Genetic mapping of paternal sorting of mitochondria in cucumber

Mitochondria are organelles that have their own DNA; serve as the powerhouses of eukaryotic cells; play important roles in stress responses, programmed cell death, and ageing; and in the vast majority of eukaryotes, are maternally transmitted. Strict maternal transmission of mitochondria makes it difficult to select for better-performing mitochondria, or against deleterious mutations in the mitochondrial DNA. Cucumber is a useful plant for organellar genetics because its mitochondria are paternally transmitted and it possesses one of the largest mitochondrial genomes among all eukaryotes. Recombination among repetitive motifs in the cucumber mitochondrial DNA produces rearrangements associated with strongly mosaic (MSC) phenotypes. We previously reported nuclear control of sorting among paternally transmitted mitochondrial DNAs. The goal of this project was to map paternal sorting of mitochondria as a step towards its eventual cloning. We crossed single plants from plant introduction (PI) 401734 and Cucumis sativus var. hardwickii and produced an F(2) family. A total of 425 F(2) plants were genotyped for molecular markers and testcrossed as the female with MSC16. Testcross families were scored for frequencies of wild-type versus MSC progenies. Discrete segregations for percent wild-type progenies were not observed and paternal sorting of mitochondria was therefore analyzed as a quantitative trait. A major quantitative trait locus (QTL; LOD >23) was mapped between two simple sequence repeats encompassing a 459-kb region on chromosome 3. Nuclear genes previously shown to affect the prevalence of mitochondrial DNAs (MSH1, OSB1, and RECA homologs) were not located near this major QTL on chromosome 3. Sequencing of this region from PI 401734, together with improved annotation of the cucumber genome, should result in the eventual cloning of paternal sorting of mitochondria and provide insights about nuclear control of organellar-DNA sorting.     

Exploiting synteny in Cucumis for mapping of Psm: a unique locus controlling paternal mitochondrial sorting

The three genomes of cucumber show different modes of transmission, nuclear DNA bi-parentally, plastid DNA maternally, and mitochondrial DNA paternally. The mosaic (MSC) phenotype of cucumber is associated with mitochondrial DNA rearrangements and is a valuable tool for studying mitochondrial transmission. A nuclear locus (Psm) has been identified in cucumber that controls sorting of paternally transmitted mitochondrial DNA. Comparative sequencing and mapping of cucumber and melon revealed extensive synteny on the recombinational and sequence levels near Psm and placed this locus on linkage group R of cucumber and G10 of melon. However, the cucumber genomic region near Psm was surprisingly monomorphic with an average of one SNP every 25 kb, requiring that a family from a more diverse cross is produced for fine mapping and eventual cloning of Psm. The cucumber ortholog of Arabidopsis mismatch repair (MSH1) was cloned and it segregated independently of Psm, revealing that this candidate gene is not Psm.     

The selection of mosaic (MSC) phenotype after passage of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) through cell culture — a method to obtain plant mitochondrial mutants

Mosaic (MSC) mutants of cucumber (Cucumis sativus L.) appear after passage through cell cultures. The MSC phenotype shows paternal transmission and is associated with mitochondrial DNA rearrangements. This review describes the origins and phenotypes of independently produced MSC mutants of cucumber, including current knowledge on their mitochondrial DNA rearrangements, and similarities of MSC with other plant mitochondrial mutants. Finally we propose that passage of cucumber through cell culture can be used as a unique and efficient method to generate mitochondrial mutants of a higher plant in a highly homozygous nuclear background.     

