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Polymorphic DNA (polymorphic + dna)

Distribution by Scientific Domains
Life Sciences72%
Medical Sciences14%
Earth and Environmental Science10%
Chemistry2%
Distribution within Life Sciences
Evolution20%
Plant Pathology16%
Plant Science13%
Applied Microbiology11%
Entomology6%
9 Other Subdomains34%

Kinds of Polymorphic DNA

  • amplified polymorphic dna
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  • random amplified polymorphic dna
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    Terms modified by Polymorphic DNA

  • polymorphic dna analysis
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  • polymorphic dna marker
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    Selected Abstracts

    Direct genotyping of the poplar leaf rust fungus, Melampsora medusae f. sp. deltoidae, using codominant PCR-SSCP markers

    FOREST PATHOLOGY, Issue 4 2005
    M. Bourassa
    Summary Two anonymous DNA markers that are revealed by single-strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono-uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono-uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re-amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono-uredinial isolates of Mmd tested. From the overall 40 mono-uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse-grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies. Résumé Deux marqueurs génétiques anonymes, révélés par analyse SSCP (Single-Strand Conformational Polymorphism) ont été développés afin de détecter des polymorphismes génétiques chez le Melampsora medusae f. sp. deltoidae (Mmd). Dans un premier temps, des isolats mono-urédiniaux ont été obtenus, puis l'ADN a été extrait à partir des urédiniospores, les produits d'amplification RAPD (Random Amplified Polymorphic DNA) ont été générés à partir de huit de ces isolats mono-urédiniaux et les résultats d'amplification ont par la suite été séparés sur gel SSCP afin d'identifier des polymorphismes entre les isolats. Les bandes sur gel SSCP représentant des loci polymorphiques putatifs entre les isolats ont été prélevées du gel, ré-amplifiées par la technique d'amplification PCR (Polymerase Chain Reaction), clonées, puis séquencées. Pour deux fragments d'ADN séquencés sur un total de trois, une paire d'amorces a été développée afin de permettre l'amplification d'un fragment de taille adéquate pour analyse SSCP (<600 pb). Chaque paire d'amorces a produit un signal d'amplification positif pour chacun des huit isolats à l'origine de ces nouvelles amorces; les produits PCR ont ensuite été analysés par la technique SSCP. Les deux loci putatifs ont révélé des polymorphismes génétiques entre les isolats. Les deux paires d'amorces ont produit un fragment d'amplification de la taille attendue pour chacun des 32 isolats mono-urédiniaux supplémentaires testés. Des 40 isolats testés, 5 et 11 allèles ont été détectés, alors que 12 et 34 isolats se sont révélés hétérozygotes (tel qu'indiqué par la présence de plus de deux bandes sur gel SSCP) pour les loci A et B, respectivement. La spécificité des deux paires d'amorces a été testée à partir de 106 isolats fongiques appartenant à différents groupes taxonomiques, incluant d'autres rouilles, de même qu'à partir de l'ADN extrait de feuilles de peupliers cultivés en serre. Un signal d'amplification positif n'a été obtenu qu'en présence d'ADN du Mmd. Les conditions d'amplification PCR ont été optimisées pour les deux paires d'amorces développées afin de permettre le génotypage directement à partir d'urédinies individuelles prélevées sur des feuilles de peuplier infectées. La possibilité de génotyper directement des urédinies individuelles permet d'éviter l'obligation de cultiver le champignon pour génotyper les individus, ce qui représente un avantage important des marqueurs génétiques développés ici, puisqu'il devient dès lors possible de traiter un grand nombre d'échantillons lors de la réalisation d'études de populations. Zusammenfassung Zum Nachweis von Polymorphismen bei Melampsora medusae f. sp. deltoidae wurden zwei anonyme DNA Marker aus einer SSCP-Analyse entwickelt. Zunächst wurden Isolate aus einzelnen Uredinien gewonnen, die DNA wurde aus den Uredosporen extrahiert und polymorphe RAPD, Amplifikationsprodukte von acht Mono-Uredinium-Isolaten wurden auf einem SSCP-Gel getrennt, um Unterschiede zwischen ihnen nachzuweisen. Banden, die bei den acht geprüften Isolaten mögliche polymorphe Loci darstellten, wurden aus dem SSCP-Gel ausgeschnitten und mit PCR reamplifiziert, dann geklont und sequenziert. Für zwei von insgesamt drei sequenzierten DNA-Fragmenten wurde ein Primerpaar entwickelt, um ein in der Grösse für die SSCP-Analyse (<600 bp) geeignetes DNA-Fragment zu amplifizieren. Jedes Primerpaar amplifizierte bei allen acht ursprünglich für ihre Entwicklung verwendeten Isolaten ein PCR-Produkt, und diese wurden anschliessend mit SSCP analysiert. Für beide putativen Loci wurden bei den Isolaten Polymorphismen festgestellt. Die beiden Primerpaare amplifizierten ein PCR-Produkt der erwarteten Grösse bei allen 32 zusätzlich geprüften Mono-Uredinium-Isolaten des Pilzes. Bei den insgesamt 40 geprüften Mono-Uredinium-Isolaten wurden für die Loci A und B 5 bzw. 11 Allele gefunden, und 12 bzw. 34 Isolate erwiesen sich als heterozygot, was durch mehr als zwei Banden auf den SSCP-Gelen angezeigt wurde. Die Spezifität der Primerpaare wurden mit 106 Pilzisolaten aus verschiedenen Taxa geprüft, darunter andere Roste sowie DNA aus gesunden Pappelblättern aus Gewächshauskulturen. DNA-Amplifikationsprodukte der erwarteten Grösse wurden nur erhalten, wenn DNA von Melampsora medusae f. sp. deltoidae präsent war. Die PCR-Amplifikations-Bedingungen mit diesen beiden Primerpaaren wurde so optimiert, dass ein Genotyping direkt bei einzelnen von infizierten Blättern entnommenen Uredinien erfolgen kann und somit eine Pilzkultur zur Charakterisierung von Individuen entfällt. Dies ermöglicht grosse Probenzahlen in Populationsstudien. [source]

    The control of sirex wood wasp using biological control agents in Victoria, Australia

