Strict sex-specific mtDNA segregation in the germ line of the DUI species Venerupis philippinarum (Bivalvia: Veneridae)

Doubly Uniparental Inheritance (DUI) is one of the most striking exceptions to the common rule of standard maternal inheritance of metazoan mitochondria. In DUI, two mitochondrial genomes are present, showing different transmission routes, one through eggs (F-type) and the other through sperm (M-type). In this paper, we report results from a multiplex real-time quantitative polymerase chain reaction analysis on the Manila clam Venerupis philippinarum (formerly Tapes philippinarum). We quantified M- and F-types in somatic tissues, gonads, and gametes. Nuclear and external reference sequences were used, and the whole experimental process was designed to avoid any possible cross-contamination. In most male somatic tissues, the M-type is largely predominant: This suggests that the processes separating sex-linked mitochondrial DNAs (mtDNAs) in somatic tissues are less precise than in other DUI species. In the germ line, we evidenced a strict sex-specific mtDNA segregation because both sperm and eggs do carry exclusively M- and F-types, respectively, an observation that is in contrast with a previous analysis on Mytilus galloprovincialis. More precisely, whereas two mtDNAs are present in the whole gonad, only the sex-specific one is detected in gametes. Because of this, we propose that the mtDNA transmission is achieved through a three-checkpoint process in V. philippinarum. The cytological mechanisms of male mitochondria segregation in males and degradation in females during the embryo development (here named Checkpoint #1 and Checkpoint #2) are already well known for DUI species; a Checkpoint #3 would act when primordial germ cells (PGCs) are first formed and would work in both males and females. We believe that Checkpoint #3 is a mere variation of the "mitochondrial bottleneck" in species with standard maternal inheritance, established when their PGCs separate during embryo cleavage.     

The Mitochondrial Genome of Soybean Reveals Complex Genome Structures and Gene Evolution at Intercellular and Phylogenetic Levels

Determining mitochondrial genomes is important for elucidating vital activities of seed plants. Mitochondrial genomes are specific to each plant species because of their variable size, complex structures and patterns of gene losses and gains during evolution. This complexity has made research on the soybean mitochondrial genome difficult compared with its nuclear and chloroplast genomes. The present study helps to solve a 30-year mystery regarding the most complex mitochondrial genome structure, showing that pairwise rearrangements among the many large repeats may produce an enriched molecular pool of 760 circles in seed plants. The soybean mitochondrial genome harbors 58 genes of known function in addition to 52 predicted open reading frames of unknown function. The genome contains sequences of multiple identifiable origins, including 6.8 kb and 7.1 kb DNA fragments that have been transferred from the nuclear and chloroplast genomes, respectively, and some horizontal DNA transfers. The soybean mitochondrial genome has lost 16 genes, including nine protein-coding genes and seven tRNA genes; however, it has acquired five chloroplast-derived genes during evolution. Four tRNA genes, common among the three genomes, are derived from the chloroplast. Sizeable DNA transfers to the nucleus, with pericentromeric regions as hotspots, are observed, including DNA transfers of 125.0 kb and 151.6 kb identified unambiguously from the soybean mitochondrial and chloroplast genomes, respectively. The soybean nuclear genome has acquired five genes from its mitochondrial genome. These results provide biological insights into the mitochondrial genome of seed plants, and are especially helpful for deciphering vital activities in soybean.     

The complete nucleotide sequence of a snake (Dinodon semicarinatus) mitochondrial genome with two identical control regions.

The 17,191-bp mitochondrial DNA (mtDNA) of a Japanese colubrid snake, akamata (Dinodon semicarinatus), was cloned and sequenced. The snake mtDNA has some peculiar features that were found in our previous study using polymerase chain reaction: duplicate control regions that have completely identical sequences over 1 kbp, translocation of tRNALeu(UUR) gene, shortened TpsiC arm for most tRNA genes, and a pseudogene for tRNAPro. Phylogenetic analysis of amino acid sequences of protein genes suggested an unusually high rate of molecular evolution in the snake compared to other vertebrates. Southern hybridization experiments using mtDNAs purified from multiple akamata individuals showed that the duplicate state of the control region is not a transient or unstable feature found in a particular individual, but that it stably occurs in mitochondrial genomes of the species. This may, therefore, be regarded as an unprecedented example of stable functional redundancy in animal mtDNA. However, some of the examined individuals contain a rather scanty proportion of heteroplasmic mtDNAs with an organization of genes distinct from that of the major mtDNA. The gene organization of the minor mtDNA is in agreement with one of models that we present to account for the concerted evolution of duplicate control regions.     

