Types Of Mitochondrial Genomes Research Articles

Linear DNA-driven recombination in mammalian mitochondria.

Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic due to its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we took advantage of a cell model with two different types of mitochondrial genomes and impaired its ability to degrade broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot and by long range PacBio® HiFi sequencing approach. Besides utilizing different electrophoretic methods, we also directly observed molecular complexes between different mtDNA haplotypes and recombination intermediates using transmission electron microscopy. We propose that the known copy-choice recombination by mitochondrial replisome could be sufficient for the needs of the small genome, thus removing the requirement for a specialized mitochondrial recombinase. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.

Assembly and marker analysis of mitochondrial genomes provide insights into origin, evolution and spread of Brassica juncea (L.) Czern. et Coss.

The release of mitochondrial genome sequences provides the basis for characterizing interspecific and intraspecific variation in Brassica mitochondrial genomes. However, few B. juncea (mustard) mitochondrial genomes have been published. We assembled the mitochondrial genomes of three B. juncea subspecies and compared them with previously published genomes. The genomes were phylogenetically classified into A, B, C, and Bna clades. Two variant sites, a transversion (C → A) at nt 79,573 and a 31-bp copy-number variation between nts 65,564 and 65,596, were identified. Based on these variant sites, mitotype-specific sequence markers were developed to characterize the variation among worldwide 558 B. juncea accessions. Three mitochondrial genome types (mitotypes MT1–MT3) were identified. In terms of geographical distribution, MT1 and MT2 accessions were distributed mainly to the north and MT3 to the south of 34°N. Root mustards carried only MT1, leaf and stem mustards carried mainly MT3, and seed mustards carried all three mitotypes, implying that the mitotypes underwent selection during B. juncea domestication. A new form of oil mustard evolved by hybridization between two gene pools in southwest China.

Nuclear and mitochondrial DNA polymorphisms suggest introgression contributed to garden beet (Beta vulgaris L.) domestication

Garden beet is the ancestor of fodder beets and sugar beets, but the origin of garden beet’s genetic potential to evolve novel beet types is debatable. In this study, we analyzed nuclear and mitochondrial DNAs in 47 garden beet accessions using DNA markers. Multiple analytical methods revealed a unified population structure with subpopulations evident in the European and Caucasian accessions. We diagnosed mitochondrial genome types (mitotypes) based on mitochondrial minisatellite loci in 541 plants from the 47 accessions, revealing a major mitotype and 11 minor mitotypes in garden beets from Europe and the Caucasus region that were also present in endemic leaf beets and wild beets. Our data indicate that European and Caucasian garden beets include genetically differentiated subpopulations. Provided that the occurrence of minor mitotypes is a vestige from crosses with leaf beets and wild beets, the notion that introgression contributed to increasing the genetic diversity in the garden beet gene pool is substantiated at the molecular level.

The complete mitochondrial genome of Dapulian pig (Sus scrofa) and a phylogenetic study

The two types of mitochondrial genomes of Dapulian pig has been sequenced and annotated completely. The entire genome is 16,610 bp in length with an A + T content of 60.6% (GenBank accession No. MG009445, MG009446). This mitochondrial genome consisted of 13 protein-coding genes, 2 ribosomal RNA subunits and 22 transfer RNAs, and 1 D-loop control regions. A phylogenetic tree with the near-complete mitochondrial genome sequences of two types of Dapulian pigs together with 17 other Chinese pig breeds was constructed. The results can be subsequently used to provide information for pig phylogenetic and insights into the evolution of genomes. The mitochondrial genome would play an important role in population genetics and phylogenetic analysis of Sus scrofa.

Comparative mitochondrial genome analysis reveals the evolutionary rearrangement mechanism in Brassica.

