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Amplified Polymorphic DNA (amplified + polymorphic_dna)
Kinds of Amplified Polymorphic DNA Terms modified by Amplified Polymorphic DNA Selected AbstractsDirect genotyping of the poplar leaf rust fungus, Melampsora medusae f. sp. deltoidae, using codominant PCR-SSCP markersFOREST PATHOLOGY, Issue 4 2005M. 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] Spatial autocorrelation and linkage of Mendelian RAPD markers in a population of Picea abies KarstMOLECULAR ECOLOGY, Issue 3 2002Gabriele 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] Genetic Diversity and Tests of the Hybrid Origin of the Endangered Yellow LarkspurCONSERVATION BIOLOGY, Issue 6 2001Jason 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 BrazilACTA ZOOLOGICA, Issue 2 2010Maria 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 ArcticECOGRAPHY, Issue 4 2000Ingibjörg 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 categoryENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 3 2001Kiriaki 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 AfricaENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 1 2001M.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 conditionsENVIRONMENTAL MICROBIOLOGY, Issue 5 2006Hervé 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 pressureENVIRONMENTAL MICROBIOLOGY, Issue 3 2001Johannes 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 polymorphismENVIRONMENTAL MICROBIOLOGY, Issue 1 2000Luigi 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] Integration of genotoxicity and population genetic analyses in kangaroo rats (Dipodomys merriami) exposed to radionuclide contamination at the Nevada Test Site, USAENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2001Christopher 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] New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatusFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2001Camile 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 2001Bart 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 markersFOREST PATHOLOGY, Issue 4 2005M. 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 conesFOREST PATHOLOGY, Issue 1 2003E. 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] A preliminary linkage map of the hard tick, Ixodes scapularisINSECT MOLECULAR BIOLOGY, Issue 2 2003A. 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] Isolation of microsatellite loci in the pollinating fig wasp of Ficus hispida, Ceratosolen solmsiINSECT SCIENCE, Issue 4 2008Hao 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 cisplatinINTERNATIONAL JOURNAL OF CANCER, Issue 4 2005Tapasya 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 analysisINTERNATIONAL JOURNAL OF CANCER, Issue 2 2002Sarantos 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] Multiple molecular approaches yield no evidence for sex-determining genes in lake sturgeon (Acipenser fulvescens)JOURNAL OF APPLIED ICHTHYOLOGY, Issue 6 2008C. 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] Use of RAPD and killer toxin sensitivity in Saccharomyces cerevisiae strain typingJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2005L. Corte Abstract Aims:, Two different strain characterization techniques, random amplified polymorphic DNA (RAPD) and killer toxin sensitivity (KTS), were compared to assess their typing performance using a set of 30 certified Saccharomyces cerevisiae strains. Methods and Results:, A sequential random resampling procedure was employed to subdivide the 32 descriptors in eight sets, in order to compare the differential performances of the two techniques with diverse number of characters. Results showed that RAPD performs better than killer, although the complete differentiation of the strains under study could be obtained only by combining profiles from the two techniques Conclusions:, The combination of different typing techniques was useful when discriminating similar organisms. In such cases, the introduction of a second typing technique can be more advantageous than increasing the number of characters obtained with a single method. Significance and Impact of the Study:, The distribution of among-strains pairwise distances and the relative performance of the two techniques has implications for the study of biodiversity, taxonomy and microbial ecology. [source] Genetic heterogeneity and functional properties of intestinal bifidobacteriaJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2004J. Mättö Abstract Aims:, The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. Methods and Results:, A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR,ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. Conclusions:, Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. Significance and Impact of the Study:, Applicability of ribotyping with the automated RiboPrinter® System for identification and genotyping of bifidobacteria was shown in the present study. [source] Genotypic and phenotypic characteristics of antibiotic-producing soil Streptomyces investigated by RAPD-PCRJOURNAL OF BASIC MICROBIOLOGY, Issue 1 2003Raad Gharaibeh Random amplified polymorphic DNA (RAPD) analysis has been used to determine the relatedness of 73 antibiotic-producing soil Streptomyces isolates that were recovered from different soil habitats in Jordan based on their RAPD-PCR fingerprints. Genetic polymorphisms between these isolates showed three common bands of 2777, 800 and 250 bp shared by approximately (95%) of them. Some specific