Evidence for extreme sequence divergence between the male‐ and female‐transmitted mitochondrial genomes in the bivalve mollusc,Modiolus modiolus (Mytilidae)

Marine mussels of the family Mytilidae, as well as a number of other bivalves, have a unique system of mitochondrial DNA inheritance called doubly uniparental inheritance (DUI). DUI is characterized by the presence of an ‘F’ mitochondrial genome that is transmitted through mothers to daughters and sons, and an ‘M’ mitochondrial genome that is transmitted only from fathers to sons. In this paper, we demonstrate that DUI exists in the horse mussel, Modiolus modiolus (Linnaeus, 1758) and compare the pattern of molecular evolution of the M and F types in this species. Total DNA was isolated from M. modiolus male and female gonad tissues, as well as from spawned sperm cells. From these DNA samples, partial mitochondrial DNA fragments were amplified from both cytochrome c oxidase subunit I (cox1), and 16S ribosomal RNA (rrnL) genes. Based on cox1 and rrnL sequences, heteroplasmy was observed in M. modiolus and characterized by the resolution of two mitotypes: an F mitotype present in tissues of both males and females, and an M mitotype present in spawned sperm. Using standardized p‐distance and Tamura‐Nei values, M. modiolus is found to display the highest M/F conspecific sequence divergence for any member of the family Mytilidae (i.e. 38% M/F sequence divergence, which is 9% higher than any other intraspecific M/F comparison for the family Mytilidae when standardized using p‐distances across all taxa observed). Sequence analysis also indicated that the M. modiolus M mitotype evolves significantly faster than its conspecific F type. The findings discussed herein broaden the range of mytilid species known to exhibit DUI and they also establish a new threshold for the genetic divergence of male mytilid mitochondrial genomes.     

The largest unassigned regions of the male- and female-transmitted mitochondrial DNAs in Musculista senhousia (Bivalvia Mytilidae)

Musculista senhousia is a marine mussel with doubly uniparental inheritance (DUI) of mitochondria. In this study we analyzed the largest unassigned region (LUR) of its female- and male-transmitted mitochondrial genomes, described their fine characteristics and searched for shared features. Our results suggest that both LURs contain the control region of their respective mitochondrial genomes. The female-transmitted control region is duplicated in tandem, with the two copies evolving in concert. This makes the F-mtDNA of M. senhousia the first Bivalve mitochondrial genome with this feature. We also compared M. senhousia control regions to that of other Mytilidae, and demonstrated that signals for basic mtDNA functions are retained over evolutionary times even among the fast-evolving mitochondrial genomes of DUI species. Finally, we discussed how similarities between female and male LURs may be explained in the context of DUI evolution and if the duplicated female control region might have influenced the DUI system in this species.     

Footprints of unconventional mitochondrial inheritance in bivalve phylogeny: Signatures of positive selection on clades with doubly uniparental inheritance

The doubly uniparental inheritance (DUI) of some bivalve mollusks is the major exception to the common maternal inheritance of mitochondria in animals. DUI involves two mitochondrial lineages with paternal and maternal transmission routes, and it appears as a complex phenomenon requiring both nuclear and mitochondrial adaptations. DUI distribution seems to be scattered among the Bivalvia, and there are several clues for its multiple origins. In this paper, we investigate whether the incipient DUI systems had left possible selective signatures on mitochondrial genomes. Alongside the outstanding divergence of amino acid sequences, we confirmed strong purifying selection to act on mitochondrial genes. However, we found evidence that distinct episodes of intense directional pressure are associated with the origins of different DUI systems: We interpret these signals as footprints of the coevolution with the nuclear genome that ought to take place at the base of a DUI clade. Six genes (atp6, cox1, cox2, cox3, nad4L, and nad6) seem to be more commonly linked to the appearance of DUI. We also identified few putative DUI‐specific mutations, thus extending support to the hypothesis of multiple independent origins of this complex phenomenon.     

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).     

Comparative analysis of gender-associated complete mitochondrial genomes in marine mussels (Mytilus spp.)

Marine mussels of the genus Mytilus have an unusual mode of mitochondrial DNA (mtDNA) transmission termed doubly uniparental inheritance (DUI). Female mussels are homoplasmic for the F mitotype, which is inherited maternally, while males are usually heteroplasmic, carrying a mixture of the maternal F mitotype and the paternally inherited M genome. Two classes of M genomes have been observed: "standard" M genomes and "recently masculinized" M genomes. The latter are more similar to F genomes at the sequence level but are transmitted paternally like standard M genomes. In this study we report the complete sequences of two standard male M. edulis and one recently masculinized male M. trossulus mitochondrial genome. A comparative analysis, including the previously sequenced M. edulis F and M. galloprovincialis F and M mtDNAs, reveals that these genomes are identical in gene order, but highly divergent in nucleotide and amino acid sequence. The large amount (>20%) of nucleotide substitutions that fall in coding regions implies that there are several amino acid replacements between the F and M genomes, which likely have an impact on the structural and functional properties of the mitochondrial proteome. Correlation of the divergence rate of different protein-coding genes indicates that mtDNA-encoded proteins of the M genome are still under selective constraints, although less highly than genes of the F genome. The mosaic F/M control region of the masculinized F genome provides evidence for lineage-specific sequences that may be responsible for the different mode of transmission genetics. This analysis shows the value of comparative genomics to better understand the mechanisms of maintenance and segregation of mtDNA sequence variants in mytilid mussels.     

