scholarly journals Randomly Amplified Polymorphic DNA PCR as a Tool for Assessment of Marine Viral Richness

2008 ◽  
Vol 74 (9) ◽  
pp. 2612-2618 ◽  
Author(s):  
Danielle M. Winget ◽  
K. Eric Wommack
Keyword(s):  
Chesapeake Bay ◽  
Spatial Dynamics ◽  
Sequence Information ◽  
Randomly Amplified Polymorphic Dna ◽  
Banding Patterns ◽  
Hybridization Probes ◽  
Community Profiling ◽  
Rapd Pcr ◽  
Pcr Products ◽  
Template Dna

ABSTRACT Recent discoveries have uncovered considerable genetic diversity among aquatic viruses and raised questions about the variability of this diversity within and between environments. Studies of the temporal and spatial dynamics of aquatic viral assemblages have been hindered by the lack of a common genetic marker among viruses for rapid diversity assessments. Randomly amplified polymorphic DNA (RAPD) PCR bypasses this obstacle by sampling at the genetic level without requiring viral isolation or previous sequence knowledge. In this study, the utility of RAPD-PCR for assessing DNA viral richness within Chesapeake Bay water samples was evaluated. RAPD-PCR using single 10-mer oligonucleotide primers successfully produced amplicons from a variety of viral samples, and banding patterns were highly reproducible, indicating that each band likely represents a single amplicon originating from viral template DNA. In agreement with observations from other community profiling techniques, resulting RAPD-PCR banding patterns revealed more temporal than spatial variability in Chesapeake Bay virioplankton assemblages. High-quality hybridization probes and sequence information were also easily generated from single RAPD-PCR products or whole reactions. Thus, the RAPD-PCR technique appears to be practical and efficient for routine use in high-resolution viral diversity studies by providing assemblage comparisons through fingerprinting, probing, or sequence information.

scholarly journals Seasonal Dynamics and Metagenomic Characterization of Estuarine Viriobenthos Assemblages by Randomly Amplified Polymorphic DNA PCR

2009 ◽  
Vol 75 (8) ◽  
pp. 2259-2265 ◽  
Author(s):  
Rebekah R. Helton ◽  
K. Eric Wommack
Keyword(s):  
Chesapeake Bay ◽  
Oligonucleotide Primer ◽  
Viral Diversity ◽  
Randomly Amplified Polymorphic Dna ◽  
Aquatic Sediments ◽  
Banding Patterns ◽  
Rapd Pcr ◽  
Viral Sequences ◽  
Dna Pcr

ABSTRACT Direct enumeration and genetic analyses indicate that aquatic sediments harbor abundant and diverse viral communities. Thus far, synecological analysis of estuarine sediment viral diversity over an annual cycle has not been reported. This oversight is due in large part to a lack of molecular genetic approaches for assessing viral diversity within a large collection of environmental samples. Here, randomly amplified polymorphic DNA PCR (RAPD-PCR) was used to examine viral genotypic diversity within Chesapeake Bay sediments. Using a single 10-mer oligonucleotide primer for all samples, RAPD-PCR analysis of sediment viral assemblages yielded unique banding patterns across spatial and temporal scales, with the occurrence of specific bands varying among the sample set. Cluster analysis of RAPD-PCR amplicon banding patterns indicated that sediment viral assemblages changed with season and to a lesser extent with geographic location. Sequence analysis of RAPD-PCR amplicons revealed that 76% of sediment viral sequences were not homologous to any sequence in the GenBank nonredundant protein database. Of the GenBank sequence homologs, the majority belonged to viruses within the Podoviridae (24%) and Myoviridae (22%) viral families, which agrees with the previously observed frequencies of these morphological families in Chesapeake Bay sediments. Furthermore, the majority of the sediment viral sequences homologous to GenBank nonredundant protein sequences were phages or prophages (57%). Hence, RAPD-PCR proved to be a reliable and useful approach for characterization of viral assemblages and the genetic diversity of viruses within aquatic sediments.

scholarly journals Characterization of Alstroemeria Species using Random Amplified Polymorphic DNA (RAPD) Analysis

