mtDNA Restriction Patterns Research Articles

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage “salchichón”

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products.

Efficiency of mitochondrial DNA restriction analysis and RAPD-PCR to characterize yeasts growing on dry-cured Iberian ham at the different geographic areas of ripening

The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)(4) and (GAC)(5) was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening.

Mitochondrial DNA restriction-fragment-length monomorphism in the Italian wolf (Canis lupus) population

Mitochondrial-DNA (mtDNA) restriction patterns were studied in 22 wolves (Canis lupus) sampled in central-northern Italy. A total of 60 restriction sites were detected, encompassing about 2 % of the mitochondrial genome of canids. All wolves showed the same restriction pattern. Therefore, a single mtDNA haplotype was detected in the Italian wolf population. Historical information on peninsular isolation and demographic decline suggest that low genetically effective population size and random drift may have strongly reduced the mtDNA variability of wolves in Italy over the last 100–200 years. A different mtDNA restriction pattern in feral dogs sampled from a wolf range in central Italy was detected. These findings suggest that the hybridization and introgression of female dog genomes into the Italian wolf population may be rare or absent.

Restriction Analysis of Mitochondrial DNA: A Powerful Tool for Genetic Control of Industrial Fermentations

ABSTRACTIn order to facilitate the mitochondrial DNA-RFLP analysis, a simplified procedure for isolation of mtDNA was developed. It reduces the time for analysis and could be applied for specific detection of genetic changes in the genome of industrially important S. cerevisiae strains. Furthermore, genetic stability control of brewing S. carlsbergensis strains was performed. Upon applying of PCR fingerprinting, chromosomal karyotyping, and mtDNA-RFLP techniques, only two very similar characteristic DNA profiles were observed in yeast strains isolated from different sources. These data confirmed that mtDNA-RFLP analysis is as reliable as other conventionally used molecular methods, giving same high discriminative results when genome rearrangements were analyzed. Contrary to the data, obtained from the analysis of brewing Saccharomyces strains, a difference between the PCR-profile and mtDNA restriction patterns was observed when distillery strains were studied. Although mtDNA-RFLP profiles of the ethanol producing strains were highly similar, slight divergence between them was observed, which was probably due to the damaging effect of ethanol and acetaldehyde on the mtDNA. Application of this technique, contrary to whole genome typing methods, could detect the presence even of small divergence in the tested strains and could be used as highly specific tool for genetic control of industrial fermentations.

Comparative analysis of organelle DNAs in acid citrus grown in Japan using PCR-RFLP method

PCR-RFLP analyses of three regions for each of chloroplast DNA (cpDNA; rbcL-ORF106, trnD-trnT, trnH-trnK) and mitochondrial DNA (mtDNA; nad7/exon2-exon3, nad7/exon3-exon4, 18S-5S) were performed in 26 cultivars of acid citrus grown in Japan to identify polymorphisms and classify them. The polymorphisms were compared with those of three true Citrus species, i.e., mandarin, pummelo and citron. Ichang papeda (C. ichangensis) was also included in this study to find its relationship with Yuzu. Inter-species cpDNA variation was recognized and the acid citrus were divided into three groups, namely; I (‘Yuzukichi’ and ‘Kinkoyu’), II [sour oranges (‘Kaiseito’, ‘Daidai’ and ‘China daidai’), ‘Nansho daidai’, ‘Kiku daidai’, C. sudachi (‘Mushi yukaku’, ‘Yushi yukaku’ and ‘Yushi mukaku’), C. sphaerocarpa (‘Kabosu’ and ‘Aka kabosu’), C. kizu (‘Taninaka kizu’, ‘Kinosu’ and ‘Kizu’), ‘Zanbo’, ‘Mochiyu’, ‘Jabara’ and ‘Naoshichi’], and III [Yuzu (‘Tetraploid’, ‘Tochikei yuzu’ and ‘Yamanekei yuzu’), ‘Matsuda sudachi’, ‘Zuishoyu’, ‘Hanayu’ and ‘Yuko’]. CpDNA restriction patterns of the three true Citrus species differed from each other as well as from those of ichang papeda. CpDNA restriction patterns of group I of the acid citrus were identical to those of mandarins. Group II showed the same as pummelos. CpDNA restriction patterns of group III were differed from those of the three true Citrus species in the three regions. This group was differed from ichang papeda after digestion of trnH-trnK PCR products with TaqI, HinfI and AluI, while they showed identical restriction patterns in two regions, rbcL-ORF106 and trnD-trnT. Citrons and ichang papeda were placed in groups IV and V, respectively. Based on mtDNA restriction patterns, the acid citrus were divided into three groups; i, ii and iii. In groups i and ii accessions of groups I and II of cpDNA were placed with mandarins and pummelos, respectively. In group iii accessions of group III of cpDNA were placed with ichang papeda. Citrons were placed in a distinct group, iv.

