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Results for “Random Amplified Polymorphic DNA Technique”

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PubMed2001

Molecular differentiation of the Old World Culicoides imicola species complex (Diptera, Ceratopogonidae), inferred using random amplified polymorphic DNA markers.

Samples of seven of the 10 morphological species of midges of the Culicoides imicola complex were considered. The importance of this species complex is connected to its vectorial capacity for African horse sickness virus (AHSV) and bluetongue virus (BTV). Consequently, the risk of transmission may vary dramatically, depending upon the particular cryptic species present in a given area. The species complex is confined to the Old World and our samples were collected in Southern Africa, Madagascar and the Ivory Coast. Genomic DNA of 350 randomly sampled individual midges from 19 populations was amplified using four 20-mer primers by the random amplified polymorphic DNA (RAPD) technique. One hundred and ninety-six interpretable polymorphic bands were obtained. Species-specific RAPD profiles were defined and for five species diagnostic RAPD fragments were identified. A high degree of polymorphism was detected in the species complex, most of which was observed within populations (from 64 to 76%). Principal coordinate analysis (PCO) and cluster analysis provided an estimate of the degree of variation between and within populations and species. There was substantial concordance between the taxonomies derived from morphological and molecular data. The amount and the different distributions of genetic (RAPD) variation among the taxa can be associated to their life histories, i.e. the abundance and distribution of the larval breeding sites and their seasonality.

Africa↗

PubMed2001

Comparison of restriction fragment length polymorphism, microsatellite length polymorphism, and random amplification of polymorphic DNA analyses for fingerprinting Aspergillus fumigatus isolates.

Aspergillus fumigatus fingerprints generated by random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) upon hybridization with repeated DNA sequences, and PCR detection of microsatellite length polymorphism (MLP) were compared among 67 isolates. In contrast to RAPD, RFLP and MLP gave discriminating and significantly concordant genotyping results.

Aspergillosis↗

PubMed2006

Renewal of genetic composition of a lancelet, Branchiostoma belcheri, in the Seto Inland Sea, Japan.

We studied renewal of genetic composition of a lancelet, Branchiostoma belcheri, at a station in the Seto Inland Sea, Japan, using random amplified polymorphic DNA (RAPD) technique. We detected a total of 49 DNA bands from 27 individuals of 5 seasonal samples. Among these, some were specific to individual or seasonal sample. There were no bands common to all individuals. We calculated the Band Sharing Index (BSI) values for each combination of individuals. BSI was in a wide range of 0.00-0.97 and the average BSI between the individuals of a sample was remarkably different from sample to sample (0.37-0.83). In cluster analysis on the basis of BSI, largely 3 clusters were constructed, which finally connected to a single cluster at as low as about 0.2 of BSI. Besides, many clusters were constructed exclusively by a seasonal sample. These results suggest a large gene pool of lancelets in this area, the remarkable differences in gene pool size among the samples and a seasonal renewal of genetic composition at a habitat.

Animals↗

PubMed2000

Existence of two geographically-linked clonal lineages in the bacterial fish pathogen Photobacterium damselae subsp. piscicida evidenced by random amplified polymorphic DNA analysis.

In this work, we applied the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida), an important pathogen for different marine fish. Regardless of the oligonucleotide primer employed, the 29 isolates of Ph. damselae subsp. piscicida tested were separated into two groups, the RAPD-PCR analysis differentiated the European strains from the Japanese strains. The similarity between both groups estimated on the basis of the Dice coefficient was 75-80%. These results show that European and Japanese isolates of Ph. damselae subsp. piscicida, regardless of their host fish species, belong to two different clonal lineages. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.

Animals↗

PubMed2000

Frequency and biodiversity of 2,4-diacetylphloroglucinol-producing bacteria isolated from the maize rhizosphere at different stages of plant growth.

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.

Antimetabolites↗

PubMedDate not supplied

Genetic diversity and population structure of Lamiophlomis rotata (Lamiaceae), an endemic species of Qinghai-Tibet Plateau.

