Effect of DNA preparation from laver (Porphyra yezoensis) thalli on reproducibility of RAPD (random amplified polymorphic DNA) patterns

Laver (Porphyra yezoensis) DNAs were extracted from thalli with five different procedures and used for RAPD (random amplified polymorphic DNA) analysis as templates. Restriction enzyme-digestive DNAs were obtained with all procedures examined. However, RAPD patterns generated with these DNAs appeared highly irreproducible and were considerably different from each other. When DNAs purified with CsCl gradient centrifugation were used for RAPD analysis as templates, highly reproducible RAPD patterns were obtained, suggesting that unpurified DNAs extracted from thalli with all five extraction procedures contained an excess of RNA, polysaccharides and/or other materials which affected the RAPD reproducibility. Thus, results indicated that purification of DNA is essential to produce reproducible RAPD patterns of Porphyra DNA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Using RAPDs to study phylogenetic relationships in Rosa

Nineteen species of rose (Rosa sp.) were analysed using Random Amplified Polymorphic DNA markers (RAPD). Each 10-base-long arbitrary primer produced a specific DNA banding pattern that grouped plants belonging to the same species and botanical sections as predicted from their genetic background. One hundred and seventy-five amplification products were examined by cluster analysis to assess the genetic relationships among species and their genetic distances. All of the accessions belonging to 1 species grouped together before branching to other species. Dendrograms constructed for intra- and inter-specific studies showed a good correlation with previous classifications by different authors based on morphological and cariological studies. Our results show that the RAPD technique is a sensitive and precise tool for genomic analysis in rose, being useful in assigning unclassified accessions to specific taxonomic groups or else allowing accessions classified by traditional criteria to be re-classified.     

Suppression of randomly primed polymerase chain reaction products (random amplified polymorphic DNA) in heterozygous diploids

In a study of genetic polymorphism in the gypsy moth Lymantria dispar we observed the aberrant inheritance of a random amplified polymorphic DNA (RAPD) fragment designated H11-589. This fragment was present in amplification products of F1 progeny of different crosses although it was not amplified from either parental DNA. DNA-mixing experiments revealed that the presence of DNA containing a template for another product (H11-746), amplified with the same primer, suppressed the synthesis of H11-589. The templates for both RAPD products were highly repetitive and scattered throughout the L. dispar genome. Southern hybridization and sequence analysis of H11-746 and H11-589 revealed an extensive sequence homology and an internal repetitive motif of 17 nucleotides present in both products. Interactions between templates for H11-746 and H11-589 are expected to occur during the polymerase chain reaction (PCR), offering an explanation for the suppression of the amplification of H11-589. The role of the internal repetitive motif and of the copy number of both templates in the suppression effect are discussed. Our results corroborate doubts regarding the suitability of the RAPD technique for quantitative genetic analysis, in particular where mixed populations are concerned.     

Analysis of clinical isolates of Propionibacterium acnes by optimised RAPD

Random amplification of polymorphic DNA (RAPD) was evaluated as a genotypic method for typing clinical strains of Propionibacterium acnes. RAPD can suffer from problems of reproducibility if parameters are not standardised. In this study the reaction conditions were optimised by adjusting template DNA concentration and buffer constituents. All isolates were typeable using the optimised RAPD protocol which was found to be highly discriminatory (Simpson's diversity index, 0.98) and reproducible. Typing of P. acnes by optimised RAPD is an invaluable tool for the epidemiological investigation of P. acnes for which no other widely accepted method currently exists.     

不同固定剂保存动物组织标本对RAPD反应的影响

为解决野外采集动物标本时,有效地保存好标本,并方便地带回实验室用于ＲＡＰＤ分析的难题。该研究以同一个体的冻存组织为对照,比较了从４种不同固定剂保存的组织标本中提取ＤＮＡ,并用于ＲＡＰＤ扩增。     

26种冬青属植物遗传多样性分析

以26种冬青属植物种质资源为研究材料,利用RAPD和AFLP技术对基因组DNA进行扩增,以研究其物种间遗传多样性以及亲缘关系.结果表明:在RAPD分析中,从400条10个碱基的寡核苷酸引物中筛选出反应稳定、扩增性强、重复性好的引物20个,共扩增出312条多态性条带,多态率为95.41%;聚类分析显示26种冬青属植物间,布利奥特夫人枸骨叶冬青和黄果在AFLP分析中,10对选择性引物组合均扩增出了丰富的多态性片段,共扩增出350条谱带,其中336条具有多态性,占95.96%.综合RAPD和AFLP聚类结果,枸骨、无刺枸骨和日拉斯纳尔逊枸骨的亲缘关系较近,钝齿冬青、金宝石钝齿冬青和龟甲冬青三者的亲缘关系较近,可为冬青属植物的杂交育种与种质创新提供理论依据.     

