Arbitrary Primers Research Articles

Inter- and intra-specific genetic diversity among cyclamen accessions investigated by RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to characterize genetic diversity of 26 Cyclamen persicum and Cyclamen com accessions. Eighty-four arbitrary primers tested, among which nine primers showed reliable polymorphic banding patterns and yielded 104 polymorphic markers. Jaccard's similarity coefficient among accessions ranged from 0.99 to 0.08. At a similarity of 68%, accessions were divided into three clusters. The cophenetic correlation coefficient between the similarity matrix and the dendrogram was relatively high ( r = 0.9) showing the goodness of fit of the dendrogram. The RAPD analysis offered a rapid and reliable tool for the estimation of inter- and intra-specific variability in cyclamens. The wide genetic variation observed for cyclamens within Iran guarantees a promising future of breeding.

Evaluation of Genetic Diversity in Aegilops geniculata Roth Accessions using Morphological and RAPD Markers

Thirteen Aegilops geniculata Roth (geniculate goat grass) accessions from collection of the North and Central Tunisian (Cap-Bon, Mogodses, Kroumiry and the Dorsal areas) were used to assess its genetic diversity by morphological and Random Amplified Polymorphic DNA (RAPD) data and to evaluate relationship between morphological and RAPD markers. Nineteen morphological traits were analyzed on all accessions using Principal Analysis Component (PCA) and clusters were constrained based on median joining distances. Nineteen arbitrary universal primers were used for the amplification of random DNA sequences and generated 212 bands ranging from 0.5 to 3 kb with 71.27% polymorphism across the 13 accessions. Both RAPD and morphological data classified accessions in two main groups. Both methods were used to compare how morphological traits and RAPD molecular markers described accessions relationship and showed a high degree of variation among analyzed accessions, indicating an important source of genetic diversity that can be used in future breeding programs. Morphological PCA traits and cluster indicated climatic stage. In fact, they grouped Ae. geniculata accessions according to genetic criteria such as earliness and high kernel yield. Comparison of morphological and molecular data using the Mantel test indicated a non significant correlation (r = -0.268). Nevertheless, RAPD and selected morphological characters appear as useful and complementary techniques for evaluation of genetic diversity in Ae. geniculata.

Haplorchis taichui, Witenberg, 1930: Development of a HAT-RAPD marker for the detection of minute intestinal fluke infection

Specific primers to determine the presence of an intestinal fluke, Haplorchis taichui, were investigated using the high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR, and 18 arbitrary primers (Operon Technologies), to generate different polymorphic DNA profiles. Thirteen kinds of parasites were used to compare fingerprints. A 256 bp HAT-RAPD marker, generated from the OPP-11 primer, was found to be H. taichui-specific, and this marker was cloned, transformed, and sequenced. From the sequence data, a pair of primers were designed with Genetyx–MAC ver.11 and indicated as: Hap-t F 5′-GGC CAA CGC AAT CGT CAT CC-3′ and Hap-t R 5′-GCG TCG GGT TTC AGA CAT GG-3′. These specific primers were tested for efficacy and specificity by amplifying them with all 13 parasites DNAs in PCR reaction. A 256 bp amplicon was generated, which was shown to have a positive result, only for H. taichui DNA. It revealed no cross-reaction with any of the other tested parasite species. The minimum DNA template, needed for detection by PCR, was 0.1 picogram (pg). The successful development of H. taichui-specific primers is expected to be beneficial for epidemiological studies and for prevention and control of these parasitic infections.

Assessment of genomic instability in blood and tissues from prostate cancer patients by random amplified polymorphic DNA analysis

All genetic alterations in the genome that predispose to the development of cancer are utmost importance in gaining a complete understanding of the exact molecular events involved in the development of tumorigenesis. In this study, the random amplified polymorphic DNA (RAPD) technique was used for assessing genomic instability in prostate cancer patients. DNAs were obtained from blood and tumor tissues of five prostate cancer patients, then amplified individually by RAPD with five different 10-mer arbitrary primers. The ability to detect genomic instability in five cancer tissues by each primer ranged from 20 to 100 percent. Change in the genome that revealed by RAPD technique included deletion or insertion and allelic losses or gains. The most important finding is that deletions were also observed in blood DNAs, while the corresponding fragments were present in the tumor DNA. Our results display an insertion of a 1093 bp fragment in 2 of 5 tumor samples using primer OPA-03. A deletion of the same 1093 bp fragment was observed in a blood sample using the primer OPA-03. These results confirm other reports that RAPD technique is useful for assessing genomic damage in cancer