Lamiophlomis rotata (Lamiaceae), a perennial medicinal herb, is endemic to the Qinghai-Tibet Plateau. A total of 188 individuals from eight natural populations of L. rotata in Qinghai-Tibet Plateau (four from Tibet, two from Yunnan, and two from Qinghai) were analyzed using intersimple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) techniques. Our results revealed that the level of genetic variation in L. rotata was relatively high (P = 94.85%, I = 0.440 +/- 0.220, H(T) = 0.289 +/- 0.028). Three genetic groups corresponding to the three geographic regions were detected, suggesting significant geographic structure. Our results suggest that the highly structured geographic pattern found in L. rotata might represent diverging evolutionary processes associated with the uplifting of the Qinghai-Tibet Plateau and Quaternary climatic oscillations. These findings imply that as many populations as possible should be preserved in situ for the conservation of this species. Given their genetic variability and peripheral distribution, Qinghai and Yunnan populations should be assigned priority for conservation. Optimal harvesting strategies, domestication and tissue culture of L. rotata should be developed as soon as possible to guarantee its sustainable use.

China↗

PubMed2004

Genetic and epigenetic evaluations of citrus calluses recovered from slow-growth culture.

The embryogenic callus of "Red Marsh" grapefruit was stored in vitro by slow growth culture method for one year, and survived with a significant weight increment over that period. The survivers regenerated somatic embryos more easily than the controls. Eight callus lines were used for genetic analyses. Although chromosome number variations were verified by cytological examination both in the controls and the stored samples, the ploidy level remained relatively stable during the storage period. Randomly amplified polymorphic DNA (RAPD) analysis was performed to detect DNA sequence variation. No difference in RAPD pattern was found with the 102 primers used. However, a methylation sensitive amplified polymorphism (MSAP) assay showed DNA methylation changes in the stored samples compared with the controls.

Citrus↗

PubMedDate not supplied

Individual DNA bands obtained by RAPD analysis of canine genomic DNA often contain multiple DNA sequences.

The random amplified polymorphic DNA (RAPD) technique has been widely applied for genetic studies of plants, insects, and fungi, and recently has been used for studies in animals including dogs. To convert the RAPD marker into a classical PCR marker, the RAPD-PCR products are size-separated in an agarose gel and a specific DNA band is selected for potential association with a trait of interest. The DNA fragments present in the desired band are then cloned and sequenced using primers specific to the cloning vector, and the sequence is used to design a pair of classical PCR primers. Often a "positive clone" is identified based solely on a match of the size of the insert in the clone with the uncharacterized DNA band originally selected. We observed that single DNA bands obtained from RAPD-PCR using canine genomic DNA often contain DNA fragments of similar size but of different sequences. Based on this observation, we report here a modification of the protocol for RAPD analysis which will ensure that a promising RAPD marker selected based on initial screening is not lost for lack of a comprehensive investigation in the later experimental analysis.

Animals↗

PubMed2006

Genotypic diversity in a localized population of Ralstonia solanacearum as revealed by random amplified polymorphic DNA markers.

AIMS: To assess genotypic diversity within Ralstonia solanacearum isolates of a single field. METHODS AND RESULTS: A total of 44 field isolates and 22 in vitro generated clones of R. solanacearum were studied for genotypic diversity by random amplified polymorphic DNA (RAPD) technique. Genomic DNA of these isolates and clones was extracted by proteinase-K-SDS lysis mini-prep method. RAPD analysis was done with 30 decamer primers. The data were analysed using NTSYSpc 2.02h software. Forty-two out of 44 field isolates and all the clonal isolates were identified as distinct genotypes at 70% similarity level. CONCLUSION: Very high level of genome variability was observed within the field and clonal isolates of R. solanacearum. This might be a reason for the wide host range of this bacterium and for quick breakdown of wilt resistance in host plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that it would be difficult to design specific diagnostic protocol for R. solanacearum even for a localized population and to breed cultivars with broad-spectrum resistance.

DNA, Bacterial↗

PubMed2005

Amplified fragment length polymorphism (AFLP) versus randomly amplified polymorphic DNA (RAPD) as new tools for inter- and intra-species differentiation within Bordetella.