Differentiation of Brucella species by random amplified polymorphic DNA analysis

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.     

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。     

Predigestion of DNA template improves the level of polymorphism of random amplified polymorphic DNAs in wheat

Random amplified polymorphic DNA (RAPD) analysis in wheat has proven to be poor in its levels of both reproducibility and polymorphism. By digesting the template, prior to performing PCR, with frequently cutting restriction enzymes, the level of polymorphism was improved. RAPD profiles from certain primers were not affected by this pretreatment of the template, but other primers produced distinct profiles from each of several restriction enzymes assayed. Some polymorphisms were specific to one or more restriction digests, but none involved the simple loss of bands from the unrestricted template profile. Genotypic comparisons enabled the selection of primer-restriction enzyme combinations that enabled polymorphic and mappable patterns to be produced both between wheat varieties and between wheats with and without chromosomal segments deriving from related species.     

Preparation of DNA from Silica Gel Dried Mini-amount of Leaves of Oryza rufipogon for RAPD Study and Total DNA Bank Construction

Gene resources of Oryza rufipogon Griff. play a crucial role in rice breeding, and hence to study their conservation is of utter importance. The authors describe a method for preparation of DNA from mini- amotmt of the silica-gel-dried leaves of Oryza rufipogon. The high molecular weight DNAs of 1 168 individuals representing 44 populations have been obtained with high yields, which could be used for RAPD PCR and construction of total DNA bank of this species. The template DNA from silica-gel-dried leaves stored for one year at room temperature gave the same RAPD results as that from the newly prepared silica-gel-dried leaves. The optional template DNA concentrations for amplification ranged from 3.1 ng to 50 ng. In addition, the quality and quantity of the template DNAs that affect RAPD results are also discussed.     

Evaluation of three methods for DNA fingerprinting of Corynebacterium pseudotuberculosis strains isolated from goats in Poland

Phenotypic approaches based on metabolic and biological characteristics of Corynebacterium pseudotuberculosis have been limited due to insufficient discrimination between closely related isolates. In this paper we present performance and convenience of three molecular typing methods: BOX-PCR, random amplification of polymorphic DNA (RAPD) and amplification of DNA fragments surrounding rare restriction site (ADSRRS-fingerprinting) in genome analysis of these bacteria. Among examined 61 strains there were distinguished four, eight and 10 different genotypes by BOX-PCR, RAPD and ADSRRS-fingerprinting, respectively. The value of discrimination index was the lowest for BOX-PCR (D = 0.265), much bigger for RAPD (D = 0.539) and the highest for ADSRRS-fingerprinting (D = 0.604). The good discriminatory ability and reproducibility of RAPD and ADSRRS-fingerprinting indicates that those techniques may be particularly applied for epidemiological studies of C. pseudotuberculosis isolates. We found that ADSRRS-fingerprinting is a rapid method offering good discrimination power, excellent reproducibility and may be applied for epidemiological studies of intraspecific genetic relatedness of C. pseudotuberculosis strains.     

从普通野生稻硅胶干燥的小量叶片中制备DNA用于RAPD分析和总DNA库的建立(英文)

普通野生稻（ Ｏｒｙｚａｒｕｆｉｐｏｇｏｎ Ｇｒｉｆｆ．）的基因资源对水稻的育种起着至关重要的作用。报道了从其硅胶干燥的小量叶片中制备ＤＮＡ的方法。用此方法制备的ＤＮＡ分子量大（４０～４５ ｋｂ） ,产率也较高（５０ ～２００ μｇ／ｇ） ,且成功地进行了ＲＡＰＤ扩增。用制备的４４ 个居群,１１６８ 个个体的总ＤＮＡ 建立了中国普通野生稻的总ＤＮＡ 库作长期冷冻保存,可用于基于ＰＣＲ 的ＤＮＡ水平上的各种目的的研究。根据实验结果,从在室温下贮存１ 周、３ 个月、６ 个月、１ 年的硅胶干燥的叶片中提取的ＤＮＡ 用于ＲＡＰＤ扩增所得的扩增产物没有差异;模板ＤＮＡ浓度在３ ．１ ～５０ ｎｇ 的范围内均得到很好的ＲＡＰＤ扩增结果。这说明了从硅胶干燥的叶片中提取的普通野生稻的ＤＮＡ 用于ＲＡＰＤ扩增的产物很稳定,将其用于群体遗传分析具有很好的可比性和可靠性。同时也讨论了模板ＤＮＡ的纯度和浓度对ＲＡＰＤ扩增的影响     