Maternal inheritance of mitochondrial genomes and complex inheritance of chloroplast genomes in Actinidia Lind.: evidences from interspecific crosses

The inheritance pattern of chloroplast and mitochondria is a critical determinant in studying plant phylogenetics, biogeography and hybridization. To better understand chloroplast and mitochondrial inheritance patterns in Actinidia (traditionally called kiwifruit), we performed 11 artificial interspecific crosses and studied the ploidy levels, morphology, and sequence polymorphisms of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of parents and progenies. Sequence analysis showed that the mtDNA haplotypes of F1 hybrids entirely matched those of the female parents, indicating strictly maternal inheritance of Actinidia mtDNA. However, the cpDNA haplotypes of F1 hybrids, which were predominantly derived from the male parent (9 crosses), could also originate from the mother (1 cross) or both parents (1 cross), demonstrating paternal, maternal, and biparental inheritance of Actinidia cpDNA. The inheritance patterns of the cpDNA in Actinidia hybrids differed according to the species and genotypes chosen to be the parents, rather than the ploidy levels of the parent selected. The multiple inheritance modes of Actinidia cpDNA contradicted the strictly paternal inheritance patterns observed in previous studies, and provided new insights into the use of cpDNA markers in studies of phylogenetics, biogeography and introgression in Actinidia and other angiosperms.     

Analysis of paternal transmission of mitochondrial DNA in Drosophila

It has previously been shown that paternal mitochondrial DNA (mtDNA) can be detected in later generations in Drosophila. To further analyze the paternal transmission of mtDNA, the progeny of two intraspecific and three interspecific crosses were examined in the frequency of the paternal transmission of mtDNA, using closely related species of the melanogaster species subgroup. Types of mtDNA in the progeny of the individual backcrosses of F(1) females were analyzed by selective amplification of paternal mtDNA. More than 100 F(1) females were examined for each backcross. The same type of mtDNA as that of the paternal mtDNA was detected in approximately 20-60% of the backcrosses. The present results indicate that paternal leakage occurs in the intraspecific crosses as well as in the interspecific crosses in Drosophila.     

The identification of quantitative trait loci that control the paternal inheritance of a mitochondrial plasmid in rapeseed (Brassica napus L.)

Some varieties of Brassica napus (rapeseed) and B. rapa contain a liner mitochondrial plasmid that is unique in that it can be inherited from the male parent through the pollen. We found that two rapeseed cultivars, Norin 16 and Westar, showed different rates of plasmid inheritance from the paternal parent (78.8% and 27.5%, respectively). To identify nuclear genes controlling the inheritance of the plasmid, we carried out quantitative trait locus (QTL) analyses using F(2) populations derived from a cross between these two cultivars. The F1 plants transmitted the plasmid from the paternal plant at a frequency of approximately 60%; the transmission rates of the F2 lines varied greatly, from 0 to 100%, with an average of 68.2%. A genetic map was constructed based on the segregation of 175 loci in the 102 F2 plants. A total of 22 linkage groups were obtained, all of which could be assigned to the 19 rapeseed chromosomes. The total map length was 1374.7 cM, with an average distance of 7.9 cM between the markers. We found that three quantitative trait loci for plasmid paternal transfer, qPpt1, qPpt2 and qPpt3, located on chromosomes A5, C2 and C9, respectively, were significantly linked to the transmission frequency, whose the logarithm of odds (LOD) score were 4.97, 3.49 and 3.57, respectively. Their explained phenotypic variances were 25.0%, 22.2% and 37.1%, respectively. These results suggest that the paternal inheritance of the mitochondrial plasmid is controlled by a relatively small number of nuclear genes.     

mtDNA inheritance in the mosquitoes of Anopheles stephensi

The inheritance of mtDNA was tested in malaria vector mosquitoes of Anopheles stephensi strains using PCR-RFLP analysis for its utility in addressing epidemiological questions related to the transmission and spread of malaria. Reciprocal crosses were made between two haplotypes with distinct mtDNA restriction fragment length polymorphism (RFLP) profiles through 20 consecutive generations. All of the progenies produced by these crosses had the mtDNA haplotype of the female parent suggesting that, if it occurs, paternal inheritance of mtDNA in An. stephensi is rare.     

