Genes for ribosomal proteins S3, L16, L5 and S14 are clustered in the mitochondrial genome of Brassica napus L.

A PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), was used for assessing genomic variability among a wide range of culture collection strains of black Aspergilli and related species. The performance of this technique is compared with that of the two other genetic techniques most commonly used, namely restriction fragment length polymorphisms on rDNA and isozyme analysis. The eight main groups as assigned by RFLP were also distinguished by RAPD patterns. On the basis of 122 polymorphic RAPD products using six random primers, the 17 collection strains examined could be subdivided into 15 distinct sub-groups. We suggest that the RAPD method is a quick and reliable tool for establishing the amount of genetic variability in closely related species. Our study indicates that the complex group of black Aspergilli is characterized by a high degree of genetic differentiation. This is also apparent from the considerable karyotype variation present in the group.     

Genetic variation in <Emphasis Type="Italic">Opisthorchis viverrini</Emphasis> (Trematoda: Opisthorchiidae) from northeast Thailand and Laos PDR based on random amplified polymorphic DNA analyses

Genetic variation in Opisthorchis viverrini adults originating from different locations in northeast Thailand and Laos, People’s Democratic Republic (PDR), was examined using random amplified polymorphic DNA (RAPD) analyses. In an initial analysis, the genomic DNA of one fluke from each of ten localities was amplified using 15 random primers (10-mers); however, genetic variation among O. viverrini specimens was detected reliably for only four primers. A more detailed RAPD analysis using these four primers was conducted on ten individuals from nine localities. Considerable genetic variation was detected among O. viverrini from different geographical areas and among some individuals from the same collecting locality. Comparison of the RAPD profiles revealed that O. viverrini adults from Laos PDR were genetically distinct from those from northeast Thailand. The taxonomic significance of this finding needs to be explored in more detail. The RAPD markers established in the present study provide opportunities to examine the biology and epidemiology of O. viverrini and fish-borne trematodes within the region. Additionally, application of these markers in such studies could have important implications in relation to the prevalence of cholangiocarcinoma in different regions of Asia.     

Genetic heterogeneity in the Trypanosoma brucei gambiense genome analysed by random amplification of polymorphic DNA

 The random amplification of polymorphic DNA (RAPD) technique has the potential to produce large amounts of characterisation data very quickly and simply, using far less DNA than conventional restriction-fragment-length polymorphism (RFLP) analysis. In the present study we assessed genetic heterogeneity among 34 Trypanosoma brucei gambiense isolates from various endemic areas in Africa by the RAPD technique using 8 arbitrary primers and compared the results with those obtained previously from RFLP analysis of polymorphisms in 5 variant surface glycoprotein (VSG) genes. The isolates were compared both among themselves and with 3 T. b. non-gambiense isolates. Most of the primers produced RAPD profiles specific for T. b. gambiense, with 4 primers showing marked polymorphisms between T. b. gambiense and non-gambiense stocks. These primers also showed minor variations between the T. b. gambiense stocks, and 2 revealed differences between Cameroonian stocks. These results were comparable with those produced by RFLP analysis, where certain polymorphisms are characteristic of T. b. gambiense. Numerical analysis showed a high correlation between the RAPD and RFLP data, with genetic variation being detected at a finer level by RAPD analysis. We conclude that RAPD analysis provides a simple and accurate method for the characterisation of T. b. gambiense. Received: 15 February 1996 / Accepted: 15 March 1996     

Detection of RAPD Markers Correlated with Chloroquine Resistance in Plasmodium falciparum

We described the use of the random amplified polymorphic DNA (RAPD) technique on Plasmodium falciparum DNA to detect genetic markers for chloroquine-resistant strains. Fourteen RAPD primers were tested, three of which generated banding patterns correlated with chloroquine resistance. To measure this correlation, the RAPD profiles were analyzed using the Nei and Li similarity coefficient. Detection of distinctive RAPD bands allowed us to synthesize specific PCR primers to be used on whole-blood samples. Two primer sets were synthesized and tested on sensitive and resistant strains for their ability to amplify the DNA fragment corresponding to the RAPD marker. These results suggest that RAPD and PCR techniques can be used as powerful tools for the detection of genetic markers associated with drug resistance.     