Technical aspects of random amplified polymorphic DNA (RAPD) technique in genotyping of bacterial strains

Random Amplified Polymorphic DNA (RAPD) method became widely applied for sensitive, efficient and fast distinguishing of different isolates of a given species, if pure culture is available. Problems with reproducibility and discriminatory power, frequently cited in the literature, can be overcome by precise optimization procedure allowing to achieve reliable conditions for each species analysed. Basing on two examples of different species, H. pylori and E. faecium, particular parameters of RAPD fingerprinting were evaluated with respect to selection of best working primers generating medium-complex profiles, using only high quality DNA samples and evaluation optimum for every reaction reagent. Stable and informative amplification patterns were obtained with different best working primers which could discriminate between all H. pylori and E. faecium strains tested. For both analysed species different optima were found, suggesting species-specific need of precise RAPD conditions evaluation. This study proved high sensitivity and efficiency of optimized RAPD profiling applicable for searching the epidemiology traces for both species.     

Rapid identification of white-Engelmann spruce species by RAPD markers

Fragments of random amplified polymorphic DNA (RAPDs) were used as markers to distinguish Picea glauca (Moench) Voss (white spruce) and Picea engelmannii Parry (Engelmann spruce). These species and their putative hybrids are difficult to differentiate morphologically and are collectively known as interior spruce. Four oligodeoxynucleotide decamer primers showed species-specific amplification products between white spruce and Engelmann spruce. These fragments are highly conserved among seed lots and individual trees of each species from diverse geographic origins. The consistency and reproducibility of these species-specific amplification products were tested in more than two amplification reactions. Therefore, RAPD markers can provide genetic markers for easy and rapid identification of the specific genetic entry of these spruce species and their reported putative hybrids. According to the frequencies of the species-specific RAPD markers, it is possible to estimate the hybrid fraction, indicative of true introgression between the two species. These results are useful for quick identification of both species and their hybrid swarms at any stage in the sporophyte phase of the life cycle, for determining the occurrence and the magnitude of introgressive hybridization in an overlap zone between the two species, and for certification purposes in operational re-forestation and tree-improvement programs.     

Genetic Mapping of Soybean [Glycine max (L.) Merr.] Using Random Amplified Polymorphic DNA (RAPD)

Random amplified polymorphic DNA (RAPD) is based on DNA amplification by polymerase chain reaction (PCR) of random DNA segments using single arbitrary nucleotide sequences. We have adapted the assay to soybeans by using Stoffel Fragment DNA polymerase and by optimizing the reaction conditions. To increase the percentage of RAPD polymorphisms, the DNA template was digested with restriction enzymes before amplification. The combination of twenty-four primers and five DNA template treatments (Undigested, DraI, EcoRI, HindIII, and TaqI digested) revealed 94 polymorphic DNA fragments differing between soybean lines PI437654 and BSR101. Many polymorphic DNA bands were found unreliable or non-scoreable after re-screening of primers and verification of marker-allele segregation with 20 recombinant inbred lines (RILs). However, 28 RAPD markers were consistently polymorphic between the parental lines and followed Mendelian expectations. The use of DNA templates digested with DraI, EcoRI, HindIII or TaqI increased three times the number of RAPD markers compared to undigested DNA template alone. The 28 RAPD markers obtained were further screened with 72 RILs and placed on an existing RFLP map.     

RAPD技术在芦荟属植物分类研究中的应用

采用CTAB法提取11个芦荟材料的基因组DNA为模板,以50个随机引物进行RAPD分析。结果表明：大部分引物可以在不同模板上扩增出条带,但仅有6个引物可以同时在11个芦荟材料的DNA上扩增出条带,对11个芦荟种变种的RAPD结果进行聚类分析,结果表明基本符合传统分类观点。对RAPD技术在芦荟属植物分类研究中的问题进行了讨论。     

Reproducibility of the differential amplification between leaf and root DNAs in soybean revealed by RAPD markers

