Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.     

Segregation analysis of random amplified polymorphic DNA (RAPD) markers in Picea abies Karst.

The reliability of arbitrarily primed amplification products was tested. The segregation analysis of 266 amplification products obtained using 17 different 10-mer oligonucleotides in 34 megagametophytes from a single tree of Picea abies was carried out. Fifty-four out of the 165 variable bands fit the 1:1 segregation ratio expected for Mendelian traits. The segregation ratio of a subset of six RAPD markers in five other individuals from the same population confirmed their genetic nature. Our results strengthen the evidence previously reported that RAPDs markers can be considered Mendelian traits useful in the detection of genetic variability among both different individuals and populations.     

Direct evidence for biased gene diversity estimates from dominant random amplified polymorphic DNA (RAPD) fingerprints

The relevance of using dominant random amplified polymorphic DNA (RAPD) fingerprints for estimating population differentiation was investigated when typically small population sample sizes were used. Haploid sexual tissues were first used to determine genotypes at RAPD loci for 75 eastern white pines ( Pinus strobus L.) representing five populations. Dominant RAPD fingerprints were then inferred from genotypic data for each individual at each locus, and gene diversity estimates from both sources of data were compared. Genotypic information at RAPD loci indicated little or no differentiation among populations, similar to allozyme loci. However, estimates of population differentiation derived from dominant RAPD fingerprints according to various common methods of analysis were generally inflated, especially when all fragments were considered. Simulations showed that an increase in loci sampling and population sample sizes did not significantly alleviate the biases observed.     

The clonal and population structure of a rare endemic plant, Wyethia reticulata (Asteraceae): allozyme and RAPD analysis

Genetic structure arises when limited gene flow between populations favours the development of distinct arrays of genetic characters within each population. Determining the spatial scale at which this differentiation occurs is critical to our understanding of population biology and microevolution of species. The genetic structure and spatial pattern of genetic variation in an endemic, clonal perennial, Wyethia reticulata E. Greene, was investigated using random amplified polymorphic DNA (RAPD) markers and allozyme alleles. Large stands (250–360 m²) were found to contain few genetic individuals. Despite the small population sizes and endemism of the species, W. reticulata was highly diverse genetically, with most of the variation (75–81%) distributed within populations. A population structure in full agreement with spatially defined populations was achieved only by combining RAPD and allozyme markers. Analysis using both types of markers appeared to provide estimates of genetic similarity between individuals that were most consistent with empirical data on plant distributions. We postulated that large, long-lived clones dominated genetic relationships within populations but also provided opportunities for gene flow between populations on a longer time scale. The two marker types yielded different estimates of between-individual similarity and revealed disparate patterns of population structure. This result will arise because allozymes and random DNA segments have dissimilar evolutionary dynamics with respect to mutation and selection.     

Applications of random amplified polymorphic DNA (RAPD) in molecular ecology

Molecular genetic markers have been developed into powerful tools to analyse genetic relationships and genetic diversity. As an extension to the variety of existing techniques using polymorphic DNA markers, the Random Amplified Polymorphic DNA (RAPD) technique may be used in molecular ecology to determine taxonomic identity, assess kinship relationships, analyse mixed genome samples, and create specific probes. Main advantages of the RAPD technology include (i) suitability for work on anonymous genomes, (ii) applicability to problems where only limited quantities of DNA are available, (iii) efficiency and low expense.     

Population genetic structure in the dioecious pioneer plant species Hippophae rhamnoides investigated by random amplified polymorphic DNA (RAPD) markers

Hippophae rhamnoides is an outcrossing pioneer plant species with a severely fragmented distribution. Random amplified polymorphic DNA (RAPD) marker variation was analysed in 10 populations of ssp. rhamnoides and in one population of ssp. mongolica to estimate the amount and distribution of genetic variability. No less than 89.7% of the scorable markers were polymorphic, but few of these were fixed and populations consequently differed mainly by frequency variation of individual markers. Within-population gene diversity was somewhat low for an outcrossing plant species: 0.192 or 0.159 for ssp. rhamnoides , depending on whether it was based on all 156 polymorphic RAPDs or on only those 63 RAPDs that fulfilled the 3/ N criterion. Analysis of molecular variance applied to the ssp. rhamnoides showed only 15% between-population variability, indicating a relatively restricted population differentiation as expected in outcrossing species and shown in several other AMOVA studies. The tendency for island populations to be somewhat more differentiated, and to have less within-population diversity than mainland populations, may indicate an effect of population fragmentation. Genetic distance estimates among populations, obtained with and without pruning of RAPD loci on the basis of the 3/ N criterion, were generally in very good agreement. Cluster analyses and principal coordinate analyses showed populations of ssp. rhamnoides to be rather close, but quite isolated from the single ssp. mongolica population. Genetic and geographical distances between the ssp. rhamnoides populations were not associated, indicating that large-scale geographical and ecotypic differentiation was not reflected in the RAPD profiles.     