bands were also observed. Further analysis of RAPD patterns with the UPGMA resulted in clustering the tested isolates into two main super clusters. Super cluster I was more homogenous than super cluster II and contained all the reference strains. However, super cluster II consists of unrelated isolates within five small groups. As RAPD fingerprints of the tested isolates linked to their phenotypes, differentiation between isolates with different cultural properties was observed. [source] Phenotypic, serological and genetic characterization of Flavobacterium psychrophilum strains isolated from salmonids in ChileJOURNAL OF FISH DISEASES, Issue 4 2009S Valdebenito Abstract Characterization of 20 Flavobacterium psychrophilum strains isolated from farmed Atlantic salmon and rainbow trout in Chile was done using phenotypic, antigenic and genetic techniques. Experimental infections were also performed to assess the virulence of two representative isolates and of the type strain. Biochemical and physiological analyses showed that Chilean F. psychrophilum strains, regardless of the host species, constitute a phenotypically very homogeneous group matching with previous descriptions of this pathogen. However, serological assays indicated the existence of antigenic heterogeneity with four patterns of serological reactions. The first group contained most (14 of 20) of the F. psychrophilum isolates showing cross-reaction with the antisera obtained against Atlantic salmon and rainbow trout isolates. Group 2 corresponded to four other rainbow trout isolates (1658, 1731, 1762 and 29009) that did not agglutinate with anti-1150 serum. Two minor serological groups were identified for the remaining isolates (Groups 3 and 4). Marked homogeneity was also revealed by genetic studies including 16S rRNA alleles, random amplified polymorphic DNA and REP-PCR showing that a major genetic group of F. psychrophilum may be dominant in disease outbreaks in farms. Restriction fragment length polymorphism of PCR analysis showed that gyrase genotypes B-S or B-R were found in Chilean isolates from rainbow trout and Atlantic salmon, whereas genotype A was not found. Virulence assays using Atlantic salmon indicated no relationship between the degree of pathogenicity and the host origin of the F. psychrophilum strains. [source] Assessment of Genetic Variation Within Indian Mustard (Brassica juncea) Germplasm Using Random Amplified Polymorphic DNA MarkersJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 4 2008Muhammad Ayub Khan Abstract Genetic diversity among 45 Indian mustard (Brassica juncea L.) genotypes comprising 37 germplasm collections, five advance breeding lines and three improved cultivars was investigated at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Fifteen primers used generated a total of 92 RAPD fragments, of which 81 (88%) were polymorphic. Of these, 13 were unique to accession ,Pak85559'. Each primer produced four to nine amplified products with an average of 6.13 bands per primer. Based on pairwise comparisons of RAPD amplification products, Nei and Li's similarity coefficients were calculated to evaluate the relationships among the accessions. Pairwise similarity indices were higher among the oilseed accessions and cultivars showing narrow ranges of 0.77,0.99. An unweighted pair-group method with arithmetic averages cluster analysis based on these genetic similarities placed most of the collections and oilseed cultivars close to each other, showing a low level of polymorphism between the accessions used. However, the clusters formed by oilseed collections and cultivars were comparatively distinct from that of advanced breeding lines. Genetically, all of the accessions were classified into a few major groups and a number of individual accessions. Advanced breeding lines were relatively divergent from the rest of the accessions and formed independent clusters. Clustering of the accessions did not show any pattern of association between the RAPD markers and the collection sites. A low level of genetic variability of oilseed mustard was attributed to the selection for similar traits and horticultural uses. Perhaps close parentage of these accessions further contributed towards their little diversity. The study demonstrated that RAPD is a simple and fast technique to compare the genetic relationship and pattern of variation among the gene pool of this crop. [source] Use of Random Amplified Polymorphic DNA Analysis for Economically Important Food CropsJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 12 2007Halima Hassan Salem Abstract The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA (RAPD) technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and validity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation. Also we discuss the advantages and limitations of RAPD. Molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics. [source] Characterization of Wheat Random Amplified Polymorphic DNA Markers Associated with the H11 Hessian Fly Resistance GeneJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 8 2006Dhia Bouktila Abstract In Tunisia, the Hessian fly Mayetiola destructor Say is a major pest of durum wheat (Triticum durum Desf.) and bread wheat (T. aestivum L.). Genetic resistance is the most efficient and economical method of control of this pest. To date, 31 resistance genes, designated H1,H31, have been identified in wheat. These genes condition resistance to the insect genes responsible for virulence. Using wheat cultivars differing