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.     

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.     

The postglacial history of Scots pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial DNA variation

The geographical structure of mitochondrial (mt)DNA variants (mitotypes) was investigated in 38 western European populations of Scots pine Pinus sylvestris using restriction fragment length polymorphism (RFLP) analysis of total DNA and a homologous cox1 probe. Three major mitotypes (designated a, b and d ) were detected. Within Spain all three major mitotypes were found, gene diversity was high, H_T = 0.586, and this diversity was distributed predominantly among rather than within populations (F_ST(M) = 0.813 for the seven Spanish populations). Mitotype d was present only in the most southerly population from the Sierra Nevada . Elsewhere in Europe, populations showed little or no mtDNA diversity within regions, but there were marked differences between regions. Italian populations were fixed for mitotype b ; populations from northern France, Germany, Poland, Russia and southern Sweden were fixed for mitotype a ; while populations in northern Fennoscandia were fixed for mitotype b . The isolated Scottish populations were predominantly of mitotype a , but mitotype b was present in three of the 20 populations scored. In Scotland, UK gene diversity (H_T = 0.120) and genetic differentiation among populations (F_ST(M) = 0.37) was much lower than in Spain. When interpreted in the light of complementary data from pollen analysis and nuclear genetic markers, the results suggest that present-day populations of P. sylvestris in western Europe have been derived from at least three different sources after glaciation.     

Evidence of doubly uniparental inheritance of the mitochondrial DNA in Polititapes rhomboides (Bivalvia,Veneridae): Evolutionary and population genetic analysis of F and M mitotypes

Doubly uniparental inheritance (DUI) is a particular mitochondrial DNA inheritance mode reported in a number of bivalves. DUI species show two types of mtDNA, one transmitted from females to daughters and sons (F mitotype) and another one from males to sons (M mitotype). In Veneridae, the existence of DUI has been investigated in several species but it was found in only two of them. In this study, we obtained partial sequences of rrnL, cytb and cox1 genes of males and females of Polititapes rhomboides from NW Spain and we demonstrated the existence of heteroplasmy in males, as expected under DUI. F and M mitotypes showed a taxon-specific phylogenetic pattern and similar evolutionary rates. We focused on cox1 for population genetic analysis, examining separately F and M mitotypes, but also F mitotypes from females (F_♀) and males (F_♂). In all cases, cox1 bears signs of strong purifying selection, with no apparent evidence of relaxed selection in the M genome, while the divergence between F and M genomes is in agreement with the neutral model of evolution. The cox1 polymorphism, higher at the M than at the F genome, also shows clear footprints of genetic hitchhiking with favourable mutations at other mtDNA loci, except for F_♂. In terms of population structure, results suggest that the pattern depends on the examined mitotype (F, F_♀, F_♂ or M).     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. I. The origin and distribution of Ogura male-sterile cytoplasm in Japanese wild radishes (Raphanus sativus L.) revealed by PCR-aided assay of their mitochondrial DNAs

Ogura male-sterile cytoplasm was surveyed in common Japanese radish cultivars and in wild radishes growing in various localities in Japan. Mitochondrial (mt) DNA rearrangement involving the atp6 gene was used as a molecular marker. To detect the mtDNA rearrangement, polymerase chain reactions (PCR) were designed to amplify the upstream region of the atp6 gene. The oligonucleotides homologous to the following three regions were synthesized: (1) trnfM, (2) ORF105 and (3) atp6. PCRs were conducted with a pair of the first and the third primers to detect normal mtDNA, and with the second and the third primers for Ogura-type mtDNA. All 15 Japanese cultivars yielded an amplification product which was the same as that of normal mtDNA, whereas some wild radishes gave the product specific to Ogura mtDNA. Twenty-four populations of wild radish were classified into three groups according to the frequency of Ogura-type mtDNA: (1) in ten populations, all four plants analyzed per population had normal type mtDNA, (2) in five populations, only plants with Ogura-type mtDNA were found, and (3) nine populations included both normal and Oguratype mtDNAs. There were no geographical restrictions and no cline in the distribution of the plants with Ogura-type mtDNA. These results suggested that the Ogura-type male-sterile cytoplasm originated in wild radishes.     