A total DNA characterization in Proteocephalus exiguus and P. percae (Cestoda: Proteocephalidae): random amplified polymorphic DNA and hybridization techniques

 Two morphologically similar fish tapeworms, Proteocephalus exiguus and P. percae, were differentiated by the random amplified polymorphic DNA (RAPD) marker and Southern-blot hybridization techniques. Four geographic isolates of P. exiguus and two of P. percae were studied using eight arbitrary decamer oligonucleotides as primers. Species-specific RAPD fragments enabled reliable separation of the taxa studied. Generally, 1–3 fragments specific for P. percae and 1–4 P. exiguus-specific fragments were detected using individual primers. Intraspecific differences in P. percae depended on the primer used. The profiles generated by the primers OPA 04 and OPA 12 showed an intraspecific variation, whereas six other primers tested revealed indistinguishable banding patterns. On the other hand, intraspecific variability of P. exiguus, detected among four geographic isolates, was evident using all primers. Additionally, the utilization of two species-specific RAPD markers as effective probes was demonstrated. Distinct differences between P. exiguus and P. percae were revealed using each of the restriction enzyme/probe combinations. Received: 28 January 1996 / Accepted: 17 May 1996     

Characterization of Giardia lamblia groups A and B from North India by isoenzyme and random amplified polymorphic DNA analysis

Giardia lamblia ( syn. G. intestinalis) infection in young adults leads to acute/chronic diarrhea in some individuals and is asymptomatic in others. Recently, G. lamblia strains have been characterized as group A (symptomatic) and group B (asymptomatic or control) by advanced isoenzyme and molecular biology studies. In the present brief pilot study, ten G. lamblia isolates obtained from five symptomatic (group A) and five asymptomatic (group B) persons were characterized by isoenzyme and random amplified polymorphic DNA (RAPD) analysis. Isoenzyme analysis demonstrated remarkable homogeneity in seven enzyme patterns, the exception, being that of phosphoglucomutase, for which two zymodemes (I and III) were observed. In contrast, RAPD analysis showed homogeneity for eight primers; exceptions were two primers, A₀₂ and B₀₅, which separated group A G. lamblia isolates into two rapdemes (A_R1 and A_R2) and group B G. lamblia isolates into four rapdemes (B_R1, B_R2, B_R3 and B_R4). Further phenetic analysis showed average genetic distances of 0.105 within group A and 0.121 within group B G. lamblia isolates according to Jaccord's distance scale, which suggests that both lineages appear to consist of a range of variants with no significant (P < 0.05) genetic diversity. The two techniques demonstrated a positive association with regard to differentiation between group A and group B G. lamblia isolates. These very preliminary results indicate that RAPD analysis could be a potentially useful substitute for isoenzyme analysis. Received: 4 December 1998 / Accepted: 21 December 1998     

用RAPD技术分析2型糖尿病的遗传多态性

为探讨2型糖尿病和非糖尿病群体间的遗传多态性差异，寻找2型糖尿病的致病相关基因。本文应用随机扩增多态性DNA技术，从140个随机引物中筛选了25个扩增满意的引物，进行PCR扩增，研究2型糖尿病患者和非糖尿病者各10例。结果：1）5个RAPD图谱表现多态性改变；2）仅K－17的RAPD普的多态性在两组间存在显著性差异（P＜0.001)。表明K－17扩增的RAPD片段与糖尿病的遗传背景有关，可能是与2型糖尿病相关的RAPD标记。     

Use of random amplified polymorphic DNA for identification of ruminant trichostrongylid nematodes

The aim of this work was to evaluate random amplified polymorphic DNA (RAPD) as a source of markers for species identification and phylogenetic analysis of ruminant trichostrongylid nematodes. As these nematodes are often polymorphic, species identification may be difficult. We tested eight species and several of their morphs:Haemonchus contortus (three vulvar morphotypes: flap, smooth, and knobbed),Teladorsagia circumcincta, Ashworthius gagarini, Spiculopteragia boehmi, Ostertagia leptospicularis (and its morphOstertagia kolchida), Cooperia oncophora (and its morphC. surnabada), Trichostrongylus colubriformis, andT. vitrinus. With five chosen 10-mer primers, genetic variations were assessed among individuals of each species or morphotype. In trichostrongylid nematodes, the identification of species is possible by means of RAPD on adult or larva DNA extracts, although the variability observed within species was very important for most species studied. The use of RAPD in phylogenetics studies is conversely questionable for this superfamily of parasitic nematodes. The interspecific distances were always larger than the intraspecific ones and did not vary much (between 0.8 and 0.9); they would not be of much use in the construction of a phylogenetic tree, at least for the species and the primers involved in this study.     