Analysis of yeast population during spontaneous alcoholic fermentation: Effect of the age of the cellar and the practice of inoculation

An ecological study of spontaneous fermentation in red wine carried out in two wineries located in the same village of the D.O.Ca. Rioja appellation (Spain). These two wineries were very different: one was very old and the other commenced its operations with the 2000 vintage. The study was conducted over seven consecutive years (1997–2003); the first 3 years in the old winery; and the other 4 years in the new one. Another difference between the wineries was the use of commercial strains: no starters were ever used in the old winery and two known strains (A and B) were employed only in a white must vat in the new winery. The number of different Saccharomyces cerevisiae strains detected for each vintage and the frequency of their appearance varied between the old winery and the new winery. There was no one clearly dominant clone in the fermentations studied in the old winery, whereas in the vinifications monitored in the new winery, a clear majority strain was detected. The same S. cerevisiae clone took over the spontaneous fermentation of all the vintages studied in this new winery. Analysis of the mtDNA restriction pattern of this strain revealed that it was identical to one of the commercial strain used (starter A), despite the fact that the fermentation studied was spontaneous. Microbial analysis conducted on winery equipment and winery surfaces prior to the 2003 vintage revealed that starter A had become a resident of the winery with a percentage of 50% among all S. cerevisiae. The presence of non- Saccharomyces yeasts in the early stages of vinification also differed between the old and new wineries.

Genetic structure of honeybee populations from southern Brazil and Uruguay

Apis mellifera scutellata was introduced to Brazil in 1956 and Africanized honeybee populations have now spread from Argentina to the southwestern United States. Temperate climatic restrictions seem to be a natural limit to Africanized honeybee expansion around parallels 35° to 40° SL. We used allozyme loci (Mdh-1 and Hk-1) and mtDNA haplotypes to characterize honeybee populations in southern Brazil and Uruguay and define a possible transition area between Africanized and European bees. Samples of 194 bee colonies were collected from ten localities between 30°-35° SL and 52°-59° WL. The mtDNA restriction patterns of these colonies were obtained through digestion of the mitochondrial genome by Eco RI, or by digestion by Bgl II and Xba I of the cytochrome B locus and the COI-COII intergenic region, respectively. The distribution limit of African bee colonies, i.e., those populations with only the African mtDNA haplotype and with a high proportion of African genes as shown by allozyme analysis, is located in northern Uruguay, with a hybridization zone located farther south in Uruguay. A gradual cline from north to south was observed, confirmed by mtDNA, racial admixture, and genetic distance analyses. No evidence of either gametic disequilibrium between nuclear markers or cytonuclear disequilibrium among the nuclear and mtDNA genotypes was detected, suggesting that the hybridization process has been completed.

Study of the authenticity of commercial wine yeast strains by molecular techniques

mtDNA restriction analysis has been carried out with 45 different commercial Saccharomyces wine yeast strains. The analysis with Hinf I provided unique profiles for 17 of the 45 strains and can therefore be considered as individual strains. Nevertheless, among the remaining 28 strains, only eight mtDNA restriction patterns appeared. These strains were subjected to electrophoretic karyotyping and PCR amplification of δ sequences. We concluded that the maximum discriminatory power was obtained when the results of the three techniques were combined, giving 13 different composite patterns for the 28 strains under study. The results showed evidence of mistakes during production or fraudulent practices by yeast producers, since only 30 individual strains have been identified among the 45 Saccharomyces wine yeast strains commercialised by different companies. Additionally, commercial starters of Saccharomyces uvarum and Saccharomyces bayanus have been re-identified as Saccharomyces cerevisiae.

