Amplified fragment length polymorphisms reveal genetic differentiation among strains of Xanthomonas albilineans

Xanthomonas albilineans, the causative agent of leaf scald disease (LSD), colonizes the vascular system of sugarcane (Saccharum spp. hybrids). In this study X. albilineans strains from 28 countries were differentiated by using two methods of amplified fragment length polymorphism (AFLP). In the manual procedure, AFLP reactions were performed on 57 X. albilineans strains and after selective amplification using radiolabelled primers, the resulting products were separated using polyacrylamide gel electrophoresis. The autoradiographs were analyzed using GelCompar version 4.1 software (Applied Maths) to construct dendograms from similarity matrices. Fluorescent AFLP (FAFLP) was also employed on 52 X. albilineans strains using three fluorescently labelled primer combinations (automated AFLP). The FAFLP data was converted to a binary format using the Genemapper Software 3.7 (Applied Biosystems). Thereafter, dendograms were generated using the NTSYSpc. Software (USA). Distinct AFLP profiles were produced for the majority of the strains and were found to be useful in differentiating X. albilineans strains from various geographical locations. Fingerprints unique to each strain were reproducibly obtained and may be used to create a database for use in the identification of the various X. albilineans strains. It can be also concluded from the results obtained that the FAFLP has considerable technical advantages compared with the manual AFLP and also that the FAFLP is more sensitive than AFLP using radiolabelled primers in differentiating X. albilineans.     

Genetic relationship between Polish and Chinese strains of the mushroom Agaricus bisporus (Lange) Sing., determined by the RAPD method

The genetic relationship between twenty-six strains of Agaricus bisporus were analysed by the RAPD (random amplified polymorphic DNA) method. DNA amplification was performed with the use of twelve arbitrary 10-mer primers. Four primers, which gave polymorphic band patterns were chosen for RAPD analysis. In total, they gave 24 distinguishable bands, of which nine were polymorphic. The conducted research showed that there is a great genetic similarity among the examined strains. Low polymorphism of the strains may be a proof of a limited genetic pool used in the cultivation of those strains.     

Random sequence oligonucleotide primers detect polymorphic DNA products which segregate in inbred strains of mice

The random amplification of polymorphic DNA (RAPD) using primers of arbitrary nucleotide sequence has been extremely valuable in identifying heritable markers in a variety of systems. The present studies examined whether the RAPD technique can identify large numbers of polymorphisms that can be used to construct genetic maps in inbred strains of mice. By screening the inbred mouse strains C57BL/6J and DBA/2J with 481 random 10-mer oligonucleotide primers, we identified 95 polymorphisms and mapped 76 of these by use of the BXD series of recombinant inbred (RI) strains. The results clearly demonstrate that the RAPD technique allows for the identification of large numbers of DNA-based polymorphisms that distinguish these two inbred strains of mice,and that such markers can readily be used to construct molecular genetic linkage maps.     

Characterization of Erwinia amylovora strains using random amplified polymorphic DNA fragments (RAPDs)

The genetic diversity among 16 strains of Erwinia amylovora, chosen to represent different host plant origins and geographical regions, was investigated by RAPD analysis. One strain of Erwinia herbicola and one of Agrobacterium vitis were used as outgroups. Ninety-eight different RAPD fragments were produced by polymerase chain reaction amplification with six different 10-mer primers. RAPD banding profiles were found that enabled the Erw. amylovora strains to be distinguished from one another. Cluster analysis based on the number of RAPD fragments shared between strains showed that strains of Erw. amylovora isolated from subfamily Pomoideae formed a single group, whereas two strains from Rubus (subfamily Rosoideae) formed a second group. Two strains isolated from Asian pear on Hokkaido, Japan, formed a third group. Sets of RAPD fragments were identified that enabled each of the two host-range groups and one geographical region (Hokkaido) of Erw. amylovora strains to be unambiguously distinguished from one another and from the outgroups. This study shows that strains of Erw. amylovora exhibit genetic diversity detectable by RAPD analysis, and that molecular and statistical analysis of RAPD fragments can be used both to distinguish between strains and to determine relatedness between them.     

Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

The use of RAPD genomic fingerprinting to study relatedness in strains of Acidithiobacillus ferrooxidans

Twelve strains of Acidithiobacillus ferrooxidans were recovered from acid mine drainage (AMD) sites from three different geographical locations: Copper Cliff, Ontario, Canada; Mineral City, OH, USA; and Cornwall, England. The spread-plate technique and various culture media were used to isolate and purify all strains. DNA was extracted from each purified culture and amplified using PCR and twenty, 10-mer primers. Amplification products were separated by gel electrophoresis and photographed under UV light. The RAPD (Randomly Amplified Polymorphic DNA) profiles were compared on the basis of the presence or absence of each DNA band and a data matrix was constructed. Strain diversity was calculated using the Jaccard's coefficient and UPGMA (Unweighted Pair-Group Arithmetic Average Clustering) cluster analysis. The variations in the banding patterns indicated genomic variability among the twelve A. ferrooxidans strains tested. The primers used in this study grouped the twelve strains into five major groups. Similarities between the strains ranged from 5.49% to 85.14%. These results show that the strains have a high degree of genomic diversity and that the RAPD procedure is a powerful technique to assess strain variability in this bacterium.     