    AGRICULTURAL AND FOREST ENTOMOLOGY, Issue 3 2009
    N. G. Collett
    Abstract 1,The sirex woodwasp, Sirex noctilio has been a significant pest of radiata pine plantations in Victoria since 1961. Outbreaks are usually associated with susceptible trees being under some form of stress including the effects of drought and overcrowding. 2,This paper reviews the spread of sirex and the history and efficacy of biological control programmes implemented against sirex in Victoria from 1970 to 2006. 3,Of the numerous biological control agents released, the most effective in managing sirex have been the nematode Beddingia siricidicola and the parasitic wasp Ibalia leucospoides. Several other parasitic wasps such as Schlettererius cinctipes and Megarhyssa nortoni nortoni have also established but provide only minimal control. 4,While rates of I. leucospoides parasitism have improved over time, it is unlikely that this improvement will continue substantially past current levels. 5,In recent years, issues have arisen regarding a decline in the infectivity of B. siricidicola necessitating a re-evaluation of strategies and development of techniques to overcome this problem. 6,Ongoing research using Random Amplification of Polymorphic DNA (RAPD) testing is underway to accurately determine nematode strains and associated infectivity present in plantations in the field in order to develop management strategies to re-introduce more effective strains. [source]

    Assessment of Genetic Diversity in Thai Isolates of Pyricularia grisea by Random Amplification of Polymorphic DNA

    JOURNAL OF PHYTOPATHOLOGY, Issue 4 2008
    P. Sirithunya
    Abstract One hundred and seventy-four isolates of Pyricularia grisea were collected from various hosts such as barley, rice, weed and wild rice in Thailand. Seven arbitrary decamer primers from the set of University of British Columbia were employed and nine lineages were classified. Lineages B, C and H were predominant, contributing up to 70% of total pathogens in this study. Analysis showed that the distribution of each lineage differs from the predominant lineages across Thailand in such that other lineages were restricted in particular area. For instance, lineage A was limited only in southern Thailand, whereas wide distribution of lineages B and C reflected an influence of both biological and physical effects on pathogen variation. Principal component analysis resulted in a total of four groups of blast pathogen with small distinctions between barley-, rice-, weed- and wild rice-infected blast. Bridging relationships occurred among border isolates of weed and rice blast suggesting a chance of migrations between hosts. Higher diversity was observed in northern, north-eastern and central Thailand while eastern and southern parts were rather low. Genetic diversity indices elucidated an abundance of pathogen lineages existing in northern Thailand suggesting that it should be the centre of diversity. [source]

    Spatial autocorrelation and linkage of Mendelian RAPD markers in a population of Picea abies Karst

    MOLECULAR ECOLOGY, Issue 3 2002
    Gabriele Bucci
    Abstract The spatial clustering of single- and di-locus genotypes in a natural, continuous population of Norway spruce was investigated using 69 Mendelian Random Amplified Polymorphic DNA (RAPD) markers that covered about 15% of the species' genome, and whose linkage relationships were known. Spatial autocorrelation techniques and randomization tests, applied to both single- and di-locus genotypes, revealed a weak, though significant, spatial structure at the scale 0,200 m (5% of single-locus and 7% of di-locus genotypes). To assess the relative importance of isolation by distance and linkage between markers on their spatial genetic structuring, we grouped joins between sampled trees into ,equivalence categories' expected to show similar, specific patterns of spatial distribution under isolation by distance. Results from both single- and di-locus analyses were consistent with the existence of patches of like homozygotes (about 8% and 11% of loci at the single- and di-locus level, respectively) surrounded by a mix of like heterozygotes. Similar structuring has been predicted by simulation models under isolation by distance and selective neutrality. Overall, linkage between markers accounted for an increase of spatial clumping of di-locus genotypes involving tightly linked loci with recombination fractions up to 0.1, a consequence of limited, stochastic spread of single-locus genotypes in space. Our results support the hypothesis that isolation by distance and linkage have a small, though significant, effect even within continuous forest tree populations. In general, the spatial distribution of multilocus genotypes within populations should be interpreted with caution when linkage relationships among the markers used are unknown. [source]

    Biotype stability of Candida albicans isolates after culture storage determined by randomly amplified polymorphic DNA and phenotypical methods

    MYCOSES, Issue 6 2010
    Katia Leston Bacelo
    Summary Typing methods to evaluate isolates in relation to their phenotypical and molecular characteristics are essential in epidemiological studies. In this study, Candida albicans biotypes were determined before and after storage in order to verify their stability. Twenty C. albicans isolates were typed by Randomly Amplified Polymorphic DNA (RAPD), production of phospholipase and proteinase exoenzymes (enzymotyping) and morphotyping before and after 180 days of storage in Sabouraud dextrose agar (SDA) and sterilised distilled water. Before the storage, 19 RAPD patterns, two enzymotypes and eight morphotypes were identified. The fragment patterns obtained by RAPD, on the one hand, were not significantly altered after storage. On the other hand, the majority of the isolates changed their enzymotype and morphotype after storage. RAPD typing provided the better discriminatory index (DI) among isolates (DI = 0.995) and maintained the profile identified, thereby confirming its utility in epidemiological surveys. Based on the low reproducibility observed after storage in SDA and distilled water by morphotyping (DI = 0.853) and enzymotyping (DI = 0.521), the use of these techniques is not recommended on stored isolates. [source]

    Evidence for massive clonal growth in the invasive weed Fallopia japonic a (Japanese Knotweed)

    BOTANICAL JOURNAL OF THE LINNEAN SOCIETY, Issue 4 2000
    MICHELLE L. HOLLINGSWORTH
    Clonal growth in introduced populations of Japanese Knotweed (Fallopia juponica) in Britain was assessed using RAPDs (Randomly Amplified Polymorphic DNA). A total of 150 British samples was analysed for genetic variation using ten arbitrary decamer primers, and compared with data from 16 samples of other introduced populations from Europe and the U.S.A. All samples produced an identical multi-primer RAPD profile. Accepting that RAPD profile identity need not equate to genet identity, based on the sensitivity of these markers for detecting genetic diversity in related taxa and on the absence of male fertile individuals of this species in Britain, we interpret this result as consistent with the presence of a single, exceptionally widespread clone. This clone must represent one of the world's largest vascular plants. [source]

    Genetic Diversity and Tests of the Hybrid Origin of the Endangered Yellow Larkspur

