Molecular analysis of cytoplasmic male sterility in chives (Allium schoenoprasum L.)

The mitochondria of chive plants with normal N or male-sterile S cytoplasms have been examined by restriction fragment analysis and Southern hybridizations of mitochondrial DNA (mtDNA) and in organello protein biosynthesis. Restriction fragment patterns of the mtDNA differed extensively between N-and S-cytoplasms. The percentage of fragments with different mobility varied between 44–48% depending on the restriction enzyme used. In contrast to mtDNA, the restriction fragment patterns of the chloropolast DNA from N- and S-cytoplasms were identical. The organization of the analyzed mitochondrial genes coxII, coxIII, nad1 and nad3 was different in N- and S-cytoplasms. Comparison of mitochondrial proteins analyzed by in organello translation revealed an 18-kDa protein present only in S-cytoplasm. The restorer gene X suppressed the synthesis of that protein in S-cytoplasm. Thus, the 18-kDa protein seems to be associated with the cytoplasmic male-sterile phenotype.     

Identification of cytoplasms using the polymerase chain reaction to aid in the extraction of maintainer lines from open-pollinated populations of onion

S-cytoplasm is the most common source of cytoplasmic-genic male sterility (CMS) used to produce hybrid-onion seed. Identification of the cytoplasm of a single plant takes from 4 to 8 years and is complicated by the segregation of a nuclear gene that restores fertility. Although CMS in onion may be due to an incompatibility between the mitochondrial and nuclear genomes, Southern analyses of DNA from individual plants from crosses of S- and N-cytoplasmic plants supported maternal inheritance of the chloroplast and mitochondrial DNA and, therefore, polymorphisms in the chloroplast DNA may be used to classify cytoplasms. Amplification by the polymerase chain reaction of a fragment that carries an autapomorphic 100-bp insertion in the chloroplast DNA of N-cytoplasm offers a significantly quicker and cheaper alternative to crossing or Southern analysis. Molecular characterization of N- and S-cytoplasms and frequencies of the nuclear non-restoring allele allow onion breeders to determine the proportion of plants in open-pollinated populations that maintain CMS and can significantly reduce the investment required to identify individual maintainer plants.     

Development of SCAR markers for early identification of cytoplasmic male sterility genotype in chili pepper (Capsicum annuum L.)

We previously used Southern blot analysis to detect restriction-length polymorphisms between male fertile and cytoplasmic male sterile (CMS) cytoplasms at the coxII and atp6 loci of the mtDNA of Capsicum annuum L. Two copies of atp6 were found in each male fertile and CMS pepper lines. Interestingly, one of the copies of atp6 in CMS pepper was a 3'-truncated pseudogene. The open reading frame of the coxII gene was the same in the fertile (N-) and CMS (S-) lines. However, the nucleotide sequence in the S-cytoplasm diverged from that in the N-cytoplasm 41 bp downstream of the stop codon. To develop CMS-specific sequence-characterized amplified region (SCAR) markers, inverse PCR was performed to characterize the nucleotide sequences of the 5' and 3' flanking regions of mitochondrial atp6 and coxII from the cytoplasms of male fertile (N-) and CMS (S-) pepper plants. Based on these data, two CMS-specific SCAR markers, 607 and 708 bp long, were developed to distinguish N-cytoplasm from S-cytoplasm by PCR. The CMS-specific PCR bands were verified for 20 cultivars containing either N- or S-cytoplasm. PCR amplification of CMS-specific mitochondrial nucleotide sequences will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F1 seed lots. The strategy used in this report for identifying CMS-specific markers could be adopted for many other crops where CMS is used for F1 seed production.     

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 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.     

Molecular characteristics and expression of cytoplasmic genes in cytoplasmic male sterility of pepper (Capsicum annuum L.)

