Random Amplified Polymorphic DNA Assay Research Articles

Molecular typing of, and distribution of genetic markers among, Burkholderia cepacia complex isolates from Brazil.

PCR tests were used to assign genomovar status to 39 non-cystic fibrosis (non-CF) and 11 CF Burkholderia cepacia complex isolates from patients in hospitals in Recife, Brazil. Non-CF isolates were assigned to genomovar IIIA (71.8%), genomovar I (15.4%), B. vietnamiensis (7.7%), and B. multivorans (5.1%). CF isolates were assigned to genomovar IIIA (18.2%), B. vietnamiensis (18.2%), and genomovar I (9.1%). Six CF isolates sharing recA PCR-restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) patterns could not be assigned to a genomovar. 16S rDNA sequence obtained from these isolates indicated a closest relationship to B. anthina, but the recA sequence was equally divergent from several genomovars. PCR screening indicated the presence of cblA in only two isolates, whereas the B. cepacia epidemic strain marker was found in 22 of 28 genomovar IIIA isolates. A type III secretion gene was detected in all but genomovar I isolates. RAPD and PCR-RFLP assays, targeting both recA and fliC, indicated a large amount of genetic variability among the isolates, with many novel patterns being observed. Nine genomovar IIIA isolates from different non-CF patients and clinical sources had identical genotypes, indicating the presence of a common clone.

Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F1 and F2 progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933.

Amplification-Based Nucleic Acid Scanning Techniques to Assess Genetic Polymorphism in Candida

Opportunistic pathogen Candida causes common fungal infections that manifest both superficially and systemically, especially in compromised patients. Although C. albicans is by far the main etiological agent of candidosis, the frequency of isolation of other non-albicans species such as C. glabrata and C. krusei is increasing at an alarming rate. Therefore, the epidemiology, pathogenicity, and diagnosis of infections due to these organisms are of great importance. Of a variety of genotyping methods utilized for strain delineation of these Candida species, amplification-based techniques such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction digestion-mediated PCR (RFLP-PCR), and single-stranded conformational polymorphism (SSCP) and microsatellite PCR (interrepeat PCR, IR-PCR) are the most popular and widely used. In the last decade or so these techniques have helped unravel the clinical epidemiological features of pathogenicity, diversity, microevolution, and natural heterozygosity in Candida species. Here we review in detail the basic principles of RAPD, the nature of the primer and factors influencing its selection, and the limitations of RAPD assays as well as analysis and interpretation of banding profiles generated using the software programs. In addition, the principles of other RAPD-based amplification techniques (AFLP, RFLP-PCR, SSCP, and IR-PCR) and their application in molecular epidemiologic studies of Candida species in particular and other fungi in general are also reviewed. It is concluded that these methods have wide applicability in genotyping fungi, although they differ greatly in their resolution and have advantages and drawbacks depending on the task in question.

Genetic analysis of two common carp broodstocks by RAPD and microsatellite markers

The whole broodstock of two Hungarian common carp farms—80 and 196 individuals—was analyzed by using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. Ten polymorphic RAPD markers and four microsatellites were selected to genotype both of the stocks. As expected, microsatellite analysis revealed more detailed information on genetic diversities than RAPD assay. Results obtained with both types of DNA markers showed lack of major differences between the genetic structure of the two stocks: heterozygosity values and allele frequencies were very similar. Dendrograms created from both sets of data did not show grouping of individuals according to stocks. Genotypes from the two stocks were also compared to those from a limited number of samples collected from other hatcheries and two rivers. Allele frequencies in the groups were similar, with the exception of wild carps. An interesting observation was that three private microsatellite alleles were found in the eight wild carp individuals, compared to the seven detected in the rest of the samples tested (372 individuals).

Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations

The random amplified polymorphic DNA (RAPD) assay and related techniques like the arbitrarily primed polymerase chain reaction (AP-PCR) have been shown to detect genotoxin-induced DNA damage and mutations. The changes occurring in RAPD profiles following genotoxic treatments include variation in band intensity as well as gain or loss of bands. However, the interpretation of the molecular events responsible for differences in the RAPD patterns is not an easy task since different DNA alterations can induce similar type of changes. In this study, we evaluated the effects of a number of DNA alterations on the RAPD profiles. Genomic DNA from different species was digested with restriction enzymes, ultrasonicated, treated with benzo[a]pyrene (B[a]P) diol epoxide (BPDE) and the resulting RAPD profiles were evaluated. In comparison to the enzymatic DNA digestions, sonication caused greater changes in the RAPD patterns and induced a dose-related disappearance of the high molecular weight amplicons. A DNA sample substantially modified with BPDE caused very similar changes but amplicons of low molecular weight were also affected. Appearance of new bands and increase in band intensity were also evident in the RAPD profiles generated by the BPDE-modified DNA. Random mutations occurring in mismatch repair-deficient strains did not cause any changes in the banding patterns whereas a single base change in 10-mer primers produced substantial differences. Finally, further research is required to better understand the potential and limitations of the RAPD assay for the detection of DNA damage and mutations.

Randomly Amplified Polymorphic DNA of Trichoderma isolates and antagonism against Rhizoctonia solani

Random Amplified Polymorphic DNA (RAPD) procedure was used to examine the genetic variability among fourteen isolates of Trichoderma and their ability to antagonize Rhizoctonia solani using a dual-culture assay for correlation among RAPD products and their hardness to R. solani. Seven oligodeoxynucleotide primers were selected for the RAPD assays which resulted in 197 bands for 14 isolates of Trichoderma. The data were entered into a binary matrix and a similarity matrix was constructed using DICE similarity (SD) index. A UPGMA cluster based on SD values was generated using NTSYS (Numerical Taxonomy System, Applied Biostatistics) computer program. A mean coefficient of similarity obtained for pairwise comparisons among the most antagonics isolates was around 40%. The results presented here showed that the variability among the isolates of Trichoderma was very high. No relationship was found between the polymorphism showed by the isolates and their hardness, origin and substrata.

Identification of sex in Carica papaya L. using RAPD markers

Brazil is currently the worlds largest producer of papaya (Carica papayaL.), producing fruits for both the domestic market and export. Only fruits from hermaphrodite plants are marketed because they have the necessary commercial characteristics, i.e. they are pear-shaped and have thicker flesh and a smaller internal cavity. Increased papaya yield has been limited mainly by the ratio of female to hermaphrodite (1: 2) plants normally occurring in orchards. This ratio causes great losses to papaya producers and the identification of the sex of seedlings during the nursery stage would be an important advance. In our study random amplified polymorphic DNA (RAPD) analysis was used to differentiate between the sexual forms of three commercial C. papaya cultivars belonging to the Solo group. RAPD assays using the BC210 primer were able to detect hermaphrodites in all of the cultivars tested. The BC210438molecular marker was much better at papaya sex differentiation than other markers described in the literature.

Morphological characterization and genetic analysis of Drechslera teres isolates

Net blotch, caused by the phytopathogen Drechslera teres, is a common disease of barley (Hordeum vulgare L) and is responsible for large economic losses in some barley growing areas. In this study the morphology and genetic variability of eight D. teres isolates from different regions of the Brazilian state of Rio Grande do Sul were investigated. Colony morphology was studied on potato-dextrose-agar (PDA) and genetic variability investigated using the random amplified polymorphic-DNA (RAPD) technique. 27 commercially available primers were tested of which 16 were selected for use in polymorphic analysis due to their good resolution and reproducibility. Similarity coefficients were used to construct dendrograms based on colony morphology and RAPD data showing the relationship between the eight isolates studied. Colony morphology showed variability between the isolates while RAPD assays showed high similarity coefficients, but grouping of the isolates according to the geographic origins of the seeds from which they were isolated was not possible.

Clonality in wild rice (Oryza rufipogon, Poaceae) and its implications for conservation management

Correlations were examined between habitat characters and clonal structures determined by the RAPD (random amplified polymorphic DNA) assay in five populations of Oryza rufipogon in China. Nine of 175 decameric primers were used in the study because they reproducibly amplified polymorphisms. The extent of clonality together with the clonal and sexual reproductive strategies varied greatly among the five populations and correlated with the habitats where they occur. The populations under serious disturbance or seasonal drought tended to have small clones with relatively high clonal diversity caused by sexual reproduction, whereas the populations with little disturbance and sufficient supply of water were prone to have large clones with relatively low clonal variation and low sexual reproduction. Therefore, the dynamics of sexual vs. clonal reproduction of this species depended mainly on environmental factors, such as external disturbance and water supply, rather than latitudes indicated by previous study. These results have important implications for in situ conservation of O. rufipogon. Adequate external disturbance and water supply control are essential for maintaining high clone diversity of in situ conserved populations. According to the extent of clonality of the populations examined, we recommend that an interval of >12 m should be required for collecting samples for ex situ conservation and for population genetic studies to capture possible genetic diversity for O. rufipogon in China.

