mtDNA Restriction Patterns Research Articles

Restriction Fragment Length Polymorphism Analysis of the Mitochondrial DNA of Fusarium oxysporum f. sp. ciceris

AbstractSeven isolates of Fusarium oxysporum f. sp. ciceris, representing pathogenic races 1, 2, 3, and 4 from India and 0, 5, and 6 from Spain, were assayed for restriction fragment length polymorphisms (RFLPs) in the mitochondrial DNA,(mt DNA). The mt DNA fraction of total fungal DNA was purified and digested with the restriction endonucleases Bam HI, Bgl II, Eco RI. Kpn I, Sac I, Sal I, Sma I, and Xho I. The mt DNA is a circular molecule of 40.5 kb. No RFLP in the mt DNA was detected among the seven races of F. o. ciceris. The identical restriction patterns of mt DNA indicates an extensive conservation in the gene composition of mt DNA without sequence variation, and suggests that mt DNA of F. o. ciceris may not be responsible for pathogenic diversity. The restriction map of mt DNA from the race 6 isolate Fo 8272 was constructed by digestion of the mt DNA with five restriction enzymes: Eco RI, Kpn I, Sac I, Sal I, and Xho I, either singly or in selected pairs.

Detection of the source of infection of apple trees by Cylindrocarpon heteronema using DNA polymorphisms

Restriction fragment length polymorphisms (RFLPs) of the ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of isolates of Cylindrocarpon heteronema were analysed using rDNA from Saccharomyces carlsbergensis and mtDNA from C. heteronema as probes. These analyses revealed intraspecific heterogeneity; four rDNA and six mtDNA restriction pattern categories were observed among the isolates tested. The two main rDNA RFLP categories, both of which subdivided into two mtDNA categories, detected within the UK isolates could not be associated with the localities from which they were obtained, but the majority of isolates originating from trees produced in the same nursery, irrespective of where they were finally planted, belonged to the same category.

Genetic characterization ofColletotrichum gloeosporioidesisolates obtained from mango

Abstract Most isolates of Colletotrichum gloeosporioides (>80%) obtained from anthracnose lesions on mango leaves, inflorescences and fruits from different regions in Sri Lanka showed the same ribosomal DNA (rDNA) and very similar mitochondrial DNA (mtDNA) restriction fragment banding patterns when genomic DNA was digested with endonuclease EcoR I. This rDNA restriction fragment banding pattern was the same as that revealed by C. gloeosporioides isolates from mango in other tropical countries. Isolates obtained from inflorescences all showed the same rDNA and mtDNA restriction patterns but a few isolates from fruits and leaves from mango grown in mixed orchards in areas of Sri Lanka with two monsoons and year‐round rainfall had rDNA restriction patterns associated with isolates from other fruit crops. This suggested that some cross‐infection onto mango occurs. However, it is suggested that there is a strain of C. gloeosporioides specific to mango and the clonal nature of this strain implies that it has di...

Restriction Fragment Length Polymorphisms of Mitochondrial and Nuclear DNAs among Korean Races of Magnaporthe grisea

AbstractThree KJ‐races of Magnaporthe grisea (virulent to only japonica type rice cultivars) and seven KI‐races (virulent to either indica or japonica type cultivars) isolated from various rice‐growing areas of Korea were assayed for restriction fragment length polymorphisms (RFLPs) in mitochondrial and nuclear DNAs of the fungus. The size of mitochondrial DNA of M. grisea was estimated to be approximately 39. 8 kb. No RFLP in the mt DNA was detected among the 10 Korean races, indicating an extensive conservation in the gene composition of mt DNA without sequence variation. The identical restriction patterns of mt DNA also suggest that mt DNA of M. grisea may not be responsible for pathotypic diversity and variability. Southern blot analysis with five single‐copy DNA probes showed considerable polymorphisms. Much diversity was detected in the three KI‐races predominated during 1978–1985 in Korea. In contrast, no genetic variation was detected between other four KI‐races and three KJ‐races. RFLPs in nuclear DNA were correlated to some extent with the prevailing races in Korea. However, relationship between RFLPs in nuclear DNA and virulence of M. grisea races was inconclusive.

Molecular analysis of plants regenerated from embryogenic cultures of hybrid sugarcane cultivars (Saccharum spp.)