This study compared the sequence variations and expressions of 12 chloroplastic and 8 mitochondrial genes in three pepper (Capsicum annuum L.) cytoplasmic male sterile (CMS) lines, their maintainers and two control cultivars. The results showed that the three CMS lines were highly similar in chloroplastic and mitochondrial fragment sequences, with average similarities of 96.81 and 98.73?%, respectively, and their chloroplastic trnH?CpsbA intergenic spacer, photosystem II 47?kDa protein (psbB) genes, mitochondrial apocytochrome b (cob) and RNAD fragments have 1, 9, 8 and 7 distinct sites from the maintainer lines, respectively, and could be used as informative sites to distinguish CMS lines from the maintainer lines. Meanwhile, the expressions of mitochondrial cob, RNAD and pvs in the reproductive organs (flowers) of CMS lines are different from those of the maintainer lines, but their expressions in the vegetative organs (roots and leaves) are similar. The results indicate that cytoplasmic DNA polymorphisms are rare in CMS lines, and mitochondrial cob, RNAD and pvs genes are closely related to pollen abortion.     

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.     

Determination of cytoplasmic male sterile factors in onion plants (Allium cepa L.) using PCR-RFLP and SNP markers

We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker that can distinguish male-fertile (N) and male-sterile (S) cytoplasm in onions. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm-containing varieties with the restriction enzyme MspI revealed that N-cytoplasm plants have a functional MspI site (CCGG), whereas the S-cytoplasm plants has a substitution in that site (CTGG), and thus no MspI target. The results obtained using this PCR-RFLP marker to distinguish between cytoplasmic male sterile factors in 35 onion varieties corresponded with those using a CMS-specific sequence-characterized amplified region (SCAR) marker. Moreover, the PCR-RFLP marker can identify N- ot S-cytoplasms in DNA sample mixtures in which they are in up to a 10-fold minority, indicating that use of the marker has high diagnostic precision. We also demonstrated the usefulness of the SNP detected in the psbA gene for high-throughput discrimination of CMS factors using Real-time PCR and a TaqMan probe assay.     

Development of a SCAR marker for early identification of S-cytoplasm based on mitochondrial SRAP analysis in pepper (Capsicum annuum L.)

Molecular markers, coxII SCAR, atp6-2 SCAR and accD-U, have been used for marker-assisted selection of cytoplasmic male sterility (CMS) in pepper. However, the presence of these markers at the sub-stoichiometric level in maintainer lines affects the reliable selection of male sterile (S-) cytoplasm. This study aimed to develop a new CMS-specific molecular marker, SCAR₁₃₀, for reliable identification of S-cytoplasm in pepper, while the new and three previous molecular markers were used to determine the cytoplasm types of pepper lines. Based on mitochondrial genome sequence related amplified polymorphism (SRAP) analysis of the CMS lines and the maintainer lines, SCAR₁₃₀ was developed from a 10-bp deletion at the SRAP primer binding site in the CMS line (130 bp) compared with that in the maintainer line (140 bp). S-cytoplasm could be unambiguously selected from the pepper lines by the different length of the marker bands. Application of the four molecular markers to various pepper lines revealed that SCAR₁₃₀ is more reliable than the other three previous markers, orf507, ψatp6-2 and accD-U. Homology alignment with BLAST showed that the marker was located between trnE and trnS in the Nicotiana tabacum mitochondrial genome. Furthermore, expression of the marker-linked gene was significantly higher at the pollen abortive stage in the CMS line (HW203A) than in the maintainer line, which indicated that the marker was closely related to male sterility. Hence, factors other than orf507 and ψatp6-2 may exist for the regulation of male sterility in pepper.     

Organizational differences between cytoplasmic male sterile and male fertile Brassica mitochondrial genomes are confined to a single transposed locus.

