Random Amplified Polymorphic DNA Method Research Articles

Primary measurement of pollen flow inLiriodendron chinense using RAPD markers

Techniques of tracking movements of pollen grains are essential for many topics in pollination biology. Pollen transfer ofLiriodendron chinense (Hemsl.) Sarg., a hermaphroditic plant, may be both interfloral and intrafloral pollination. Observations on pollinator movements and flower emasculated experiments in natural populations showed that most of pollen grains deposited on stigmas in a flower are foreign. But it is not certain whether cross pollination or long distance pollen transfer occurs in this insect-pollinated plant. The authors attempt to use random amplified polymorphic DNA (RAPD) loci for paternity analysis to mark pollen dispersal. The results indicated pollen via gene transportation reached at least 100 m away from the pollen source in a selected population. The primary study suggests a larger effective population size in this endangered plant. With its rapid and simple characters, RAPD methods can be a suitable technique for marking pollen flow and able to be widely used in various taxa.

An evaluation of RAPD fragment reproducibility and nature.

Random amplified polymorphic DNA (RAPD) fragment reproducibility was assayed in three animal species: red deer (Cervus elaphus), wild boar (Sus scrofa) and fruit fly (Drosophila melanogaster). Ten 10-mer primers (Operon) were tested in two replicate reactions per individual under different stringency conditions (annealing temperatures of 35 degrees C or 45 degrees C). Two estimates were generated from the data: autosimilarity, which tests the reproducibility of overall banding patterns, and band repeatability, which tests the reproducibility of specific bands. Autosimilarity (the similarity of individuals with themselves) was lower than 1 for all three species ranging between values of 0.66 for Drosophila at 45 degrees C and 0.88 for wild boar at 35 degrees C. Band repeatability was estimated as the proportion of individuals showing homologous bands in both replicates. The fraction of repeatable bands was 23% for deer, 36% for boar and 26% for fruit fly, all at an annealing temperature of 35 degrees C. Raising the annealing temperature did not improve repeatability. Phage lambda DNA was subjected to amplification and the pattern of bands compared with theoretical expectations based on nucleotide sequence. Observed fragments could not be related to expected ones, even if a 2 bp mismatch is allowed. Therefore, the nature of genetic variation uncovered by the RAPD method is unclear. These data demonstrate that prudence should guide inferences about population structure and nucleotide divergence based on RAPD markers.

Randomly amplified polymorphic DNA analysis of starved and viable but nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is an estuarine bacterium capable of causing a rapidly fatal infection in humans. Because of the low nutrient levels and temperature fluctuations found in the organism's natural habitat, the starvation state and viable but nonculturable (VBNC) state are of particular interest. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed previously for the detection of V. vulnificus strains grown in rich media and has been applied to starved and VBNC cells of V. vulnificus in the present study. As cells were subjected to starvation in artificial seawater, changes in the RAPD profile were detected as early as 15 min into the starvation period. Most noticeable was a uniform loss of RAPD amplification products. By 4 h of starvation, the cells were undetectable by the RAPD method. Cells that had been starved for up to 1 year again became detectable by the RAPD method when nutrients were added to the starvation microcosm. The same loss of signal, but at a lower rate, was also seen as cells entered the VBNC state. VBNC cells were resuscitated by a temperature upshift and were once again detectable by the RAPD method. The addition of chloramphenicol prevented the RAPD signal from being lost in both the starvation and VBNC states. This suggests that DNA binding proteins produced during starvation and entrance into the VBNC state may be responsible for the inability of the RAPD method to amplify V. vulnificus DNA in these states.