The distribution of random amplified polymorphic DNA (RAPD) diversity amongst populations of Isotoma petraea (Lobeliaceae)

RAPDs were generated from plants of six populations of Isotoma petraea F. Muell. The species occurs on rock outcrops in southern and western Australia, with populations exhibiting different breeding systems, including complete autogamy, varying levels of outbreeding and complex hybridity. Non-metric multidimensional scaling (nMDS) analysis of the random amplified polymorphic DNA (RAPD) data set clearly resolved all populations. The Pigeon Rock population, which is home to both complex hybrid and structural homozygote plants, was divided into those two groups by the nMDS analysis. There was little diversity in highly autogamous populations, but levels were higher in the outbred Yackeyackine population. All complex hybrid populations and plants possessed numerous genetic system-specific RAPDs, some of which were shown to be held in fixed heterozygosity. Estimating G _ST using RAPDs has been problematical due to their dominance, and analytical methods usually rely on knowledge of the selfing rate or assume Hardy–Weinberg equilibrium. This assumption does not hold when populations exhibit fixed heterozygosity, and an alternative method, Shannon's Index, was used to partition genetic diversity. The distribution of genetic diversity fit expectations for an inbreeding species, with most of the variation (87.5%) occurring between populations. This compares to an average RAPD-based G _ST of 59.6% for inbreeding species generally and 15.5% for outbreeding species.     

Features of DNA fragments obtained by random amplified polymorphic DNA (RAPD) assays

Random amplified polymorphic DNA (RAPD) fragments were prepared from samples of Calonectris diomedea (Cory's shearwater, Aves) and Haemonchus contortus (Nematoda) DNA by polymerase chain reaction (PCR) using decamers containing two restriction enzyme sites as primers. Six of 19 studied RAPD fragments probably originated from traces of commensal microorganisms. Many rearranged fragments, absent in the original genomic DNA, were synthesized and amplified during the processing of all the DNA samples, indicating that interactions occur within and between strands during the annealing step of PCR. The model of interactions between molecular species during DNA amplification with a single arbitrary oligonucleotide primer was modified to include nested primer annealing and interactions within and between strands. The presence of these artefacts in the final RAPD have a major effect on the interpretation of polymorphism studies.     

Extending a RFLP-based genetic map of rye using random amplified polymorphic DNA (RAPD) and isozyme markers

RFLP-based genetic map of rye, developed previously using a cross of lines DS2×RXL10 (F₂ generation), was extended with 69 RAPD and 12 isozyme markers. The actual map contains 282 markers dispersed on all seven chromosomes and spans a distance of 1,140 cM. The efficiency of mapping RAPD markers was close to ten loci per 100-screened arbitrary primers. A strong selection of polymorphic, intensive and reproducible fragments was necessary to reveal individual marker loci that could be assigned to rye chromosomes. Newly mapped markers cover a substantial part of the rye genome and constitute a valuable tool suitable for map saturation, marker-aided selection and phenetic studies. A specific nomenclature for the RAPD loci mapped on individual rye chromosomes, which could be helpful in managing of accumulating data, is proposed. Received: 8 May 2000 / Accepted: 17 October 2000     

Genetic diversity study of Chromobacterium violaceum isolated from Kolli Hills by amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD)

Aim: Chromobacterium are saprophytes that cause highly fatal opportunistic infections. Identification and strain differentiation were performed to identify the strain variability among the environmental samples. We have evaluated the suitability of individual and combined methods to detect the strain variations of the samples collected in different seasons. Methods and Results: Amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD) profiles were obtained using four different restriction enzyme digestions (AluI, HaeIII, MspI and RsaI) and five random primers. A matrix of dice similarity coefficients was calculated and used to compare these restriction patterns. ARDRA showed rapid differentiation of strains based on 16S rDNA, but the combined RAPD and ARDRA gave a more reliable differentiation than when either of them was analysed individually. Conclusion: A high level of genetic diversity was observed, which indicates that the Kolli Hills’C. violaceum isolates would fall into at least three new clusters. Significance and Impact of the Study: Results showed a noteworthy bacterial variation and genetic diversity of C. violaceum in the unexplored, virgin forest area.     

Use of random amplified polymorphic DNA (RAPD) markers in the discrimination and verification of genotypes in Eucalyptus

We carried out four separate studies using random amplified polymorphic DNA (RAPD) markers to analyse samples of Eucalyptus supplied by several different organisations. The objective was to examine the reproducibility of the RAPD technique and its ability to discriminate between individual genotypes for verification of clonal identities. We found that RAPD profiles that are unique to a genotype can be generated reliably and simply and that even closely related genotypes can be distinguished. In addition, in each of the four studies, we detected cases where the plant material studied had been mis-sampled or mis-labelled (i.e. the RAPD profiles were not consistent with the identification numbers): (1) ramets of a Eucalyptus grandis clone were found to be derived from 2 different clones; (2) ramets labelled as 2 different Eucalyptus hybrid clones were found to be the same clone, owing to a mis-planted clonal hedge; (3) samples supplied as a single progeny of a controlled E. nitens cross were derived from two crosses involving different pairs of parents; (4) mis-labelling was detected for ramets of 4 of a set of 10 clones of E. grandis and E. camaldulensis. For three of the four studies, the detection of genotype mis-identifications was unexpected, suggesting that labelling or sampling errors during the handling of plant material are a frequent occurrence, with potentially serious economic consequences.     