우리나라에서 분리한 Xanthomonas arboricola pv. pruni의 유전적 다양성

Xanthomonas arboricola pv. pruni, the causal agent of bacterial shot holes in stone fruits, was known to have a low population diversity. To investigate the genetic characteristics of X. arboricola pv. pruni isolated in Korea, three strains which have identical 16S rDNA sequences - including type strain (LMG852), Japanese isolate (MAFF301420) and Korean isolate (XWD1) - were analysed based on the nucleotide sequences of three DNA regions and RAPD pattern. No sequence diversity among the three strains was found within the ITS, glnA and atpD gene sequences. However, five of 756 nucleotides of the atpD gene determined (accession number FJ429319) were different from those of the French strain available from the Genbank database. RAPD analyses performed with 40 different arbitrary primers revealed that two strains isolated from Korea and Japan showed similarity in their band patterns distinguished by type strain. These results suggest that Korean and Japanese strains are very close and belong to a population with a low genetic diversity, and might have a different origin from strains found in West Europe.

Cobalt Chloride-Induced Downregulation of Puromycin-Sensitive Aminopeptidase Suppresses the Migration and Invasion of PC-3 Cells

Cobalt chloride (CoCl(2)) treatment of cells in vitro has been shown to induce cellular changes that are similar to those seen following hypoxia. To identify genes that are differentially expressed in response to treatment with CoCl(2), we compared the mRNA expression profiles of PC-3 cells that were treated with CoCl(2) with those of untreated PC-3 cells, using specific arbitrary primers and two anchored oligo(dT) primers provided in the ACP-based GeneFishing kits. The results of this study demonstrated that the puromycin-sensitive aminopeptidase (PSA) gene was downregulated in PC-3 cells that were treated with CoCl(2). This downregulation of PSA expression, in turn, suppressed the proliferation, migration, and invasion of PC-3 cells, as well as the secretion and expression of matrix metalloproteinase-9 (MMP-9).

Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis

Random amplified polymorphic DNA (RAPD) and high performance liquid chromatography (HPLC) were applied to investigate genetic and chemical variations of 2 natural C. sinensis, 16 fungal strains isolated from C. sinensis, and 2 fungal strains of C. militaris. Five of the 68 arbitrary decamer primers were available for discrimination of the investigated samples. As a result, 20 investigated samples were divided into three main clusters according to the genetic distance, and some fungal strains isolated from natural C. sinensis were obviously different. But according to the contents of nucleosides, including uracil, uridine, hypoxanthine, inosine, guanosine, adenosine, adenine, and cordycepin, natural and cultured Cordyceps were in two individual sub-groups, which suggested that chemical characteristics among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one.

Genetic relatedness among Tunisian plum cultivars by random amplified polymorphic DNA analysis and evaluation of phenotypic characters

Analysis of differentiation among local and introduced plum cultivars using morphological, pomological and RAPD markers revealed considerable genetic diversity. The phenotypic analysis shows that pomological and tree growth traits were permitted to evaluate morphological variability of plum cultivars. Ten arbitrary primers used to assay 27 cultivars revealed 143 RAPD markers. The percentage of polymorphic bands (97.28) and the resolving power (Rp) (82) showed the efficiency of used primers. The revealed RAPD markers permit to distinguish all considered cultivars and resolve homonymy problem. Cluster and principal component analysis were performed to assess patterns of diversity among cultivars and showed unstructured genetic diversity. In fact, the cultivars distribution occurs undependably of their geographic origin. The overall distribution pattern of molecular variation suggests that about 94.79% of the total variance was accounted within group component of variance. The remaining 5.21% of the variation was hierarchically structured between components due to differentiation among groups. A significant correlation ( r = 0.30; P = 0.00) was verified between morphological descriptors distance and distance generated by molecular data. These results proved the importance of both morphological and RAPD markers to elucidate in part denomination problems and elucidate relationships among cultivars. Wide phenotypic and molecular diversity found in local plum germplasm indicates a considerable potential for improving this crop. The combined analysis of phenotypic and molecular markers was a valuable tool for assessing the genetic diversity in plums.