Comparison of the physical maps of male fertile (cam) and male sterile (pol) mitochondrial genomes of Brassica napus indicates that structural differences between the two mtDNAs are confined to a region immediately upstream of the atp6 gene. Relative to cam mtDNA, pol mtDNA possesses a 4.5 kb segment at this locus that includes a chimeric gene that is cotranscribed with atp6 and lacks an approximately 1kb region located upstream of the cam atp6 gene. The 4.5 kb pol segment is present and similarly organized in the mitochondrial genome of the common nap B.napus cytoplasm; however, the nap and pol DNA regions flanking this segment are different and the nap sequences are not expressed. The 4.5 kb CMS-associated pol segment has thus apparently undergone transposition during the evolution of the nap and pol cytoplasms and has been lost in the cam genome subsequent to the pol-cam divergence. This 4.5 kb segment comprises the single DNA region that is expressed differently in fertile, pol CMS and fertility restored pol cytoplasm plants. The finding that this locus is part of the single mtDNA region organized differently in the fertile and male sterile mitochondrial genomes provides strong support for the view that it specifies the pol CMS trait.     

Organization of the mitochondrial Cob 2 pseudogene in different lines of rice

In the fertile rice line IR 36 there are two copies of the apocytochrome b (cob) gene: a functional copy, cob 1, and a pseudogene, cob 2 (Kaleikau et al. 1992). In a survey of diverse rice lines, we found that cob 2 was absent in the wild abortive(WA)-type cytoplasmic male-sterile cytoplasm, but was present in the fertile lines. While cob 1 was conserved among all the lines, fertile and sterile, the cob 2 region was different in the fertile lines tested. The 5′ regions of most cob 2 loci were similar to cob 1 (about 4 kb of the flanking region and most of the coding region), but the 3′ region varied among different fertile lines. The point of divergence, the break-point, from the cob 1 sequence was conserved in all the cob 2 regions tested. In all the cob 2 regions, this break-point seems to be linked to the variable region of cob 2 through a conserved 192-bp segment, which is not a part of cob 1. It is proposed that the cob 2 regions could have been produced by recombination or insertion events involving cob 1 and the 192-bp segment which is present at different locations in the mitochondrial genomes of the various rice lines.     

Somaclonal variation of the mitochondrial ATPase subunit 6 gene region in regenerated triticale shoots and full-grown plants

Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.     

Molecular diversity of male sterility inducing and male-fertile cytoplasms in the genus Helianthus

The organisation of mtDNA was investigated for 28 sources of cytoplasmic male sterility (CMS) and a fertile line (normal cytoplasm) of Helianthus annuus by Southern hybridisation. In addition to nine known mitochondrial genes (atp6, atp9, cob, coxI, coxII, coxIII, 18S, 5S and nd5) three probes for the open reading frames in the rearranged area of PET1, orfH522, orfH708 and orfH873, were used. Genetic similarities of the investigat-ed cytoplasms varied between 0.3 and 1. Cluster analyses using the UPGMA method allowed the distinction of ten mitochondrial (mt) types between the 29 investigated cytoplasms. Most mitochondrial types comprise two or more CMS sources, which could not be further separated, like the PET1-like CMS sources (with the exception of ANO1 and PRR1), or ANN1/ANN2/ANN3, ANN4/ ANN5, ARG3/RIG1, BOL1/EXI1/PEF1/PEP1 and GIG1/ PET2. ANL1, ANL2 and the fertile cytoplasms are also regarded as one mitochondrial type. Unique banding patterns were only observed for ANT1 (atp6), MAX1 (atp6, orfH522 and orfH708) and PRR1 (coxII). However, four of the mitochondrial types showed unique hybridisation signals: ANN4/ANN5 had characteristic bands for atp6 and orfH708, PEF1/PEP1/EXI1/BOL1 for atp6 and coxII, and PET2/GIG1 for atp9. The PET1-like cytoplasms all shared the same patterns for orfH522, orfH708 and cob (except ANO1). It could be demonstrated that CMS sources, like, e.g., PET2 and PEF1, are different from PET1 in mtDNA organisation and the CMS mechanism. Therefore, these CMS sources represent interesting candidates for the development of new hybrid breeding systems based on new CMS mechanisms. Received: 20 April 2001 / Accepted: 3 August 2001     

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.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-561) contains supplementary material, which is available to authorized users.