    CONSERVATION BIOLOGY, Issue 6 2001
    Jason A. Koontz
    The total number of individuals in these two populations is estimated to be <100. We used allozyme and random amplified polymorphic DNA ( RAPD) markers to (1) assess levels and patterns of genetic diversity in one wild population and two cultivated populations and (2) test the hypothesis that D. luteum is of hybrid origin between D. decorum and D. nudicaule. These data will be used to aid in developing a management plan to conserve the species. The wild population maintains high levels of genetic diversity. Genetic data indicate that both cultivated populations, especially the north Sonoma population, have several allozymes and RAPD markers not found in the wild population and could be used to establish new populations of D. luteum or to enhance the diversity and size of the wild population. The allozyme data did not reveal any fixed differences between D. decorum and D. nudicaule, although allele frequencies of the putative parental populations differed. At these loci, D. luteum resembled D. nudicaule more than D. decorum . Many unique RAPD markers distinguish each of the three species. The diagnostic markers from populations of D. nudicaule and D. decorum were not additive in the putative hybrid, and these data indicate that D. luteum is not of recent hybrid origin. Conservation of the yellow larkspur should include strategies that use the cultivated populations of D. luteum, but hybridizing D. decorum and D. nudicaule to "recreate"D. luteum is not recommended. Resumen:Delphidium luteum ( Ranunculaceae), un delfinio en peligro de extinción, está restringido a dos poblaciones silvestres cerca de Bodega Bay, California. Se estima que el total de individuos en estas dos poblaciones es de <100. Utilizamos marcadores de alozimas y RAPD para (1) evaluar los niveles y patrones de diversidad genética en una población silvestre y dos poblaciones cultivadas y (2) probar la hipótesis de que D. luteum es de origen híbrido entre D. decorum y D. nudicaule. Estos datos serán utilizados para ayudar a desarrollar un plan de manejo para conservar la especie. La población silvestre mantiene altos niveles de diversidad genética. Los datos genéticos indican que ambas poblaciones cultivadas, especialmente en la población de Sonoma norte, tienen varias alozimas y marcadores RAPD que no se encuentran en poblaciones silvestres y podrían utilizarse para establecer nuevas poblaciones de D. luteum o reforzar la diversidad y tamaño de la población silvestre. Los datos de alozimas no revelaron diferencias fijas entre D. decorum y D. nudicaule, aunque las frecuencias alélicas de las poblaciones parentales putativas fueron diferentes. En estos loci, D. luteum fue más semejante a D. nudicaule que a D. decorum. Muchos marcadores RADP únicos distinguen a cada una de las tres especies. Los marcadores diagnóstico de poblaciones de D. decorum y D. nudicaule no fueron aditivos en el híbrido putativo, y estos datos indican que D. luteum no es de origen híbrido reciente. La conservación del delfinio amarillo debería incluir estrategias que usen las poblaciones cultivadas de D. luteum; sin embargo, no se recomienda la hibridación de D. decorum y D. nudicaule para "recrear" a D. luteum. [source]

    A prime inference on genetic diversity (RAPDs) in the marine fish Atherinella brasiliensis (Teleostei, Atherinopsidae) from Southern Brazil

    ACTA ZOOLOGICA, Issue 2 2010
    Maria Cristina Da Silva Cortinhas
    Abstract Da Silva Cortinhas, M. C., Glienke, C., Prioli, A. J., Noleto, R. B., Matoso, D. A. and Cestari, M. M. 2010. A prime inference on genetic diversity (RAPDs) in the marine fish Atherinella brasiliensis (Teleostei, Atherinopsidae) from Southern Brazil. ,Acta Zoologica (Stockholm) 91: 242,248 As a result of the importance of Atherinella brasiliensis in estuarine environments, random amplified polymorphic DNA (RAPD) markers were used to verify the genetic diversity in A. brasiliensis from two different places in Paranaguá Bay (Paraná State) and one from the Conceição Lagoon (Santa Catarina State). Cytogenetic data have shown a high karyotypic diversity in some populations, although in others this peculiarity demonstrates rearrangements such as heterochromatinization. In the present study, a low level of genetic structuring between the samples from Conceição Lagoon compared with the others was observed through principal coordinate analysis (PCO), analysis of molecular variance and Mantel test according to 79 RAPD markers. As this specie does not perform horizontal migration and the individuals of Conceição Lagoon are isolated, three hypotheses are proposed to explain the results: (i) similar environments may show homogeneous populations not depending on the geographical distance, (ii) because vicariant events that formed the bays occurred in a recent period, the fragmentation effects over the structuring of the genetic diversity may still be low and not totally detectable by the RAPD technique and (iii) the isolation time or the number of generations may not be enough to promote a possible differentiation and genetic structuring between the specimens of these three places. The specimens of these places present a low level of differentiation and genetic structuring so we can consider them as a unique homogeneous population. [source]

    Genet age in marginal populations of two clonal Carex species in the Siberian Arctic

    ECOGRAPHY, Issue 4 2000
    Ingibjörg S. Jónsdóttir
    During a Swedish-Russian expedition to northern Siberia 1994, we sampled two marginal populations of two Carex species at two high arctic sites (C, stans Drej. on Faddeyevsky Island and C. ensifolia V. Krecz ssp. arctisibirica Jurtz. at north-eastern Taymyr Peninsula), both north of previously documented localities in that areas for the two species. These populations were composed of a few distinct patches of ramet colonies, some of them shaped like fairy rings with dead centres. We measured the size of all colonies and collected samples for detailed morphometric analyses of rhizome growth. By using RAPD (random amplified polymorphic DNA) analysis we established that the largest colony at each site consisted of a single genet, based on 41 polymorphic bands amplified with three primers. Pooled samples from each of two additional colonies of C. stans on Faddeyevsky Island were analysed and showed that clones of the same species at the same site were relatively dissimilar (Dice's similarity index 0.26 0,43), We then assumed that each ramet colony represented a single genet. Based on the morphometric data, we developed a deterministic growth model that simulates the clonal growth of these species and enabled estimates of the time since establishment of the genets. The estimated age of the five C. Stans clones varied from 17 to 154 yr and the age of the two C. ensifolia ssp, arctisibirica clones was well over 3000 yr. [source]

    Genetic variation in Myzus persicae populations associated with host-plant and life cycle category

    ENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 3 2001
    Kiriaki Zitoudi
    Abstract Random amplified polymorphic DNA (RAPD) analysis was applied on 96 clones of Myzus persicae (Sulzer) (Homoptera: Aphididae) representing seven populations collected from different host-plants and regions of Greece. Ten decamer random primers were used to evaluate genetic variation among the examined samples. Despite the variability found between clones, no specific RAPD marker was detected to discriminate the different populations. A significant finding was that aphids from peach and pepper, which were collected far away from tobacco-growing regions, especially those from peach, showed genetic divergence from the tobacco-feeding clones. Moreover, data analysis revealed a significant genetic divergence between holocyclic and anholocyclic populations from tobacco. Lastly, holocyclic clones showed higher level of estimated heterozygosity than the nonholocyclic (anholocyclic, androcyclic and intermediate) ones. [source]