Automated amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) techniques with fluorescently labelled primers were used to track differences among isolates of the eight known species of the Bordetella genus. Eighty-one representative strains of these species from international and Polish bacterial collections were genotyped according to RAPD protocols using primer 1254 or 1247, and AFLP involving EcoRI/MseI or newly designed SpeI/ApaI restriction/ligation/amplification procedures. By comparing AFLP and RAPD data, it was concluded that the discriminatory power of AFLP is higher in comparison with RAPD for both intra- and inter-species differentiation of isolates of the Bordetella genus. The most precise level of inter-species discrimination and the highest level of intra-species discrimination of the Bordetella isolates of the eight species were observed in the AFLP EcoRI/MseI and SpeI/ApaI sets, respectively. Both techniques might provide alternative tools for the identification of Bordetella at the genomic species and strain levels, and thus may be valuable in human and veterinary diagnostics as well as in epidemiology. By applying the AFLP technique presented in this article, more precise data on the emergence of newly acquired and/or on expanded clones and transmission routes of isolates of the Bordetella genus in the human and animal environments might be obtained.

Animals↗

PubMed2004

[Development of hybrid strains from geographic isolates of Eimeria tenella and their immunoprotection].

OBJECTIVE: To cultivate hybrid strains from three geographic isolates of Eimeria tenella and to explore the possibility of developing vaccine candidates. METHODS: Three parental strains were selected from five geographic isolates of E. tenella through immune experiment, and hybrid strains were cultivated. The genomic DNA of the three parental strains and their filial generation were analyzed by random amplified polymorphic DNA (RAPD) technique with 30 optimization primers screened from 200 primers, the hybrid strains were isolated from the filial generation by RAPD. Chicken were inoculated with hybrid strains, and challenged with different strains to compare the immunogenicity and immunoprotection. RESULTS: Immunogenicity and immunoprotection of the three strains isolated from Guangzhou, Baoding and Changchun were stronger than those of other strains. Hybridization was performed to cultivate hybrid strain. Two hybrid strains were isolated from Changchun x Baoding and Guangzhou x F1 by RAPD. The result of immune experiment proved that immunoprotecion of F1 and F2 were higher than their parental strains. CONCLUSION: Two hybrid strains have been cultivated from the three geographic isolates of E. tenella, with the immunogenicity of their parental strains. Chicken immunized by F2 strain have shown strong resistance against the infection of the geographic strains, with an average protection rate of 84%.

Animals↗

PubMed1998

An ecological study of lactococci isolated from raw milk in the camembert cheese registered designation of origin area.

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of "Camembert de Normandie" cheese.

Animals↗

PubMed2004

Clonal diversity in the ancient asexual ostracod Darwinula stevensoni assessed by RAPD-PCR.

As Darwinulidae (Ostracoda) are considered to be ancient asexuals with a wide geographical and ecological distribution, they are expected to have accumulated mutations during a long timeframe. However, previous studies on genetic variability suggested a low genetic divergence within the darwinulid species Darwinula stevensoni. Here, overall genotopic diversity of D. stevensoni is estimated with the Random Amplified Polymorphic DNA (RAPD) technique. Using six primers revealing 47 consistently scorable polymorphic loci, substantial clonal diversity within this species is detected. Five of the seven surveyed populations are multiclonal. Moreover, the seven populations have a different clonal composition with almost all of the observed clonal genotypes being restricted to single populations, indicating the absence of a single widespread 'clone'. The observed clonal diversity seems to refute the existence of a widespread general purpose genotype for D. stevensoni. However, in light of previously detected uniformity at functional loci, we reconsider the definition of a GPG. We suggest that it need not imply a genome-wide fixed genotype, but rather consists of a set of ecologically relevant genes.

Animals↗

PubMed1996

Identification, mapping and linkage analysis of randomly amplified DNA polymorphisms in Tetrahymena thermophila.

Using the random amplified polymorphic DNA (RAPD) technique and exploiting the unique genetics of Tetrahymena thermophila, we have identified and characterized 40 DNA polymorphisms occurring between two inbred strains (B and C3) of this ciliated protozoan. These RAPD markers permit the PCR amplification of a DNA species using template DNA from SB1969 (B strain) but fail to do so using DNA from C3-368-5 (C3 strain). Polymorphisms were mapped to chromosomes using a panel of monosomic strains constructed by crossing B strain-derived nullisomic strains to inbred strain C3. They map to all five chromosomes and appear to be evenly distributed throughout the genome. Chromosomal groups were then analyzed for linkage using meiotic segregants; four linkage groups were identified in chromosomes 1R 2L, 3 and 5. The RAPD method appears useful for the construction of a genetic map of the Tetrahymena genome based on DNA polymorphisms.

