RFLP Analysis Of Mitochondrial DNA Research Articles

New Balkan records of Bombus subterraneus (Linnaeus 1758) and Bombus cryptarum (Fabricius 1775) from Greece

From the study of the Greek bumblebee fauna (Hymenoptera: Apidae, Bombini), species lists have been published based on both literature records and original data from collected bees. Since 1995 a special effort to confirm with newly collected bees all bumblebee species reported in literature records for Greece has been in progress. Although numerous specimens have been collected and examined and in some instances yielding new Bombus species for the Greek insect fauna, some species, mainly those reported in older references, have not yet been found. Recently, identification of bumblebees collected in the Florina Prefecture - Northwest Macedonia, during the years 2006 and 2007 yielded information for two “literature cited” species, Bombus subterraneus (Linnaeus 1758) and Bombus cryptarum (Fabricius 1775). A B. subterraneus queen (collected at 40°47´38N, 21°26´10E on Vicia cracca) was distinguished by morphological characteristics and a worker B. cryptarum (collected at 40°41´58,7N, 21°28´18,5E on Echium spp) was revealed using mitochondrial DNA RFLP analysis of the CO1 gene. These new records from Florina are provided with comments, confirming the species presence in Greece for the first time after approximately 40 years.

Identification of Japanese Lymantria species (Lepidoptera: Lymantriidae) based on PCR–RFLP analysis of mitochondrial DNA

A polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP)-based method for species identification was applied to seven Japanese Lymantria species, including four Asian gypsy moth (AGM) species. We sequenced the partial end of the cytochrome c oxidase I (COI) gene, tRNA leucine, COII gene, and partial end of the tRNA lysine in mitochondrial DNA (mtDNA) for one individual of each of the seven species. We analyzed the recognition sites of three restriction endonucleases and constructed a scheme for Lymantria species identification using PCR–RFLP. We then applied the scheme to 291 individuals from 45 populations of seven species. We found that all seven species were correctly identified using PCR–RFLP. These results suggest that PCR–RFLP is useful for identifying Japanese Lymantria species, which may be detected at Japanese ports.

Genetic structure and variation in aggressiveness in Fusarium virguliforme in the Midwest United States

Sudden death syndrome of soybean, caused by Fusarium virguliforme, is one of the most damaging diseases affecting soybean production in the Midwestern region of the USA. To date, there has been very low genetic variation detected in F. virguliforme populations, although isolates can exhibit differing aggressiveness on soybean. To further investigate the genetics behind this variation in aggressiveness, multiple fingerprint analyses were conducted on a collection of F. virguliforme isolates from Iowa and neighbouring states, and their aggressiveness was tested using greenhouse and culture filtrate assays. Twelve RAPD primers identified 13 genotypes, while hybridization of the (CAT)5 probe to Pst I-restricted genomic DNA yielded eight genotypes. Fingerprint patterns obtained by RFLP analysis of mitochondrial DNA with the enzyme HaeIII were identical for all F. virguliforme isolates. Combined analysis of the RAPD and (CAT)5 binary data identified 25 genotypes within F. virguliforme, indicating a greater amount of genetic diversity than was previously known. Disease severity and plant growth varied significantly among isolates, but isolate aggressiveness was not associated with genetic variation. This study provides evidence for the existence of genetic variation in F. virguliforme but suggests that minor quantitative traits and environmental interactions are primarily responsible for the variation in aggressiveness found among isolates within the species.

Geographical markers for Saccharomyces cerevisiae strains with similar technological origins domesticated for rice-based ethnic fermented beverages production in North East India

Autochthonous strains of Saccharomyces cerevisiae from traditional starters used for the production of rice-based ethnic fermented beverage in North East India were examined for their genetic polymorphism using mitochondrial DNA-RFLP and electrophoretic karyotyping. Mitochondrial DNA-RFLP analysis of S. cerevisiae strains with similar technological origins from hamei starter of Manipur and marcha starter of Sikkim revealed widely separated clusters based on their geographical origin. Electrophoretic karyotyping showed high polymorphism amongst the hamei strains within similar mitochondrial DNA-RFLP cluster and one unique karyotype of marcha strain was widely distributed in the Sikkim-Himalayan region. We conceptualized the possibility of separate domestication events for hamei strains in Manipur (located in the Indo-Burma biodiversity hotspot) and marcha strains in Sikkim (located in Himalayan biodiversity hotspot), as a consequence of less homogeneity in the genomic structure between these two groups, their clear separation being based on geographical origin, but not on technological origin and low strain level diversity within each group. The molecular markers developed based on HinfI-mtDNA-RFLP profile and the chromosomal doublets in chromosome VIII position of Sikkim-Himalayan strains could be effectively used as geographical markers for authenticating the above starter strains and differentiating them from other commercial strains.