The genomic stability of tissue culture regenerants of sugarcane (Saccharum spp. hybrids, cvs 'CP721210', 'CP68-1067' and 'B43-62') was analyzed by DNA restriction fragment length polymorphism (RFLP). Plants regenerated from calli, cell suspensions, cryopreserved cell suspensions and protoplasts were used. Total DNA isolated from 19 different sources was digested with EcoRI, HindIII, BamHI, BamHI, EcoRI and PstI and probed with six known maize mitochondrial genes (coxI, coxII, atpA, atp6, atp9 and rrn18-rrn5), three random maize mitochondrial cosmid clones, two random maize chloroplast cosmid clones and a wheat Nor locus clone. Hybridization patterns indicated that the variation observed was minor and appeared only in the secondcycle regenerants. No differences were observed among the three cultivars and the regenerants from calli, suspension culture, cryopreserved suspension culture and protoplasts. Mitochondrial DNA (mtDNA) isolated from 'CP72-1210' plants and its embryogenic cell suspensions, and bulk samples from all 'CP72-1210' regenerants pooled together were digested with EcoRI, HindIII, PstI, BamHI and SalI and probed with three recombinationally active wheat mtDNA clones, K', K3 and X2. No variation in the mtDNA restriction patterns was observed between the 'CP72-1210' plants and its regenerants. However, restriction pattern variation was observed only from EcoRI digestion, and hybridization patterns of K3, K' and X2 revealed minor variations in the mtDNA of cell suspensions when compared with the DNA of the 'CP72-1210' plant. Except for a qualitative variation detected by the X2 probe and minor stoichiometric variations detected by the K3 probe, sugarcane DNAs were found to be stable after plant regeneration.

Genetic diversity of the yeast Candida utilis.

The electrophoretic karyotypes and some mtDNA restriction fragment patterns of 13 strains of Candida utilis and one strain of Hansenula jadinii were compared. PFGE separations revealed remarkable chromosome length polymorphisms between two groups of strains suggesting that perhaps they do not belong to the same species. However, all strains had the same or similar EcoRI, HindIII and BamHI mtDNA restriction patterns. The mtDNA genomes had an average size range of 55 kb. These results support the supposition that C. utilis is a yeast with a highly variable electrophoretic karyotype as already known for another imperfect yeast species, Candida albicans.

An analysis of allozyme, mitochondrial DNA and morphological variation in mussel (Mytilus galloprovincialis) populations from Greece

Three populations ofM. galloprovincialis from northern Greece were investigated using isozyme analysis, discriminant analysis of morphological characteristics and analysis of restriction fragments of mtDNA. For all three types of analysis significant intra- and interpopulation differentiation was found. This differentiation is very noticeable at the mtDNA genotype frequencies. Furthermore, the restriction patterns of mtDNA were different from those reported for Atlantic populations of this species.

Extensive nuclear influence on mitochondrial transcription and genome structure in male-fertile and male-sterile alloplasmic Nicotiana materials

Nuclear influences on mitochondrial transcription and genome organization were analysed in six different male-fertile and male-sterile alloplasmic Nicotiana cultivars. The alloplasmic materials were compared with the corresponding nuclear species (N. tabacum) and cytoplasmic donor species (N. debneyi, N. rapanda or N. suaveolens) in Northern and Southern analyses using twelve different mitochondrial genes as probes. The investigation revealed that the nucleus exerts extensive influence on the expression and structure of the mitochondrial genome. For the majority of the probes, changes in both mitochondrial transcription and DNA patterns in alloplasmic cultivars were detected. Even though changes in transcription patterns, which correlated with male sterility, were detected for three of the probes (atpA, orf25 and coxII), the changes were not consistent for all the male-sterile materials. Likewise, no consistent association between mtDNA restriction patterns and cytoplasmic male sterility (CMS) was detected.

Mitochondrial DNAs and plasmids as taxonomic characteristics in Trichoderma viride

Mitochondrial DNA (mtDNA) was purified from 12 isolates of the Trichoderma viride aggregate and found to be, on the average, 32.7 kb in size. Plasmids were present in the mtDNA preparations from 8 of 12 strains of T. viride examined. Plasmids in four of the strains produced ladderlike banding patterns on gels, and these plasmids were studied in detail. The ladderlike patterns were produced by single molecules that were supercoiled to various degrees. Plasmids from two of the strains do not have homology with the mtDNA but do have a limited amount of homology with each other. No phenotype could be associated with the presence of a plasmid. Restriction endonuclease digestion of the mtDNAs produced patterns in which the presence or absence of certain fragments correlated with the classification of the strains into T. viride group I or II. Phenetic cluster analysis and parsimony analysis of the fragment patterns produced groups that corresponded to T. viride groups I and II. The fragment patterns were very diverse, with nearly all strains having a unique pattern. However, two strains of T. viride group I from widely different geographical locations did have identical restriction patterns for all the enzymes used in this study. This result indicates that it may not be possible to use mtDNA restriction patterns alone to identify Trichoderma strains.