Differential transmission of the Cucumis organellar genomes

 Although plants generally show maternal transmission of the organellar genomes, previous research has demonstrated that the mitochondrial (mt) genome of cucumber is paternally transmitted. In this study, we identified RFLPs in the organellar genomes of melon, squash, and watermelon to establish organellar DNA transmission. Serial dilutions of DNA demonstrated that our hybridizations revealed the presence of a polymorphic cytoplasm when it represented at least 1% of the DNA sample. At this level of sensitivity, the chloroplast genomes of melon, squash, and watermelon were maternally transmitted. The mitochondrial genomes of squash and watermelon were maternally transmitted; however, melon, like cucumber, showed paternal transmission of the mitochondrial genome. Because most angiosperms and the related genera Cucurbita and Citrullus show maternal transmission of the mtDNA, paternal transmission in Cucumis is likely the derived state. The Cucumis mitochondrial genomes are several-fold larger than those of other cucurbits. Based on 55 probe-enzyme combinations, mtDNA size differences could not be explained by duplication of the entire genome or partial duplication of regions hybridizing with the mitochondrial probes. Because the chloroplast, mitochondrial, and nuclear genomes of Cucumis are differentially transmitted, this genus is an excellent system to study the role of intergenomic transfer in the evolution of extremely large mitochondrial genomes. Received: 20 November 1997 / Accepted: 30 December 1997     

Inheritance of Mitochondrial DNA and Plasmids in the Ascomycetous Fungus, Epichloe Typhina

We analyzed the inheritance of mitochondrial DNA (mtDNA) species in matings of the grass symbiont Epichloe typhina. Eighty progeny were analyzed from a cross in which the maternal (stromal) parent possessed three linear plasmids, designated Callan-a (7.5 kb), Aubonne-a (2.1 kb) and Bergell (2.0 kb), and the paternal parent had one plasmid, Aubonne-b (2.1 kb). Maternal transmission of all plasmids was observed in 76 progeny; two progeny possessed Bergell and Callan-a, but had the maternal Aubonne-a replaced with the related paternal plasmid Aubonne-b; two progeny lacked Callan-a, but had the other two maternal plasmids. A total of 34 progeny were analyzed from four other matings, including a reciprocal pair, and in each progeny the plasmid transmission was maternal. The inheritance of mitochondrial genomes in all progeny was analyzed by profiles of restriction endonuclease-cleaved mtDNA. In most progeny the profiles closely resembled those of the maternal parents, but some progeny had nonparental mtDNA profiles that suggested recombination of mitochondrial genomes. These results indicate that the fertilized stroma of E. typhina is initially heteroplasmic, permitting parental mitochondria to fuse and their genomes to recombine.     

Heteroplasmy and paternally oriented shift of the organellar DNA composition in barley-wheat hybrids during backcrosses with wheat parents.

Mitochondrial (mt) and chloroplast (ct) genome inheritance was studied in barley-wheat hybrids, as were their progenies obtained from backcrosses with different common wheat cultivars, by monitoring the composition of 4 mtDNA (coxI, a 5'-flanking region of cob, nad3-orf156, and 5'-upstream region of 18S/5S) and 2 ctDNA (simple-sequence repeat locus downstream of trnS and a 3'-flanking region of rbcL) loci. In male sterile F1 and BC1 plants, maternal barley mtDNA fragments were mainly detected and very low levels of paternal wheat fragments were occasionally detected by PCR in coxI, a 5'-flanking region of cob and nad3-orf156, whereas a 5'-upstream region of 18S/5S showed clear heteroplasmy, containing both maternal and paternal copies, with maternal copies prevailing. Plants showing such heteroplasmic mtDNA composition remained either semisterile or became completely sterile in the later backcross generations. Only maternal ctDNA copies were detected in these plants. In 3 stable, self-fertile, and vigourous lines obtained in the advanced backcross generations and possessing recombinant wheat nuclear genome, however, only mt- and ctDNA copies of wheat parents were detected; thus, the original alloplasmic condition appeared to be lost. Our results suggest that transmission followed by selective replication of the paternal wheat organellar DNA leads to a paternally oriented shift of the organellar DNA composition in barley-wheat hybrids, which correlates with the restoration of fertility and plant vigour. These 2 processes seem to be related to nucleocytoplasmic compatibility and to be under the control of the nuclear genome composition.     