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains.

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.

Detection of somatic DNA alterations in azoxymethane-induced F344 rat colon tumors by random amplified polymorphic DNA analysis.

Colon carcinogenesis induced in rats by azoxymethane (AOM) is a useful experimental model as it mimics the human adenoma-carcinoma sequence and allows the study of dietary variation and of the effects of chemopreventive substances. Alterations of specific oncogenes and tumor suppressor genes (APC and K-ras) play roles at different stages of this carcinogenesis process. Recently, it has been suggested that genomic instability is the necessary step for the generation of multiple mutations underlying the occurrence of cancer. We studied the frequency of K-ras and microsatellite instability (MSI) in 30 colorectal tumors induced by AOM (30 mg/kg) in F344 rats. We also used the random amplified polymorphic DNA (RAPD) method to identify genomic alterations in chemically induced aberrant crypt foci (ACF), adenomas and adenocarcinomas. K-ras mutations were identified in 16.7% of the cases (5/30; 9% in adenomas and 37.5% in adenocarcinomas) and MSI in 20% (6/30) of the tumors (only one sample exhibited instability at more than one locus). Of 21 primers used for the RAPD assay, six were very informative. All the analyzed tumors (16/16) showed at least one RAPD profile with lost or additional bands compared with the normal mucosa. A lower level of genomic alteration was present in the ACF analyzed (7/10). In conclusion, K-ras and MSI are not often involved in the AOM carcinogenesis in the rat, whereas extensive genomic instability is always present and can be detected using the RAPD analysis.

Use of randomly amplified polymorphic DNA (RAPD) analysis for identification of Proteus penneri.

The RAPD assay applied in our study confirmed the taxonomic position of P. penneri bacteria. The low annaealing temperature and the 22- oligonucleoide primers used in our study resulted in PCR patterns highly specific for P. penneri species. The analysis of a number of Proteus clinical isolates as well as other Gram-negative bacteria showed that P. penneri strains composed a clonally related group which can be distinguished from other bacteria including P. mirabilis and P. vulgaris. Moreover, the RAPD assay performed by us could be useful in one step PCR based identification of P. penneri strains.

Molecular epidemiological investigation using a randomly amplified polymorphic DNA assay of Burkholderia cepacia isolates from nosocomial outbreaks.

We experienced two Burkholderia cepacia outbreaks over a 1-year period. During this period, 28 B. cepacia isolates were obtained from clinical specimens, and 2 were obtained from environmental specimens (i.e., from a nebulizer solution and a nebulizer tube). These 30 isolates were subjected to the PCR-based randomly amplified polymorphic DNA (RAPD) assay as well as to pulsed-field gel electrophoresis (PFGE). In the first outbreak, in which eight patients hospitalized in the Trauma and Critical Care Center were involved, the RAPD assay revealed that all 20 isolates obtained from clinical specimens and the 2 isolates from environmental specimens had identical DNA profiles. These RAPD data enabled us to pinpoint a possible source and to take countermeasures to prevent further spread of the epidemic-causing strain. In the second outbreak, two consecutive B. cepacia infection/colonization cases were seen in the surgery ward. The RAPD profiles of four isolates obtained were again identical, but they were distinct from those seen in the first outbreak, clearly indicating that the second outbreak was not related to the first. Thus, our experience demonstrated that the RAPD assay is a useful and reliable tool for epidemiological studies of B. cepacia isolates from nosocomial outbreaks. Since the RAPD assay could provide discriminatory potential and reproducibility comparable to those of the widely used PFGE assay with less complexity and in a shorter time, the introduction of the RAPD assay into hospital microbiology laboratories as a routine technique may help prevent nosocomial outbreaks.