Genetic hitchhiking,mitonuclear coadaptation,and the origins of mt DNA barcode gaps

DNA barcoding based on mitochondrial (mt) nucleotide sequences is an enigma. Neutral models of mt evolution predict DNA barcoding cannot work for recently diverged taxa, and yet, mt DNA barcoding accurately delimits species for many bilaterian animals. Meanwhile, mt DNA barcoding often fails for plants and fungi. I propose that because mt gene products must cofunction with nuclear gene products, the evolution of mt genomes is best understood with full consideration of the two environments that impose selective pressure on mt genes: the external environment and the internal genomic environment. Moreover, it is critical to fully consider the potential for adaptive evolution of not just protein products of mt genes but also of mt transfer RNAs and mt ribosomal RNAs. The tight linkage of genes on mt genomes that do not engage in recombination could facilitate selective sweeps whenever there is positive selection on any element in the mt genome, leading to the purging of mt genetic diversity within a population and to the rapid fixation of novel mt DNA sequences. Accordingly, the most important factor determining whether or not mt DNA sequences diagnose species boundaries may be the extent to which the mt chromosomes engage in recombination.     

Analysis of the complete human mtDNA genome: methodology and inferences for human evolution

The analysis of mitochondrial DNA (mtDNA) sequences has been a potent tool in our understanding of human evolution. However, almost all studies of human evolution based on mtDNA sequencing have focused on the control region, which constitutes less than 7% of the mitochondrial genome. The rapid development of technology for automated DNA sequencing has made it possible to study the complete mtDNA genomes in large numbers of individuals, opening the field of mitochondrial population genomics. Here we describe a suitable methodology for determining the complete human mitochondrial sequence and the global mtDNA diversity in humans. Also, we discuss the implications of the results with respect to the different hypotheses for the evolution of modern humans.     

The mitogenome of Paphia euglypta (Bivalvia: Veneridae) and comparative mitogenomic analyses of three venerids

Extraordinary variation has been found in mitochondrial (mt) genome inheritance, gene content and arrangement among bivalves. However, only few bivalve mt genomes have been comparatively analyzed to infer their evolutionary scenarios. In this study, the complete mt genome of the venerid Paphia euglypta (Bivalvia: Veneridae) was firstly studied and, secondly, it was comparatively analyzed with other venerids (e.g., Venerupis philippinarum and Meretrix petechialis) to better understand the mt genome evolution within a family. Though several common features such as the AT content, codon usage of protein-coding genes, and AT/GC skew are shared by the three venerids, a high level of variability is observed in genome size, gene content, gene order, arrangements and primary sequence of nucleotides or amino acids. Most of the gene rearrangement can be explained by the "tandem duplication and random loss" model. From the observed rearrangement patterns, we speculate that block interchange between adjacent genes may be common in the evolution of mt genomes in venerids. Furthermore, this study presents several new findings in mt genome annotation of V. philippinarum and M. petechialis, and hence we have reannotated the genome of these two species as: (1) the ORF of the formerly annotated cox2 gene in V. philippinarum is deduced by using a truncated "T" codon and a second cox2 gene is identified; (2) the trnS-AGN gene is identified and marked in the mt genome of both venerids. Thus, this study demonstrated a high variability of mt genomes in the Veneridae, and showed the importance of comparative mt genome analysis to interpret the evolution of the bivalve mt genome.     

The Complete Female- and Male-Transmitted Mitochondrial Genome of Meretrix lamarckii

Bivalve mitochondrial genomes show many uncommon features, like additional genes, high rates of gene rearrangement, high A-T content. Moreover, Doubly Uniparental Inheritance (DUI) is a distinctive inheritance mechanism allowing some bivalves to maintain and transmit two separate sex-linked mitochondrial genomes. Many bivalve mitochondrial features, such as gene extensions or additional ORFs, have been proposed to be related to DUI but, up to now, this topic is far from being understood. Several species are known to show this unusual organelle inheritance but, being widespread only among Unionidae and Mytilidae, DUI distribution is unclear. We sequenced and characterized the complete female- (F) and male-transmitted (M) mitochondrial genomes of Meretrix lamarckii, which, in fact, is the second species of the family Veneridae where DUI has been demonstrated so far. The two mitochondrial genomes are comparable in length and show roughly the same gene content and order, except for three additional tRNAs found in the M one. The two sex-linked genomes show an average nucleotide divergence of 16%. A 100-aminoacid insertion in M. lamarckii M-cox2 gene was found; moreover, additional ORFs have been found in both F and M Long Unassigned Regions of M. lamarckii. Even if no direct involvement in DUI process has been demonstrated so far, the finding of cox2 insertions and supernumerary ORFs in M. lamarckii both strengthens this hypothesis and widens the taxonomical distribution of such unusual features. Finally, the analysis of inter-sex genetic variability shows that DUI species form two separate clusters, namely Unionidae and Mytilidae+Veneridae; this dichotomy is probably due to different DUI regimes acting on separate taxa.     

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.