Genetic differentiation of cercariae infrapopulations of the avian schistosome <Emphasis Type="Italic">Trichobilharzia szidati</Emphasis> based on RAPD markers and mitochondrial <Emphasis Type="Italic">cox1</Emphasis> gene

Avian schistosome Trichobilharzia szidati is a member of the largest genus within the family Schistosomatidae (Trematoda). Population genetic structure of Trichobilharzia spp. schistosomes, causative agents of cercarial dermatitis in humans, has not been studied yet. The knowledge of the genetic structure of trichobilharzian populations is essential for understanding the host–parasite coevolutionary dynamics and epidemiology strategies. Here we examined genetic diversity in three geographically isolated local populations of T. szidati cercariae inhabiting Russia based on nuclear (randomly amplified polymorphic DNA, RAPD) and mt (cox1) markers. We analyzed T. szidati cercariae shed from seven naturally infected snails of Lymnaea stagnalis. Using three random primers, we demonstrated genetic variation among populations, thus posing genetic structure across geographic sites. Moreover, T. szidati cercariae have been genetically structured among hosts (infrapopulations). Molecular variance analysis was performed to test the significance of genetic differentiation within and between local populations. Of total parasitic diversity, 18.8% was partitioned between populations, whereas the higher contribution (48.9%) corresponds to the differences among individual cercariae within infrapopulations. In contrast to RAPD markers, a 1,125-bp fragment of cox1 mt gene failed to provide any significant within-species structure. The lack of geographic structuring was detected using unique haplotypes which were determined in the current work for Moscow and Western Siberian local populations as well as obtained previously for European isolates (Czech Republic and Germany). All T. szidati/Trichobilharzia ocellata haplotypes were found to be mixed across their geographical origin.     

Genetic variability in Gibberella fujikuroi and some related species of the genus Fusarium based on random amplification of polymorphic DNA (RAPD)

One of the most important rice pathogens is Fusarium moniliforme (perfect stage: Gibberella fujikuroi), the causal agent of the super-elongation (bakanae) disease. Thirty-seven strains of this species from different geographical regions were analyzed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). All GA-producing isolates showed nearly identical RAPD patterns using 51 oligonucleotide nona- and deca-mers as arbitrary primers. On the other hand, large differences between GA-nonproducing isolates were obtained. Comparison of the RAPD patterns with those of the tester strains of the six known mating populations (A,B,C,D,E,F) of G. fujikuroi showed that all producer strains belong to mating population C and all nonproducer isolates to other mating populations. Evidence for the usefulness of the RAPD technique to distinguish between mating populations was provided by sexual crossings. Consensus phylogenetic trees based on RAPDs were constructed by the Phylogenetic Analysis Using Parsimony (PAUP) system. In combination with morphological analysis, RAPD can distinguish between different species of the genus Fusarium. These investigations may find an application in the diagnosis of unknown Fusarium spp. and in distinguishing isolates of G. fujikuroi within the section Liseola.     

Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens

The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains. Received: 9 June 2000 / Accepted: 5 October 2000     

Species and strain differentiation ofEimeria spp. of the domestic fowl using DNA polymorphisms amplified by arbitrary primers

Eimeria spp. from the domestic fowl were examined for genetic relatedness by the random amplified polymorphic DNA (RAPD) assay. Nine different oligonucleotide decamers with arbitrary DNA sequences were tested as primers to amplify DNA from sixEimeria species infecting chickens. Two strains each ofE. acervulina andE. tenella were used. Depending on the species/strain-primer combination, between 1 and 12 DNA segments ranging in size from 0.16 to 4.95 kb were amplified. The two strains ofE. acervulina showed minor and major differences in their amplified DNA patterns, giving a similarity coefficient of 61%. The two strains ofE. tenella seemed to be more closely related, yielding a similarity coefficient of 98%. The differences observed between species were greater than those found between strains with every primer used, indicating that the RADP assay could be a useful tool for the study of relationships among these coccidia. The results obtained in this study also indicate the presence of unique, species-specific, amplified DNA segments that could be exploited to identifyEimeria species of the chicken.     