 Random amplified polymorphic DNA (RAPD) was used to determine whether such markers can be employed for detecting genomic modification during plant development or under certain stress environments. Pairwise comparisons in RAPD patterns of leaf and root DNA amplifications were studied for 11 soybean accessions representing different origins. Hydroponic culture was used for the ease of harvesting roots. From a total of 40 primers screened, it was found that 16 can detect leaf DNA polymorphism, 19 for root DNA polymorphism, while 10 show a greater consistency for detecting polymorphism between leaf and root (L/R) DNAs. Nevertheless, problems were encountered when the newly synthesized oligo-primers and different thermal cyclers were used to check the data. Several factors were then tested for their reproducibility. The results indicated that the amplified differences between root and leaf DNAs are mostly not affected by template DNA concentrations. The addition of DMSO (dimethyl sulphoxide) or TMAC (tetramethyl-ammonium chloride) also did not mask the L/R differences. However, DNA polymerase and oligo-primers synthesized from different manufacturers, as well as the thermal cyclers, reacted differently sometimes. Regardless of the general problems of reproducibility in RAPD patterns, some amplified differences remain between the L/R DNAs. The most distinct patterns involve differences in the relative intensity of amplified bands. Differential amplification might have occurred during plant leaf and root development. Southern hybridization of the eluted polymorphic bands against restriction digestion of total genomic DNA confirms their being homologous to soybean DNA fragments. Polymorphism of these specific L/R differences also exists among varieties. RAPD should be a useful tool in detecting genomic alterations during plant development or under certain stress environments, as long as the factors affecting the reproducibility of RAPD patterns can be properly controlled. An additional cycle of selection would be possible if such a type of polymorphism is proved to be correlated with certain developmental characters. Received: 7 October 1996 / Accepted: 20 May 1997     

满江红鱼腥藻与其宿主的遗传多样性和协同性的RAPD分析

从16个代表不同种属或地域来源的满江红样本中分离出共生藻并通过处理获得无藻的满江红宿主,对二者同步进行了RAPD扩增,分别得到了大量DNA多态片段。通过建立满江红鱼腥藻及其宿主的UPGMA聚类关系图,看出二者在遗传分支上存在着一定程度的协同对应关系。但在种内的不同品系间,这种协同性有所减少,发现有的品系的共生藻发生了明显的变异。 Abstract:Symbiotic Anabeana azollae and its host plant Anabeana-free Azolla were isolated from 16 Azolla accessions representing different Azolla species or geographic origins.DNA polymorphic fragments were obtained by simultaneous RAPD amplification of both symbiont and host.The UPGMA clusters of Anabeana azollae and its host Azolla were established separately based on Dice coefficient caculation and a coordinated relationship was shown between Anabeana azollae and its Azolla host along both individual genetic divergence,but this genetic homology was reduced among different strains within Azolla species while the obvious mutants of Anabeana azollae were detected in some Azolla tested strains collected from different geographic area in the same host species.     

Use of random amplified polymorphic DNA markers for the detection of Azospirillum strains in soil microcosms

Probes for the detection of Azospirillum strains were obtained from DNA fragments generated by random amplification of polymorphic DNA (RAPD) and tested to assess their specificity towards DNA extracted from pure cultures. The most specific probe, referred to as α4, produced a hybridization signal only with amplified DNA of A. lipoferum ATCC29731. This strain was inoculated, together with two other Azospirillum strains, in soil microcosms of different complexity and its presence tested with the probe α4. This probe confirmed its high specificity with amplified DNA extracted from the soil microcosm and in the presence of other A. lipoferum strains, indicating that the strategy for bacterial detection, based on RAPD markers, is useful for monitoring the presence of a particular strain under environment-like conditions. Other RAPD-derived probes, when tested on soil samples, did not show the same level of specificity as that shown on DNA from pure cultures. This result suggests that some precautions are necessary in the choice of a really specific RAPD marker. In a further development of this strategy, the α4 probe was sequenced and two pairs of “nested” primers were designed, which enabled a diagnostic polymerase chain reaction from soil samples that was specific for the A. lipoferum species. Received: 7 July 1997 / Accepted: 14 October 1997     

蹄盖蕨科DNA的提取和几种因素对RAPD分析的影响

以黑龙江省牡丹江市4种蹄盖蕨科植物为材料,对其DNA提取及RAPD分析进行了研究,分别测试了模板浓度、Taq DNA聚合酶浓度、引物浓度、Mg²⁺浓度、dN TP浓度,确定适合蕨类植物DNA提取和RAPD分析的较理想的扩增条件,为RAPD分析应用于蹄盖蕨科遗传多样性研究打下良好基础。