Biparental mitochondrial DNA inheritance in the parasitic trematode Schistosoma mansoni

The maternal inheritance of mitochondrial DNA (mtDNA) in eukaryotic organisms occurs because of the selective destruction of paternal mtDNA molecules that may be present in the zygote. The elimination of sperm mtDNA is less efficient in interspecific crosses, and biparental inheritance of mtDNA has been observed in a variety of species. Because interspecific crosses are likely to be extremely rare in nature, parental inheritance of mtDNA has been deemed of little relevance to population genetics. The mtDNA of the parasitic trematode Schistosoma mansoni was examined for its utility in addressing epidemiological questions related to the transmission and spread of schistosomiasis. Prior to embarking on such experiments, we sought to confirm the mode of inheritance of this molecule using the highly polymorphic mtDNA minisatellite as a marker. In 3 separate crosses, mtDNA apparently identical to paternal DNA was observed in some individuals of the F2 and F3 generations. These observations thus suggest the intraspecific paternal inheritance of mtDNA across multiple generations in Schistosoma mansoni.     

No evidence for presence of maternal mitochondrial DNA in the sperm of Mytilus galloprovincialis males

Species of the mussel family Mytilidae have a special mitochondrial DNA (mtDNA) transmission system, known as doubly uniparental inheritance (DUI), which consists of a maternally inherited (F) and a paternally inherited (M) mitochondrial genome. Females are normally homoplasmic for the F genome and males are heteroplasmic mosaics, with their somatic tissues dominated by the maternal and their gonads dominated by the paternal genome. Several studies have indicated that the maternal genome may often be present in the male germ line. Here we report the results from the examination of mtDNA in pure sperm from more than 30 males of Mytilus galloprovincialis. In all cases, except one, we detected only the M genome. In the sperm of one male, we detected a paternal genome with an F-like primary sequence that was different from the sequence of the maternal genome in the animal's somatic tissues. We conclude that the male germ line is protected against invasion by the maternal genome. This is important because fidelity of gamete-specific transmission of the two mitochondrial genomes is a basic requirement for the stability of DUI.     

Altered mitochondrial gene expression in a maternal distorted leaf mutant of Arabidopsis induced by chloroplast mutator.

Chloroplast mutator (chm) of Arabidopsis is a recessive nuclear mutation that causes green and white variegation in leaves and is inherited in a non-Mendelian fashion. In this study, we have identified and characterized a mutant observed in F1 and backcrossed BC1 populations from a cross between chm1-3 and ecotype Columbia. This mutant, maternal distorted leaf (MDL), grows very poorly and is distinguished by distorted rough leaves and aborted flowering organs. Electron microscopic observation showed that in MDL plants, a significant portion of mitochondria are abnormal and appear to be nonfunctional. DNA gel blot and sequence analysis of the MDL mitochondrial DNA (mtDNA) revealed rearrangements in two mtDNA fragments associated with rps3-rpl16 genes (encoding ribosomal proteins S3 and L16, respectively). One rearrangement resulted in the insertion of the rps3-rpl16 operon downstream of atp9. An independent deletion in this region had eliminated the majority of rps3. In contrast, another rearrangement deleted part of rpl16, whereas rps3 remained intact. RNA gel blot analysis indicated that expression of these genes is also altered as a consequence of the mtDNA rearrangements. Thus, a mutation at the CHM locus affects mitochondrial gene expression, and impaired mitochondrial function may result in the distorted phenotype.     