Toxoplasma gondii virulence markers identified by random amplified polymorphic DNA polymerase chain reaction

Genomic DNAs from 35 Toxoplasma gondii strains were amplified by random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) using 18 arbitrary 10-mer primers. At least four primers were found to generate DNA fragments that discriminate the 35 T. gondii strains into a genotype of virulent strains and a genotype of avirulent strains. Primer B12 was found to generate a virulence-specific fragment and primers B5, C8, and C20 were found to generate avirulence-specific fragments, which in all cases clearly identified either the virulence phenotype or the avirulence phenotype, respectively. In addition, the DNA polymorphic bands detected were analyzed by parsimony and distance analysis. A similar genetic relationship among the T. gondii strains was determined by the two phylogenetic methods, which use completely different assumptions. Consistent with the division of the 35 strains into a genotype of virulent strains and a genotype of avirulent strains, both analyses revealed 2 clonal lineages directly correlated with murine virulence. These results strongly support the hypothesis that the genus Toxoplasma may actually contain two clonal lineages correlated with virulence, which have evolved independently following their initial separation. Received: 1 October 1996 / Accepted: 15 October 1996     

Molecular arguments for splitting of Schistosoma intercalatum, into two distinct species

The taxonomic status of the two known strains of Schistosoma intercalatum, the Lower Guinea strain (originating from Edea, Cameroon) and the Zaire strain (originating from Kinshasa, Democratic Republic of Congo, formerly Zaire) was examined using random amplified polymorphic DNA (RAPD) markers. Two additional species within the S. haematobium group, S. haematobium and S. mattheei, were included in the study. DNA was extracted from four male and four female worms of each species and strain under investigation. In all, 13 primers gave reproducible and informative marker patterns; the monomorphic bands in all the males and females of each sample were scored, and 138 bands were included in the final analysis. Overall, 14 RAPD fragments were shared by all the schistosomes studied, and 19 RAPD fragments were considered to be sex markers. Only 22% (20/91) of the RAPD fragments were shared between S. intercalatum Zaire and S. intercalatum Cameroon. The mean values recorded for the Nei and Li's genetic distances between S. haematobium and S. mattheei and between S. intercalatum Zaire and S. intercalatum Cameroon were 0.546 and 0.596, respectively. A principal component analysis and one-way analysis of variance (ANOVA/MANOVA) showed a significant separation between S. intercalatum Zaire and S. intercalatum Cameroon. The data support the hypothesis that S. intercalatum Zaire and S. intercalatum Cameroon are distinct species. Additional molecular-biology studies are in progress that involve the use of nuclear and mitochondrial markers to confirm the extent of the genetic divergence prior to the establishment of final decision on the taxonomic status of the two strains of S. intercalatum. Received: 6 June 2000 / Accepted: 11 July 2000     

Homogeneity of Trypanosoma cruzi I, II, and III populations and the overlap of wild and domestic transmission cycles by Triatoma brasiliensis in northeastern Brazil

The genetic variability of 24 Trypanosoma cruzi isolates from humans (11) and triatomines (13) in northeastern Brazil was analyzed by random amplified polymorphic DNA (RAPD) and compared with taxonomic groups, host, and geographical origin of the parasite. TcI (12.5 %), TcII (45.8 %), and TcIII (41.6 %) showed a similarity coefficient (SC) of 0.74 using the mean of three primers and 0.80, 0.75, and 0.66 for λgt11-F, M13-40F, and L15996 primers, respectively. The samples were clustered according to their phylogenetic origin in two polymorphic and divergent branches: one associated with TcI and the other with two subbranches corresponding to TcII and TcIII. TcI was only identified in humans and correlated with the Id homogenous group (0.80 SC). TcII from humans and Triatoma brasiliensis showed 0.86 SC and was clustered according monoclonal or polyclonal populations with similar RAPD profiles detected among the vector and/or humans in different municipalities. TcIII was isolated exclusively in sylvatic cycles from T. brasiliensis and Panstrongylus lutzi and showed low variability (0.84 SC) and high homology mainly among isolated populations at the same locality. The homology of T. cruzi among different hosts and locations suggests the distribution of principal clones circulating and reveals an overlapping between the sylvatic and domestic cycles in this area, where T. brasiliensis infected with TcII acts as link in both environments. This species is important to maintain TcII and TcIII in wild cycles and deserves particular attention due an emergent risk of these populations being introduced into the domestic cycle; moreover, its clinical and epidemiological implications remain unknown.     