MULTIPLE ORIGINS OF GENDER-ASSOCIATED MITOCHONDRIAL DNA LINEAGES IN BIVALVES (MOLLUSCA: BIVALVIA)

Previous studies have shown that marine mussels (genus Mytilus) and a freshwater mussel (Pyganodon grandis) contain two distinct gender-associated mitotypes, which is a characteristic feature of the phenomenon of doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA). Here we present evidence for the presence of distinct male (M) and female (F) mitotypes in three other bivalve species, the mytilid Geukensia demissa, and the unionid species P. fragilis and Fusconaia flava. Nucleotide sequences of a segment of the COI gene from the M and F mitotypes from each of the three mytilid species (M. edulis, M. trossulus, G. demissa) and three unionid species (P. grandis, P. fragilis, F. flava) were used for phylogenetic analysis. The analysis suggests three independent origins of M and F mitotypes for the six species examined; one for the three unionid species, one for the two Mytilus species, and one for Geukensia. The first of these F/M divergence events, while of uncertain age, predates the divergence of the two unionid genera and is likely older than either of the two F/M divergence events in the mytilid taxa. The most parsimonious explanation of multiple F/M divergence events is that they represent independent origins of DUI. Another possibility is that, in a given taxon, an F or M mitotype assumes the role of the opposite mitotype (by virtue of a mechanism that remains to be clarified) and subsequently was fixed within its new gender. The fixation of a mtDNA lineage derived from a mitotype of switched function would reset the divergence of the gender-associated lineages to zero, thereby mimicking a de novo split of F and M lineages from a preexisting mtDNA genome that was not gender specific. Further broad-scale taxonomic studies of the occurrence of distinct M and F mitotypes may allow for the evaluation of the latter hypothesis.     

Identification of highly variable chloroplast sequences and development of cpDNA-based molecular markers that distinguish four cytoplasm types in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

Four types of cytoplasms (Ogura, DCGMS, DBRMF1, and DBRMF2) were identified in the previous studies using molecular markers based on mitochondrial genome variations in radish (Raphanus sativus L.). However, mtDNA markers have limitations in obtaining clear results due to complexity of radish mitochondrial genomes. To improve fidelity, molecular markers based on variation of chloroplast genome sequences were developed in this study. We searched for the sequence variations of chloroplast genome among the four cytoplasm types in 11 noncoding intergenic regions of ~8.7 kb. Highly variable intergenic regions between trnK and rps16 were identified, and a couple of 4–34 bp indels were used to develop a simple PCR-based marker that distinguished the four cytoplasm types based on the PCR product length polymorphism. Two additional cpDNA markers were developed by using a single nucleotide polymorphism and 17-bp insertion. Analysis of 90 accessions using both mtDNA and cpDNA markers showed the perfect match of results of both the markers, suggesting strict co-transmission of mitochondria and chloroplast in radish. Phylogenetic trees showed that two male-sterility inducing cytoplasms, Ogura and DCGMS, were closely related to DBRMF1 and DBRMF2, respectively. Analysis of 120 radish germplasms introduced from diverse countries showed that the frequency of male-sterility inducing mitotypes of Ogura and DCGMS was very low, and DCGMS was predominately detected in eastern European countries. Majority of accessions from Europe and Asia were shown to contain DBRMF2 and DBRMF1 mitotypes, respectively. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. S. Kim and Y.-P. Lee contributed equally to this work.     

Cytoplasmic male sterility in the olive (Olea europaea L.)