Identification of protoplast fusion strain Fhhh by randomly amplified polymorphic DNA

A functional strain Fhhh was constructed through protoplast fusion of three parental strains (Phanerochaete chrysosporium, Saccharomyces cerevisiae and native bacterium YZ) to improve the degradation efficiency of purified terephthalic acid wastewater. Randomly amplified polymorphic DNA (RAPD) and scanning electron microscope (SEM) analysis were applied to identify and confirm the fusant Fhhh through phenotypic and genetic relationship. The result of SEM analysis demonstrated that the cell shape of fusant Fhhh differed from all three parental strains. RAPD analysis of 40 arbitrary primers generated a total of 1,135 bands. The genetic similarity indices between Fhhh and parental strains Phanerochaete chrysosporium (PC), Saccharomyces cerevisiae (SC) and native bacterium (YZ) were 34.01%, 33.16%, and 35.97%, respectively. The targeted-gene PCR results showed that Fhhh inherited the DNA fragments of mnp and lip genes from parental strain PC and FLO1 gene fragment from parental strain SC. Our results suggested protoplast fusion technique may be considered as a promising technique in environmental pollution control.

Arbitrary Multi-gene Reference for Normalization of Real-Time PCR Gene Expression Data

Analysis of gene expression using real-time reverse transcription polymerase chain reaction (RT-PCR) requires reference genes to normalize expression values between samples. We have developed a novel reference for real-time RT-PCR using an arbitrary primer to amplify a random set of genes. The arbitrary primer amplifies over 30 genes, whose cumulative expression as measured by real-time RT-PCR closely follows that of UBQ 11, an Arabidopsis thaliana gene that is used as a reference on microarrays. The expression of arbitrary genes is also compared with potato (Solanum tuberosum spp. tubersosum) housekeeping genes and was shown to be stable during Phytophthora infestans infection.

Volatile and molecular characterization of two Portuguese endemic species: Angelica lignescens and Melanoselinum decipiens

Chemical and genetic differences of 11 individuals of Angelica lignescens and 3 individuals of Melanoselinum decipiens were studied to determine whether volatile components could be used as taxonomical markers and to examine the correlation between molecular and chemical markers. The volatiles were isolated from each individual by distillation–extraction and analyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). The same plants were analyzed by Inter Simple Sequence Repeat (ISSR) using 14 arbitrary primers. The main component of volatile fraction isolated from vegetative aerial parts of A. lignescens was limonene (57–86%) while in M. decipiens the main components detected were β-pinene (38–47%) and sabinene (1–33%). Cluster analyses based both on the chemical composition of volatile fraction and on molecular markers grouped the 14 accessions in two main groups, corresponding to each of the two species under study. Considering the species together, a moderate Pearson's correlation of r = 0.61 was obtained between the two analyses.

Antifungal Action of New Trichoderma Spp. Romanian Isolates on Different Plant Pathogens

ABSTRACTThe genus Trichoderma comprises various fungal strains that can act as biological control agents against a large diversity of plant pathogens. A number of commercial products are available but the diversity of plant pathogens, and their increased resistance to the current control products (chemical or biological products) determined the search of new strains, potentially useful for biological control. New strains of Trichoderma spp. were isolated from Romanian soils and their antagonistic activity against Phytium spp and Rhizoctonia spp. was examined. The in vitro biocontrol activity of Trichoderma spp., as well as of other antagonistic fungi (Penicillium chrysogenum Gliocladium roseum and Eppicoccum purpurescens) on the plant pathogens was increased in the presence of FeCl3. The molecular analysis realized by ITS-RFLP and PCR with specific primers allow the confirmation of previous taxonomic determination of T.harzianum and T.viride. However, an increased intraspecific molecular polymorphism was observed using several arbitrary primers (RAPD analysis). The interactions between fungal strains (plant pathogens and antagonistic strains) were also examined, in order to determine the mechanism of action of the antifungal strains. It was observed that all of the Trichoderma strains were able to produce large amount of hydrolytic enzymes (chitinase, cellulases and proteases) and to act as mycoparasites for pathogens. The involvement of fungal lectins in the interactions was also examined. Fungal extracts obtained from the best antagonists were tested for the induction of plant (soybean) resistance against pathogens. Higher levels of plant enzymes PAL, POX and chitinase were observed, and correlated with an increased resistance to artificial infection. The results obtained could be used in further experiments to establish new approaches of “rplant immunization” with microbial products.