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 482F-482 ◽  
Author(s):  
Deric D. Picton ◽  
Harrison G. Hughes
Keyword(s):  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Randomly Amplified Polymorphic Dna ◽  
Banding Patterns ◽  
Extraction Buffer ◽  
Rapd Pcr ◽  
High Degree ◽  
Species Hybrids ◽  
Color Variants

In this study, 11 species, hybrids, and color variants were characterized using randomly amplified polymorphic DNA (RAPD) analysis. Total genomic DNA was extracted using a 2% CTAB extraction buffer using fresh or frozen leaf material. The DNA was amplified using standard RAPD-PCR protocols utilizing 10-mer primers. All primers utilized exhibited a high degree of polymorphism in their banding patterns among the species and hybrids studied. The primers used produced ≈40 reproducible bands. It was possible to identify and uniquely distinguish all species and hybrids investigated using these bands.

scholarly journals Efficiency of Randomly Amplified Polymorphic DNA to Sequence Characterized Amplified Region Marker Conversion and their Comparative Polymerase Chain Reaction Sensitivity in Cucumber

1999 ◽  
Vol 124 (2) ◽  
pp. 128-135 ◽  
Author(s):  
Thomas Horejsi ◽  
Jodie M. Box ◽  
Jack E. Staub
Keyword(s):  
Rapd Markers ◽  
Scar Marker ◽  
Scar Markers ◽  
Sequence Characterized Amplified Region ◽  
Pcr Product ◽  
Rapd Pcr ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Template Dna ◽  
Pcr Conditions

The conversion of randomly amplified polymorphic DNA (RAPD) markers to sequence characterized amplified region (SCAR) markers, and the effects of differing polymerase chain reaction (PCR) conditions were studied in cucumber (Cucumis sativus L.). Attempts were made to clone and sequence 75 RAPD PCR products to produce SCAR primers (16 to 22 nucleotides) designed to amplify original RAPD PCR products. The influence of template DNA source, purity, and concentration, MgCl2 concentration, Taq polymerase source, and type of thermocycler upon RAPD and SCAR marker performance was evaluated. Conversion of RAPD to SCAR markers was not universally successful, and SCAR primers reacted differently to varying PCR conditions. Only 48 (64%) of 75 RAPD markers were successfully converted to SCAR markers and 11 (15%) of these reproduced the polymorphism observed with the original RAPD PCR product. Moreover, some SCAR primer pairs produced multiple polymorphic PCR products. The band intensity of SCAR markers were brighter (P = 0.05) than their corresponding RAPD markers with only one exception. The SCAR markers examined were less influenced (P = 0.05) by MgCl2 concentration than their corresponding RAPD markers. However, some SCAR markers were more sensitive to reaction impurities than their RAPD counterparts and SCAR markers tended to be less readily visualized (decrease in frequency of visible PCR product) with low concentrations (1 and 2 mm) of template DNA than their corresponding RAPD markers. Neither the source of Taq nor the type of thermocycler used affected the performance of SCAR and RAPD markers. These data suggest that although SCAR markers may demonstrate enhanced performance over the RAPD markers from which they are derived, careful consideration must be given to both the costs and potential benefits of SCAR marker development in cucumber.

Use of Randomly Amplified Polymorphic Dna Markers as a Tool to Study Variation in Lichen-Forming Fungi

1999 ◽  
Vol 31 (3) ◽  
pp. 257-267 ◽  
Author(s):  
G. J. Murtagh ◽  
P. S. Dyer ◽  
P. C. McClure ◽  
P. D. Crittenden
Keyword(s):  
Dna Polymerase ◽  
Dna Markers ◽  
Rapd Markers ◽  
Rapd Analysis ◽  
Pcr Amplification ◽  
Randomly Amplified Polymorphic Dna ◽  
Banding Patterns ◽  
Rapd Pcr ◽  
Key Features ◽  
Axenic Cultures

AbstractA protocol is described to enable the production of reliable genetic fingerprints of lichen-forming fungi using randomly amplified polymorphic DNA (RAPD) markers. Key features of the method are the use of mycobiont DNA extracted from axenic cultures by a phenol-chloroform procedure, and PCR amplification using DyNAzyme II DNA polymerase. RAPD-PCR fingerprints of Graphis scripta, G. elegans and Phacographis dendritica were successfully generated using this protocol and individual isolates could be identified on the basis of differences in banding patterns produced. DNA extracted from whole thalli of G. scripia was also subjected to RAPD-PCR but the fingerprints produced differed from those given by axenic cultures of the mycobiont. Therefore difficulties of interpretation may arise when whole thalli are used in RAPD analysis.