The olive tree is usually hermaphrodite but self-incompatible. In the Western Mediterranean some cultivars are totally male-sterile. Three different male-sterile phenotypes have been recognised. To infer the genetic basis of male sterility we studied its inheritance and cytoplasmic diversity in wild (oleaster) and cultivated Mediterranean olive. In the cross Olivière×Arbequina, the male-sterile trait was maternally inherited and affected all progenies. We also checked that both chloroplast and mitochondrial DNAs are maternally inherited. RFLP studies on chloroplast and mitochondrial DNAs revealed several cytotypes: two chlorotypes and four mitotypes in cultivars and oleaster (wild or feral Mediterranean olive). Furthermore, a total linkage desequilibrium between the CCK chlorotype and the MCK mitotype in cultivars and oleaster from different regions supports the fact that paternal leakage of organelles was not observed. The male sterility (ms 2) displayed by Olivière, plus six other cultivars and three oleaster was strictly associated with the CCK chlorotype and the MCK mitotype. These facts suggest that Olivière carries cytoplasmic male sterility. Male-fertile and male-sterile oleasters carrying this cytotype showed the presence of restorer alleles. This CMS might be due to a distant cross between olive taxa. The two other male-sterile phenotypes displayed by Lucques (ms 1) and Tanche (ms 3) were associated with the ME1 mitotype but we have not demonstrated CMS. Received: 26 July 1999 / Accepted: 27 August 1999     

Identification of a novel mitochondrial genome type and development of molecular markers for cytoplasm classification in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

Plant mitochondrial genomes have complex configurations resulting from the multipartite structures and highly rearranged substoichiometric molecules created by repetitive sequences. To expedite the reliable classification of the diverse radish (Raphanus sativus L.) cytoplasmic types, we have developed consistent molecular markers within their complex mitochondrial genomes. orf138, a gene responsible for Ogura male-sterility, was detected in normal cultivars in the form of low-copy-number substoichiometric molecules. In addition to the dominant orf138-atp8 Ogura mitochondrial DNA (mtDNA) organization, three novel substoichiometric organizations linked to the atp8 gene were identified in this study. PCR amplification profiles of seven atp8- and atp6-linked sequences were divided into three groups. Interestingly, the normal cytoplasm type, which had previously been considered a single group, showed two patterns by PCR amplification. The most prominent difference between the two normal mtDNAs was size variation within four short-repeat sequences linked to the atp6 gene. This variation appeared to be the result of a double crossover, mediated by these homologous, short-repeat sequences. Specific PCR amplification profiles reflecting the stoichiometry of different mtDNA fragments were conserved within cultivars and across generations. Therefore, the specific sequences detected in these profiles were used as molecular markers for the classification of diverse radish germplasm. Using this classification system, a total of 90 radish cultivars, or accessions, were successfully assigned to three different mitotypes.     

Comparative Restriction Enzyme Analysis of Mitochondrial DNA in Several Populations of Lake Resident Chars of the Genus Salvelinus

Restriction enzyme analysis was employed in studying the mitochondrial DNA (mtDNA) ATPase6/ND4L region in several Northeast Asian populations of resident lake char of the genus Salvelinus. On evidence of mitotypes, genetic similarity was assumed for populations of neiva (Ueginskoe Lake), lake resident char from the northern coast of the Sea of Okhotsk (Mak-Mak Lake and Elekchanskie lakes), and dolly varden. Mitotype AAAA proved to be common for these populations. Lake char populations of the Juliet and Maxi lakes (the basin of the Kolyma River) had mitotype DBAA, which is similar to mitotype DBAB observed earlier in Taranetz char from Chukotka. The divergence between mitotypes AAAA and DBAA was estimated at 0.3%. Different origins were assumed for the lake resident char populations from the basins of the Sea of Okhotsk and of the Kolyma River, the former originating in the Pacific and the latter, in the Arctic basins.     