Evidence for recombination of mitochondrial DNA in triploid crucian carp

In this study, we report the complete mitochondrial DNA (mtDNA) sequences of the allotetraploid and triploid crucian carp and compare the complete mtDNA sequences between the triploid crucian carp and its female parent Japanese crucian carp and between the triploid crucian carp and its male parent allotetraploid. Our results indicate that the complete mtDNA nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. Moreover, the presence of a pattern of identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents provides direct evidence that triploid crucian carp possessed the recombination mtDNA fragment (12,759 bp) derived from the paternal fish. These results suggest that mtDNA recombination was derived from the fusion of the maternal and paternal mtDNAs. Compared with the haploid egg with one set of genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily make its mtDNA fuse with the mtDNA of the haploid egg. In addition, the triple hybrid nature of the triploid crucian carp probably allowed its better mtDNA recombination. In summary, our results provide the first evidence of mtDNA combination in polyploid fish.     

Patterns of mitochondrial sorting in yeast zygotes.

Inheritance of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae is usually biparental. Pedigree studies of zygotic first buds indicate limited mixing of wild-type (p+) parental mtDNAs: end buds are frequently homoplasmic for one parental mtDNA, while heteroplasmic and recombinant progeny usually arise from medial buds. In crosses involving certain petites, however, mitochondrial inheritance can be uniparental. In this study we show that mitochondrial sorting can be influenced by the parental mtDNAs and have identified intermediates in the process. In crosses where mtDNA mixing is limited and one parent is prelabeled with the matrix enzyme citrate synthase 1 (CS1), the protein freely equilibrates throughout the zygote before the first bud has matured. Furthermore, if one parent is p0 (lacking mtDNA), mtDNA from the p+ parent can also equilibrate; intracellular movement of mtDNA is unhindered in this case. Surprisingly, in zygotes from a p0 CS1+ x p+ CS1- cross, CS1 is quantitatively translocated to the p+ end of the zygote before mtDNA movement; subsequently, both components equilibrate throughout the cell. This initial vectorial transfer does not require respiratory function in the p+ parent, although it does not occur if that parent is p-. Mouse dihydrofolate reductase (DHFR) present in the mitochondrial matrix can also be vectorially translocated, indicating that the process is general. Our data suggest that in zygotes mtDNA movement may be separately controlled from the movement of bulk matrix constituents.     

Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage.

The transmission profiles of sperm mtDNA introduced into fertilized eggs were examined in detail in F1 hybrids of mouse interspecific crosses by addressing three aspects. The first is whether the leaked paternal mtDNA in fertilized eggs produced by interspecific crosses was distributed stably to all tissues after the eggs'' development to adults. The second is whether the leaked paternal mtDNA was transmitted to the subsequent generations. The third is whether paternal mtDNA continuously leaks in subsequent backcrosses. For identification of the leaked paternal mtDNA, we prepared total DNA samples directly from tissues or embryos and used PCR techniques that can detect a few molecules of paternal mtDNA even in the presence of 10(8)-fold excess of maternal mtDNA. The results showed that the leaked paternal mtDNA was not distributed to all tissues in the F1 hybrids or transmitted to the following generations through the female germ line. Moreover, the paternal mtDNA leakage was limited to the first generation of an interspecific cross and did not occur in progeny from subsequent backcrosses. These observations suggest that species-specific exclusion of sperm mtDNA in mammalian fertilized eggs is extremely stringent, ensuring strictly maternal inheritance of mtDNA.     