for the presence of an individual Hessian fly resistance gene and random amplified polymorphic DNA (RAPD) analysis, we have identified a polymorphic 386-bp DNA marker (Xgmib1-1A.1) associated with the H11 Hessian fly resistance gene. Blast analysis showed a high identity with a short region in the wild wheat (T. monococcum) genome, adjacent to the leaf rust resistance Lr10 gene. A genetic linkage was reported between this gene (Lr10) and Hessian fly response in wheat. These data were used for screening Hessian fly resistance in Tunisian wheat germplasm. Xgmib1-1A.1-like fragments were detected in four Tunisian durum and bread wheat varieties. Using these varieties in Hessian fly breeding programs in Tunisia would be of benefit in reducing the damage caused by this fly. (Managing editor: Li-Hui Zhao) [source] Yersinia pseudotuberculosis infection in breeding monkeys: detection and analysis of strain diversity by PCRJOURNAL OF MEDICAL PRIMATOLOGY, Issue 3 2002T. Kageyama In the last three decades, several monkeys reared in outdoor/indoor,outdoor breeding colonies and cages of the Primate Research Institute, Kyoto University, died of yersiniosis caused by Yersinia pseudotuberculosis, necessitating introduction of a method to detect the bacteria rapidly and thus allow preventive measures to be undertaken. A rapid nested polymerase chain reaction (PCR) method for identification of Y. pseudotuberculosis in fecal samples and a random amplified polymorphic DNA (RAPD)-PCR approach for distinguishing between bacterial strains were therefore developed. Yersinia pseudotuberculosis isolates from monkey specimens were found to be classifiable into several types. To determine the source of infection, hundreds of fecal samples of wild rats, pigeons, and sparrows were collected from around the breeding colonies and cages, and subjected to PCR analyses. Yersinia pseudotuberculosis was detected in 1.7% of the fecal samples of wild rats. The DNA fingerprints of the bacteria revealed by RAPD-PCR were the same as that of one strain isolated from macaques, suggesting the wild rat to be a possible source of infection. [source] GENETIC DIVERGENCE CORRELATES WITH MORPHOLOGICAL AND ECOLOGICAL SUBDIVISION IN THE DEEP-WATER ELK KELP, PELAGOPHYCUS PORRA (PHAEOPHYCEAE)JOURNAL OF PHYCOLOGY, Issue 5 2000Kathy Ann Miller Pelagophycus porra (Leman) Setchell has a narrow distribution confined to deep water from the Channel Islands off the southern California coast to central Baja California, Mexico. Distinct morphotypes are consistently correlated with distinctive habitats, that is, windward exposures characterized by strong water motion and rocky substrates, and sheltered areas with soft substrates found on the lee sides of the islands. We tested the hypothesis that morphologically and ecologically distinct forms reflect genetically distinct stands. Individuals representing populations from three islands and the mainland were compared using RFLP analyses of the nuclear rDNA internal transcribed spacers (ITS1 and ITS2), chloroplast trnL (UAA) intron sequences, and random amplified polymorphic DNA (RAPDs). No variation was found in a survey of 20 restriction sites of ITS1 (ca. 320 base pair [bp]) and ITS2 (ca. 360 bp) among individuals from six populations. Likewise, comparisons of trnL intron (241 bp) sequences among nine individuals from seven populations were identical with the exception of a CATAGT insert in two adjacent stands. A RAPD analysis of 24 individuals from nine populations (4 windward and 5 leeward) using 16 primers generated 166 bands. Thirty-eight percent of the bands did not vary, 16% were unique to a given individual, and 46% were variable. Neighbor joining analysis produced a well-resolved tree with moderately high bootstrap support in which windward and leeward populations were easily distinguished. The lack of divergence in both the fast evolving nuclear rDNA-ITS and the chloroplast trnL intron does not support the morphotypes as different species. However, the compartmentalized differentiation shown in the RAPD data clearly points to isolation. This, and previous ecological studies that demonstrate habitat specificity suggest that leeward stands probably comprise a species in statu nascendi. [source] Genetic Variability Analysis and Molecular Detection of Fusarium oxysporum f.sp. eustomae Isolated from Eustoma grandiflorum in Northern ItalyJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2010Yuan Li Abstract A total of 35 isolates of Fusarium oxysporum f.sp. eustomae obtained from diseased Eustoma grandiflorum plants in northern Italy, showing typical Fusarium wilt symptoms, were analysed for their genetic variability and molecular identification. Genetic diversity of the isolates was studied by using random amplified polymorphic DNA (RAPD). This analysis clustered the isolates into three groups at a genetic similarity of 69%. Sequence analysis of RAPD fragments led to the design of a pair of specific primers that amplify a 505-bp SCAR (sequence characterized amplified region) marker (SCAR505) which was used to rapidly detect F. oxysporum f.sp. eustomae on Eustoma grandiflorum plants. In a temperature-controlled chamber, detection of the pathogen by PCR was 100% successful in root and stem samples of infected but still symptomless plants. The diagnostic procedure could be completed in 1 day and allowed rapid and reliable detection of the pathogen in asymptomatic plants in the early stages of disease development. [source] | ||||||||||||||||||||||||||||||||||||||||