    Mating compatibility, life-history traits, and RAPD-PCR variation in Bemisia tabaci associated with the cassava mosaic disease pandemic in East Africa

    ENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 1 2001
    M.N. Maruthi
    Abstract The pandemic of a severe form of cassava mosaic virus disease (CMVD) in East Africa is associated with abnormally high numbers of its whitefly vector, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). To determine whether a novel B. tabaci biotype was associated with the CMVD pandemic, reproductive compatibility, fecundity, nymphal development, and random amplified polymorphic DNA (RAPD) variability were examined in, and between, B. tabaci colonies collected from within the CMVD pandemic and non-pandemic zone in Uganda. In a series of reciprocal crosses carried out over two generations among the six CMVD pandemic and four non-pandemic zone cassava B. tabaci colonies, there was no evidence of mating incompatibility. All the crosses produced both female and male progeny in the F1 and F2 generations, which in a haplo-diploid species such as B. tabaci indicates successful mating. There also were no significant differences between the sex ratios for the pooled data of experimental crosses, between individuals from two different colonies and control crosses between individuals from the same colony. Only one instance of mating incompatibility occurred in a control cross between cassava B. tabaci from Uganda and cotton B. tabaci from India. Measures of fecundity of the pandemic and non-pandemic zone B. tabaci on four cassava varieties showed no significant differences in their fecundity, nymphal development or numbers surviving to adult eclosion. Cluster analysis of 26 RAPD bands using six 10-mer primers was concordant with the mating results, grouping the pandemic and non-pandemic zone colonies into a single large group, also including a B. tabaci colony collected from cassava in Tanzania. These results suggest that it is unlikely that the severe CMVD pandemic in East Africa is associated with a novel and reproductively isolated B. tabaci biotype. [source]

    Population dynamics of the ectomycorrhizal fungal species Tricholoma populinum and Tricholoma scalpturatum associated with black poplar under differing environmental conditions

    ENVIRONMENTAL MICROBIOLOGY, Issue 5 2006
    Hervé Gryta
    Summary Fungi combine sexual reproduction and clonal propagation. The balance between these two reproductive modes affects establishment dynamics, and ultimately the evolutionary potential of populations. The pattern of colonization was studied in two species of ectomycorrhizal fungi: Tricholoma populinum and Tricholoma scalpturatum. The former is considered to be a host specialist whereas T. scalpturatum is a generalist taxon. Fruit bodies of both basidiomycete species were mapped and collected over several years from a black poplar (Populus nigra) stand, at two different sites. Multilocus genotypes (= genets) were identified based on the analysis of random amplified polymorphic DNA (RAPD) patterns, inter-simple sequence repeat (ISSR) patterns and restriction fragment length polymorphisms (RFLPs) in the ribosomal DNA intergenic spacer (rDNA IGS). The genetic analyses revealed differences in local population dynamics between the two species. Tricholoma scalpturatum tended to capture new space through sexual spores whereas T. populinum did this by clonal growth, suggesting trade-offs in allocation of resources at the genet level. Genet numbers and sizes strongly differ between the two study sites, perhaps as a result of abiotic disturbance on mycelial establishment and genet behaviour. [source]

    The structure of a local population of phytopathogenic Pseudomonas brassicacearum from agricultural soil indicates development under purifying selection pressure

    ENVIRONMENTAL MICROBIOLOGY, Issue 3 2001
    Johannes Sikorski
    Among the isolates of a bacterial community from a soil sample taken from an agricultural plot in northern Germany, a population consisting of 119 strains was obtained that was identified by 16S rDNA sequencing and genomic fingerprinting as belonging to the recently described species Pseudomonas brassicacearum. Analysis of the population structure by allozyme electrophoresis (11 loci) and random amplified polymorphic DNA,polymerase chain reaction (RAPD,PCR; four primers) showed higher resolution with the latter method. Both methods indicated the presence of three lineages, one of which dominated strongly. Stochastic tests derived from the neutral theory of evolution (including Slatkin's exact test, Watterson's homozygosity test and the Tajima test) indicated that the population had developed under strong purifying selection pressure. The presence of strains clearly divergent from the majority of the population can be explained by in situ evolution or by influx of strains as a result of migration or both. Phytopathogenicity of a P. brassicacearum strain determined with tomato plants reached the level obtained with the type strain of the known pathogen Pseudomonas corrugata. The results show that a selective sweep was identified in a local population. Previously, a local selective sweep had not been seen in several populations of different bacterial species from a variety of environmental habitats. [source]

    Different portions of the maize root system host Burkholderia cepacia populations with different degrees of genetic polymorphism

    ENVIRONMENTAL MICROBIOLOGY, Issue 1 2000
    Luigi Chiarini
    In order to acquire a better understanding of the spatial and temporal variations of genetic diversity of Burkholderia cepacia populations in the rhizosphere of Zea mays, 161 strains were isolated from three portions of the maize root system at different soil depths and at three distinct plant growth stages. The genetic diversity among B. cepacia isolates was analysed by means of the random amplified polymorphic DNA (RAPD) technique. A number of diversity indices (richness, Shannon diversity, evenness and mean genetic distance) were calculated for each bacterial population isolated from the different root system portions. Moreover, the analysis of molecular variance ( amova) method was applied to estimate the genetic differences among the various bacterial populations. Our results showed that, in young plants, B. cepacia colonized preferentially the upper part of the root system, whereas in mature plants, B. cepacia was mostly recovered from the terminal part of the root system. This uneven distribution of B. cepacia cells among different root system portions partially reflected marked genetic differences among the B. cepacia populations isolated along maize roots on three distinct sampling occasions. In fact, all the diversity indices calculated indicated that genetic diversity increased during plant development and that the highest diversity values were found in mature maize plants, in particular in the middle and terminal portions of the root system. Moreover, the analysis of RAPD patterns by means of the amova method revealed highly significant divergences in the degree of genetic polymorphism among the various B. cepacia populations. [source]

    Evaluating wastewater-induced plant genotoxicity using randomly amplified polymorphic DNA