Analysis of chloroplast and mitochondrial segregation in three different combinations of somatic hybrids produced within Brassicaceae

Mitochondrial and chloroplast DNA were characterized in three different combinations of somatic hybrids produced between different species within Brassicaceae. The fusions were made between B. campestris and B. oleracea, B. napus and B. nigra and between B. napus and Eruca sativa. The combinations represent interspecific hybridizations, but the phylogenetic distance between the species used in each instance is different. Whereas the B. campestris (+) B. oleracea and the B. napus (+)B. nigra hybrids are both examples of intrageneric hybrids, B. campestris is more closely related to B. oleracea than B. napus is to B. nigra. The fusion of B. napus and E. sativa represents an intergeneric hybridization. Since hybrids were produced with reproducible and uniform fusion and culture methods, a comparison of chloroplast and mitochondrial segregation and mitochondrial DNA (mt-DNA) rearrangements could be made between the combinations. The segregation of both chloroplasts and mitochondria was biased in the B. napus (+)B. nigra and the B. napus (+)E. sativa combination. The nonrandom segregation of chloroplasts and mitochondria could be due to the different ploidy levels of the fusion partners and/or reflect differences in organelle replication rate. Furthermore, segregation of mitochondria was correlated to the differences in phylogenetic distance between the species used in the fusions. However, mitochondrial segregation, in contrast to chloroplast segregation, could in all combinations also have been affected by the cell type used as protoplast source in the fusions. All different chloroplast types could be established within each combination. Hybrids containing chloroplast from one parent together with mitochondria from the other parent were found in two of the combinations, although the majority of the hybrids had mt-DNA that was altered compared to the parental species. The rearranged mt-DNA found in most hybrids was an effect of the heteroplasmic state following protoplast fusion rather than of the tissue culture methods, since no mt-DNA rearrangements were found in B. napus plants regenerated from protoplast culture. The mtDNA restriction patterns of the hybrids with rearranged mt-DNA indicated that specific regions of the mt-DNA were involved in the rearrangements following protoplast fusion.

Cytoplasmic male sterility inBeta is associated with structural rearrangements of the mitochondrial DNA and is not due to interspecific organelle transfer

Chloroplast (ct) and mitochondrial (mt) DNAs from four cytoplasmic male sterile (cms) and 22 normal fertile sugar beet lines and accessions of wild beets from the genus Beta have been compared with restriction analyses and Southern hybridizations. We have used restriction analyses of ctDNA as a phylogenetic marker to confirm the taxonomic relationships between the different cytoplasms. According to the ctDNA data, all four cms cytoplasms belong to the same taxonomic section, Beta. Restriction patterns of ct and mtDNA from fertile accessions produced analogous trees of similarity and showed a close correlation between the organellar DNA diversity and the accepted taxonomic classification of the species studied. However, the mt-DNA restriction profiles of the four cms types differed dramatically from each other and from those of all fertile accessions from the genus. No indication of cytoplasmic introgression was found in any of the four investigated cms types. Southern hybridization to mtDNA revealed variant genomic arrangements in the different fertile and cms cytoplasms, indicating that rearrangement of the mitochondrial genome is a common denominator to the different cms systems in Beta. It may, indeed, be a common property to spontaneously occurring cms in all or most species.

Mitochondrial DNA as a genetic marker for brown trout, Salmo trutta L., populations

Mitochondrial DNA was examined in natural and hatchery‐reared stocks of brown trout, using different methods of restriction analysis. The methods included the development of a brown trout mt DNA hybridization probe through cloning part of the brown trout mitochondrial genome. In addition, fragments were analysed by ethidium bromide staining and end‐labelling. The relative merits of each of these methods in assessing levels of genetic relatedness between the natural and hatchery‐reared brown trout stocks were evaluated. In addition, the study revealed a diagnostic mtDNA restriction pattern which could be used as a genetic marker for the discrimination of these two groups of brown trout.

