Cytoplasmic male sterility and fertility restoration in wheat are not associated with rearrangements of mitochondrial DNA in the gene regions for cob,coxII, or coxI

In comparing the genetic organization and exploring the molecular basis of cytoplasmic male sterility (CMS) in wheat, mitochondrial DNAs (mtDNA) from Triticum aestivum, T. timopheevi, CMS alloplasmic wheat with T. aestivum nucleus and T. timopheevi mitochondria, and fertility-restored lines were compared by hybridization analysis with specific probes for three gene regions: CoxII, cob, and coxI. Minor differences between T. aestivum- and T. timopheevi-derived sources were found for gene regions for coxII and cob. For coxI, there are significant differences between T. timopheevi-derived mtDNAs and T. aestivum mtDNA extending beyond an 8 kb distance. All T. timopheevi-derived mtDNA sources have a chimeric gene region (orf256) with part of the upstream coxI gene region, including some coxI-coding region, preceding coxI. The part of orf256 that does not include any of coxI and the 3-flanking region of CMS coxI are not found in T. aestivum mtDNA. Neither orf256 nor the CMS 3-flanking region of coxI are found in T. timopheevi or T. aestivum chloroplastic or nuclear DNA. There do not appear to be DNA sequence differences for the three gene regions studied that are related to either CMS or fertility-restored states.     

A codominant molecular marker in linkage disequilibrium with a restorer-of-fertility gene (Ms) and its application in reevaluation of inheritance of fertility restoration in onions

To reveal the linkage relationship between the Ms locus, a restorer-of-fertility gene for cytoplasmic male-sterility (CMS) caused by CMS-S cytoplasm in onion (Allium cepa L.) and previously reported molecular markers linked to the Ms locus, 11 recombinants selected from 4,273 segregating plants originating from the cross between male-sterile maternal and male-fertile paternal lines were analyzed. Results showed that genotypes of a codominant marker, jnurf12, were perfectly matched with the male-fertility phenotypes in all recombinants, but that this marker was not applicable in diverse breeding lines due to multiple band patterns. For the development of more reliable markers, a 12-bp indel was identified from the sequences which were obtained by genome walking, and was used to develop a simple PCR marker which was designated jnurf13. When 104 diverse breeding lines containing CMS-S cytoplasm were analyzed with the jnurf13 marker, male-fertility phenotypes of all breeding lines were perfectly matched with marker genotypes. To our surprise, phenotypes of 153 breeding lines containing CMS-T-like cytoplasm were also matched with genotypes of the jnurf13 marker which was linked to the Ms locus for the CMS-S system. Furthermore, phenotypes of four F₂ populations containing CMS-T-like cytoplasm co-segregated perfectly with jnurf13 genotypes. Allelic segregation distortion was detected in two F₂ populations using the jnurf13 maker. The results of this study were in conflict with a previous model for inheritance of fertility restoration in the CMS-T system. Therefore, we proposed a new model based on the data analyzed with the jnurf13 marker, which was in linkage disequilibrium with restorer-of-fertility genes for both CMS systems.     

A chimeric gene (orf256) is expressed as protein only in cytoplasmic male-sterile lines of wheat

Mitochondria derived from Triticum timopheevi have a chimeric gene, orf256, immediately upstream from coxI. Antibodies to a peptide corresponding to a part of the encoded amino acid sequence of orf256 detect a 7 kDa protein on western blots of mitochondrial proteins from cytoplasmic male-sterile (cms) wheat (T. aestivum nucleus, T. timopheevi mitochondria) but not in mitochondrial proteins from T. aestivum, T. timopheevi, or cms plants restored to fertility by introduction of nuclear genes for fertility restoration. The 7 kDa protein appears to serve as a marker for cms wheat. Its occurrence as an integral protein of the inner membrane may indicate a cms effect through an influence on mitochondrial membrane function.     