    ENVIRONMENTAL TOXICOLOGY, Issue 1 2008
    K. M. Swaileh
    Abstract Wastewater often contains genotoxic substances that can resist different stages of the treatment process. In the present study, randomly amplified polymorphic DNA technology was applied to evaluate the genotoxic effects of wastewater (treated and raw) irrigation on oat plants (Avena sativa). RAPD profiles obtained showed that both treated and raw wastewater (RWW) were having genotoxic effects on oat plants. This was apparent by the appearance/disappearance of bands in the treatments compared with the control plants. From the 15 primers used, 186 bands were obtained with an average of 12.4 bands per primer. Irrigating plants with RWW caused 51 new bands to appear and 19 to disappear. Treated wastewater (TWW) caused only 16 new bands and the loss of 17 bands. This makes TWW less genotoxic than RWW. The Euclidean distances shown on the dendrogram, revealed the presence of two clusters according to dissimilarity values. One cluster contained the control plants and those irrigated with TWW, whereas the second contained the plants irrigated with RWW. Similarity indices calculated between the treatments and the control plants showed that the control and the plants irrigated with TWW had a similarity index of 0.87, the control and plants irrigated with RWW 0.73 and between the treatments 0.75. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source]

    Integration of genotoxicity and population genetic analyses in kangaroo rats (Dipodomys merriami) exposed to radionuclide contamination at the Nevada Test Site, USA

    ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2001
    Christopher W. Theodorakis
    Abstract We examined effects of radionuclide exposure at two atomic blast sites on kangaroo rats (Dipodomys merriami) at the Nevada Test Site, Nevada, USA, using genotoxicity and population genetic analyses. We assessed chromosome damage by micronucleus and flow cytometric assays and genetic variation by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (mtDNA) analyses. The RAPD analysis showed no population structure, but mtDNA exhibited differentiation among and within populations. Genotoxicity effects were not observed when all individuals were analyzed. However, individuals with mtDNA haplotypes unique to the contaminated sites had greater chromosomal damage than contaminated-site individuals with haplotypes shared with reference sites. When interpopulation comparisons used individuals with unique haplotypes, one contaminated site had greater levels of chromosome damage than one or both of the reference sites. We hypothesize that shared-haplotype individuals are potential migrants and that unique-haplotype individuals are potential long-term residents. A parsimony approach was used to estimate the minimum number of migration events necessary to explain the haplotype distributions on a phylogenetic tree. The observed predominance of migration events into the contaminated sites supported our migration hypothesis. We conclude the atomic blast sites are ecological sinks and that immigration masks the genotoxic effects of radiation on the resident populations. [source]

    Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

    FEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2002
    Teresa R Motta
    Abstract Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection. [source]

    New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus

    FEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2001
    Camile Pizeta Semighini
    Abstract In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical. [source]

    Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLPTm), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE)

    FEMS YEAST RESEARCH, Issue 2 2001
    Bart Theelen
    Abstract Three molecular tools, amplified fragment length polymorphism (AFLPTm), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples. [source]

    Direct genotyping of the poplar leaf rust fungus, Melampsora medusae f. sp. deltoidae, using codominant PCR-SSCP markers

    FOREST PATHOLOGY, Issue 4 2005
    M. Bourassa
    Summary Two anonymous DNA markers that are revealed by single-strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono-uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono-uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re-amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono-uredinial isolates of Mmd tested. From the overall 40 mono-uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse-grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies. Résumé Deux marqueurs génétiques anonymes, révélés par analyse SSCP (Single-Strand Conformational Polymorphism) ont été développés afin de détecter des polymorphismes génétiques chez le Melampsora medusae f. sp. deltoidae (Mmd). Dans un premier temps, des isolats mono-urédiniaux ont été obtenus, puis l'ADN a été extrait à partir des urédiniospores, les produits d'amplification RAPD (Random Amplified Polymorphic DNA) ont été générés à partir de huit de ces isolats mono-urédiniaux et les résultats d'amplification ont par la suite été séparés sur gel SSCP afin d'identifier des polymorphismes entre les isolats. Les bandes sur gel SSCP représentant des loci polymorphiques putatifs entre les isolats ont été prélevées du gel, ré-amplifiées par la technique d'amplification PCR (Polymerase Chain Reaction), clonées, puis séquencées. Pour deux fragments d'ADN séquencés sur un total de trois, une paire d'amorces a été développée afin de permettre l'amplification d'un fragment de taille adéquate pour analyse SSCP (<600 pb). Chaque paire d'amorces a produit un signal d'amplification positif pour chacun des huit isolats à l'origine de ces nouvelles amorces; les produits PCR ont ensuite été analysés par la technique SSCP. Les deux loci putatifs ont révélé des polymorphismes génétiques entre les isolats. Les deux paires d'amorces ont produit un fragment d'amplification de la taille attendue pour chacun des 32 isolats mono-urédiniaux supplémentaires testés. Des 40 isolats testés, 5 et 11 allèles ont été détectés, alors que 12 et 34 isolats se sont révélés hétérozygotes (tel qu'indiqué par la présence de plus de deux bandes sur gel SSCP) pour les loci A et B, respectivement. La spécificité des deux paires d'amorces a été testée à partir de 106 isolats fongiques appartenant à différents groupes taxonomiques, incluant d'autres rouilles, de même qu'à partir de l'ADN extrait de feuilles de peupliers cultivés en serre. Un signal d'amplification positif n'a été obtenu qu'en présence d'ADN du Mmd. Les conditions d'amplification PCR ont été optimisées pour les deux paires d'amorces développées afin de permettre le génotypage directement à partir d'urédinies individuelles prélevées sur des feuilles de peuplier infectées. La possibilité de génotyper directement des urédinies individuelles permet d'éviter l'obligation de cultiver le champignon pour génotyper les individus, ce qui représente un avantage important des marqueurs génétiques développés ici, puisqu'il devient dès lors possible de traiter un grand nombre d'échantillons lors de la réalisation d'études de populations. Zusammenfassung Zum Nachweis von Polymorphismen bei Melampsora medusae f. sp. deltoidae wurden zwei anonyme DNA Marker aus einer SSCP-Analyse entwickelt. Zunächst wurden Isolate aus einzelnen Uredinien gewonnen, die DNA wurde aus den Uredosporen extrahiert und polymorphe RAPD, Amplifikationsprodukte von acht Mono-Uredinium-Isolaten wurden auf einem SSCP-Gel getrennt, um Unterschiede zwischen ihnen nachzuweisen. Banden, die bei den acht geprüften Isolaten mögliche polymorphe Loci darstellten, wurden aus dem SSCP-Gel ausgeschnitten und mit PCR reamplifiziert, dann geklont und sequenziert. Für zwei von insgesamt drei sequenzierten DNA-Fragmenten wurde ein Primerpaar entwickelt, um ein in der Grösse für die SSCP-Analyse (<600 bp) geeignetes DNA-Fragment zu amplifizieren. Jedes Primerpaar amplifizierte bei allen acht ursprünglich für ihre Entwicklung verwendeten Isolaten ein PCR-Produkt, und diese wurden anschliessend mit SSCP analysiert. Für beide putativen Loci wurden bei den Isolaten Polymorphismen festgestellt. Die beiden Primerpaare amplifizierten ein PCR-Produkt der erwarteten Grösse bei allen 32 zusätzlich geprüften Mono-Uredinium-Isolaten des Pilzes. Bei den insgesamt 40 geprüften Mono-Uredinium-Isolaten wurden für die Loci A und B 5 bzw. 11 Allele gefunden, und 12 bzw. 34 Isolate erwiesen sich als heterozygot, was durch mehr als zwei Banden auf den SSCP-Gelen angezeigt wurde. Die Spezifität der Primerpaare wurden mit 106 Pilzisolaten aus verschiedenen Taxa geprüft, darunter andere Roste sowie DNA aus gesunden Pappelblättern aus Gewächshauskulturen. DNA-Amplifikationsprodukte der erwarteten Grösse wurden nur erhalten, wenn DNA von Melampsora medusae f. sp. deltoidae präsent war. Die PCR-Amplifikations-Bedingungen mit diesen beiden Primerpaaren wurde so optimiert, dass ein Genotyping direkt bei einzelnen von infizierten Blättern entnommenen Uredinien erfolgen kann und somit eine Pilzkultur zur Charakterisierung von Individuen entfällt. Dies ermöglicht grosse Probenzahlen in Populationsstudien. [source]

