Molecular Characterization of the Plant Pathogen Verticillium dahliae Kleb. Using RAPD-PCR and Sequencing of the 18SrRNA-Gene

Thirty-four isolates of Verticillium dahliae Kleb. from nine different genera of dicotyledonous host plants and a broad range of geographic regions were analysed genotypically, Random amplified polymorphic DNA (RAPD) markers were used for the estimation of the genetic variability within the species. Using four primers for the analysis, 79 distinct fragments were obtained. The derived phenogram clustered the isolates in two main groups; one consisted almost entirely of V. dahliae isolates from oilseed rape ( Brassica napus napus ), the other group comprised isolates from a wide range of host plants. No correlation between geographic location of the isolates and the RAPD-pattern was observed.
Sequencing of the gene for the 18SrRNA and calculation of the phylogenetic tree integrated the deuteromycetous fungus V. dahliae into the sexual system of the filamentous ascomycetes.     

Assessment of the genetic diversity among strains of Xanthomonas cynarae by randomly amplified polymorphic DNA analysis and development of specific characterized amplified regions for the rapid identification of X. cynarae

The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.     

Primer R108 performs best in the RAPD strain typing of threeAspergillus species frequently isolated from patients

We evaluated the suitability of various primers for the RAPD (random amplified polymorphic DNA) accurate species identification and strain typing of Aspergillus clinical isolates. Five primers described previously were tested for their discriminatory power in three Aspergillus species (A. fumigatus, A. niger agg. and A. flavus - 23 clinical isolates and 2 reference strains). Clustering of RAPD fingerprints corresponded well with the identification based on morphological features. All isolates were resolved as different strains using the primer R108 and the RAPD protocol optimized for a Robocycler thermal cycler. RAPD with the primer R108 thus can be considered to be a valuable, simple and powerful tool for identification and strain delineation of Aspergillus spp.     

利用RAPD研究桂林桂花品种间的亲缘关系

采用随机扩增多态性DNA(RAPD)技术,从100个随机引物中筛选出扩增效果较好的20个引物,分 析桂林市23个桂花品种的基因组多态性。20个随机引物共检测到193个位点,其中多态位点114个,占 59.1%。并进行了聚类分析,构建出树状聚类图,将这些品种划分为4个品种群,与传统分类学结果一致。结 果表明,以基因型而不是以表现型为基础,分析桂花品种间的区别是可能的。该技术为解决桂林市的桂花品 种分类问题提供了重要依据。     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究(英文)

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

Molecular analysis of Japanese isolates of Verticillium dahliae and V. albo-airum

Sixteen isolates of different pathogenicity groups of the plant pathogen Verticillium dahliae and four isolates of V. albo-atrum from Japan were analysed by means of an RAPD (random amplified polymorphic DNA) method using a PCR (polymerase chain reaction). Verticillium dahliae and V. albo-atrum could be distinguished by RAPD analysis. Four pathogenicity groups of V. dahliae could also be classified to a certain extent by this method. Similarities and differences in banding patterns obtained by RAPD may be a useful molecular tool in phylogenetic studies of the pathogenicity groups.     

Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm, Bombyx mori, using random amplification of polymorphic DNA: morphological and ultrastructural characterization

Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultrastructural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to be more virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmission in the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8-16 coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded a total of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whose presence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of band sharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dice coefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g, NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were found to be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microsporidian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect polymorphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in this process have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy.     