Cytochrome c oxidase deficiency in Leigh's syndrome

Although an apparently generalized defect of cytochrome c oxidase (COX) occurs in many patients with subacute necrotizing encephalomyelopathy (Leigh's syndrome), the mode of inheritance in this disorder is not known. We transformed COX-deficient fibroblasts from a child with Leigh's syndrome with simian virus 40 to obtain cells with an infinite life span. These cells were still COX-deficient, grew normally in HAT medium, and were ouabain-sensitive. We fused these cells with a HAT-sensitive, ouabain-resistant variant of HeLa cells (HeLacot) and isolated surviving hybrid clones in ouabain-containing HAT medium. Prolonged cultivation of the hybrids was accompanied by preferential loss of HeLacot mitochondrial DNA (mtDNA), as determined by mtDNA restriction patterns of parental and hybrid cell DNA with the restriction endonuclease HaeII. COX activity was normal or higher than normal in hybrids, including the progeny of cell clones that had lost almost all the HeLacot mtDNA. These data demonstrate that COX deficiency in this Leigh's syndrome patient's cells was corrected by a nuclear DNA-encoded factor from the HeLacot parent and ruled out an mtDNA mutation as the basis for COX deficiency. This system can be used to determine whether different generalized mitochondrial disorders are due to mutations of nuclear or mtDNA.

Mitochondrial DNA evolution in theDrosophila nasuta subgroup of species

The Drosophila nasuta group consists of about 12 closely related species distributed throughout the Indo-Pacific region. They are of great interest because of their evolutionary idiosyncrasies including little morphological differentiation, the ability to intercross in the laboratory often producing fertile offspring, and substantial chromosomal evolution. Studies of metric traits, reproductive isolation, and chromosomal and enzyme polymorphisms have failed to resolve the phylogeny of the species. We report the results of a survey of the mitochondrial DNA (mtDNA) restriction patterns of the species. The phylogeny obtained is consistent with other available information and suggests that D. albomicans may represent the ancestral lineage of the group. The amount of polymorphism in local populations (pi = 1.0% per site) is within the typical range observed in other animals, including Drosophila. The degree of differentiation between species is, however, low: the origin of the group is tentatively dated about 6-8 million years ago. This study confirms the usefulness of mtDNA restriction patterns for ascertaining the phylogeny of closely related species.

Dwarfism and male sterility in interspecific hybrids of Epilobium

Mitochondrial DNA (mtDNA) and transcriptional patterns of mitochondrial genes have been examined in dwarf, normal, fertile and male sterile Epilobium hybrids. No alterations or rearrangements of mitochondrial DNA could be detected in the developmentally disturbed hybrids. They exhibit restriction patterns of mtDNA that correspond exactly to those of their female parents. However, the transcription of at least one mitochondrial gene is significantly altered in the male sterile hybrid E. hirsutum x montanum. In normal plants, one mRNA of 1.6 kb hybridizes to the cytochrome c oxidase subunit II gene, while in male sterile plants a transcript of this size is lacking and instead a major transcript of 2.0 kb and two smaller ones occur. The transcript pattern of the F1 ATPase alpha subunit (atpA) gene exhibits slight alterations in sterile plants also. Since these hybrids have the same cytoplasm as normal plants, an incompatibility between the nuclear and the mitochondrial genotype may be responsible for the altered mitochondrial gene expression. No alteration of the transcripts of the mitochondrial genes tested could be detected in dwarf hybrids. The coincidence of male sterility with an altered transcription pattern of mitochondrial genes suggests that the mitochondria are involved in the occurrence of this phenotype.

Mitochondrial DNA variation in pearl millet and related species

Mitochondrial DNA (mtDNA) restriction endonuclease fragment patterns and patterns of mtDNA hybridized by mitochondrial gene probes were used to study phylogenetic relationships of seven Pennisetum species, including five P. americanum (pearl millet) ecotypes and a reference species from the distantly related genus, Panicum. The restriction patterns of the pearl millet ecotypes were uniform with the exception of the ecotype collected in Ethiopia. The probe hybridization method revealed more variability, with both the Rhodesian and Ethiopian ecotypes differing from the others and from each other. Considerable restriction pattern polymorphism was noted among different species of Pennisetum, and Panicum. Significant relationships were noted of Pennisetum polystachyon to P. pedicellatum and of P. purpureum to P. squamulatum using the restriction pattern method. In addition to those relationships, the hybridization method showed relationships of pearl millet to P. purpureum and to P. squamulatum. The relationships noted between species by the hybridization method agreed more closely to the cytological data than those indicated by the restriction pattern method. Therefore, the hybridization method appeared to be the preferred method for studying species relationships. The mitochondrial genome size of pearl millet was calculated to be 407 kb and the mitochondrial genome sizes of other Pennisetum species ranged from 341 to 486 kb.