On the origin and systematics of the northern African wood mouse (Apodemus sylvaticus) populations: a comparative study of mtDNA restriction patterns

Conflicting hypotheses have been formulated regarding the origin of wood mouse (Apodemus sylvaticus) populations in northern Africa. In this study, the mtDNA restriction patterns of mice (n = 28) collected in Tunisia and Morocco are compared with those of representatives from southern Europe (n = 102). The neighbour-joining tree confirms the existence of the three lineages previously found in the Mediterranean area: western, Tyrrhenian–Balkan, and Sicilian. The western group is isolated from the two others, with bootstrap values of 89 and 95%. Northern African patterns are included in the western group. Their variability is low, the same pattern being shared by five Tunisian and all Moroccan animals (n = 18), caught either in the north of the country (Cap Spartel) or in the south (Marrakech). This implies that northern African wood mouse populations have a southwestern European origin and that their presence in the region is probably recent, which corresponds to both paleontological data and the hypothesis of anthropogenic introduction.

On the origin and systematics of the northern African wood mouse (<i>Apodemus sylvaticus</i>) populations: a comparative study of mtDNA restriction patterns

Conflicting hypotheses have been formulated regarding the origin of wood mouse (Apodemus sylvaticus) populations in northern Africa. In this study, the mtDNA restriction patterns of mice (n = 28) c...

Molecular identification of wine yeasts at species or strain level: a case study with strains from two vine-growing areas of Greece.

The composition of wine yeast populations, present during spontaneous fermentation of musts from two wine-producing areas of Greece (Amyndeon and Santorini) and followed for two consecutive years, were studied using a range of molecular techniques. Internal Transcribed Spacer (ITS) ribotyping was convincingly applied for yeast species identification, proving its usefulness as a reliable tool for the rapid characterization of species composition in yeast population studies. Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (mtDNA) was shown to be a convenient criterion for the detection of intraspecies genetic diversity of both Saccharomyces and non-Saccharomyces isolate populations. Similarly, polymorphism of amplified delta interspersed element sequences provided an additional criterion for S. cerevisiae strain differentiation. Comparative analysis of S. cerevisiae genetic diversity, using mtDNA restriction patterns and delta-amplification profiles, showed a similar discriminative power of the two techniques. However, by combining these approaches it was possible to distinguish/characterize strains of the same species and draw useful conclusions about yeast diversity during alcoholic fermentation. The most significant findings in population dynamics of yeasts in the spontaneous fermentations were (i) almost complete absence of non-S.cerevisiae species from fermentations of must originating from the island Santorini, (ii) a well recorded strain polymorphism in populations of non-Saccharomyces species originating from Amyndeon and (iii) an unexpected polymorphism concerning S. cerevisiae populations, much greater than ever reported before in similar studies with wine yeasts of other geographical regions.

Variation in the mitochondrial DNA of the potential biological control agent Chondrostereum purpureum

The mitochondrial DNA (mtDNA) of Chondrostereum purpureum (Pers.:Fr.) Pouzar was extracted and purified, and the size ranged from 51.8 to 66.4 kb. One isolate each from British Columbia, Alberta, Finland, the Netherlands, and New Zealand were found to have identical BamHI mtDNA restriction patterns, resulting in a mitochondrial genome of 63.8 kb. An additional isolate from British Columbia and one from Switzerland had different banding patterns, however, resulting in mitochondrial genomes of 66.4 kb and 51.8 kb, respectively. A sequence-characterized amplified region (SCAR) assay, based on a polymerase chain reaction, was developed to rapidly screen a larger population of 84 isolates from North America, Europe, and New Zealand. Two SCARs, one encoding the NADH 4 gene (3 kb) and the second encoding the ATPase VI and cytochrome b genes (5.1 kb), were digested with 24 restriction enzymes. There were no polymorphisms in the NADH 4 containing SCAR, while a single polymorphism was detected by NsiI in the ATPase VI - cytochrome b containing SCAR. Two mitochondrial haplotypes that were distributed throughout the sample population were thus identified. The coancestry coefficient (<$Q7A0D00000010446D80BFFEFF88A524F5343905055B98C420120907B4DDA9ECB1F0>) for all subpopulations of the sample population was calculated to be 0.0353. The level of gene diversity in the mtDNA ofC. purpureum suggested that the chance introduction of novel mitochondrial genes following biological control applications of the fungus is relatively low.

Analysis and dynamics of the chromosomal complements of wild sparkling-wine yeast strains.