ANALYSIS OF GENETIC DIVERSITY OF 26 CASSAVA VARIETIES (Manihot esculenta CRANTZ) WITH DIFFERENT RESPONSES TO WATERLOGGING CONDITIONS BY USING RAPD MARKERS

2021 ◽  
Vol 66 (3) ◽  
pp. 170-179
Author(s):  
Sengsoulichan Dethvongsa ◽  
Vu Nguyen Anh ◽  
Van Tran Khanh
Keyword(s):  
Genetic Diversity ◽  
Phylogenetic Tree ◽  
Manihot Esculenta ◽  
Rapd Markers ◽  
Flood Tolerance ◽  
Randomly Amplified Polymorphic Dna ◽  
Manihot Esculenta Crantz ◽  
Rapd Pcr ◽  
Cassava Production ◽  
Cassava Varieties

RAPD (Randomly Amplified Polymorphic DNA) is an indicator for high and stable polymorphism, widely used in the study of the diversity of cassava. In this paper, the results of using 20 polymorphic primers OPK combined with the establishment of the phylogenetic tree to analyze the genetic diversity of 26 cassava varieties with different responses to waterlogging conditions by using the RAPD-PCR technique were presented. The purpose of this experiment was to show the genetic relevance of the studied cassava varieties. The results showed that the flood tolerance of cassava was not related to the polymorphism and branching characteristics of the stem. This information may be use as a basis for selecting flood-tolerant cassava varieties for cassava production, as well as the basis for selecting genetically different parents for breeding.

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp.bulgaricus and L. delbrueckii subsp.lactis

1999 ◽  
Vol 65 (10) ◽  
pp. 4351-4356 ◽  
Author(s):  
Sandra Torriani ◽  
Giacomo Zapparoli ◽  
Franco Dellaglio
Keyword(s):  
Numerical Analysis ◽  
Dairy Products ◽  
Lactobacillus Delbrueckii ◽  
Randomly Amplified Polymorphic Dna ◽  
Specific Pcr ◽  
Single Colony ◽  
Rapd Pcr ◽  
Reliable Differentiation ◽  
Dna Pcr ◽  
Proline Iminopeptidase

ABSTRACT Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp.bulgaricus and L. delbrueckii subsp.lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene ofL. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp.delbrueckii LMG 6412T, which clustered withL. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

scholarly journals Sex Determination During Inflorescence Bud Differentiation in Monoecious Pistacia chinensis Bunge

Forests ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 202 ◽  
Author(s):  
Qian Bai ◽  
Chenyi Zhu ◽  
Xia Lei ◽  
Tao Cao ◽  
Shuchai Su ◽  
...  
Keyword(s):  
Sex Determination ◽  
Early Stage ◽  
Sex Differentiation ◽  
Banding Patterns ◽  
Vegetative Period ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Sex Determination Mechanisms

Pistacia chinensis Bunge is widely acknowledged to be dioecious, but rare monoecious individuals have been found. However, the origin of monoecism and the sex differentiation of different sex types remain intriguing questions. Here, sex expressions were explored by identification of sex-associated DNA markers, determination of the sex stability after grafting, and histological characterization of inflorescence bud development using anatomical analysis. The results showed that (1) although polymorphisms among individuals existed, the banding patterns of Polymerase Chain Reaction (PCR) products for different sex types on the same monoecious tree were consistent; (2) the sex expressions of grafted trees were not consistent with those of scions, indicating that monoecism probably did not originate from a stable bud mutation; and (3) both males and females underwent a bisexual period, then the stamen primordia in female buds degenerated into the second round tepals, while the pistil primordia in male buds gradually disappeared. During the sex differentiation phase, female buds were spindle-shaped, while the male buds were full teardrop-shaped, and male buds were bigger than female buds. Taken together, no sex-associated DNA marker was found, sex expressions were unstable after grafting, and the alternative sex organs appeared in the early stage of sex differentiation, suggesting that sex determination occurred during floral development instead of the early vegetative period. These results indicated that the sex expressions may be affected by environmental factors, increasing the understanding of sex determination mechanisms in P. chinensis and other species.