Effects of grape marcs acidification treatment on the evolution of indigenous yeast populations during the production of grappa

Grappa is a typical Italian product obtained from the distillation of grape marcs, the main by-product of grape crushing. One technological treatment frequently performed on marcs is their acidification, in order to contrast the development of unwanted spoilage bacteria during the storage period needed for alcoholic fermentation. A pilot-scale experiment was set-up to study the dynamics of yeast populations during a 30-day fermentation of acidified and nonacidified Prosecco grape pomace. Saccharomyces cerevisiae population, examined after 4 and 15 days of storage by mitochondrial DNA-RFLP analysis, resulted considerably different at strain level upon acidification. In particular, although the number of different strains rescued appeared particularly high in both kind of marcs compared with what happens in must fermentation, in the acidified material such number tends to moderately decrease during storage. Results obtained evidence that the acidification treatment did not influence yeast population neither in terms of number of cells nor in terms of biodiversity at species level. Therefore, such treatment can be used in distillery without negatively influencing ethanol production. Even though some data are available on the effects of technological treatments on the chemical composition of the distillate, no microbiological studies have been published so far on the consequence of these practices on composition, biodiversity and evolution of yeast population.

Effect of the use of commercial Saccharomyces strains in a newly established winery in Ronda (Málaga, Spain)

An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin "Sierras de Málaga" (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.

PCR–RFLP analysis of mitochondrial DNA in tench Tinca tinca

Polymorphism was detected at ND1, ND6, D-loop and cyt b segments of mtDNA in 105 tench (Tinca tinca L.), using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and five composite haplotypes were identified. The diversity indices and the results of the population comparisons revealed that the identified markers provide a powerful tool for further studies on this species.

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.

DNA typing methods for differentiation of yeasts related to dry-cured meat products

RFLP analysis of the ITS and 18S rDNA, RAPD-PCR using mini- and microsatellite primers and RFLP analysis of mitochondrial DNA were examined to discriminate yeasts related to dry-cured meat products at species and strain level. Seven species and 35 strains of yeasts usually found in dry-cured meat products were tested. RFLP analysis of the ITS1-5.8S rDNA-ITS2 and 18S rDNA did not allow the separation at species level of all of the species tested. RAPD with a M13 primer was found to be useful for differentiation of Rhodotorula mucilaginosa, Candida zeylanoides, Yarrowia lipolytica, Debaryomyces hansenii and Saccharomyces cerevisiae. However, no differences were observed between Debaryomyces polymorphus and Pichia carsonii. RAPD analysis with microsatellite primers (GACA) 4, (GTG) 5 and (GAC) 5 enabled discrimination at species and strain level. However, the degree of discrimination by means of RAPD-PCR depends highly on the primers used. Thus, the PCR fingerprinting with primer (GACA) 4 enabled a higher level of discrimination than primers (GAC) 5 and (GTG) 5. The RFLP analysis of mtDNA allowed the discrimination at the species and strain level except for R. mucilaginosa, where no polymorphisms were observed in the strains tested. RAPD analysis with primer (GACA) 4 and the restriction analysis of mtDNA used in the present work are useful for the differentiation at species and strain level of yeasts related to dry-cured meat products.

Pathogenic free-living amoebae in Korea.

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.

RFLP analysis of mitochondrial DNA in eggplant and related Solanum species

RFLP analysis of mitochondrial DNA (mtDNA) was performed by the Southern hybridization method using total DNA extracted from eggplant, Solanum melongena L., and six related Solanum species, S. surattense Burm. (i.e. S. virginianum L.), S. torvum Swartz, S. gilo Raddi (i.e. S. aethiopicum), S. integrifolium Poir. (i.e. S. aethiopicum), S. indicum auct. non L. (i.e. S. violaceum Ort.) and S. sanitwongsei Craib. Forty-one fragments were detected by the analysis using 12 combinations of four restriction enzymes and three probes of mtDNA clones from rice. Thirty-four out of the 41 fragments were polymorphic among the species, whereas the other seven were monomorphic. This RFLP analysis of mtDNA is demonstrated to be appropriate for assessing phylogenetic relationships in eggplant and related Solanum species at the interspecific level.

Acanthamoeba sohi, n. sp., a pathogenic Korean isolate YM-4 from a freshwater fish.