Detection of genetic diversity using RAPD-PCR and sugar analysis in watermelon [Citrullus lanantus (Thunb.) Mansf.] germplasm

RAPD (random amplified polymorphic DNA) markers generated by 15 arbitrary decamers were used to determine the frequency of DNA polymorphism in 39 watermelon [Citrullus lanantus (Thunb.) Mansf.] germplasms. Of the 15 primers tested, all except 1 (primer 275) directed the amplification of polymorphic products. A total of 162 amplification products were generated across all 39 genotypes. Among the 162 fragments, 35 (21%) appeared to be reliable polymorphic markers. The mean value by marker difference in this comparison was 0.24, and the highest, 0.69. Eight RAPD markers could be utilized in the unique variety discrimination 8 watermelon genotypes. From the phenograms constructed by UPGMA based on the comparison of RAPD markers, four clusters were resolved. Each group was also characterized and identified with morphological and genetic characteristics for each genotype. The free sugars of the edible parts of watermelons were analyzed by HPLC (high-performance liquid chromatography). Results from the phylogenetic analysis of band sharing data were consistent with sweetness as measured by HPLC. In conclusion, RAPD assays can be used for providing alternative markers for identifying genotypes and quantitative characteristics in watermelon.     

Differentiation of isolates of Discula umbrinella (Teleomorph: Apiognomonia errabunda) from beech, chestnut, and oak using randomly amplified polymorphic DNA markers.

Genetic variation in 30 isolates of Discula umbrinella derived from beech, chestnut, and oak was assessed using randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphic markers. Polymerase chain reaction amplifications with 17 primers produced 134 different DNA fragments. Three RAPD fragments were subsequently used for Southern hybridization. By these techniques up to four different individuals could be detected in the same leaf. The presence of several individuals within a single leaf indicates a finely tuned balance between the endophyte and its host. Cluster analysis of all arbitrary primed amplified DNA fragments showed that the isolates could be placed into four groups corresponding to their host origin. The high percentage of private RAPD variants within groups is consistent with low gene flow.     

利用RAPD分子标记对三组三系杂交水稻及亲本的遗传分析和鉴定

利用RAPD技术,从248个随机寡聚核苷酸（10bp）中筛选出13个引物能在供试的三组三系杂交水稻及亲本间扩增出43条稳定性较好的多态性片段,其中6个引物能在供试材料间扩增出20个强的多态性标记。利用这些标记能有效地区分各组合中不育系、保持系、恢复系和F1,并能看出各组合中不育系与保持系、不育系与恢复系、F1与亲本间的遗传关系。 Abstract:A total of 248 arbitrary 10-mer oligonucleotide primers were screened using RAPD (random amplified polymorphic DNA) techniques with the genome DNA of three groups of three-line hybrid rice and their parents.Thirteen primers produced 43 polymorphism fragments.Six primers of them produced 20 obviously repeatable polymorphic markers among rice lines tested.Using this RAPD markers,the hybrid rice combinations (sterile-line,maintainer-line,restorer-line and F1)can be effectively identified,and the genetic relationship among them can be shown.     

Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures

An aberrant random amplified polymorphic DNA (RAPD) marker in genomic DNA of tissue culture plantlets was frequently observed during a comparison of DNA fingerprints derived from potato germplasm grown in tissue culture and the field. The RAPD marker was cloned, sequenced and determined to be of bacterial origin. A bacterial contaminant was isolated from the tissue culture plants and identified as a Bacillus pumilus. A set of sequence characterised amplified region (SCAR) primers were designed from the sequence of the cloned fragment and tested for the specific detection of B. pumilus. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) were also used to generate B. pumilus profiles specific to our isolate in order to test and confirm the sequence homology of amplified markers generated from a range of DNA samples isolated from tissue culture plants and pure isolates of B. pumilus-like bacteria.     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by PCR-RAPD based fingerprinting

Seventy isolates of Bacillus thuringiensis were isolated from soil samples collected from cotton fields. These isolates were characterized by randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern based on their source of origin. Different random decamer primers were used for RAPD amplification, which generated a total of 1935 fragments; of these 1865 were polymorphic and 68 monomorphic. The primers OPA03, OPA08, OPD14, OPD19, OPD20, OPE17 and OPD19 produced 100% polymorphic fragments, whereas primers OPC06, OPC20 and OPD17 produced 20, 31 and 17 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 70 isolates fell into two separate clusters, cluster I and cluster II, which includes 26 and 44 B. thuringiensis isolates, respectively. These two main clusters were further divided into four subclusters at Eucledian distance of 150 and 80% similarity index. All primers showed amplification and indicated the good diversity of B. thuringiensis isolates. The RAPD pattern showed 4–10 bands per isolate, with MWt in the range of 0.4–3.5 Kb and an average of 193.5 fragments were produced per primer. The primer OPE17 was found to be the most discriminatory as it produced 286 polymorphic bands.     