Use of random amplified polymorphic DNA (RAPD) markers in the study of the parasitic weed Orobanche

Despite the tremendous economic impact of broomrapes (Orobanche spp.) on agriculture in many countries little is known of the pattern of genetic variation within this group of parasitic weeds. The present paper describes the use of RAPD markers for the study of five Orobanche species in agricultural fields in Israel. Pronounced genetic differentiation was found between the species, and RAPD markers were raised for the identification of each of them. Southern-hybridization patterns of RAPD products of the various species were used to confirm the interpretation. The same markers were valid both for broomrapes collected in agricultural fields and for those collected in natural habitats. The validity of the markers found for O. cumana and O. crenata was confirmed on plants of the same species that were collected in Spain. Parsimony analysis of 86 RAPD characters produced a tree that clearly distinguishes between the five studied Orobanche species, separates the two Orobanche species belonging to sect. Trionychon from those belonging to sect. Osproleon, and supports the separation of O. cumana from O. cernua and of O. aegyptiaca from O. ramosa.     

A review of random amplified polymorphic DNA (RAPD) markers in fish research

During the last 15 years, molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The ease and simplicity of RAPD (randomly amplified polymorphic DNA) techniques make it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics. This review summarizes the use of RAPD markers in fish research. The advantages and disadvantages of the RAPD approach will be discussed.     

Amplification of bacterial DNA bound on clay minerals by the random amplified polymorphic DNA (RAPD) technique

Abstract: Chromosomal DNA from Bacillus subtilis , bound on the clay minerals, montmorillonite (Wyoming (W) and Apache County (Ap)) and kaolinite (K), was subjected to the random amplified polymorphic DNA (RAPD) technique. DNA bound on the clays was not amplified with 0.625, 1.875, 6.25, and 12.5 U of Taq DNA polymerase, but amplification occurred when the clay-DNA complexes were diluted 10- and 20-fold or when 21 U of Taq DNA polymerase was added. DNA desorbed from the Ap-DNA and K-DNA equilibrium complexes was amplified with 0.625 U of Taq DNA polymerase, whereas amplification of DNA desorbed from the W-DNA complex occurred only after a 10-fold dilution or when 1.875 U of Taq DNA polymerase was used. These observations indicate that clay minerals differentially affect the amplification process, probably by inhibiting the activity of Taq DNA polymerase.     

Genome relationships among Lotus species based on random amplified polymorphic DNA (RAPD)

The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lotus was evaluated for several geographically dispersed accessions of four diploid Lotus species, L. tennis Waldst. et Kit, L. alpinus Schleich., L. japonicus (Regel) Larsen, and L. uliginosus Schkuhr and for the tetraploid L. corniculatus L., in order to ascertain whether RAPD data could offer additional evidence concerning the origin of the tetraploid L. corniculatus. Clear bands and several polymorphisms were obtained for 20 primers used for each species/accession. The evolutionary pathways among the species/accessions presented in a cladogram were expressed in terms of treelengths giving the most parsimonious reconstructions. Accessions within the same species grouped closely together. It is considered that L. uliginosus which is most distantly related to L. corniculatus, may be excluded as a direct progenitor of L. corniculatus, confirming previous results from isoenzyme studies. Lotus alpinus is grouped with accessions of L. corniculatus, which differs from previous studies. With this exception, these findings are in agreement with previous experimental studies in the L. corniculatus group. The value of the RAPD data to theories on the origin of L. corniculatus is discussed.     

Genomic fingerprinting of Clostridium difficile isolates by using a random amplified polymorphic DNA (RAPD) assay

Abstract This study describes the use of a new and easy method called random amplfied polymorphic DNA (RAPD) assay to distinguish strains of C. difficile . We used two single short primers (AP4 and AP5) with arbitrary nucleotide sequences in a polymerase chain reaction to amplify genomic DNA. The profiles observed after electrophoretic separation were able to distinguish 20 reference C. difficile strains previously serotyped by Delmées method. The fingerprints of 11 epidemiologically unrelated C. diffiile strains clearly yielded a DNA polymorphism between all the strains. Latterly, RAPD profiles of 11 C. difficile strains isolated from 2 independant suspected outbreaks showed, in each case, a predominant banding pattern correponding to an epidemic strain. These results suggest that RAPD assay could be a valuable tool for epidemiological studies.