Species Recognition of Congeneric Acanthocephalans in Slider Turtles by Random-Amplified Polymorphic DNA (RAPD) Markers

Species recognition of acanthocephalans of the genus Neoechinorhynchus (Hamann, 1892) found in the freshwater turtle Trachemys scripta (Wied, 1838) has previously been based primarily on female body and egg morphology. Observed morphological plasticity within and among species may lead to the misclassification of female specimens and leaves males of different species completely indistinguishable. Here, random-amplified polymorphic DNA (RAPD) analysis was used to genetically characterize samples of Neoechinorhynchus pseudemydis (Cable and Hopp, 1954), Neoechinorhynchus emydis (Leidy, 1851), and Neoechinorhynchus emyditoides (Fisher, 1960). Amplifications performed with 3 decamer oligonucleotides showed banding patterns with a few diagnostic fragments that allowed the recognition of N. pseudemydis specimens from those of the N. emydis-N. emyditoides group. No primer gave a species-specific locus that allowed the differentiation of N. emydis from N. emyditoides specimens, suggesting that they could belong to a sole taxon. The species assignment of females of uncertain classification and of males is fully reliable using RAPD markers. Thus, identification of acanthocephalan species by RAPD in the helminth infracommunities could potentially be very useful to determine community structure. RAPD and other polymerase chain reaction-based methods have some practical advantages over multilocus discriminant analysis, such as the ability to use ethanol-stored specimens and small tissue samples of parasites.

Determination of Genetic Diversity and Paternity in the Gray-Tailed Vole (Microtus canicaudus) by RAPD-PCR

Genetic relatedness of gray-tailed voles (Microtus canicaudus) was determined by random amplified polymorphic DNA (RAPD). This work is the first reported use of the RAPD method for pedigree analysis of M. canicaudus and demonstrates the feasibility of RAPD for assessing paternity and genetic similarity between inbred and outbred individuals. Amplified polymorphisms and two statistical approaches were used to evaluate genetic similarities between individual voles. DNA profiles of wild-caught (outbred) voles were significantly less similar than were inbred voles, using two different primers (P ≤ 0.03 and P ≤ 0.003). In pedigree analyses using three unrelated males in a discrimination test, indication of paternity was based upon bands shared by the progeny and the male in question. Non-sire males produced bands that were not seen in the probable father or offspring and, conversely, bands were found in the progeny that were apparently inherited only from the probable sire. The RAPD procedure may have wide applications in identifying genetic relationships among individuals to estimate genetic heterogeneity, determine parentage, and address questions concerning gene flow and kinship in feral vole populations.

Identification and genetic characterization of yeasts isolated from freshwater prawns, Macrobrachium rosenbergii de Man, in Taiwan

Since 1987, farmers in southern Taiwan have reported significant disease-related mortalities in freshwater prawns. Most mortalities have occurred during the winter, and usually almost all the adult prawns die within 4–5 days after clinical signs appear. Histopathological studies show that cells in hepatopancreatic ducts and tubules are vacuolized and degenerated. Large numbers of membrane-bound yeast aggregates are observed in the affected tissues. Two hundred and seventeen yeast isolates were obtained from diseased prawns, pond water and sediments from six areas. DNA restriction fragment length polymorphism (RFLP) was used to analyse the yeast genomes and to categorize them into five groups. Conventional biochemical and metabolic methods were then used to identify the yeasts of each group. The results showed that two strains of Candida sake (I and II), Pichia anomala, Endomyces fibuliger and Candida famata were present. In addition, the RAPD (random amplified polymorphic DNA) method was used to determine their genomic similarities. Two strains (I and II) of C. sake were the most similar (72%). C. sake I appears to be the primary causative agent of disease, based on frequencies of occurrence of the yeasts found in the diseased prawns.