Genetic diversity among microsporidian isolates from the silkworm, Bombyx mori, as revealed by randomly amplified polymorphic DNA (RAPD) markers

2011 ◽  
Vol 56 (4) ◽  
Author(s):  
B. Surendra Nath ◽  
W. Hassan ◽  
S. Nageswara Rao ◽  
N. Vijaya Prakash ◽  
S. Gupta ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Type Species ◽  
Rapd Markers ◽  
Randomly Amplified Polymorphic Dna ◽  
Chain Reaction ◽  
Rapd Pcr ◽  
Random Amplification ◽  
Major Cluster ◽  
Sources Of Infection ◽  
Polymerase Chain

AbstractRandom amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was carried out to assess the genetic diversity of five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworms. A type species, NIK-1s_mys was used as control for comparison. Differences in the spore shape, length and width were observed. Of the 30 decamer random primers tested, 22 primers gave repeatable RAPD profiles and yielded a total of 143 fragments, of which 78 were polymorphic (55%). The resulting data was used to derive genetic similarity values for constructing a dendrogram. The neighbour joining method based on Dice coefficients indicate a major cluster comprising NIK-1s_mys, NIWB-11bp and NIWB-12n, whereas NIWB-13md, NIWB-14b and NIWB-15mb appear to be different from each other as well from the major cluster mentioned above which includes the type species (NIK-1s_mys). Based on the reproducibility of RAPD profiles, we are able to identify these microsporidians as different isolates. The RAPD technique may be useful in detecting sources of infection of this economically important domestic insect.

scholarly journals A Pseudomonas syringae Diversity Survey Reveals a Differentiated Phylotype of the Pathovar syringae Associated with the Mango Host and Mangotoxin Production

2013 ◽  
Vol 103 (11) ◽  
pp. 1115-1129 ◽  
Author(s):  
José A. Gutiérrez-Barranquero ◽  
Víctor J. Carrión ◽  
Jesús Murillo ◽  
Eva Arrebola ◽  
Dawn L. Arnold ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Randomly Amplified Polymorphic Dna ◽  
Chain Reaction ◽  
Catabolic Diversity ◽  
Rapd Pcr ◽  
Geographical Regions ◽  
Extensive Collection ◽  
Polymerase Chain ◽  
Apical Necrosis ◽  
Dna Pcr

Pseudomonas syringae pv. syringae, the causal agent of bacterial apical necrosis (BAN) in mango crops, has been isolated in different mango-producing areas worldwide. An extensive collection of 87 P. syringae pv. syringae strains isolated from mango trees affected by BAN from different countries, but mainly from Southern Spain, were initially examined by repetitive sequence-based polymerase chain reaction (rep-PCR) to analyze the genetic diversity with an epidemiological aim. rep-PCR was powerful in assessing intrapathovar distribution and also allowing clustering of the P. syringae pv. syringae strains isolated from mango, depending on the isolation area. A clear pattern of clustering was observed for all the P. syringae pv. syringae strains isolated from mango distinct from strains from other hosts, including strains for the same geographical regions as the mango isolates. For this reason, a representative group of 51 P. syringae pv. syringae strains isolated from mango and other hosts, as well as some P. syringae strains from other pathovars, were further characterized to determine their possible genetic, phenotypic, and phylogenetic relationships. Similar to the rep-PCR results, the randomly amplified polymorphic DNA PCR (RAPD-PCR) and catabolic diversity analysis using the Biolog GN2 profile grouped 90% of the mango isolates together in a unique cluster. Interestingly, the majority of P. syringae pv. syringae strains isolated from mango produced mangotoxin. The analysis of the phylogenetic distribution using the multilocus sequence typing analysis strongly supports the existence of a differentiated phylotype of the pathovar syringae mainly associated with the mango host and characterized by the mangotoxin production.

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