Development of a CMS-specific marker based on chloroplast-derived mitochondrial sequence in pepper

Molecular markers developed from the flanking sequences of two cytoplasmic male sterility (CMS)-associated genes, orf456 and ψatp6-2, have been used for marker-assisted selection of CMS in pepper. However, in practice, the presence of orf456 and ψatp6-2 at substoichiometric levels even in maintainer lines hampers reliable selection of plants containing the CMS gene. In this study, we developed a novel CMS-specific molecular marker, accD-U, for reliable determination of CMS lines in pepper, and used the newly and previously developed markers to determine the cytoplasm types of pepper breeding lines and germplasms. This marker was developed from a deletion in a chloroplast-derived sequence in the mitochondrial genome of a CMS pepper line. CMS pepper lines could be unambiguously determined by presence or absence of the accD-U marker band. Application of orf456, ψatp6-2 and accD-U to various pepper breeding lines and germplasms revealed that accD-U is the most reliable CMS selection marker. A wide distribution of orf456, but not ψatp6-2, in germplasms suggests that the pepper cytoplasm containing both orf456 and ψatp6-2 has been selected as CMS cytoplasm from cytoplasm containing only orf456. Furthermore, factors other than orf456 may be required for the regulation of male sterility in pepper.     

Complete mitochondrial genome sequence and identification of a candidate gene responsible for cytoplasmic male sterility in radish (Raphanus sativus L.) containing DCGMS cytoplasm

A novel cytoplasmic male sterility (CMS) conferred by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its restorer-of-fertility gene (Rfd1) was previously reported in radish (Raphanus sativus L.). Its inheritance of fertility restoration and profiles of mitochondrial DNA (mtDNA)-based molecular markers were reported to be different from those of Ogura CMS, the first reported CMS in radish. The complete mitochondrial genome sequence (239,186 bp; GenBank accession No. KC193578) of DCGMS mitotype is reported in this study. Thirty-four protein-coding genes and three ribosomal RNA genes were identified. Comparative analysis of a mitochondrial genome sequence of DCGMS and previously reported complete sequences of normal and Ogura CMS mitotypes revealed various recombined structures of seventeen syntenic sequence blocks. Short-repeat sequences were identified in almost all junctions between syntenic sequence blocks. Phylogenetic analysis of three radish mitotypes showed that DCGMS was more closely related to the normal mitotype than to the Ogura mitotype. A single 1,551-bp unique region was identified in DCGMS mtDNA sequences and a novel chimeric gene, designated orf463, consisting of 128-bp partial sequences of cox1 gene and 1,261-bp unidentified sequences were found in the unique region. No other genes with a chimeric structure, a major feature of most characterized CMS-associated genes in other plant species, were found in rearranged junctions of syntenic sequence blocks. Like other known CMS-associated mitochondrial genes, the predicted gene product of orf463 contained 12 transmembrane domains. Thus, this gene product might be integrated into the mitochondrial membrane. In total, the results indicate that orf463 is likely to be a casual factor for CMS induction in radish containing the DCGMS cytoplasm.     

A comparison of cytoplasmic revertants to fertility from different CMS-S maize sources

Summary The mitochondrial genome organizations of a number of independent culture-derived fertile CMS-S revertants with the nuclear genotype W182BN were compared to spontaneous field revertants with the genotypes WF9, M825/Oh07 and 38-11. Regions of the genome around sequences homologous to the terminal repeats of the linear S1 and S2 episomes characteristic of CMS-S mitochondria were used as hybridization probes on Southern blots of BamHI and SalI digested mitochondrial DNA. The results obtained suggest that the nuclear, not the cytoplasmic, genotype of the parent plant affects the type of novel mitochondrial DNA organization found in the revertant. The DNA reorganization during reversion from CMS-S in tissue culture appears to be similar to that observed in spontaneous revertants obtained during the normal plant life-cycle. Unlike the situation for reversion from CMS-T, no common DNA sequence or reading frame appeared to be lost or disrupted in revertants.     

5′ Untranslated region of the Hsp12 gene contributes to efficient translation in Aspergillus oryzae

We describe a 5′ untranslated region (5′UTR) that dramatically increases the expression level of an exogenous gene in Aspergillus oryzae. Using a series of 5′UTR::GUS (uidA) fusion constructs, we analyzed the translation efficiency of chimeric mRNAs with different 5′UTRs at different temperatures. We found that the 5′UTR of a heat-shock protein gene, Hsp12, greatly enhanced the translation efficiency of the chimeric GUS mRNA at normal temperature (30°C). Moreover, at high temperature (37°C), the translation efficiency of the mRNA containing the Hsp12 5′UTR was far superior to that of mRNAs containing nonheat-shock 5′UTRs, resulting in much more efficient expression of GUS protein (about 20-fold higher GUS activity compared to the control construct). This 5′UTR can be used in combination with various strong promoters to enhance the expression of foreign proteins in A. oryzae.     