We isolated Saccharomyces cerevisiae yeast strains that are able to carry out the second fermentation of sparkling wine from spontaneously fermenting musts in El Penedès (Spain) by specifically designed selection protocols. All of them (26 strains) showed one of two very similar mitochondrial DNA (mtDNA) restriction patterns, whereas their karyotypes differed. These strains showed high rates of karyotype instability, which were dependent on both the medium and the strain, during vegetative growth. In all cases, the mtDNA restriction pattern was conserved in strains kept under the same conditions. Analysis of different repetitive sequences in their genomes suggested that ribosomal DNA repeats play an important role in the changes in size observed in chromosome XII, whereas SUC genes or Ty elements did not show amplification or transposition processes that could be related to rearrangements of the chromosomes showing these sequences. Karyotype changes also occurred in monosporidic diploid derivatives. We propose that these changes originated mainly from ectopic recombination between repeated sequences interspersed in the genome. None of the rearranged karyotypes provided a selective advantage strong enough to allow the strains to displace the parental strains. The nature and frequency of these changes suggest that they may play an important role in the establishment and maintenance of the genetic diversity observed in S. cerevisiae wild populations.

Construction of Physical Maps of Mitochondrial DNAs in Fusarium Species Causing Fusarium Head Blight

RFLPs in Mitochondrial (mt) DNAs were analyzed using 21 strains from seven Fusarium species that cause fusarium head blight in wheat, including F. graminearum, F. culmorum, F. avenaceum, F. acuminatum, F. sporotrichioides, F. nivale, and F. tricinctum. MtDNA was purified by CsCl ultracentrifugation in the presence of bisbenzimide. Molecular sizes of mtDNAs were determined after digestion with restriction enzymes. A large interspecific variation was observed in mtDNA size: the largest one was 116 Kb in strain ARC-2125-1 of F. culmorum, while the smallest one was 49 kb in strain SUF-1121 of F. avenaceum. A considerable intraspecific size variation was also detected. All the species were identified by their mtDNA restriction patterns and assigned to individual clusters by UPGMA analysis, except for one strain of F. sporotrichioides. A Pstl clone library was constructed from strain KU-1615 of F. graminearum that is highly pathogenic and produces a large amount of nivalenol. Using this library, physical maps of F. gramineomm and F. culmorum were constructed. The mtDNA library and physical maps would facilitate species identification and further study of mtDNA polymorphisms in Fusarium species.

New CMS types in Plantago lanceolata and their relatedness

Mitochondrial variation in Plantago lanceolata was used to detect new CMS types. Directional reciprocal crosses were made between plants which differed in mtDNA restriction patterns. Differential segregation of male steriles in reciprocal crosses indicated that the parents differed in CMS type. MtDNA variation revealed nine RFLP patterns, which could be categorised according to the sex phenotype of the plants as MS1 (brown-anther type), MS2 (petaloid flower type) and MS3 (more yellow anthers than MS1). A single mtDNA pattern was found within MS1, six mtDNA patterns were found within the MS2 group, and two mtDNA patterns were found within hermaphrodites which segregated MS3 in the crosses. MS1 and MS2 are known to represent different CMS types, CMSI and CMSII. In reciprocal crosses between plants with different mtDNA patterns within the MS2 group, different ratios of male steriles segregated in the crosses, indicating that the parents differed in CMS type. Within the MS2 group two CMS types were found, designated CMSIIa and b. Finally, the sex phenotype H/MS3 turned out to be a different type. From previous studies it was known that the MS3 phenotype can also occur in CMSI and CMSII types, hence it is unclear whether MS3 is diagnostic for CMSIII. Since the data in this study cannot distinguish between the new type being a fully restored new CMS type or a ‘Normal’ cytoplasm, it was denoted as CMSIII. In total, four CMS types were found in the material studied. CtDNA variation was screened and three chloroplast haplotypes were identified. Two haplotypes were associated with CMSI plants, and one haplotype with the other CMS types. The ctDNA variation indicated that the CMSI type is widespread within the species, due to migration rather than to recurrent mutation. This may lead to the conclusion that only a limited number of CMS types are successful.