Mitochondrial DNA variation in a species with two mitochondrial genomes: the case of Mytilus galloprovincialis from the Atlantic, the Mediterranean and the Black Sea

We have examined mitochondrial DNA (mtDNA) variation in samples of the mussel Mytilus galloprovincialis from the Black Sea, the Mediterranean and the Spanish Atlantic coast by scoring for presence or absence of cleavage at 20 restriction sites of a fragment of the COIII gene and at four restriction sites of the 16S RNA gene. This species contains two types of mtDNA genomes, one that is transmitted maternally (the F type) and one that is transmitted paternally (the M type). The M genome evolves at a higher rate than the F genome. Normally, females are homoplasmic for an F type and males are heteroplasmic for an F and an M type. Occasionally molecules from the F lineage invade the paternal transmission route, resulting in males that carry two F-type mtDNA genomes. These features of the mussel mtDNA system give rise to a new set of questions when using mtDNA variation in population studies and phylogeny. We show here that the two mtDNA types provide different information with regard to amounts of variation and genetic distances among populations. The F genome exhibits higher degrees of diversity within populations, while the M genome produces higher degrees of differentiation among populations. There is a strong differentiation between the Atlantic and the Black Sea. The Mediterranean samples have intermediate haplotype frequencies, yet are much closer to the Black Sea than to the Atlantic. We conclude that in this species gene flow among the three Seas is restricted and not enough to erase the combined effect of mutation and random drift. In one sample, that from the Black Sea, the majority of males did not contain an M mtDNA type. This suggests that a molecule of the maternal lineage has recently invaded the paternal route and has increased its frequency in the population to the point that the present pool of paternally transmitted mtDNA molecules is highly heterogeneous and cannot be used to read the population's history. This liability of the paternal route means that in species with doubly uniparental inheritance, the maternal lineage provides more reliable information for population and phylogenetic studies.     

Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans

Maternal inheritance is one of the hallmarks of animal mitochondrial DNA (mtDNA) and central to its success as a molecular marker. This mode of inheritance and subsequent lack of heterologous recombination allows us to retrace evolutionary relationships unambiguously down the matriline and without the confounding effects of recombinant genetic information. Accumulating evidence of biparental inheritance of mtDNA (paternal leakage), however, challenges our current understanding of how this molecule is inherited. Here, using Drosophila simulans collected from an East African metapopulation exhibiting recurring mitochondrial heteroplasmy, we conducted single fly matings and screened F1 offspring for the presence of paternal mtDNA using allele-specific PCR assays (AS–PCR). In all, 27 out of 4092 offspring were identified as harboring paternal mtDNA, suggesting a frequency of 0.66% paternal leakage in this species. Our findings strongly suggest that recurring mtDNA heteroplasmy as observed in natural populations of Drosophila simulans is most likely caused by repeated paternal leakage. Our findings further suggest that this phenomenon to potentially be an integral part of mtDNA inheritance in these populations and consequently of significance for mtDNA as a molecular marker.     

Complete elimination of maternal mitochondrial DNA during meiosis resulting in the paternal inheritance of the mitochondrial genome in Chlamydomonas species

Summary. The non-Mendelian inheritance of organellar DNA is common in most plants and animals. In the isogamous green alga Chlamydomonas species, progeny inherit chloroplast genes from the maternal parent, as paternal chloroplast genes are selectively eliminated in young zygotes. Mitochondrial genes are inherited from the paternal parent. Analogically, maternal mitochondrial DNA (mtDNA) is thought to be selectively eliminated. Nevertheless, it is unclear when this selective elimination occurs. Here, we examined the behaviors of maternal and paternal mtDNAs by various methods during the period between the beginning of zygote formation and zoospore formation. First, we observed the behavior of the organelle nucleoids of living cells by specifically staining DNA with the fluorochrome SYBR Green I and staining mitochondria with 3,3′-dihexyloxacarbocyanine iodide. We also examined the fate of mtDNA of male and female parental origin by real-time PCR, nested PCR with single zygotes, and fluorescence in situ hybridization analysis. The mtDNA of maternal origin was completely eliminated before the first cell nuclear division, probably just before mtDNA synthesis, during meiosis. Therefore, the progeny inherit the remaining paternal mtDNA. We suggest that the complete elimination of maternal mtDNA during meiosis is the primary cause of paternal mitochondrial inheritance. Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 901-0213, Japan.