-I comprising the A₁, A₂, A₃ CMS lines and the male-fertile line, and cluster-II comprising the A₄ and A₅ CMS lines.     

Unusual Mitochondrial Genome Organization in Cytoplasmic Male Sterile Common Bean and the Nature of Cytoplasmic Reversion to Fertility

Spontaneous reversion to pollen fertility and fertility restoration by the nuclear gene Fr in cytoplasmic male sterile common bean (Phaseolus vulgaris L.) are associated with the loss of a large portion of the mitochondrial genome. To understand better the molecular events responsible for this DNA loss, we have constructed a physical map of the mitochondrial genome of a stable fertile revertant line, WPR-3, and the cytoplasmic male sterile line (CMS-Sprite) from which it was derived. This involved a cosmid clone walking strategy with comparative DNA gel blot hybridizations. Mapping data suggested that the simplest model for the structure of the CMS-Sprite genome consists of three autonomous chromosomes differing only in short, unique regions. The unique region contained on one of these chromosomes is the male sterility-associated 3-kb sequence designated pvs. Based on genomic environments surrounding repeated sequences, we predict that chromosomes can undergo intra- and intermolecular recombination. The mitochondrial genome of the revertant line appeared to contain only two of the three chromosomes; the region containing the pvs sequence was absent. Therefore, the process of spontaneous cytoplasmic reversion to fertility likely involves the disappearance of an entire mitochondrial chromosome. This model is supported by the fact that we detected no evidence of recombination, excision or deletion events within the revertant genome that could account for the loss of a large segment of mitochondrial DNA.     

RFLP analysis of mitochondrial DNA from cytoplasmic male-sterile lines of pearl millet

Mitochondrial DNA (mtDNA) from 13 cytoplasmic male-sterile (cms) lines from diverse sources were characterized by Southern blot hybridization to pearl millet and maize mtDNA probes. Hybridization patterns of mtDNA digested with PstI, BamHI, SmaI or XhoI and probed with 13.6-, 10.9-, 9.7- or 4.7-kb pearl millet mtDNA clones revealed similarities among the cms lines 5141 A and ICMA 1 (classified as the S-A1 type of cytoplasm based on fertility restoration patterns), PMC 30A and ICMA 2. The remaining cms lines formed a distinct group, within which three subgroups were evident. Among the maize mitochondiral gene clones used, the coxI probe revealed two distinct groups of cytoplasms similar to the pearl millet mtDNA clones. The atp9 probe differentiated the cms line 81 A4, derived from P. glaucum subsp. monodii, while the coxII gene probe did not detect any polymorphism among the cms lines studied. MtDNA digested with BamHI, PstI or XhoI and hybridized to the atp6 probe revealed distinct differences among the cms lines. The maize atp6 gene clone identified four distinct cytoplasmic groups and four subgroups within a main group. The mtDNA fragments hybridized to the atp6 gene probe with differing intensities, suggesting the presence of more than one copy of the gene in different stoichiometries. Rearrangements involving the coxI and/or rrn18-rrn5 genes (mapped within the pearl millet clones) probably resulted in the S-A1 type of sterility. Rearrangements involving the atp6 gene (probably resulting in chimeric form) may be responsible for male sterility in other cms lines of pearl millet.     

Extensive structural variations between mitochondrial genomes of CMS and normal peppers (Capsicum annuum L.) revealed by complete nucleotide sequencing

Background

Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp.

Results

We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (ψatp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and ψatp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes.

Conclusions

Although large portion of sequence context was shared by mitochondrial genomes of CMS and male-fertile pepper lines, extensive genome rearrangements were detected. CMS candidate genes located on the edges of highly-rearranged CMS-specific DNA regions and near to repeat sequences. These characteristics were detected among CMS-associated genes in other species, implying a common mechanism might be involved in the evolution of CMS-associated genes.

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