Genetic diversity of Iranian clinical isolates of Mycobacterium tuberculosis

In the current study we aimed to execute a rather less complicated molecular tying method, i.e. the random amplification of polymorphic DNA (RAPD) analysis to find the heterogeneity of Iranian strains of Mycobacterium tuberculosis. The isolates comprised a total of 96 strains of M. tuberculosis collected from clinical specimens of patients in Isfahan and Tehran. The isolates were assigned to the species M. tuberculosis by the key conventional and molecular methods. They were then subjected to RAPD analysis by four arbitrary primers, namely, the primers 27F, 1525R, MS- GF and INS-2. They were then evaluated for the number and intensity of the band patterns. The RAPD profiles of the Iranian isolates showed a degree of heterogeneity which varied based on the primer used. However, analysis of the isolates by primer INS-2 revealed the highest degree of diversity yielding 31 distinguishable RAPD types. RAPD analysis provides a rapid and easy means of identifying heterogeneity among the M. tuberculosis isolates. This typing system might be considered a valuable alternative molecular typing for countries with limited resources provided that the reproducibility and reliability of the method is carefully assured.     

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.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

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.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

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.     

毛木耳种质资源的RAPD分析

利用22个随机引物对来源不同的56个木耳菌株进行了RAPD分析。结果表明,所有引物的扩增产物DNA片段均表现出明显的多态性,供试菌株总共扩增出164条多态性片段,占总扩增片段的99%;供试菌株两两间的遗传相似系数变化较大(平均GS值0.2143￣0.8764)。采用系统聚类法中的类平均法,对供试的所有菌株两两间相似系数进行聚类,可将它们分为四大类,各大类的类间和类内菌株的遗传变异程度较大,以IV类内各菌株间的最高(平均GS值0.3891),II和III类间的最低(平均GS值0.5887),表明遗传变异也较丰富(总平均GS值0.4918)。将RAPD技术应用于不同菌株间遗传差异的研究,具有反应迅速、不受外界环境条件影响、能从DNA分子水平上揭示菌株间遗传差异等优点,是一种快速准确评估木耳种质资源的有效方法。     

Genetic variability among Alexandrium tamarense and Alexandrium minutum strains studied by RAPD banding pattern analysis

We performed random amplification of polymorphic DNA (RAPD) analysis on five strains of Alexandrium tamarense and nine strains of Alexandrium minutum. Arbitrary 10-mer oligonucleotides were used as primers for the PCR. Electrophoresis on denaturing acrylamide gels improved RAPD reproducibility and increased the band number. Eight of the 20 primers assayed gave reproducible results and the band profiles generated by them were used for constructing a similarity matrix. Analyses were performed independently for the strains of each species and jointly for all the strains of both species. Results for A. tamarense showed the highest similarity for two distinct clones isolated from the same water sample in the Baltic Sea during a bloom (KAC01 and KAC02). The highest similarity among A. minutum clones was found for three strains (AL1V, AL2V and AL3V) isolated in the Ria de Vigo in NW Spain. The results show a high genetic diversity within a single species. We have shown the potential of the RAPD technique to discriminate between two conspecific strains, as well as for establishing similarities that are related to the biogeographic origin of the strains.     

稗草致病菌——尖角突脐孢菌菌株RAPD指纹图谱的分析

以我国主要稻区的稗草植株上分离的17株尖角突脐孢菌菌株为试验材料,采用改良的SDS法提取其基因组DNA,并运用优化的RAPD分析体系对其进行了分子标记遗传差异研究。从25个随机引物中筛选出20个扩增效果好的引物,对全部试验材料进行了RAPD扩增,共得到239条有效带,其中多态性带229条（占95.8%）。依据扩增结果建立了17株尖角突脐孢菌基因型的DNA指纹图谱并对其进行了有效区分。根据RAPD分析结果计算了菌株间的遗传距离,分析了它们的遗传差异并进行了聚类分析,结果表明,RAPD分子标记技术是能够用于杂草致病菌资源的鉴定的,并可以进一步应用于特定性状的基因标记研究。     

Random-amplified polymorphic DNA (RAPD) analysis shows intraspecies differences among Xanthomonas albilineans strains

Five strains of Xanthomonas albilineans , causal agent of leaf scald disease in sugarcane from various geographical regions, were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the DNA level. CsC1-purified genomic DNA from these strains were amplified by the polymerase chain reaction (PCR) using arbitrary 10-mer primers according to standard RAPD conditions and the amplification product profiles analysed by conventional agarose gel electrophoresis. Although most RAPD markers were common to all five strains, unique profiles for each strain were discernible using four 10-mer arbitrary primers individually. Reproducible DNA fingerprints indicate that RAPD analysis can be used to identify and differentiate the X. albilineans strains. This technique has the potential for use in monitoring the appearance of foreign strains of X. albilineans in various geographical regions and could be used for the construction of phylogenetic trees.     