Characterization of multiple isolates ofAspergillus fumigatusfrom patients: genotype, mating type and invasiveness

The possible co-existence of different genotypes of Aspergillus fumigatus in the same case was studied in five patients colonized or infected by this opportunistic mould. A total of 22 isolates were typed by random amplified polymorphic DNA (RAPD) and microsatellite analysis. Differences in the mating type and invasiveness of the isolates were also considered. The combination of four arbitrary primers used in RAPD typing differentiated all the isolates. In microsatellite analysis, at least two different genotypes were identified in four of the five patients. The 22 isolates showed elastase activity after 10 days of incubation at 37 degrees C in an elastin-containing medium. The presence of strains of compatible mating type was observed in one of the colonized patients and one of the individuals with invasive aspergillosis. Some isolates that belonged to the same genotype in microsatellite analysis were of a different mating type. Taken together, our results suggest that multiple isolates of A. fumigatus obtained from colonized or infected patients may differ not only in their genotypes, but also in their invasiveness and mating types. Furthermore, mating type determination may be of great assistance in differentiating some isolates, as two isolates of different mating type cannot be genotypically identical.

The effect of the mecA gene and its mutant form on the response of S. aureus to the most common antibiotics

Staphylococcus aureus isolates were collected from different hospitals in the Alexandria governorate in Egypt. The 100 isolates were subjected to fingerprinting based on their resistance/susceptibility to antibiotics which are commonly used in Egypt and all over the world. About 54% of the isolates showed multidrug resistance. The Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) was used to study the relationship between the examined isolates. Four arbitrary primers were used in this study and the results revealed that approximately 54 different banding patterns were obtained, dividing the isolates into five different groups. The PCR was performed for the detection of the mecA gene in all the isolates (susceptible and resistant). The mecA gene was detected in all the resistant isolates and in only one susceptible isolate (AS38). The mecA gene from the AS38 strain (Methicillin-Susceptible S. aureus or MSSA) and another one from a resistant strain (Methicillin-Resistant S. aureus or MRSA) were subjected to DNA sequencing. The sequence alignment showed that a point mutation was carried out in the nucleotide 215 (T substituted by A), which leads to the conversion of the amino acid asparagine into isoleucine. Phylogenetic analysis was carried out to study the evolution of such genes between different S. aureus strains. Also, the data revealed that there is a contentious modification for such genes.

Estudio de la estabilidad genética mediante el uso de marcadores RAPD en Ipomoea: una alternativa para la conservación de la diversidad biológica mediante técnicas moleculares

This work was conducted in the Centre of Studies of Plant Biotechnology of the Faculty of Agricultural Sciences of the University of Granma. We used tuberous roots of the sweet potato clone INIVIT B 93-1 and vitroplants derived from somatic embryos coming from calli obtained from leaf tissue grown in MS medium. Tissue samples from both types of plant material were randomly obtained and their genetic stability analyzed by molecular analysis of the ADN (RAPD), using ten arbitrary primers (OPF y OPA) and evaluating the bands in terms of presence and absence. Such a molecular monitoring is required for any study of genetic diversity conservation to be conducted in different localities, regions and countries. As a result, seven primers produced an acceptable amplification, indicated by the intensity of the bands found in the donor and the somatic embryo-derived vitroplants. The high monomorphism appearing between both materials gives more accurate information for the establishment of strategies and procedures for the conservation and diffusion of the biodiversity.