Determination of relationships in Pseudocercosporella herpotrichoides by analysis of mitochondrial DNA

Relatedness between isolates of Pseudocercosporella herpotrichoides, the causal fungus of eyespot disease of cereals, was investigated by a quantitative analysis of restriction fragment length polymorphisms of mitochondrial DNAs. Restriction patterns of mtDNA for all nine enzymes used in this study were different between the two groups of P. herpotrichoides, identified on the basis of cultural characteristics on PDA: fast-growing, even-edged (FE) and slow-growing, feathery or uneven-edged (SF). The nucleotide sequence divergence of mtDNA between these two groups was considerably higher than that between isolates belonging to the same taxa of fungi. Within each group, minor or no polymorphisms were observed among isolates from diverse geographical origins. An isolate of pathotype C could not be distinguished from the other FE isolates of P. herpotrichoides. Major nucleotide sequence divergence of mtDNA was observed among isolates of both groups of P. herpotrichoides, P. anguioides and P. aestiva, indicating there are no close relationships among these species. This study suggests that isolates of P. herpotrichoides consist of two genetically distinct taxonomic groups, corresponding to separate species.

Distinguishing Fall Armyworm (Lepidoptera: Noctuidae) Strains Using a Diagnostic Mitochondrial DNA Marker

The fall armyworm, Spodoptera frugiperda (J. E. Smith), includes morphologically indistinguishable corn and rice strains. The two strains were surveyed for diagnostic restriction patterns in mitochondrial DNA (mtDNA) using 25 restriction endonucleases. Polymorphic mtDNA restriction patterns were identified for BstNI, HinfI and MspI. The MspI pattern was the most distinctive since the molecular size of each DNA fragment differed between the two strains. Analyses of laboratory and field-collected insects showed the MspI mtDNA pattern to be a diagnostic marker for corn and rice strain insects. Strain identification by the MspI mtDNA profile correlated exactly with nuclear DNA markers. Since no HaeIII sites are present in fall armyworm mtDNA, a double-digest of total fall armyworm DNA using HaeIII and MspI allowed the direct detection of mtDNA restriction fragments from total DNA on a stained agarose gel. In contrast to conventional techniques utilizing mtDNA markers, this rapid and simple procedure does not require the isolation of mtDNA, or avoids the use of DNA blots and labeled mtDNA.

Characterization of Wine Yeast Strains of the Saccharomyces Genus on the Basis of Molecular Markers: Relationships Between Genetic Distance and Geographic or Ecological Origin

We identify and characterize 31 Saccharomyces strains from different wine regions, deposited at the Spanish Type Culture Collection, according to mtDNA restriction patterns and chromosomal profiles. By using this kind of information we analyze the correlation between genetic distances and ecological or geographical factors by means of a cluster analysis, assessed by an analysis of the molecular variance (AMOVA). From these analyses, red wine strains are significantly grouped according to their geographic origin, independently of the wine type and the grapevine cultivar, and white wine strians according to ecological factors (wine type of grapevine cultivars). This study also confirms the usefulness of the analysis of interpopulation genetic polymorphism, such as that resulting from mtDNA restriction fragment analysis of wine yeast strains, to determine the distribution of variation in natural populations of Saccharomyces cerevisiae .

Restriction endonuclease analysis of mitochondrial DNA of three farm animal species: Cattle, sheep and goat

Five restriction endonucleases (HindIII, BgIII, EcoRI, EcoRV and BamHI) were employed to analyse mitochondrial DNA of cattle, sheep and goat. The results showed completely different restriction patterns of mtDNA among the three bovid species. A total of 11, 16, and 17 restriction fragments in cattle, sheep and goat respectively, were detected by the five restriction endonucleases. Average total sizes of mtDNA of cattle, sheep and goat were found to be 16.49 ± 0.18, 16.30 ± 0.25 and 16.44 ± 0.08 kb, respectively. The mtDNA cleavage patterns were identical for all seven individuals belonging to two cattle breeds and for 10 individuals from one sheep breed.

Are mitochondria inherited paternally in Ascaris?

Mitochondrial DNA (mtDNA) is finding increasing usage as a tool for studying the systematics, population genetics and epidemiology of parasitic helminths, and is generally assumed to be inherited maternally. Yet two features of Ascaris biology—fertilization by large amoeboid sperm and some novel aspects of sperm mitochondria—suggest a paternal component to mitochondrial inheritance in this organism. In this study, we compare mtDNA restriction patterns of parental worms with those of their progeny but find no evidence for paternal inheritance. We suggest that sperm-derived mitochondria are actively destroyed or outcompeted by maternal organelles in the zygote.