Development of a multiple primer RAPD assay as a tool for phylogenetic analysis in Listeria spp. strains isolated from milkproduct associated epidemics, sporadic cases of listeriosis and dairy environments

A total of 82 Listeria strains comprising four species were examined by amplification with a multiple primer random amplified polymorphic DNA (RAPD) assay. It was the objective of the study to set up a procedure suitable for analysis of the relationships among strains from milkproduct-associated epidemics, strains from sporadic cases of listeriosis and field strains from dairy products and dairy environments. In a preliminary study, 205 primers, each 10 bp long, were screened for suitability as primers and 44 primers showing reliable and reproducible RAPD patterns at a defined reaction condition were selected. The 82 strains were assigned to 54 RAPD groups positioned in 13 major clusters. Strains isolated during milk product-associated epidemics were found to belong to a single cluster I. Human isolates from sporadic cases of listeriosis predominantly were assigned to four separate clusters. It was found that strains of clinical origin were mainly assigned to other clusters than strains of non-clinical origin.

Molecular Marker Analysis of Leymus flavescens and Chromosome Pairing in Leymus flavescens Hybrids (Poaceae: Triticeae)

Leymus flavescens (Scribner & Smith) Pilger, yellow wild rye, is a long‐lived, strongly rhizomatous, tetraploid (2n=4x=28) perennial grass of the tribe Triticeae distributed throughout central Washington, eastern Oregon, and the Snake River plains of Idaho. Our objectives were (1) to describe chromosome pairing and fertility in F1 hybrids between L. flavescens and North American tetraploids (2n=4x=28) L. triticoides and L. cinereus and Eurasian tetraploids L. secalinus, L. racemosus, and L. alaicus subsp. karataviensis and (2) to utilize genome‐specific random amplified polymorphic DNA (RAPD) markers to verify the genomic composition of L. flavescens. The hybrids L. flavescens × L. triticoides (NsNsXmXm), L. flavescens × L. secalinus (NsNsXmXm), L. flavescens × L. racemosus (NsNsXmXm), L. flavescens × L. cinereus (NsNsXmXm), and L. flavescens × L. alaicus subsp. karataviensis (NsNsXmXm) averaged 13.9, 13.8, 13.6, 13.1, and 11.9 bivalents per cell, respectively. Genome‐specific RAPD assay indicates that L. flavescens has the Ns genome but lacks the St genome from the genus Pseudoroegneria and the H genome from the genus Hordeum. On the basis of the bivalent chromosome pairing frequency in the F1 hybrids of L. flavescens, the genomic formula of L. flavescens is NsNsXmXm. The presence of the Ns genome was verified by molecular characterization.

Evaluating alterations of genetic diversity in sunfish populations exposed to contaminants using RAPD assay

Bioindicators of pollutant exposure can be more sensitive and ecologically relevant than simply measuring levels of pollutants in the environment and are applied here as a tool for assessing environmental stress on aquatic organisms. DNA polymorphisms, detected by using the randomly amplified polymorphic DNA (RAPD) technique, are used as biomarkers to assess genetic diversity and genetic distance among populations of redbreast sunfish ( Lepomis auritus) residing in reference streams and a contaminated stream (East Fork Poplar Creek, EFPC). The RAPD technique uses the polymerase chain reaction (PCR) with short oligonucleotide primers under reduced stringency to produce DNA fragments which, when analyzed by gel electrophoresis, form banding patterns similar to DNA fingerprints. A total of 13 primers were used which produced 45 polymorphic bands among all populations of fish tested. Using this technique, only slight differences in genetic diversity were detected among populations of sunfish within EFPC although the diversity of all EFPC populations analyzed together did differ from the reference populations. Fish populations in EFPC were consistently less genetically distant from each other than they were from each of the reference sites. Differences in genetic distance between populations may be due to selection pressure of pollutants on fish in EFPC which is supported by the finding that frequencies of certain unique genotypes in EFPC sunfish were correlated with the downstream pollutant gradient. These results are discussed in relation to implications of RAPD assays and recent remediation efforts within EFPC.