48个烟草品种遗传多样性的RAPD分析

利用RAPD分子标记技术对48个烟草品种进行遗传多样性研究。从200个10bp的随机引物用RAPD方法筛选获得28个多态性引物,然后对48份烟草种质资源的基因组DNA进行扩增,共获得184条DNA扩增片断带。其中多态性带86条,平均多态检出率为46.7%。48份材料的遗传距离为0~7.81,采用UPGMA法聚类分析,可将其分为两大类群,即黄花烟草群与普通烟草群,后者又可分为4组。     

RAPD analysis of Campylobacter isolates: DNA fingerprinting without the need to purify DNA

A method was developed to obtain reproducible DNA fingerprints from Campylobacter by PCR-based amplification, without the need to isolate total DNA. Randomly amplified polymorphic DNA (RAPD) profiles were generated with three randomly designed 10-mers, using each separately as an amplification primer. A range of C. jejuni serotypes could be typed by RAPD analysis. Depending on the primer, the analysis of RAPD profiles resulted in different levels of discrimination between the strains. Clear correlations were observed between results of RAPD analysis and serotyping. Two of the primers tested generated RAPD profiles which allowed discrimination of strains within given Penner and Lior serotypes.     

Detection of genetic polymorphism by RAPD-PCR among isolates of Bacillus thuringiensis.

Sixteen isolates of Bacillus thuringiensis recovered from different Jordanian habitats were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the molecular level. Total genomic DNA from each isolate and three reference strains were amplified using 10-mer primers. Electrophoretic analysis of the amplification products revealed the incidence of polymorphism among the isolates. Pair-wise comparisons of polymorphic products were used to construct a dendrogram applying the cluster analysis. Fifteen of the isolates were all in one major cluster which was divided into six small groups. Such analysis showed some regional variation among the isolates, but did not indicate a clearly defined habitat locational pattern of the DNA polymorphism.     

Random amplification of polymorphic DNA (RAPD) of Salmonella: strain differentiation and characterization of amplified sequences

Random amplification of polymorphic DNA (RAPD) was evaluated for its ability to differentiate Salmonella strains from various sources. Under defined conditions RAPD using a 10-mer primer (1254) produced a series of amplification products able to reproducibly distinguish strains representing 20 different serotypes of Salmonella. Primer 1254 also proved capable of discrimination between some but not all isolates of Salm. ser. Enteritidis and Salm. ser. Typhimurium, phage typing proving to be most discriminatory for the latter serotype. Cloning of fragments into a vector allowed sequencing and database searching for identification of fragments and an indication of criteria for primer template interaction in RAPD. Southern blotting using a digoxigenin-labelled probe allowed identification of related bands between RAPD profiles. These observations demonstrate the potential of rapid molecular typing by RAPD for the genomic typing of Salmonella strains.     

Use of RAPD-PCR fingerprinting to detect genetic diversity of soil Streptomyces isolates

Random amplified polymorphic DNA (RAPD) was used for identification and assessment of genetic diversity between isolates of Streptomyces from soil. Genomic DNA from 18 Streptomyces isolates and 2 reference strains were amplified using four different 10-mer primers. Different DNA fingerprinting patterns were obtained for all the isolates. Electrophoretic and cluster analysis of the amplification products revealed incidence of polymorphism among the isolates and none of them was identical to the reference strains although there were some common amplification bands. Two highly divergent groups were determined among the isolates. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.     

Development of DNA markers for identifying chrysanthemum cultivars generated by ion-beam irradiation

Four chrysanthemum cultivars were generated through (carbon) ion-beam irradiation of the original ‘Jimba’ (Chrysanthemum morifolium Ramat.). The new cultivars had acquired a number of superior cultivation traits, while remaining identical to the commercially available ‘Jimba’ in appearance. In this study, polymerase chain reaction (PCR) assays were used to detect the mutated region of each strain, thereby allowing clear identification at the molecular level. PCR assays were performed with 446 primer sets, including random amplified polymorphic DNA (RAPD) primer sets (10-mer RAPD), arbitrarily primed (AP)-PCR primers based on retrotransposon-like sequences and modified RAPD primers (15-mer RAPD). 15-mer RAPD primers generated a 1.49-fold increased band number at high annealing temperatures compared with the original 10-mer RAPD primers and could thus be effective for detection of polymorphic patterns. Our results provide information on the mutated regions of these ion-beam-irradiated chrysanthemum cultivars. Thus, specific DNA markers could be used to improve identification of new cultivars of chrysanthemum as well as other clonal cultivars of horticultural and agricultural crops.     

Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa

Seventeen virulent bacteriophages specific to Pseudomonas aeruginosa strains were isolated by screening various environmental samples. These isolated bacteriophages were grouped based on results obtained from restriction fragment analysis of phage genomes, random amplification of polymorphic DNA (RAPD) typing, morphology observations under transmission electron microscope, and host range analysis. All 17 bacteriophages are double-stranded DNA viruses and can be divided into 5 groups based on DNA restriction profiles. A set of 10-mer primers was used in RAPD typing of phages, and similar conclusions were obtained as for restriction fragment analysis. One phage was randomly selected from each of the 5 groups for morphology observations. Four of them had an icosahedral head with a long contractile tail, belonging to the Myoviridae family, and one phage had an icosahedral head with a short tail, thereby belonging to the Podoviridae family. Host range experiments were conducted on 7 laboratory strains and 12 clinical strains of P.?aeruginosa. The results showed that 13 phages had the same infection profile, killing 8 out of 19 tested P.?aeruginosa strains, and the remaining 4 phages had different and unique infection profiles. This study highlights the diversity of bacteriophages specific to P.?aeruginosa in the environment.     

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.     

Development of a random amplification of polymorphic DNA typing method for Listeria monocytogenes.

The 10-mer primer OPM-01 (5'-GTT GGT GGC T-3') was used to generate random amplification of polymorphic DNA (RAPD) profiles by polymerase chain reaction for 91 strains of Listeria monocytogenes from raw milk, food, and veterinary, medical, and food-environmental sources. The profiles obtained contained 1 to 10 bands within the molecular size range of 0.5 to 5.0 kbp. Reproducibility was enhanced by annealing at low stringency and introducing a 1-min ramp time between annealing and extension temperatures. Thirty-three RAPD profiles were observed, with specific profiles being observed for strains from each source. RAPD profiles allowed discrimination within serogroups, although five RAPD profiles which were not confined to one serotype were found. Within food strains, one RAPD profile was more common than others, suggesting this to be a common type among strains from this source.     

Genetic variation in Drechslera teres populations as indicated by RAPD markers

Random amplified polymorphic DNA (RAPD) was used to assess genetic variation among 48 isolates of Drechslera teres originating from different sites in Finland. RAPD profiles were generated with five arbitrary 10-mer primers and revealed polymorphisms suitable for screening differentiation in this fungal population. Using UPGMA clustering analysis, a similarity coefficient of approximately 63% was observed between all D. teres isolates studied. The variation was, however, distributed on a small scale as different genotypes were found from the same plant. The isolates could not be grouped according to geographic origin, aggressiveness, growth rate or morphological features, indicating that the primers used in this study were neutral markers for these characters. The primers were, however, able to differentiate between isolates of Helminthosporium species (D. teres, Drechslera graminea and Bipolaris sorokiniana).     

Simultaneous molecular subtyping and shiga toxin gene detection in Escherichia coli using multiplex polymerase chain reaction

A robust random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) protocol was developed for the combined epidemiological typing and shiga toxin detection of clinical shiga toxin-producing O157 and non-O157 Escherichia coli isolates. Using shiga toxin gene-specific primers, combined with two short 10-mer primers, in a multiplex shiga toxin/RAPD-PCR the fingerprints generated allowed differentiation between epidemiologically unrelated strains and allowed identification of a band amplified from the shiga toxin gene(s). Hybridization with a digoxigenin-labelled probe specific for stx1 and stx2 confirmed its identity. The combination of primers in this way allows valuable additional information to be gained from discriminatory RAPD profiles, with further benefits of time and cost savings over tests performed individually.     

Genetic diversity assessment in Portugal accessions of <Emphasis Type="Italic">Olea europaea</Emphasis> 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.     

Characterization of Bacillus licheniformis with the RAPD technique (randomly amplified polymorphic DNA)

RAPD technique (randomly amplified polymorphic DNA) was used for epidemiological subtyping of Bacillus licheniformis and other Bacillus spp. Within 46 isolates of B. licheniformis , up to 10 strain types could be determined when two 10-mer primers were used. RAPD patterns, which were found in eight further strains of Bacillus spp., clearly differed from those of B. licheniformis. Thus RAPD technique proved to be a promising tool for characterization of Bacillus spp.     

Arbitrary Primer Based RAPD — A Useful Genetic Marker for Species Identification in Morels

Internal transcribed spacer (ITS) regions of 5.8S ribosomal DNA gene of 46 monospore cultures representing different species of family Morchellaceae were sequenced. Eight putative species namely Morchella esculenta, M. crassipes, M. angusticeps, M. conice, Mitrophora semilibera, Morchella spongiola, M. vulgaris and Verpa conica were designated to the sequenced cultures analyzed based on similarity with the best aligned sequence of the BLAST search. We recorded interspecific polymorphism in ITS regions of the 5.8S r-DNA gene amongst putative species of morels. However no intraspecific ITS polymorphism could be visualized amongst monospores from single and or different ascocarps collected from various geographical regions. PCR RAPDamplified profiles of different monospores were identical at intraspecific levels and could not be distinguished by eight arbitrary primers tested. RAPD profiles exhibited significant interspecific polymorphism distinguishing all the eight putative species from each other by primer OPP-6 (5’- GTG GGT TGA C- 3’) and a custom primer (5’-CGC ACC GCA G-3’). RAPDprofiles generated using aforesaid arbitrary primers can serve as useful genetic markers for species identification and to improve morel systematics. Present study is the first report on exploitation of random primers in species differentiation in morels.