A new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Acanthamoeba sp. YM-4 (Korean isolate YM-4). The trophozoites were 11.0-23.0 micrometer in length and had hyaline filamentous projections. Cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Acanthamoeba YM-4 can survive at 40 degrees C, and its generation time was 19.6 hr, which was longer than that of A. culbertsoni. In terms of the in vitro cytotoxicity of lysates, Acanthamoeba YM-4 was weaker than A. culbertsoni, but stronger than A. polyphaga. On the basis of the mortality of experimentally infected mice, Acanthamoeba YM-4 was found to be highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. An anti-Acanthamoeba YM-4 monoclonal antibody, McAY7, was found to react only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster on the basis of phylogenetic distances. Thus the Acanthamoeba Korean isolate YM-4 was identified as a new species, and assigned as Acanthamoeba sohi.

Mitochondrial DNA diversity in wild and cultured populations of Brycon opalinus (Cuvier, 1819) (Characiformes, Characidae, Bryconinae) from the Paraı́ba do Sul Basin, Brazil

RFLP analysis of mitochondrial DNA was carried out to investigate the population genetic structure of cultured and wild populations of Pirapitinga-do-sul— Brycon opalinus (Cuvier, 1819) (Characiformes, Characidae, Bryconinae) from the Paraı́ba do sul basin, Brazil. mtDNA samples were collected from 257 specimens sampled in seven rivers of the Paraı́ba basin and in a hatchery used for restocking. An initial screening with 24 restriction enzymes revealed six informative enzymes, which generated 27 haplotypes. Haplotype diversities were high both in the hatchery broodstock ( h=0.75) and in the samples from the wild ( h=0.60). Nucleotide diversity among the 27 B. opalinus haplotypes was 0.825%. Analysis of molecular variance (AMOVA) showed the highest variance within populations (70.48%) while 28.16% of the total diversity was due to interpopulation variance, suggesting the existence of genetic differentiation among B. opalinus populations.

Intra- and interspecific genetic variability and relationships among mud crabs, Scylla spp.(Decapoda: Portunidae), demonstrated by RFLP analysis of mitochondrial DNA.

Intra- and interspecific genetic variability and relationships among mud crabs, Scylla spp.(Decapoda: Portunidae), demonstrated by RFLP analysis of mitochondrial DNA.

Analysis of yeast populations during alcoholic fermentation: A six year follow-up study

Wine yeasts were isolated from fermenting Garnatxa and Xarel.lo musts fermented in a newly built and operated winery between 1995 and 2000. The species of non-Saccharomyces yeasts and the Saccharomyces cerevisiae strains were identified by ribosomal DNA and mitochondrial DNA RFLP analysis respectively. Non-Saccharomyces yeasts, particularly Hanseniaspora uvarum and Candida stellata, dominated the first stages of fermentation. However Saccharomyces cerevisiae was present at the beginning of the fermentation and was the main yeast in the musts in one vintage (1999). In all the cases, S. cerevisiae took over the process in the middle and final stages of fermentation. The analysis of the S. cerevisiae strains showed that indigenous strains competed with commercial strains inoculated in other fermentation tanks of the cellar. The continuous use of commercial yeasts reduced the diversity and importance of the indigenous S. cerevisiae strains.

Genetic Introgression by the Rose Bitterling, Rhodeus ocellatus ocellatus, into the Japanese Rose Bitterling, R. o. kurumeus (Teleostei: Cyprinidae)

The Japanese rose bitterling, Rhodeus ocellatus kurumeus, is an endangered cyprinid species. The main reason of drastic diminution of R. o. kurumeus has been suggested to be due to hybridization between this subspecies and R. o. ocellatus introduced from China. Both RFLP analysis of mitochondrial DNA and RAPD-PCR analysis of genomic DNA were performed to elucidate the genetic introgression of R. o. ocellatus into R. o. kurumeus. The two subspecies were distinguished in the D-loop region of mtDNA by six restriction endonucleases. Rhodeus o. kurumeus had eleven subspecies-specific RAPD markers and R. o. ocellatus had two. Except some R. o. kurumeus populations, most of the populations of R. ocellatus in Japan were hybrids, equipped with subspecies-specific RAPD markers for the two subspecies, respectively. The genetic constitution of these markers in hybrids, however, greatly differed among populations. The R. o. ocellatus mtDNA was predominantly observed in hybrid populations, except two populations with mtDNAs of the two subspecies. Judging from the genetic dominance of morphological and physiological characters of R. o. ocellatus against R. o. kurumeus, hybrids probably have the same ecological dominance as R. o. ocellatus against R. o. kurumeus. Therefore, it is considered that R. o. kurumeus not only has its genetic property spoiled by hybridization with R. o. ocellatus, but also is expelled by R. o. ocellatus and hybrids. The replacement of mtDNA and genomes of R. o. kurumeus with those of R. o. ocellatus in hybridization might be accelerated by the backcross between hybrids and R. o. ocellatus.