Comparisons among two fertile and three male-sterile mitochondrial genomes of maize

We have sequenced five distinct mitochondrial genomes in maize: two fertile cytotypes (NA and the previously reported NB) and three cytoplasmic-male-sterile cytotypes (CMS-C, CMS-S, and CMS-T). Their genome sizes range from 535,825 bp in CMS-T to 739,719 bp in CMS-C. Large duplications (0.5-120 kb) account for most of the size increases. Plastid DNA accounts for 2.3-4.6% of each mitochondrial genome. The genomes share a minimum set of 51 genes for 33 conserved proteins, three ribosomal RNAs, and 15 transfer RNAs. Numbers of duplicate genes and plastid-derived tRNAs vary among cytotypes. A high level of sequence conservation exists both within and outside of genes (1.65-7.04 substitutions/10 kb in pairwise comparisons). However, sequence losses and gains are common: integrated plastid and plasmid sequences, as well as noncoding "native" mitochondrial sequences, can be lost with no phenotypic consequence. The organization of the different maize mitochondrial genomes varies dramatically; even between the two fertile cytotypes, there are 16 rearrangements. Comparing the finished shotgun sequences of multiple mitochondrial genomes from the same species suggests which genes and open reading frames are potentially functional, including which chimeric ORFs are candidate genes for cytoplasmic male sterility. This method identified the known CMS-associated ORFs in CMS-S and CMS-T, but not in CMS-C.     

Fungal origin by horizontal transfer of a plant mitochondrial group I intron in the chimeric coxI gene of Peperomia

We present phylogenetic evidence that a group I intron in an angiosperm mitochondrial gene arose recently by horizontal transfer from a fungal donor species. A 1,716-bp fragment of the mitochondrial coxI gene from the angiosperm Peperomia polybotrya was amplified via the polymerase chain reaction and sequenced. Comparison to other coxI genes revealed a 966-bp group I intron, which, based on homology with the related yeast coxI intron aI4, potentially encodes a 279-amino-acid site-specific DNA endonuclease. This intron, which is believed to function as a ribozyme during its own splicing, is not present in any of 19 coxI genes examined from other diverse vascular plant species. Phylogenetic analysis of intron origin was carried out using three different tree-generating algorithms, and on a variety of nucleotide and amino acid data sets from the intron and its flanking exon sequences. These analyses show that the Peperomia coxI gene intron and exon sequences are of fundamentally different evolutionary origin. The Peperomia intron is more closely related to several fungal mitochondrial introns, two of which are located at identical positions in coxI, than to identically located coxI introns from the land plant Marchantia and the green alga Prototheca. Conversely, the exon sequence of this gene is, as expected, most closely related to other angiosperm coxI genes. These results, together with evidence suggestive of co-conversion of exonic markers immediately flanking the intron insertion site, lead us to conclude that the Peperomia coxI intron probably arose by horizontal transfer from a fungal donor, using the double-strand-break repair pathway. The donor species may have been one of the symbiotic mycorrhizal fungi that live in close obligate association with most plants. Correspondence to: J.C. Vaughn     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

An alloplasmic male-sterile line of Brassica oleracea harboring the mitochondria from Diplotaxis muralis expresses a novel chimeric open reading frame, orf72

Nuclear so-called fertility-restorer genes reverse the pollen sterility of cytoplasmic male-sterile (CMS) plants caused by disturbed mitochondrial-nuclear interactions. We identified a CMS-associated chimeric mitochondrial gene in an alloplasmic CMS line of Brassica oleracea in the 'mur' system. This novel chimeric gene, orf72, was found in the mitochondrial genome of donor cytoplasm. It was located downstream of normal rps7 and contained part of atp9 (atp9-b). It was expressed specifically on the nuclear background of CMS B. oleracea, partially suppressed in the fertility-restored line and entirely suppressed in the cytoplasmic donor.