The genus Brassica has many species that are important for oil, vegetable and other food products. Three mitochondrial genome types (mitotype) originated from its common ancestor. In this paper, a B.nigra mitochondrial main circle genome with 232,407bp was generated through de novo assembly. Synteny analysis showed that the mitochondrial genomes of B.rapa and B.oleracea had a better syntenic relationship than B.nigra. Principal components analysis and development of a phylogenetic tree indicated maternal ancestors of three allotetraploid species in Us triangle of Brassica. Diversified mitotypes were found in allotetraploid B.napus, in which napus-type B.napus was derived from B.oleracea, while polima-type B.napus was inherited from B.rapa. In addition, the mitochondrial genome of napus-type B.napus was closer to botrytis-type than capitata-type B.oleracea. The sub-stoichiometric shifting of several mitochondrial genes suggested that mitochondrial genome rearrangement underwent evolutionary selection during domestication and/or plant breeding. Our findings clarify the role of diploid species in the maternal origin of allotetraploid species in Brassica and suggest the possibility of breeding selection of the mitochondrial genome.

Mitochondrial SSRs and their utility in distinguishing wild species, CMS lines and maintainer lines in pigeonpea (Cajanus cajan L.)

AbstractAnalysis of the pigeonpea mitochondrial genome sequence identified 25 SSRs. Mononucleotide SSR motifs were the most abundant repeats followed by dinucleotide and trinucleotide repeats. Primer pairs could be designed for 24 SSRs, 23 of which were polymorphic amongst the 22 genotypes consisting of cytoplasmic male sterile (CMS or A) line, maintainer or B line and wild Cajanus species representing six different CMS systems viz., A1, A2, A4, A5, A6 and A8. These markers amplified a total of 107 alleles ranging from 2 to 10 with an average of 4.65 alleles per locus. The polymorphic information content for these markers ranged from 0.09 to 0.84 with an average of 0.52 per marker. Hence, the present study adds a novel set of 24 mitochondrial SSR markers to the markers repository in pigeonpea, which would be useful to distinguish the genotypes based on mitochondrial genome types in evolutionary and phylogenetic studies.

Presence of two mitochondrial genomes in the mytilid Perumytilus purpuratus: Phylogenetic evidence for doubly uniparental inheritance.

This study presents evidence, using sequences of ribosomal 16S and COI mtDNA, for the presence of two mitochondrial genomes in Perumytilus purpuratus. This may be considered evidence of doubly uniparental mtDNA inheritance. The presence of the two types of mitochondrial genomes differentiates females from males. The F genome was found in the somatic and gonadal tissues of females and in the somatic tissues of males; the M genome was found in the gonads and mantle of males only. For the mitochondrial 16S region, ten haplotypes were found for the F genome (nucleotide diversity 0.004), and 7 haplotypes for the M genome (nucleotide diversity 0.001), with a distance Dxy of 0.125 and divergence Kxy of 60.33%. For the COI gene 17 haplotypes were found for the F genome (nucleotide diversity 0.009), and 10 haplotypes for the M genome (nucleotide diversity 0.010), with a genetic distance Dxy of 0.184 and divergence Kxy of 99.97%. Our results report the presence of two well-differentiated, sex-specific types of mitochondrial genome (one present in the male gonad, the other in the female gonad), implying the presence of DUI in P. purpuratus. These results indicate that care must be taken in phylogenetic comparisons using mtDNA sequences of P. purpuratus without considering the sex of the individuals.

The complete mitochondrial genome from an unidentified Phalansterium species.

We describe the complete sequence and organization of the mitochondrial genome from an unidentified species of Phalansterium. This is the first sequenced mitochondrial genome of a member of Variosea clade (Amoebozoa, Conosa). The sequence was assembled from shotgun reads of DNA from a mixed culture containing the euglenid Monomorphina aenigmatica and an amoebozoan that we demonstrate here is closely related to Phalansterium (in nuclear SSU rRNA phylogenies, it branches between two sequences from described species of Phalansterium). Sequence assembly resulted in two distinct mitochondrial genome types, one fragmented and euglenid-like, and the second a single circular- mapping contig of 53,614 bp with an amoebozoan-like set of genes. The Phalansterium sp. mitochondrial genome is gene-rich and densely packed, with a large number of tRNAs and an unusually low ratio of identifiable protein-coding genes to unidentified ORFs. These ORFs potentially encode ribosomal proteins exhibiting a divergent character at the sequence level, and whose identification may be hindered by the presence of RNA editing in Phalansterium mitochondria, as inferred from numerous acceptor stem mis-matches typical of amoebozoan tRNA 5' editing.