Random amplified polymorphic DNA (RAPD) analysis ofPenicillium marneffei strains isolated from AIDS patients in Thailand

Results of random amplified polymorphic DNA (RAPD) analysis using three different primers showed that 16 strains ofPenicillium marneffei isolated from AIDS patients in Thailand belonged to a genetically homogenous group, but different slightly from an isolate from bamboo rat in China. Six PCR fragments (from about, 200 to 600 bp) that were commonly observed in the RAPD fingerprint of all strains were extracted and sequented. Usefulness of this sequence information for identification ofP. marneffei is discussed.     

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.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

RAPD typing for distinguishing species and strains in the genus Listeria

The randomly amplified polymorphic DNA (RAPD) technique was employed in the development of a typing protocol for Listeria isolates, particularly Listeria monocytogenes strains. A single strain of L. monocytogenes was used and 200 random decamer primers were screened for their discriminatory abilities by visualizing the amplification products electrophoretically. Three candidate primers displaying potentially useful banding patterns were selected and tested against 52 L. monocytogenes strains, encompassing 11 serotypes, and 12 other strains representing five other Listeria spp. Thirty-four banding profiles were obtained with one particular primer. RAPD analysis allowed differentiation between Listeria spp. and was found to further subdivide strains of the same serotype. Where only one primer was used strains from different serotypes were occasionally found to produce identical banding profiles. RAPD analysis, which in our hands proved to be reproducible, shows much promise as a molecular alternative to traditional L. monocytogenes typing protocols.     

羊肚菌栽培菌株遗传多样性分析及种特异性RAPD-SCAR标记开发

近年来中国的羊肚菌Morchella spp.栽培技术取得了长足进步,但基础研究薄弱影响其稳产和高产,国内外尚无羊肚菌栽培菌株种质资源遗传多样性的研究报道。本文对来自全国12省份的36个羊肚菌栽培菌株进行了ITS系统发育分析,并采用RAPD进行了遗传多样性评价。结果表明,结合有效的参考菌株序列,通过ITS序列分析可以将供试菌株进行区分和鉴定,在36个菌株中,26个菌株属于梯棱羊肚菌Morchella importuna,其他10个菌株属于六妹羊肚菌M. sextelata;将自40条RAPD引物中筛选出的14条用于供试菌株遗传多样性分析,共扩增出124条多态性条带;UPGMA聚类可将供试菌株分为两大类群,分别对应于ITS系统发育分析中的梯棱羊肚菌和六妹羊肚菌两个物种,梯棱羊肚菌种内菌株多态性高于六妹羊肚菌。OPA17引物和OPA18引物分别在AA02和AA15菌株中扩增出具有唯一性的特征条带,对两个特征条带进行回收测序后,设计出两个特异性SCAR的引物,它们能有效地从36个供试菌株群体中将菌株AA02和AA15鉴别出来。本文首次全面系统地采用ITS分析鉴别了我国羊肚菌栽培菌株的种性,采用RAPD分子标记系统地评价了羊肚菌栽培菌株的遗传多样性,并验证了RAPD分子标记转化为菌株特征性SCAR标记的可行性。     

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.     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.     

银杏DNA提取及RAPD分析

采用SDS裂解液和苯酚/氯仿/异戊醇提取液从银杏叶中提取银杏总DNA,并进行DNA样品分光光度测定和琼脂糖凝胶电泳分析。通过引物筛选和反应参数优化,选用3个随机引物对DNA样品进行RAPD扩增,获得较为清晰并有一定多态性差异的扩增谱带,初步摸索出适合于以银杏叶为材料的DNA提取方法和RAPD扩增程序,为研究银杏遗传多态性及种质资源研究提供一种实用的分析方法。     

Relationship among acidophilic bacteria from diverse environments as determined by randomly amplified polymorphic DNA analysis (RAPD)

Summary Random amplification of polymorphic DNA (RAPD), a PCR-based technique was applied to evaluate genomic diversity among three strains of Acidithiobacillus thiooxidans, five strains of Acidithiobacillus ferrooxidans and one acidophilic moderate thermophile strain, using 45 random primers of five different series. More than 2200 bands were observed, with an average of 45 bands per primer. Primer OPC-3 produced the maximum number of fragments whereas minimum numbers of fragments were produced with primer OPA-5. A dendrogram was generated using cluster analysis by the unweighted pair group method of arithmetic means (UPGMA). The dendrogram showed three groups with similarity ranging from 29 to 85%. The maximum similarity (85%) was observed between the strains T.t₁ and T.t₂ of Acidithiobacillus thiooxidans.     

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.