Genetic Similarity Among Broccoli Inbreds “Essentially Derived” from Commercial Cultivars

DNA markers can assess close genetic relationships between individuals of a crop as when one variety is developed (i.e., “essentially derived”) from another. An acceptable threshold, based on empirical results, should be established for a crop to indicate what constitutes an “essentially derived” variety in the absence of clear pedigree information. Empirical data could help settle infringements of intellectual property rights, but appropriate data are not being generated for most crops. Thus, our objectives were to characterize genetic relationships among broccoli varieties “essentially derived” from known parents using random amplified polymorphic DNA (RAPD) markers as a measure of genotype and to provide an empirical basis for threshold levels in this crop. Six F1 broccoli hybrids and three inbred lines (doubled-haploids) developed from each of the hybrids (24 entries) were evaluated by RAPD analysis. RAPD assays were conducted using 23 different oligonucleotide 10-mers. Of 179 RAPD bands scored, 94 were polymorphic among the entries. Similarity indices were computed from RAPD data, and a multi-dimensional scaling (MDS) plot was constructed. Similarity indices for all pairwise comparisons ranged from 0.40 to 0.90. `High Sierra' and it's derived lines were the most closely related group with indices from 0.81 to 0.90. With `High Sierra', `Sultan', and `Marathon', the three derived lines were more closely related to their respective parental hybrids than were any other entries. The hybrids `Futura', `Everest', and `Viking' were more genetically similar to other entries than to their derived lines. A threshold level based on data from `High Sierra', `Marathon', `Sultan', and their derived lines would not identify “essentially derived” lines developed from other hybrids.

Genomic diversity among Streptococcus agalactiae isolates detected by a degenerate oligonucleotide-primed amplification assay.

A random-amplified polymorphic DNA assay using partially degenerate oligonucleotides as primers was used for the characterization of 78 epidemiologically related and unrelated clinical isolates of Streptococcus agalactiae belonging to different serotypes. Thirty distinct amplification profiles were obtained among 52 unrelated S. agalactiae isolates assigned to nine groups by serotyping (including 3 nontypeable strains), uncovering the extent of genomic heterogeneity existent within serotypes. This method was particularly useful in providing evidence for or against vertical transmission of a given clone of this microorganism, as well as for relapsing or reinfection in related cases, and suggested clonal relatedness between unrelated S. agalactiae isolates associated with some invasive infections. Thus, this simple methodology represents a suitable tool for the epidemiologic study of S. agalactiae infections.

Genetic relationships of avocado (Persea americana Mill.) using RAPD markers

RAPD (randomly amplified polymorphic DNA) analysis was carried out on 16 accessions representing the three ecological races of avocado (Persea americana Mill.), and one accession of P. schiedeana Nees. Twenty two preselected primers produced 133 polymorphic DNA fragments in the RAPD assay of the avocado accessions. One primer was identified which could differentiate each of the avocado accessions. Potentially race-specific markers for each of the Mexican, Guatemalan, and West Indian races, have been detected. A Jaccard's similarity coefficient matrix was generated and a dendrogram constructed using UPGMA (unweighted pair-group method of arithmetic averages) cluster analysis. Percentage similarity between avocado accessions ranged from 46% to 85%. The lowest similarity (between 22% and 29%) was revealed between P. schiedeana and any P. americana accession. Average similarity within races of avocado was 75% for the Mexican race, 71% for the West Indian race and 73% for the Guatemalan race. Average similarity between races ranged from 53% to 58%. The dendrogram identified three groups, representing the races of avocado. These results are in concordance with the present classification of avocado into three subspecies (varieties) of P. americana, namely drymifolia, americana, and guatemalensis, corresponding to the Mexican, West Indian and Guatemalan races, respectively, and confirm the separate species status of P. schiedeana. We conclude that RAPD markers may be useful for the classification of avocado and for the assessment of genetic diversity of avocado germplasm.

Agronomic performance and molecular assessment of tissue culture‐derived barley lines

AbstractIn order to assess the genetic variability among regenerants. field trials were conducted with 64 barley lines derived from tissue culture (TCD lines) of four Bulgarian barley varieties. The results indicate that the agronomic performance was altered by tissue culture regeneration and the frequency of variation was cultivar dependent. Seven TCD lines showing desirable agronomic characters were selected in the SC16 progeny. Molecular markers (protein, restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) have used applied to determine the variability induced by tissue culture. Polymorphism was detected in sequences coding for C‐hordeins in line 70 derived from cv.‘Jubiley’Two lines from cv.‘Ruen’exhibited polymorphic bands after hybridization with a mitochondrial DNA probe. RAPD assays have been carried out using 20 different l0‐mer primers. Heritable polymorphisms in several TCD lines have been observed.