Animals↗

PubMed2003

Molecular fingerprinting of fish-pathogenic Lactococcus garvieae strains by random amplified polymorphic DNA analysis.

In this work, we used the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Lactococcus garvieae, an important pathogen for fish. Fifty-seven strains with different hosts and geographical origins, including Japan and several countries of the Mediterranean area such as Spain, Portugal, France, Italy, England, and Turkey, were analyzed. Two primers, oligonucleotides 5 and 6 (Pharmacia Biotech) were utilized; primer 5 was the most discriminative, since allowed us to differentiate 10 RAPD -types related to the origin of the strains. Regardless of the oligonucleotide primer employed, the 57 isolates of L. garvieae studied were separated into three genetic groups, composed of the Spanish, Portuguese, English, and Turkish strains (group A), the Italian and French strains (group B), and the Japanese strains (group C). The similarity of isolates within each group, estimated on the basis of the Dice coefficient, ranged from 75 to 100%. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.

Animals↗

PubMed1996

Use of RAPD for the detection of genetic variation in the human blood fluke, Schistosoma japonicum, from mainland China.

A random amplified polymorphic DNA (RAPD) technique using 16 decamer oligonucleotide primers was employed to characterize isolates of Schistosoma japonicum from seven geographical locations (Sj1: Zhejiang; Sj2: Anhui; Sj3: Jiangxi; Sj4: Hunan; Sj5: Hubei; Sj6: Sichuan; Sj7: Yunnan) of the People's Republic of China. Distinct differences between some isolates were reproducibly detected in RAPD patterns produced using five of the primers. The analyses showed that both Sj6 and Sj7 were quite distinct genetically from Sj1-Sj5 based on the presence/absence of particular bands (A10-200 bp, A9-220 bp, B17-520 bp, P205-680 bp and P235-930 bp). These findings are in line with previous reports on the biological, biochemical, immunological and chemotherapeutic differences of S. japonicum from Sichuan and Yunnan compared with other geographical regions. The present study showed, based on RAPD profiles, that genetic differences exist within S. japonicum from mainland China. This finding may have important implications for studying the population biology, epidemiology and clinical forms of the disease in China, as well as for developing vaccines and diagnostic test systems.

Animals↗

PubMed1999

A molecular marker that segregates with sorghum leaf blight resistance in one cross is maternally inherited in another.

Leaf blight-resistant sorghum accession SC326-6 was crossed to the susceptible cultivar BTx623 to analyze the genetic basis for resistance. Field scoring of inoculated F2 progeny revealed that resistance was transmitted as a dominant single-gene trait. By combining the random amplified polymorphic DNA (RAPD) technique with bulked-segregant analysis, it was possible to identify PCR amplification products that segregated with disease response. Primer OPD12 amplified a 323-bp band (D12R) that segregated with resistance. Creation of longer primers, or SCARs (sequence characterized amplified regions) for D12R resulted in the amplification of a single major band of the predicted size from all the resistant F2 progeny and the resistant parent SC326-6, but not from BTx623 or 24 of 29 susceptible F2 progeny. The SCAR primers also amplified a single band with DNA from IS3620C, the female parent in a cross with BTx623 that has been used to produce a recombinant inbred population for RFLP mapping. An equivalent band was amplified from all 137 recombinant inbred progeny, indicating that organelle DNA is the amplification target in this cross.

Base Sequence↗

PubMed2004

Molecular ecology and pathogenic potential of Fonsecaea species.

The genus Fonsecaea is revised on the basis of ribosomal DNA internal transcribed spacer (ITS) sequence data. Two species are recognized, F. pedrosoi and the new defined F. monophora. The distinction between these species does not correspond with the classical distinction of F. pedrosoi and F. compacta. The latter appears to be no more than a morphological variant. Both species recognized in this study are agents of human chromoblastomycosis; however, in F. pedrosoi a strict association with this disease is noted, while F. monophora is a more general opportunist. Subspecific randomly amplified polymorphic DNA (RAPD) typing revealed a high degree of strain diversity, although clonal reproduction is also likely to occur. Most strains with Fonsecaea-like morphology isolated from environments to which symptomatic human patients were exposed were found to be more closely related to species of Cladophialophora than to Fonsecaea.

Ascomycota↗