    Association of Sphaeropsis sapinea with insect-damaged red pine shoots and cones

    FOREST PATHOLOGY, Issue 1 2003
    E. Feci
    Summary The association of the shoot blight and canker pathogen Sphaeropsis sapinea with red pine (Pinus resinosa) shoots and cones damaged by insects (especially Dioryctria sp.) was investigated. Samples from a single plantation approximately 35 years old, in Sauk Co., Wisconsin and also from three plantations, between approximately 40 and 50 years old, located in an area of pine shoot moth activity in the preceding year in Adams Co., Wisconsin were visually examined. Samples were arbitrarily collected from trees felled in the first plantation in May. Pycnidia of S. sapinea and insect damage were observed on 56 of 91 (62%) of closed cones and 17 of 165 (7%) of previous year's shoots. In the absence of insect damage, pycnidia of the pathogen were identified only on eight of 91 (9%) closed cones and never on previous year's shoots. In each of the other three plantations, 10 trees were located at intervals along transects in mid-June; one branch from the lower half of the crown per tree was pruned off, and both current and previous year's shoots were examined. Insect damage and S. sapinea pycnidia were too rare on current year's shoots to draw any conclusions. Insect damage occurred on 20,40% of over 2000 previous year's shoots that were examined, but pycnidia of the pathogen were identified on only about 5%. Although infrequent, S. sapinea was identified in association with insect-damaged previous year's shoots from these three plantations three times more frequently than those without insect damage. Random amplified polymorphic DNA (RAPD) markers from eight randomly selected isolates were consistent with the A group of S. sapinea, which can be aggressive on red pine. This ability to exploit insect-damaged shoots may facilitate long-term persistence of S. sapinea at low disease incidence and severity. The potential role of insect wounds as infection courts and insects as vectors of this important pathogen of pines deserves further study. Résumé L'étude a porté sur l'association entre le parasite de pousses et agent de chancre Sphaeropsis sapinea, et les pousses et cônes de Pinus resinosa endommagés par des insectes (surtout Dioryctria sp.). Des échantillons ont été examinés visuellement; ils provenaient d'une plantation d'environ 35 ans à Sauk Co., Wisconsin, et de trois plantations âgées d'environ 40 et 50 ans situées dans une zone où les insectes des pousses avaient été actifs l'année précédente à Adams Co., Wisconsin. Dans la première plantation, les échantillons ont été prélevés arbitrairement sur des arbres abattus en mai. Des pycnides de S. sapinea et des dégâts d'insectes ont été observés sur 56/91 (62%) des cônes fermés et sur 17/165 (7%) des pousses de l'année précédente. En l'absence de dégâts d'insectes, les pycnides n'ont été trouvées que sur 8/91 (9%) des cônes fermés, et jamais sur les pousses de l'année précédente. Dans chacune des trois autres plantations, 10 arbres ont été choisis à la mi-juin le long de transects ; sur chaque arbre une branche a été coupée dans la moitié inférieure de la couronne, et les pousses de l'année en cours et de l'année précédente ont été examinées. Sur les pousses de l'année, les dégâts d'insectes et les pycnides de S. sapineaétaient trop rares pour pouvoir en tirer des conclusions. Parmi plus de 2000 pousses de l'année précédente examinées, les dégâts d'insectes étaient présents sur 20,40% des pousses, mais les pycnides n'ont été trouvées que sur environ 5% d'entre elles. Bien que peu fréquent chez ces trois plantations, S. sapinea a été trouvé associé aux pousses de l'année précédente, 3 fois plus fréquemment chez celles endommagées par les insectes que chez les non endommagées. Pour huit isolats pris au hasard, les marqueurs RAPD ont indiqué leur appartenance au groupe A de S. sapinea qui peut être agressif sur P. resinosa. Cette aptitude de S. sapineaà utiliser les pousses endommagées par les insects peut faciliter sa persistance à long terme à des niveaux bas d'abondance et de dégâts. Le rôle potentiel des blessures d'insectes comme voies d'infection, et des insectes comme vecteurs du champignon parasite mérite d'être étudié. Zusammenfassung Es wurde die Assoziation zwischen Sphaeropsis sapinea (Erreger von Triebsterben und Rindennekrosen) und Schädigung an Trieben und Zapfen von Pinus resinosa untersucht, durch Insekten (vorwiegend Dioryctria sp.) untersucht. Proben von einer ca. 35 Jahre alten Plantage in Sauk Co., Wisconsin und von drei 40-50jährigen Plantagen mit Dioryctria -Befall im Vorjahr in Adams Co., Wisconsin wurden makroskopisch untersucht. Die Proben am ersten Standort wurden von Bäumen entnommen, die im Mai gefällt wurden (willkürliche Auswahl). Pyknidien und Schädigung durch Insekten wurden an 56/91 (62%) der geschlossenen Zapfen und an 17/165 (7%) der vorjährigen Triebe beobachtet. An Organen ohne Schädigung durch Insekten wurden die Pyknidien des Pathogens nur bei 8/91 (9%) der geschlossenen Zapfen und in keinem Fall an den vorjährigen Trieben nachgewiesen. In den anderen drei Plantagen wurden Mitte Juni je 10 Bäume entlang von Transekten untersucht; pro Baum wurde aus dem unteren Kronenbereich ein Ast abgeschnitten und sowohl die diesjährigen als auch die vorjährigen Triebe wurden untersucht. An den diesjährigen Triebabschnitten waren sowohl Schädigungen durch Insekten als auch Pyknidien von S. sapinea zu selten, um daraus Schlüsse zu ziehen. An den vorjährigen Triebabschnitten kamen Insektenschäden an 20,40% von über 2,000 untersuchten Objekten vor, aber Pyknidien des Pathogens wurden nur in 5% der Fälle nachgewiesen. Trotz des geringen Vorkommens wurde S. sapinea auf den vorjährigen und von Insekten beschädigten Trieben dreimal häufiger nachgewiesen als an Trieben ohne Beschädigung. Acht zufällig ausgewählte Isolate wurden anhand von RAPD Markern der Gruppe A von S. sapinea zugeordnet, die auf P. resinosa agressiv sein kann. Die Fähigkeit von S. sapinea, durch Insekten beschädigte Triebe zu nutzen, kann das Überdauern des Pilzes auf einem niedrigen Befallsniveau erleichtern. Die Bedeutung von Wunden, die durch Insekten verursacht werden, als Infektionspforten und die mögliche Rolle von Insekten als Vektoren dieses wichigen Pathogens sollte in weiteren Untersuchungen geklärt werden. [source]