A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.)

BackgroundPlant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138.ResultsOgura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura-type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3 kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences.ConclusionsOgura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.

Discovery of a novel cytoplasmic male-sterility and its restorer lines in radish (Raphanus sativus L.)

A male-sterile (MS) radish (Raphanus sativus L.) was found in an accession collected from Uzbekistan. Unlike Ogura MS radishes in which no pollen grain is typically visible during anthesis, a small number of pollen grains stuck together in the dehiscing anthers was observed in the newly identified MS radish. Fluorescein diacetate tests and scanning electron micrographs showed that pollen grains in the new MS radish were severely deformed and non-viable. Cytological examination of pollen development stages showed a clear difference in the defective stage from that seen in Ogura male-sterility. Reciprocal cross-pollination with diverse male-fertile lines indicated that pollen grains of the new MS radish were completely sterile, and the female organs were fully fertile. When the new MS radish and Ogura MS lines were cross-pollinated with a set of eight breeding lines, all F1 progeny originating from crosses with the new MS radish were male-sterile. In contrast, most of the F1 progeny resulting from crosses with Ogura MS lines were male-fertile. These results demonstrated that factors associated with induction of the newly identified male-sterility are different from those of Ogura male-sterility. The lack of restorer lines for the newly identified male-sterility led us to predict that it might be a complete cytoplasmic male-sterility without restorer-of-fertility genes in nuclear genomes. However, cross-pollination with more diverse radish germplasm identified one accession introduced from Russia that could completely restore fertility, proving the existence of restorer-of-fertility gene(s) for the new male-sterility. Meanwhile, the PCR amplification profile of molecular markers for the classification of radish mitochondrial genome types revealed that the new MS radish contained a novel mitotype.

Investigation of the origin and transmission of linear mitochondrial plasmid based on phylogenetic analysis in Japanese rapeseed varieties

A linear mitochondrial plasmid is present in some varieties of rapeseed. To elucidate its origin and transmission the author investigated types of mitochondrial genome and the presence of plasmid in 78 rapeseed varieties and landraces in Japan and carried out a comparative analysis using the breeding history of Japanese rapeseed varieties. The mitochondrial genome of rapeseed was classified roughly into 2 types, type I (nap) and type II (cam). Type II rapeseed mitochondria closely resembles that of Brassica rapa, which is a related species of rapeseed. In this study, the author found that all varieties with type II mitochondria originated from interspecific crosses between rapeseed (B. napus) and B. rapa. This indicates that type II cytoplasm was introduced to rapeseed through a breeding program. The presence of plasmid was limited to B. rapa landraces and rapeseed varieties that arose by interspecific crosses between B. napus and B. rapa. The results suggest that mitochondrial plasmid is of B. rapa origin and that it has been introduced into rapeseed by interspecific crosses in a modern breeding program, as in the case of the mitochondrial genome. Phylogenetic study of Japanese rapeseed varieties suggests the participation not of the mitochondrial genome but, rather, the nuclear genome for the perpetuation of plasmid in progeny varieties.

MITOCHONDRIAL BIOCHEMICAL ACTIVITIES AND HETEROPLASMY EVOLUTION IN ESTABLISHED D. SUBOBSCURA CELL LINE

A mutant strain of drosophila (D. subobscura) has two types of mitochondrial genomes: a small population (20%) identical to that of the wild strain (15.9 kb) and a predominant population (80%) which has undergone a 5-kb deletion affecting more than 30% of the coding zone. Two cell lines were established from homogenates of embryos from mutant and wild strains. The activities of the respiratory complexes measured in the different cell lines are much lower than in the flies, indicating a glycolytic metabolism. Various modifications of the medium composition did not change this metabolic pathway. The mutant cell line has two types of populations of mitochondrial genomes and the heteroplasmy is equivalent to that measured in the mutant strain. However, the biochemical characteristics differ from those observed in the flies (i.e., the decrease of complex I and III activities), and the various systems of compensation for the consequences of the deletion that are showed in the mutant strain are no longer observed. Furthermore, in contrast with observations made on mutant flies, the heteroplasmy appears unstable in the mutant cell lines: after 60 or so generations, it progressively decreases until it disappears completely. The limited importance of mitochondrial energy metabolism in cells may explain the low impact of the mutation on the established cell line, in contrast to what is seen in the mutant strain.