New approach to the mitotype classification in black honeybee <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> and Iberian honeybee <Emphasis Type="Italic">Apis mellifera iberiensis</Emphasis>

The black honeybee Apis mellifera mellifera L. is today the only subspecies of honeybee which is suitable for commercial breeding in the climatic conditions of Northern Europe with long cold winters. The main problem of the black honeybee in Russia and European countries is the preservation of the indigenous gene pool purity, which is lost as a result of hybridization with subspecies, A. m. caucasica, A. m. carnica, A. m. carpatica, and A. m. armeniaca, introduced from southern regions. Genetic identification of the subspecies will reduce the extent of hybridization and provide the gene pool conservation of the black honeybee. Modern classification of the honeybee mitotypes is mainly based on the combined use of the DraI restriction endonuclease recognition site polymorphism and sequence polymorphism of the mtDNA COI–COII region. We performed a comparative analysis of the mtDNA COI–COII region sequence polymorphism in the honeybees of the evolutionary lineage M from Ural and West European populations of black honeybee A. m. mellifera and Spanish bee A. m. iberiensis. A new approach to the classification of the honeybee M mitotypes was suggested. Using this approach and on the basis of the seven most informative SNPs of the mtDNA COI–COII region, eight honeybee mitotype groups were identified. In addition, it is suggested that this approach will simplify the previously proposed complicated mitotype classification and will make it possible to assess the level of the mitotype diversity and to identify the mitotypes that are the most valuable for the honeybee breeding and rearing.     

Phylogeography of Pinus tabulaeformis Carr. (Pinaceae), a dominant species of coniferous forest in northern China

How coniferous trees in northern China changed their distribution ranges in response to Quaternary climatic oscillations remains largely unknown. Here we report a study of the phylogeography of Pinus tabulaeformis, an endemic and dominant species of coniferous forest in northern China. We examined sequence variation of maternally inherited, seed‐dispersed mitochondrial DNA (mtDNA) (nad5 intron 1 and nad4/3–4) and paternally inherited, pollen‐ and seed‐dispersed chloroplast DNA (cpDNA) (rpl16 and trnS‐trnG) within and among 30 natural populations across the entire range of the species. Six mitotypes and five chlorotypes were recovered among 291 trees surveyed. Population divergence was high for mtDNA variation (G_ST = 0.738, N_ST = 0.771) indicating low levels of seed‐based gene flow and significant phylogeographical structure (N_ST > G_ST, P < 0.05). The spatial distribution of mitotypes suggests that five distinct population groups exist in the species: one in the west comprising seven populations, a second with a north–central distribution comprising 15 populations, a third with a southern and easterly distribution comprising five populations, a fourth comprising one central and one western population, and a fifth comprising a single population located in the north‐central part of the species’ range. Each group apart from the fourth group is characterized by a distinct mitotype, with other mitotypes, if present, occurring at low frequency. It is suggested, therefore, that most members of each group apart from Group 4 are derived from ancestors that occupied different isolated refugia in a previous period of range fragmentation of the species, possibly at the time of the Last Glacial Maximum. Possible locations for these refugia are suggested. A comparison of mitotype diversity between northern and southern subgroups within the north‐central group of populations (Group 2) showed much greater uniformity in the northern part of the range both within and between populations. This could indicate a northward migration of the species from a southern refugium in this region during the postglacial period, although alternative explanations cannot be ruled out. Two chlorotypes were distributed across the geographical range of the species, resulting in lower levels of among‐population chlorotype variation. The geographical pattern of variation for all five chlorotypes provided some indication of the species surviving past glaciations in more than one refugium, although differentiation was much less marked, presumably due to the greater dispersal of cpDNA via pollen.     