    Different Helicobacter pylori Strains Colonize the Antral and Duodenal Mucosa of Duodenal Ulcer Patients

    HELICOBACTER, Issue 2 2000
    Ann-Catrin E. Thoreson
    Background. We have investigated the possibility that the same patients may be colonized by Helicobacter pylori strains of different genotypes or phenotypes in the antrum as compared to in the duodenum. The strains were typed for DNA fingerprints, different lipopolysaccharides (LPS), and Lewis antigen expression on the O,side chains of LPS. Materials and Methods. Polymerase chain reaction (PCR) amplifications using primer sequences (i.e., the Enterobacterial Repetitive Intergenic Consensus [ERIC]) and randomly amplified polymorphic DNA (RAPD) elements were performed to asses chromosomal DNA diversity between H. pylori strains. The expression of different LPS types and Lewis antigens in the various H. pylori isolates were determined by whole bacterial enzyme-linked immunosorbent assays using monoclonal antibodies. Results. Duodenal ulcer patients had different H. pylori genotypes in the duodenum as compared to in the antrum as shown by ERIC-PCR (44%) and by RAPD-PCR (75%). Different DNA patterns were found among the strains that were isolated from various regions of the duodenum in 4 of 16 patients (25%) as shown by ERIC-PCR and in 8 of 16 patients (50%) as shown by RAPD-PCR. Sixty-three percent of the duodenal ulcer patients had H. pylori strains with a different Lewis antigen phenotype in the duodenum as compared to in the antrum, and 3 of 16 patients (19%) had strains with different Lewis antigens expressed by strains from different duodenal biopsies from the same patient. Conclusion. The results suggest that a mixed population of different H. pylori strains with marked variation, both genotypically and phenotypically, colonize the same patient. [source]

    A preliminary linkage map of the hard tick, Ixodes scapularis

    INSECT MOLECULAR BIOLOGY, Issue 2 2003
    A. J. Ullmann
    Abstract A linkage map of the Ixodes scapularis genome was constructed, based upon segregation amongst 127 loci. These included 84 random amplified polymorphic DNA (RAPD) markers, 32 Sequence- Tagged RAPD (STAR) markers, 5 cDNAs, and 5 microsatellites in 232 F1 intercross progeny from a single, field-collected P1 female. A preliminary linkage map of 616 cM was generated across 14 linkage groups with one marker every 10.8 cM. Assuming a genome size of , 109 bp, the relationship of physical to genetic distance was found to be , 300 kb/cM in the I. scapularis genome. [source]

    Molecular identification and population dynamics of two species of Pemphigus (Homoptera: Pemphidae) on cabbage

    INSECT SCIENCE, Issue 2 2009
    Naiqi Chen
    Abstract The poplar petiole gall aphid, Pemphigus populitransversus Riley, has been one of the major pests on cruciferous vegetable in the Rio Grande Valley (LRGV) of Texas since the late 1940s. It normally migrates from poplar trees to cruciferous vegetables in the fall, and migrates back to the trees in early spring of the coming year. Some root-feeding aphids were found on cruciferous vegetables in late spring and early summer in 1998 and the following years. Those aphids have been identified as Pemphigus obesinymphae Moran. This discovery completely changed the current knowledge about the root-feeding aphids on cruciferous vegetables in the LRGV. Due to their small size, morphological and feeding similarities between P. populitransversus and P. obesinymphae, their identification and distinction are difficult. In this study, random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to distinguish these two species over a period of time when the two species occurred together, or separately, in cabbage fields. The two species occurred on cabbage at different times of the year, and overlapped from October to June. From May to October, both species migrated to their primary hosts. The apterous aphids found on cabbage in winter contained mainly P. obesinymphae, whereas in early spring more apterous P. populitransversus were recovered. The root-feeding aphids would feed on cabbage plants as long as this host was available even during the hot, dry summer in the LRGV, although their populations were generally low. Both RAPD and AFLP techniques were efficient in discriminating the two species that showed obviously genetic variability. These molecular techniques confirmed the existence of the two aphid species in apterous samples collected from the soil in cabbage fields in the LRGV, and the results performed by RAPD were confirmed by AFLP. Furthermore, the results suggest that RAPD technique was a better choice despite its reproducibility problem, as it was less time-consuming and required less technology, labor and expense than AFLP. [source]

    Isolation of microsatellite loci in the pollinating fig wasp of Ficus hispida, Ceratosolen solmsi

    INSECT SCIENCE, Issue 4 2008
    Hao Yu
    Abstract Microsatellite loci were isolated for Ceratosolen solmsi, pollinator of the dioecious Ficus hispida. We developed nine polymorphic microsatellite loci based on the method of polymerase chain reaction isolation of microsatellite arrays (PIMA). Enrichment of genomic libraries was performed by random amplified polymorphic DNA (RAPD). A subset of 38 positive clones was sequenced; 15 clones showed microsatellite loci. We tested 15 designed primer pairs and nine of them produced polymorphic amplification in 48 individual wasps collected from different fruits of the dioecious host fig Ficus hispida in China. Among the 48 individuals, 49 alleles were obtained at the nine loci. The observed heterozygosity ranged between 0.357 and 0.634. [source]