Assessment of the Genetic Diversity among Strains of Xanthomonas cynarae by Randomly Amplified Polymorphic DNA Analysis and Development of Specific Characterized Amplified Regions for the Rapid Identification of X. cynarae

The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.     

Variation revealed by RAPD‐PCR profiles of microsymbiont Anabaena azollae strains from four N2 fixing Azolla cultures

Nitrogen fixing Anabaena azollae strains isolated from four different Azolla cultures were characterized based on their total protein profile and RAPD profile to study the existing variation among them. As expected, the isolates showed almost similar protein banding patterns, but exhibited differences in 40–70 KDa protein subunits. Polymerase chain reaction of the DNA of the isolates, using four different primers, amplified specific sequences of DNA and showed clear polymorphism among the isolates. The RAPD profile generated the fingerprinting pattern characteristic of each strain based on the sequence of the primers used. Common band sharing observed between the strains A. azollae‐RS‐KK‐SK‐AM and A. azollae‐RS‐KK‐SK‐RP probably represents maternal inheritance of DNA to the progeny. The polymorphic bands were generated specifically for the isolates A. azollae‐RS‐KK‐SK‐RP and A. azollae‐RS‐KK‐SK‐AM with primers numbered 2 and 4, respectively, which could be developed as possible markers for these isolates.     

Use of RAPD to investigate the epidemiology of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> infection in Malaysian hospitals

Summary Randomly ampli.ed polymorphic DNA (RAPD) with four different decamer oligonucleotide primers was performed on 50 clinical Staphylococcus aureus isolates obtained from di.erent hospitals in Malaysia. All the four primers generated polymorphisms in all 50 isolates of S. aureus studied, revealing DNA markers with sizes ranging from 100 to 7000 bp. The dendrogram generated from the RAPD analysis revealed two major groups (Groups I-II) with three clusters each in one group. S. aureus strains isolated from the same hospital were found to be genetically closely related and most of them were placed in the same cluster. In addition RAPD di.erentiated between MRSA and non-MRSA based on the clustering, where all MRSA and non-MRSA were placed in their respective clusters. The RAPD analysis showed that there could be four to .ve clones of S. aureus spreading around Malaysia, of which two clones may be MRSA. The overall genetic distances ranged from 0.088235 to 0.954545 among the isolates. This technique was found to be a simple and e.ective method for epidemiological investigation. Because of these e.cient features, this technique may have more general application for the study of S. aureus infections in hospitals and the community.     

DNA Polymorphism among Rice Somaclones

Molecular markers were used to detect the influence of high concentrations of 2,4 dichlorophenoxyacetic acid (2,4-D) in the callusing media on DNA variations in regenerated rice plants. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) based RFLP analysis were carried out on 12 somaclones of Oryza sativa L. cv. B-370. In vitro culture induced DNA variations were detected in the regenerated plants but the effect of high auxin concentration in the medium could not be revealed. In a second study, fingerprinting of 15 semi-dwarf, high yielding somaclones of B-370 was carried out using RAPD technique. Amplification using 20 random primers produced a total of 167 DNA bands out of which 97 bands were polymorphic. A total of 32 unique DNA bands were detected across all the somaclones and they could be grouped based on their similarity to B-370. RAPD analysis helped to reveal similarity or differences among the somaclones while fingerprinting using additional RAPD markers was not successful.