Identification and genetic characterization of yeasts isolated from freshwater prawns, Macrobrachium rosenbergii de Man, in Taiwan

Since 1987, farmers in southern Taiwan have reported significant disease-related mortalities in freshwater prawns. Most mortalities have occurred during the winter, and usually almost all the adult prawns die within 4-5 days after clinical signs appear. Histopathological studies show that cells in hepatopancreatic ducts and tubules are vacuolized and degenerated. Large numbers of membrane-bound yeast aggregates are observed in the affected tissues. Two hundred and seventeen yeast isolates were obtained from diseased prawns, pond water and sediments from six areas. DNA restriction fragment length polymorphism (RFLP) was used to analyse the yeast genomes and to categorize them into five groups. Conventional biochemical and metabolic methods were then used to identify the yeasts of each group. The results showed that two strains of Candida sake (I and II), Pichia anomala, Endomyces fibuliger and Candida famata were present. In addition, the RAPD (random amplified polymorphic DNA) method was used to determine their genomic similarities. Two strains (I and II) of C. sake were the most similar (72%). C. sake I appears to be the primary causative agent of disease, based on frequencies of occurrence of the yeasts found in the diseased prawns.

Detection of a specifically amplified DNAfragment in Brucella abortus by arbitrarilyprimed polymerase chain reaction

Randomly amplified polymorphic DNA (RAPD) profiles of Brucella and non-Brucella DNA were established after polymerase chain reaction (PCR) amplification. Five arbitrary oligonucleotide primers were screened to generate Brucella-specific DNA fingerprints. The arbitrary primer OPB-01 (5′-GTTTCGCTCC-3′) produced DNA bands specific to Brucella. Amplification conditions must be optimized for reproductibility. Accordingly, we optimized and established the conditions, which included Mg2+, enzyme (DNA polymerase), primer, template and deoxyribonucleoside triphosphate (dNTP) concentrations as well as the optimum number of thermal cycles to produce OPB-01 directed Brucella DNA fingerprints. The optimized RAPD method can produce a 1.3 kb DNA fragment specific to Brucella. This DNA fragment was common to eight biovars of B. abortus and one biovar of B. melitensis. The fragment was not detected in genetically related species such as Ochrobactrum anthropi and other non-Brucella organisms associated with farm animals. We anticipate the use of this fragment as a possible probe for the detection of Brucella organisms.

Pedigree and RAPD‐Based DNA Analysis of Commercial U.S. Rice Cultivars

Pedigree information has traditionally been used to obtain estimates of genetic variation, predict hybrid performance, and select parents for crossing in crop improvement programs. We have generated and compared pedigree and randomly amplified polymorphic DNA (RAPD) data obtained from 26 elite cultivars and lines rice (Oryza sativa L.) recommended for commercial production in Louisiana and elsewhere in the USA. For the RAPD analysis, 220 commercially available primers (10‐mers) were evaluated for their ability to distinguish pairwise combinations of the cultivars‐lines that included four sets of full sibs. A total of 69 primers produced 92 polymorphic bands, 0.25 to 3.5 kb in size. Seven primers were identified that could differentiate all cultivars‐lines. Genetic distances obtained from RAPD data were correlated with estimated kinship coefficients. Within the RAPD or pedigree method, similar mean genetic distances were detected for long‐ and medium‐grain cultivars. However, mean genetic distances based on pedigrees were significantly greater than those based on RAPD data for both grains types. Cultivars with the same maturity group or grain type were generally placed together in cluster analyses based on either RAPD or known pedigree data. RAPD information can be used to establish genetic relationships among commercial U.S. rice cultivars with unknown or unrelated pedigrees.

Inheritance of RAPD markers and detection of interspecific hybridization with brown trout and Atlantic salmon

Genetic variation and inheritance of random amplified polymorphic DNA (RAPD) markers were studied in 20 intra- and interspecific crosses between nonanadromous salmon ( Salmo salar) and brown trout ( S. trutta). Altogether 21 parent fish and 166 F 1 individuals were studied. Three of the 40 primers tested produced screenable and reproducible DNA markers which separated nonanadromous salmon and brown trout. The inheritance of RAPD markers was strictly Mendelian and dominant in all types of crosses. Nonparental bands were not generated from DNAs of F 1 individuals and it is suggested that the frequency of nonparental bands should be less than 0.03% of all bands. The amount of genetic variation was generally low since nonanadromous salmon were totally monomorphic and in brown trout three primers produced six polymorphic DNA fragments. Mean similarity ( s ̄ ) among 11 unrelated specimens of brown trout was 0.941 and between species ( s ̄ ) was 0.338. Among parents, 11 out of 27 and 14 out of 24 DNA fragments were unique for nonanadromous salmon and brown trout, respectively. It is concluded that the RAPD method is one of the most time-saving and the least expensive noninvasive method for detection of interspecific hybridization.