    Inter- alu PCR detects high frequency of genetic alterations in glioma cells exposed to sub-lethal cisplatin

    INTERNATIONAL JOURNAL OF CANCER, Issue 4 2005
    Tapasya Srivastava
    Abstract Increased genomic instability contributes to higher frequency of secondary drug resistance and neoplastic progression in tumors as well as in cells exposed to sub-lethal concentrations of chemotherapeutic agents. We have used PCR based DNA fingerprinting techniques of randomly amplified polymorphic DNA (RAPD) and inter- alu PCR to study this phenomenon in the tumor genome. The choice of the primer, either random (for RAPD) or specific (inter- alu PCR) can determine the nature of alterations being assessed. We have compared the inter- alu PCR and RAPD profiles of U87MG glioblastoma cells exposed to sequentially increasing low doses of cisplatin for 24 passages to that of untreated controls. Inter- alu PCR, with 2 primers, demonstrated a number of alterations in the treated cells, in the form of loss / gain and changes in the intensity of bands. No changes were observed by RAPD analysis with 5 primers, however, indicating a preferential increase in the alu mediated recombination frequency in the treated cells (p = 1.866 × 10,4). The number of changes observed with respect to the corresponding leucocyte DNA in the inter- alu PCR profile of 26 primary tumors (Grade II = 13; Grade IV = 13), resected before chemotherapy, for the 2 inter- alu primers was very small. We present a novel application of the inter- alu PCR in detecting alterations in long term cultured cells at low dose exposure to a chemotherapeutic agent. Our results suggest that alu mediated recombination may be important in cells exposed to sub-lethal doses of cisplatin but not in the genesis of primary glioma. © 2005 Wiley-Liss, Inc. [source]

    Assessment of genomic instability in breast cancer and uveal melanoma by random amplified polymorphic DNA analysis

    INTERNATIONAL JOURNAL OF CANCER, Issue 2 2002
    Sarantos Papadopoulos
    Abstract Some types of cancer have been associated with abnormal DNA fingerprinting. We used random amplified polymorphic DNA (RAPD) to generate fingerprints that detect genomic alterations in human breast cancer. Primers were designed by choosing sequences involved in the development of DNA mutations. Seventeen primers in 44 different combinations were used to screen a total of 6 breast cancer DNA/normal DNA pairs and 6 uveal melanoma DNA/normal DNA pairs. Forty-five percent of these combinations reliably detected quantitative differences in the breast cancer pairs, while only 18% of these combinations detected differences in the uveal melanoma pairs. Fourteen (32%) and 12 (27%) primers generated a smear or did not produce any band patterns in the first and second cases, respectively. Taking into account the ability of RAPD to screen the whole genome, our results suggest that the genomic damage in breast cancer is significantly higher than in uveal melanoma. Our study confirms other reports that the molecular karyotypes produced with random priming, called amplotypes, are very useful for assessing genomic damage in cancer. © 2002 Wiley-Liss, Inc. [source]

    Prevalence and characterisation of Bacillus cereus in vacuum packed potato puree

    INTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 8 2006
    Andreja Rajkovic
    Summary Refrigerated processed foods of extended durability (REPFED) potato puree was analysed for Bacillus cereus contamination along the production line and during the product shelf-life. Isolated B. cereus strains were tested for their psychrotrophic character and the ability to produce enterotoxins. Bacillus cereus contamination during four subsequent productions was in the range of 2.3,4.0 log cfu g,1. Productions five and six were significantly less contaminated with B. cereus (,1 log cfu g ,1). All B. cereus isolates from the first four productions were able to grow at 7 ° and 10 °C, whereas the majority of the isolates from productions five and six did not. No B. cereus isolates grew at 4 °C. randomly amplified polymorphic DNA (RAPD) fingerprinting showed that the most of B. cereus contamination originated from one source. In total, 30.4% of isolates expressed enterotoxic character. The present study points out the necessity to prevent an ,in house' colonisation and contamination during food processing in order to accomplish the safety of REPFED throughout the shelf-life. It also indicates the most critical steps in the production line of ready-to-eat potato puree and impact of failures regarding the food safety. The data provided can be used for risk assessment studies regarding B. cereus in REPFED. [source]

    Multiple molecular approaches yield no evidence for sex-determining genes in lake sturgeon (Acipenser fulvescens)

    JOURNAL OF APPLIED ICHTHYOLOGY, Issue 6 2008
    C. R. McCormick
    Summary Common DNA-based sexing assays have been widely used for the conservation and management of mammals and birds. However, many fishes do not have genetic sex determination and in those that do, the plasticity of the genes involved means that species-specific assays are normally required. Such DNA-sexing markers would be especially valuable in lake sturgeon (Acipenser fulvescens) because of their sexual monomorphism, delayed sexual maturity, and conservation status. We tried to identify genetic differences between male and female lake sturgeon using several different molecular genetic methods, including randomly amplified polymorphic DNA, representational difference analyses, subtractive hybridization, and a candidate gene approach. Ultimately, a number of genes were identified but none was sex-specific. Although the ultimate mechanism of sex determination is yet unknown, it is possible that sex determination is environmental in lake sturgeon, especially since recent studies have also failed to identify sex determination genes in other sturgeon species. [source]

    Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2007
    C. Miot-Sertier
    Abstract Aims:, In recent years, Brettanomyces/Dekkera bruxellensis has caused increasingly severe quality problems in the wine industry. A typing method at the strain level is needed for a better knowledge of the dispersion and the dynamics of these yeasts from grape to wine. Methods and Results:, Three molecular tools, namely random-amplified polymorphic DNA, PCR fingerprinting with microsatellite oligonucleotide primers and SAU-PCR, were explored for their relevance to typing strains of Brettanomyces bruxellensis. The results indicated that discrimination of each individual strain was not possible with a single PCR typing technique. We described a typing method for B. bruxellensis based on restriction enzyme analysis and pulse field gel electrophoresis (REA-PFGE). Results showed that electrophoretic profiles were reproducible and specific for each strain under study. Conclusions:, Consequently, REA-PFGE should be considered for the discrimination of B. bruxellensis strains. This technique allowed a fine discrimination of B. bruxellensis, as strains were identified by a particular profile. Significance and Impact of the Study:, This study constitutes a prerequisite for accurate and appropriate investigations on the diversity of strains throughout the winemaking and ageing process. Such studies will probably give clearer and more up-to-date information on the origin of the presence of Brettanomyces in wine after vinification when they are latent spoilage agents. [source]