MITOCHONDRIAL DNA AND PROTEIN DIFFERENTIATION BETWEEN HYBRIDIZING CYTOTYPES OF THE WHITE-FOOTED MOUSE, PEROMYSCUS LEUCOPUS.

Restriction-enzyme analysis of mitochondrial DNA and protein electrophoresis were used to document patterns of gene flow across a hybrid zone between chromosomal races of Peromyscus leucopus. Chromosomal markers (three inversions) are such that individuals can be classified as potential F1 's, backcrosses, or parental types. Allozymic characterization of the hybrid zone is congruent with the chromosomal data (Stangl, 1986) and indicates an assymetrical distribution of markers, with the northeastern markers being distributed at a higher frequency into southwestern populations. Restriction patterns of mtDNA suggest that the two cytotypes may have had different evolutionary histories, and the distribution of haplotypes is concordant with other genetic markers used to identify the hybrid zone. Concordant changes in chromosomes, allozymes, and mtDNA suggest that the most viable hypothesis for the origin of the zone is secondary contact. A unique aspect of this study is that the same individuals were used for protein electrophoresis, mtDNA analysis, and chromosomal analysis. Thus, patterns of genetic variation can be interpreted free of any historical bias associated with samples collected at different times.

Chondriome analysis in sexual progenies of Nicotiana cybrids

We studied the chondriomes (the mitochondrial genomes) of sexual-progeny plants derived from eleven Nicotiana cybrids which resulted from donor-recipient protoplast fusions. The recipients were either N. tabacum or N. sylvestris and the donor (of the cytoplasm) was N. bigelovii. The chondriomes were characterized by the mitochondrial DNA (mtDNA) restriction-patterns. The differences in mtDNA restriction patterns were revealed after Sal I digestions and probing the respective Southern-blots with three mtDNA fragments. The hybridization patterns of mtDNAs from 35 second-generation plants (i.e. the sexual progeny derived from the cybrid plants) indicated only minor variations between plants derived from the same cybrid but pronounced variations among sibs derived from different cybrids. The mtDNA of 32 second-generation plants varied from both original fusion partners but the mtDNA of one (male-sterile) plant was apparently identical with the mtDNA of one of the original donor (N. bigelovii) and the mtDNA of two other (male-fertile) plants was apparently identical to the mtDNA of an original recipient (N. sylvestris). Generally, the mtDNAs of male-fertile, second-generation plants were similar to the mtDNAs of the original recipients while the mtDNAs of the male-sterile second-generation plants were similar to the mtDNA of the donor (N. begelovii). The analyses of mtDNAs from the thirdgeneration plants indicated stabilization of the chondriomes; no variations were detected between the mtDNAs of plants derived from a given second-generation plant.

Differential fate of plastid and mitochondrial genomes in Petunia somatic hybrids

The chloroplast (cp) and mitochondrial (mt) DNAs of Petunia somatic hybrid plants, which were derived from the fusion of wild-type P. parodii protoplasts with albino P. inflata protoplasts, were analyzed by endonuclease restriction and Southern blot hybridization. Using (32)P-labelled probes that distinguished the two parental cpDNAs at a BamH1 site and at a HpaII site, only the P. parodii chloroplast genome was detected in the 10 somatic hybrid plants analyzed. To examine whether cytoplasmic mixing had resulted in rearrangement of the mitochondrial genome in the somatic hybrids, restriction patterns of purified somatic hybrid and parental mtDNAs were analyzed. Approximately 87% of those restriction fragments which distinguish the two parental genomes are P. inflata-specific. Restriction patterns of the somatic hybrid mtDNAs differ both from the parental patterns and from each other, suggesting that an interaction occurred between the parental mitochondrial genomes in the somatic fusion products which resulted in generation of the novel mtDNA patterns. Southern blot hybridization substantiates this conclusion. In addition, somatic hybrid lines derived from the same fusion product were observed to differ in mtDNA restriction pattern, reflecting a differential sorting-out of mitochondrial genomes at the time the plants were regenerated.

Extrachromosomal genetics of Claviceps purpurea

In order to establish a system for gene cloning in the ergot fungus Claviceps purpurea mitochondrial DNA was purified from two wild strains and four descendants of production strains. A characterization of the mt DNA with respect to buoyant density, molecular weight (45 kb corresponding to acontour lenght of 14 μm) showed no differences. However, considerable differences were detected in the restriction pattern, with the exception of two commercial strains of possibly common origin. From this it follows that restriction patterns of mt DNA may be used as finger prints to identify related strains.