MOLECULAR CHARACTERIZATION AND GENETIC STRUCTURE OF OLIVE GERMPLASM COLLECTION IN CONSERVATOIRE BOTANIQUE NATIONAL MÉDITERRANÉEN DE PORQUEROLLES USING NUCLEAR RAPD MARKERS AND RFLP OF MITOCHONDRIAL DNA

With more than 500 trees and about one hundred accessions, the olive collection in the Conservatoire Botanique National Mediterraneen de Porquerolles (CBNMP) forms the major part of the national French germplasm. We performed a molecular characterization and genetic structure analysis of the olive germplasm collection of CBNMP using nuclear RAPDs and RFLPs of mitochondrial DNA. Using four primers, 32 markers were determined allowing the identification of 114 multilocus profiles. Each of the 78 denominations were identified by one specific multilocus profile. Among the remaining 36 multilocus profiles, we noted several homonyms, synonymies and mislabeling. A dendrogram was constructed according to the Ward algorithm and Euclidean distance based on a FCA coordinate matrix. This dendrogram showed 8 clusters of olive profiles. Oil cultivars were present in all clusters, whereas cultivars used for oil and canned olive were restricted to clusters 1, 2, 3 and 4. This suggested that the selection for genotypes with large olive has been performed in a more restricted genetic pool. RFLP analysis of mitochondrial DNA showed that 85 % out of 114 olive profiles were characterized by the mitotype ME1, which is characteristic of the Eastern Mediterranean. The remaining profiles were characterized by mitotypes MOM and MCK specific of the Western Mediterranean. AMOVA analysis, based on RAPD data, showed no genetic differentiation between groups of genotypes defined according to the four mitotypes. These results showed that French olive germplasm has been derived from different gene pools with a predominance of the Eastern group. The absence of genetic differentiation suggests that some cultivars have originated from hybrids between plants from different origins.

OLIVE CULTIVAR IDENTIFICATION USING NUCLEAR RAPDS AND MITOCHONDRIAL RFLPS

One hundred and thirteen olive accessions obtained from different collections and various orchards around the Mediterranean Basin were characterised using RAPD-PCR. The polymorphic bands obtained using a combination of only three RAPD primers enabled us to distinguish between one hundred and two of the accessions. We estimate that the probability of obtaining the same RAPD profile for two different trees was between 6.75 × 10 -5 and 4.82 × 10 -14 . Many of the RAPD profiles were correlated with the country or region of origin of the accessions, suggesting that cultivar selection occurred within different genetic pools and within different areas. Furthermore, RFLP analysis of mitochondrial DNA revealed four mitotypes. Mitotype ME1 (72 %) was by far more frequent than mitotypes ME2 (10 %), MOM (9 %) and MCK (9 %). Accessions that were genetically similar, based on the RAPD analysis, occurred in either the eastern (ME1 or ME2) or the western (MOM or MCK) group of the Mediterranean. This finding suggests that nuclear and cytoplasmic DNAs have been combined due to crosses between trees from different origins.

RFLP Analysis of Mitochondrial DNA in Wakegi Onion.

Wakegi onion is a natural hybrid between Japanese bunching onion and shallot. To reveal the donor of mitochondrial DNA (mtDNA) in wakegi onion, RFLPs of mtDNA using probes and PCR-RFLPs of V7 region of srRNA gene were analyzed in the three species. In both methods, interspecific polymorphisms were detected between Japanese bunching onion and shallot. All the clones of wakegi onion had the same RFLP patterns as Japanese bunching onion. These results insist that many clones of wakegi onion originated from the natural hybridization between Japanese bunching onion as the maternal parent and shallot as the paternal parent, which supports the previous results based on cpDNA analyses in wakegi onion.

Genetic differentiation and phylogenetic relationships among Greek Chub Leuciscus cephalus L. (Pisces, Cyprinidae) populations as revealed by RFLP analysis of mitochondrial DNA

Abstract The phylogenetic and systematic relationships among 12 populations of Greek chub ( Leuciscus cephalus L.) were investigated using RFLP analysis of two PCR-amplified mtDNA segments (D-loop and cytochrome b). Three out of eight, and nine out of ten restriction enzymes were polymorphic for D-loop and cytochrome b, respectively. Twenty-one different haplotypes were detected. Estimated nucleotide sequence divergence among the 21 genotypes ranged from 0.313 to 6.79%; the mean value of sequence divergence among populations was 3.421±0.00%. The calculated N ST value of 0.94, indicates high interpopulation diversity. Dendrograms based on haplotypes as well as those of populations, support the previous recognition of different subspecies, but these mtDNA data are not concordant with previous allozyme data among these fish. Our results suggest that to clarify phylogenetic relationships multiple genetic systems need to be examined.