Trypanosoma cruzi: Evaluation of a RAPD Synapomorphic Fragment as a Species-Specific DNA Probe

A methodological approach is proposed to select rapidly DNA sequences characterized by a well defined specificity and potentially interesting to be used as diagnostic probes or as taxonomic and phylogenetic markers. A fragment amplified from a diversified sample of Trypanosoma cruzi stocks by the RAPD (random amplified polymorphic DNA) method with the A8 primer, previously found to be monomorphic in all stocks, was separated in 2 fragments using polyacrylamide electrophoresis. RFLP (restriction fragment length polymorphism) analysis of the 750-bp fragment common to all stocks revealed some sequence heterogeneity within the T. cruzi species, whereas hybridization experiments showed a high homology between fragments amplified from different T. cruzi stocks. These results suggest that sequence analysis will allow the design of internal primers to be used as probes to target specific taxonomic levels (clone, family of related clones, or species) and for diagnosis.

RAPD data suggest the Gamkapoort Aloe to be of hybrid origin

The recent discovery of a population of Aloe with an unusual morphology in the Gamkapoort Nature Reserve has raised some interesting issues concerning its origin. The intermediate morphology of these plants suggested that they were of hybrid origin, the hypothesized parent species being A. ferox and A. claviflora, both of which are found in the region. In this study, the hypothesis that the Gamkapoort population is a hybrid originating from these two species is corroborated by data obtained using the RAPD (random amplified polymorphic DNA) method.

Application of random amplified polymorphic DNA to study genetic diversity in Paspalum scrobiculatum L. (Kodo millet, Poaceae)

Genetic diversity and patterns of geographic variation among collections of Paspalum scrobiculatum (kodo millet) and P. polystachyum were studied using molecular markers generated through the random amplified polymorphic DNA (RAPD) method. A high level of polymorphism in RAPD markers was observed among the individual accessions, demonstrating the high genetic diversity of the crop. The markers obtained from the RAPD method were analyzed with the cluster analysis, principal coordinates and minimum spanning tree methods. Three major groups were resolved, one representing the African accessions, and two for the Indian accessions. The accessions of the north African kodo millet and P. polystachyum (considered conspecific with P. scrobiculatum) were quite distinct. The Australian kodo millet showed higher affinity to the African types. The study demonstrated that the RAPD technique can be applied to resolving degrees and patterns of genetic variation at the population and species levels, identifying cultivars, and defining gene pools of this crop.

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

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

Assessing population genetic structure and variability with RAPD data: Application to Vaccinium macrocarpon (American Cranberry)

AbstractA method for estimating and comparing population genetic variation using random amplified polymorphic DNA (RAPD) profiling is presented. An analysis of molecular variance (AMOVA) is extended to accomodate phenotypic molecular data in diploid populations in Hardy‐Weinberg equilibrium or with an assumed degree of selfing. We present a two step strategy: 1) Estimate RAPD site frequencies without preliminary assumptions on the unknown population structure, then perform significance testing for population substructuring. 2) If population structure is evident from the first step, use this data to calculate better estimates for RAPD site frequencies and sub‐population variance components. A nonparametric test for the homogeneity of molecular variance (HOMOVA) is also presented. This test was designed to statistically test for differences in intrapopulational molecular variances (heteroscedasticity among populations). These theoretical developments are applied to a RAPD data set in Vaccinium macrocarpon (American cranberry) using small sample sizes, where a gradient of molecular diversity is found between central and marginal populations. The AMOVA and HOMOVA methods provide flexible population analysis tools when using data from RAPD or other DNA methods that provide many polymorphic markers with or without direct allelic data.