Characterization of Ornithobacterium rhinotracheale field isolates from Hungary

Ornithobacterium rhinotracheale is a widely distributed rod-shaped Gram-negative bacterium that infects several avian species including chickens and turkeys. It is associated with respiratory signs, growth retardation, mortality, and reduced egg production, thus causing severe economic losses to the poultry industries. In this study, 37 field isolates of O. rhinotracheale, collected from various locations in Hungary between 1997 and 2015, were identified and characterized by the analysis of partial 16S rRNA gene sequences, enterobacterial repetitive intergenic consensus (ERIC)-PCR, and random amplified polymorphic DNA (RAPD) PCR assays with the OPG11, OPH19, and M13 primers. Most of the field isolates were serotype A, one was serotype B, and four were serotype D. One isolate could not be typed with antisera against serotypes A–E. In a phylogenetic analysis of the 16S rRNA sequences, the isolates formed two clusters. Thirteen distinct patterns were identified with ERIC-PCR, and the RAPD assay with the M13 primer assigned the isolates to 10 different patterns. The other two RAPD assays were unsuitable for distinguishing and grouping the isolates. Neither ERIC type nor RAPD pattern correlated with the place or year of isolation. However, the strains isolated from chickens were more heterogeneous on ERIC-PCR than the isolates recovered from turkeys. In this study, ERIC-PCR was the most discriminatory method for investigating the genetic diversity of O. rhinotracheale isolates.     

Random amplified polymorphic DNA profiles of Trypanosoma cruzi isolates from chagasic patients with different clinical forms

The genetic variability of 61 Trypanosoma cruzi isolates from 47 chronic chagasic patients of Minas Gerais state was analyzed by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) using M13-40, λgt11-F, and L15996 primers. Cluster analysis by unweighted pair group method analysis was applied to RAPD profiles, and cluster analysis used to verify a possible correlation among different clinical forms of the disease from these patients. The T. cruzi isolates showed distinct grouping on tree topology, with the isolates not being possible to establish a correlation to the clinical forms of Chagas’ disease. These data showed that the T. cruzi isolates from these patients would compose a group of populations well correlated genetically.     

Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA

Summary We have used a PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), to assess genome variability between 21 isolates from F. solani f. sp. cucurbitae races 1 and 2. Based on RAPD marker patterns the isolates fell into two distinct groups corresponding to mating populations MPI and MPV. Four isolates that could not be assigned to one or other mating population by traditional means were distinguished by RAPD patterns. Seven polymorphic RAPD products were used to probe Southern blots of MPI and MPV genomic DNA. Six of the seven probes hybridized to single-copy sequences and five of the seven probes showed specificity for one or other mating population. We suggest that not only is the technique a rapid and reliable tool for isolate-typing of fungi but it also provides a rapid method for obtaining species- or racespecific hybridization probes.     

Study of the inter- and intraspecific variation ofEimeria spp. from the rabbit using random amplified polymorphic DNA

A genetic polymorphism study was performed in coccidia from the rabbit. A comparative analysis of the RAPD (random amplified polymorphic DNA)-generated fingerprints, using 11 arbitrary primers, was carried out (1) in nineEimeria species (E. intestinalis, E. magna, E. piriformis, E. flavescens, E. vejdovskyi, E. coecicola, E. perforans, E. exigua, andE. media) and (2) in two strains ofE. intestinalis and four strains ofE. media originating from different geographic areas. For each of these four strains ofE. media, three lines deriving from the multiplication of a single oocyst were compared. All the primers tested yielded about ten amplified fragments. The profiles obtained differed considerably according to the species; thus, it was not possible to establish a phylogeny. On the other hand, species-specific fingerprints were observed, showing that RAPD assays might be useful for diagnosis. InE. media, analysis of the RAPD products showed weak differences between each of the four strains but nevertheless allowed differentiation of the lines deriving from the multiplication of one oocyst. Similar results were obtained with three methods of analysis: correspondence analysis, the hierarchical unweighted pair-group method with arithmetic averages (UP-GMA), and parsimony analysis. RAPD proved to be a useful technique for these intraspecific studies.     

Rapid genotyping ofEscherichia coli O157 isolates by random amplification of polymorphic DNA

Although several typing methods have been described for Shiga-like toxin-producingEscherichia coli O157, the methods are somewhat cumbersome. Using 22 isolates ofEscherichia coli O157 and five otherEscherichia coli isolates, two primers (M13 core sequence and 970-11) were found to give excellent differentiation between isolates using random amplified polymorphic DNA (RAPD). Using only the presence or absence of variable bands, a matrix of 20 variable characters was identified. From these characters, similarity coefficients were calculated and a phenogram constructed. All of theEscherichia coli O157 isolates were easily distinguished from the non-O157Escherichia coli isolates. Using a 95% similarity cutoff, we found 13 RAPD types among the 22Escherichia coli O157 isolates. Isolates thought to be identical by toxin and phage typing as well as by epidemiological association were distinguished, and others thought to be distinct by lack of epidemiological association were identical. RAPD using M13 and 970-11 primers is a potentially useful typing tool forEscherichia coli isolates of serotype O157 and possibly otherEscherichia coli isolates.