Evidence for multi-copy Mega-NUMTs in the human genome

The maternal mode of mitochondrial DNA (mtDNA) inheritance is central to human genetics. Recently, evidence for bi-parental inheritance of mtDNA was claimed for individuals of three pedigrees that suffered mitochondrial disorders. We sequenced mtDNA using both direct Sanger and Massively Parallel Sequencing in several tissues of eleven maternally related and other affiliated healthy individuals of a family pedigree and observed mixed mitotypes in eight individuals. Cells without nuclear DNA, i.e. thrombocytes and hair shafts, only showed the mitotype of haplogroup (hg) V. Skin biopsies were prepared to generate ρ° cells void of mtDNA, sequencing of which resulted in a hg U4c1 mitotype. The position of the Mega-NUMT sequence was determined by fluorescence in situ hybridization and two different quantitative PCR assays were used to determine the number of contributing mtDNA copies. Thus, evidence for the presence of repetitive, full mitogenome Mega-NUMTs matching haplogroup U4c1 in various tissues of eight maternally related individuals was provided. Multi-copy Mega-NUMTs mimic mixtures of mtDNA that cannot be experimentally avoided and thus may appear in diverse fields of mtDNA research and diagnostics. We demonstrate that hair shaft mtDNA sequencing provides a simple but reliable approach to exclude NUMTs as source of misleading results.     

Novel Repetitive Structures, Deviant Protein-Encoding Sequences andUnidentified ORFs in the Mitochondrial Genome of the BrachiopodLingula anatina

Complete sequence determination of the brachiopod Lingula anatina mtDNA (28,818 bp) revealed an organization that is remarkably atypical for an animal mt-genome. In addition to the usual set of 37 animal mitochondrial genes, which make up only 57% (16,555 bp) of the entire sequence, the genome contains lengthy unassigned sequences. All the genes are encoded in the same DNA strand, generally in a compact way, whereas the overall gene order is highly divergent in comparison with known animal mtDNA. Individual genes are generally longer and deviate considerably in sequence from their homologues in other animals. The genome contains two major repeat regions, in which 11 units of unassigned sequences and six genes (atp8, trnM, trnQ, trnV, and part of cox2 and nad2) are found in repetition, in the form of nested direct repeats of unparalleled complexity. One of the repeat regions contains unassigned repeat units dispersed among several unique sequences, novel repetitive structure for animal mtDNAs. Each of those unique sequences contains an open reading frame for a polypeptide between 80 and 357 amino acids long, potentially encoding a functional molecule, but none of them has been identified with known proteins. In both repeat regions, tRNA genes or tRNA gene-like sequences flank major repeated units, supporting the view that those structures play a role in the mitochondrial gene rearrangements. Although the intricate repeated organization of this genome can be explained by recurrent tandem duplications and subsequent deletions mediated by replication errors, other mechanisms, such as nonhomologous recombinations, appear to explain certain structures more easily.     

Polymorphism of mitochondrial DNA and infection with symbiotic cytoplasmic bacterium <Emphasis Type="Italic">Wolbachia pipientis</Emphasis> in mosquitoes of the <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera,Culicidae) complex from Russia

A total of 208 mosquitoes of the Culex pipiens complex from 15 basement and terrestrial populations collected in different regions of the European part of Russia and Siberia were examined by genetic methods. Among these, two major mitotypes, M and P, were identified. These mitotypes differed by six substitutions in the 246-bp mitochondrial DNA cytochrome oxidase I gene fragment examined. Urban basement mosquitoes C. pipiens (form molestus) were characterized by the presence of mitotype M and infection with the endosymbiotic bacteria of the genus Wolbachia. Mosquitoes of the C. pipiens complex inhabiting opened biotopes harbored mitotype P, or its variety, mitotype P1, and were not infected with Wolbachia. Thus, in natural conditions marked linkage disequilibrium between cytoplasmic elements, mitochondrial DNA and Wolbachia, can be observed. Similarity of mitotypes in form molestus mosquito from different geographical localities favors the hypothesis on the common ancestry of urban mosquitoes.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 320–325.Original Russian Text Copyright © 2005 by Shaikevich, Vinogradova, Platonov, Karan, Zakharov.