Clonal and population RAPD variation of cercariae obtained from Bucephalus polymorphus sporocysts (Trematoda: Bucephalidae)

Three arbitrary primers produced 114 RAPD markers for 37 cercariae from nine Bucephalus polymorphus sporocysts obtained from three Dreissena polymorpha mollusks, which were collected in two water reservoirs of the Volga basin. Analysis of the RAPD patterns established a unique genotype for each cercaria. The topology of an UPGMA dendrogram did not reliably differentiate the cercaria according to the corresponding sporocysts. However, three groups of genotypes were isolated and corresponded to the host mollusks, indicating that each cercaria clone had a different genotype set. A within-sporocyst variation made the greatest contribution (53.0%) to the total RAPD diversity, while the contributions of within-host and between-host variations to the total diversity were equal (23.5%). Cercariae isolated from two mollusks of the Rybinsk Water Reservoir were more similar to each other than to cercariae from the geographically distant Gor'kovskoe Water Reservoir. Possible causes and distribution specifics of the observed genetic diversity of B. polymorphus are discussed.

GENETIC VARIABILITY AND POPULATION STRUCTURE OF GROUPER (Epinephelus suillus) FROM MAKASSAR STRAIT AND BONE BAY, SOUTH SULAWESI, INDONESIA

Random Amplified Polymorphic DNA (RAPD) was employed to determine the genetic variability and population structure of grouper (Epinephelus suillus) from Makassar Strait and Bone Bay, South Sulawesi, Indonesia. Genomic DNA was isolated from preserved muscle tissue using Phenol-Chloroform technique. Among 24-screened arbitrary primers, ten primers (OPA-02, OPA-06, OPA-08, OPA-10, OPA-15, OPA-16, OPA-17, OPA-18, OPA-19 and CA-05) were selected to generate RAPD fingerprinting of grouper populations. The ten primers generated a total of 212 fragments (loci) and 120 polymorphic fragments in their size ranging from 250 to 2,500bp. The high polymorphism (60%) was obtained from Makassar population followed by Bone (59%) and Pare-Pare populations (50%). Similarity index of individuals was 0.86±0.07 for Pare-Pare, 0.80±0.11 for Makassar and 0.82±0.07 for Bone population. Fifteen fragments from ten primes were identified as species-specific markers of E. suillus. The UPGMA cluster analysis showed that the dendrogram seemed to be clustered according to their geographical location, where Pare-Pare population was genetically closer to Makassar population (D=0.20) than to Bone population (D=0.24).

Evaluation of genotypic diversity of Streptococcus mutans using distinct arbitrary primers

Streptococcus mutans has been considered one of the main etiological agents of dental caries and the genotypic diversity rather than its salivary counts may be considered as a virulence factor of this bacterium. For genotyping with polymerase chain reaction (PCR) with arbitrary primers, several primers have been used in order to improve complexity and specificity of amplicon patterns. Thus, the aim of this study was to evaluate the degree of agreement of genotypic identification among AP-PCR reactions performed with 5 distinct arbitrary primers of S. mutans isolated from saliva. Stimulated saliva was collected from 11 adult volunteers for isolation of S. mutans, and a total of 88 isolates were genotyped with arbitrary primers OPA 02, 03, 05, 13 and 18. Fourteen distinct genotypes were identified in the saliva samples. Most volunteers (9 out of 11) presented only one genotype. The results of the present study suggest that primers OPA 02, 03, 05 and 13 were suitable for genotypic identification of S. mutans isolates of saliva from adult volunteers.

Genetic diversity assessment in Portugal accessions of Olea europaea by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.

Conserved Region Amplification Polymorphism (CoRAP), a Novel Marker Technique for Plant Genotyping in Salvia miltiorrhiza

Salvia miltiorrhiza (SM), a widely popular Chinese herb, is grown in various regions in China. Identifying SMs grown in different provinces of China is difficult, and therefore genotyping these collections would be highly valuable. Based on the techniques of sequence-related amplified polymorphism and target region amplified polymorphism, a novel PCR-based molecular marker technique called conserved region amplification polymorphism (CoRAP) is reported in this study to genotype SMs. The CoRAP technique is based on the use of two primers: fixed and arbitrary primers. The former is derived from target EST sequences deposited in Genbank; while, the core sequence (CACGC) of the latter is a conserved region found in most introns. In the present study, we utilized CoRAP to genotype SMs from different geographical origins. PCR amplification is performed for 30 cycles at an annealing temperature of 52°C. Each PCR reaction has generated as many as 30–50 fragments of 50 to 1,000 bp in size. The successful DNA genotyping of SMs by CoRAP was achieved. This new genotyping method is rapid, efficient, and reproducible.