Genetic diversity for RAPD markers between cultivated and wild accessions of Coffea arabica

Random amplified polymorphic DNA (RAPD) markers have been successfully employed to analyse the genetic diversity among cultivated and subspontaneous accessions of Coffea arabica. The narrow genetic base of commercial cultivars was confirmed. On the other hand, a relatively large genetic diversity was observed within the germplasm collection demonstrating the importance of collecting missions. Results suggested an East-West differentiation in Ethiopia, the primary centre of diversification of C. arabica. The large heterosis effect reported in intergroup hybrids could be related to such genetic differentiation. RAPD method appeared to be effective in resolving genetic variations and in grouping germplasm in C. arabica.

Genetic map of randomly amplified DNA polymorphisms closely linked to the mating type locus of Tetrahymena thermophila.

We have used the PCR-based randomly amplified polymorphic DNA (RAPD) method to efficiently identify and map DNA polymorphisms in the ciliated protozoan Tetrahymena thermophila. The polymorphisms segregate as Mendelian genetic markers. A targeted screen, using DNA from pooled meiotic segregants, yielded the polymorphisms most closely linked to the mat locus. A total of 10 polymorphisms linked to the mat-Pmr segment of the left arm of micronuclear chromosome 2 have been identified. This constitutes the largest linkage group described in T. thermophila. We also provide here the first crude estimate of the frequency of meiotic recombination in the mat region, 20 kb/cM. This frequency is much higher than that observed in most other eukaryotes. Special features of Tetrahymena genetics enhanced the power of the RAPD method: the ability to obtain in a single step meiotic segregants that are whole-genome homozygotes and the availability of nullisomic strains permitting quick deletion mapping of polymorphisms to micronuclear chromosomes or chromosome segments. The RAPD method appears to provide a practical and relatively inexpensive approach to the construction of a high-resolution map of the Tetrahymena genome.

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food‐borne yeasts

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.

Molecular analysis of Japanese isolates of Verticillium dahliae and V. albo-airum

Sixteen isolates of different pathogenicity groups of the plant pathogen Verticillium dahliae and four isolates of V. albo-atrum from Japan were analysed by means of an RAPD (random amplified polymorphic DNA) method using a PCR (polymerase chain reaction). Verticillium dahliae and V. albo-atrum could be distinguished by RAPD analysis. Four pathogenicity groups of V. dahliae could also be classified to a certain extent by this method. Similarities and differences in banding patterns obtained by RAPD may be a useful molecular tool in phylogenetic studies of the pathogenicity groups.

Amplification methods for epidemiologic investigations of infectious diseases

DNA restriction fragment length polymorphisms (RFLP) are extremely valuable tools for laboratory-based evaluation of hypotheses generated by epidemiologic investigations of infectious disease outbreaks. Recently, polymerase chain reaction (PCR)-based DNA amplification methods have been used to index differences between suspected etiologic agents isolated from cases and suspected source. These methods are attractive because they require minute amounts of target DNA, and can be completed in a very short time. Two types of PCR-based subtyping methods are available. The PCR-RFLP method involves the amplification of previously characterized or phylogenetically conserved targets followed by restriction endonuclease analysis to evaluate polymorphisms within the amplified sequences. The Random Amplified Polymorphic DNA (RAPD) and Arbitrarily Primed PCR (AP-PCR) methods require no prior knowledge of DNA sequences of test organisms because they rely on random amplification of target DNA by arbitrarily chosen primers. RAPD and AP-PCR do not require any restriction analysis of amplified DNA. The PCR-RFLP methods are organism-dependent and do not always provide adequate discrimination between unrelated isolates. RAPD methods often suffer from poor reproducibility because the amplification is performed by using crude target DNA under nonstringent conditions.