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

Mitochondrial genome diversity: evolution of the molecular architecture and replication strategy.

Mitochondrial genomes in organisms from diverse phylogenetic groups vary in both size and molecular form. Although the types of mitochondrial genome appear very dissimilar, several lines of evidence argue that they do not differ radically. This would imply that interconversion between different types of mitochondrial genome might have occurred via relatively simple mechanisms. We exemplify this scenario on patterns accompanying evolution of mitochondrial telomeres. We propose that mitochondrial telomeres are derived from mobile elements (transposons or plasmids) that invaded mitochondria, integrated into circular or polydisperse linear mitochondrial DNAs (mtDNAs) and subsequently enabled precise resolution of the linear genophore. Simply, the selfish elements generated a problem - how to maintain the ends of a linear DNA - and, at the same time, made themselves essential by providing its solution. This scenario implies that insertion or deletion of such resolution elements may represent relatively simple routes for interconversion between different forms of the mitochondrial genome.

The complete mitochondrial DNA sequence of Scenedesmus obliquus reflects an intermediate stage in the evolution of the green algal mitochondrial genome.

Two distinct mitochondrial genome types have been described among the green algal lineages investigated to date: a reduced-derived, Chlamydomonas-like type and an ancestral, Prototheca-like type. To determine if this unexpected dichotomy is real or is due to insufficient or biased sampling and to define trends in the evolution of the green algal mitochondrial genome, we sequenced and analyzed the mitochondrial DNA (mtDNA) of Scenedesmus obliquus. This genome is 42,919 bp in size and encodes 42 conserved genes (i.e., large and small subunit rRNA genes, 27 tRNA and 13 respiratory protein-coding genes), four additional free-standing open reading frames with no known homologs, and an intronic reading frame with endonuclease/maturase similarity. No 5S rRNA or ribosomal protein-coding genes have been identified in Scenedesmus mtDNA. The standard protein-coding genes feature a deviant genetic code characterized by the use of UAG (normally a stop codon) to specify leucine, and the unprecedented use of UCA (normally a serine codon) as a signal for termination of translation. The mitochondrial genome of Scenedesmus combines features of both green algal mitochondrial genome types: the presence of a more complex set of protein-coding and tRNA genes is shared with the ancestral type, whereas the lack of 5S rRNA and ribosomal protein-coding genes as well as the presence of fragmented and scrambled rRNA genes are shared with the reduced-derived type of mitochondrial genome organization. Furthermore, the gene content and the fragmentation pattern of the rRNA genes suggest that this genome represents an intermediate stage in the evolutionary process of mitochondrial genome streamlining in green algae.

Phylogenetic relationships of the mitochondrial genomes in the genus Glycine subgenus Soja.

Restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) of wild and cultivated soybeans were analyzed to study their phylogenetic relationships. The observed number of differences in hybridization profiles greatly varied (1-17 patterns) with both the mtDNA probes and the restriction enzymes that were used. A cladistic analysis was conducted based on the RFLP data. In the parsimonious tree, four distinct groups appeared among 20 accessions of the subgenus Soja representing 20 mitochondrial genome types. Common features with regard to geographic distributions in natural populations in East Asia were observed among the mitochondrial genome types of wild soybean that belonged to the same group: one clade consisted of genome types IIg and VIIg that are detected with very low frequencies; another clade consisted of genome types Ic, Id, Ie, and Ik whose distributions are highly biased mainly in Japan. The genome types that are widely distributed in East Asia such as IVa, IVb, and Va were not grouped into the same clade. The mitochondrial genome types IIIb, IVb, and IVc, in which two different chloroplast genome types exist, belonged to the same clade. Possible changes in mitochondrial genomes during the expansion of the distribution of wild soybeans in East Asia were discussed.

A second type of normal maize mitochondrial genome: an evolutionary link.

A new type of maize mitochondrial genome has been identified in the male fertile (normal) inbred line A188. It has been named NA (N in the A188 nuclear background). In comparison to previously described maize mitochondrial genomes, it is classified as a new type since the genome contains unique DNA sequences and unique sets of repeated sequences, and has a unique organization. This brings the number of the maize mitochondrial genome types to five of which three are the cytoplasmic male steriles cmsT, cmsC and cmsS and of which two are the male fertile types NA (in this report) and NB (the previously characterized normal genome in the B37 nuclear background).

Tissular distribution of heteroplasmy and ultrastructural studies of mitochondria from a Drosophila subobscura mitochondrial deletion mutant

A mutant strain of Drosophila subobscura possesses two mitochondrial genome types: a minority population (20%) identical to the wild strain mtDNA (15.9 kb), and a largely predominant population (80%) of shorter genomes (10.9 kb), presenting a deletion of more than 30% of its coding region. Study of tissular distribution of heteroplasmy shows it to be identical--about 80%--in the head (nervous tissue) and thorax (muscles). On the other hand, a lower percentage (64%) is observed in the ovaries. The strain is apparently unaffected despite this massive loss of genes, coding for four tRNA and for complex I and III subunits. Contrary to observations of similar situations in man, the mutant strain shows no accumulation or structurally abnormal mitochondria. Furthermore, cytochemical studies fail to detect mitochondria devoid of cytochrome oxidase activity (COX-). Finally, mitoribosome populations are identical in mitochondria from both strains. These results suggest that, in the mutant strain, there are no mitochondria containing deleted genomes only: heteroplasmy would thus be intramitochondrial.

Intraspecific heterogeneity of the Vicia faba mitochondrial genome: evidence for multiregional rearrangements in the mitochondrial chromosome associated with coxII-orf192 chimeric gene formation

Previous RFLP-analysis of mtDNA isolated from different lines and cultivars of Vicia faba with respect to variability of the coxII gene revealed two types of mitochondrial genome: one with a normal coxII gene and the other with both normal coxII and chimeric coxII-orf192 genes. In this study we analyzed other regions of these two types of mitochondrial genome and found significant differences in the arrangement of regions around the coxII, coxIII, cob, rrn26 and atpA genes. More detailed analysis of the rrn26 and atpA gene regions showed that these genes are associated with recombinationally active repeats. Restriction maps of the rrn26 and atpA gene regions in different recombinative variants are presented.

Characterization of Vicia faba mitochondrial genomes with normal coxII and coxII-orf 192 chimeric genes

Southern hybridization analysis of mitochondrial genomes from different lines and cultivars of Vicia faba, with respect to variability of the coxII gene sequence, revealed two predominant types of mitochondrial genomes. The type I mitochondrial genome contained the coxII gene sequence in a 6.5-kb BamHI fragment. Type II had two copies of the coxII sequence: the first in a 6.5-kb and the second in a 2.6-kb BamHI fragment. The second copy was represented by a coxII-orf192 chimeric gene. We found several pure lines with type I and type II mitochondrial genomes. Each type of genome was stably inherited. No chimeric gene was found in mitochondria of the male-sterile line cms447. Nucleotide sequences of Vicia faba mitochondrial DNA (mtDNA) containing normal and chimeric coxII genes are presented. The sequence of the normal coxII gene was compared to the coxII gene from mitochondria of Pisum sativum. The similarity of nucleotide sequences and of predicted amino acid sequences between these two genes was more than 98%. A very high similarity between transcription initiation and termination signals was also observed. The sequence of the chimeric gene was characterized at the 5' end by the almost complete sequence of the normal coxII gene, up to the fifth nucleotide before the termination codon. The 3' end of the chimeric gene was represented by the 3' part of an orf previously called orf128+. The full size of this orf was 576 nucleotides, and the full size of the predicted polypeptide was 192 amino acid residues. Therefore, this orf can be finally called orf192. Northern hybridization analysis showed that orf192 was actively transcribed into a 1.4-kb transcript. The chimeric gene was also transcribed into a minor transcript of about 3 kb. Comparative analysis of the normal coxII gene and orf192 supported the suggestion that the chimeric gene resulted from nonhomologous recombination.