Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species Complex

Three amplicons, appearing in a species-specific manner on the electrophoregrams of RAPD reactions that were obtained with primer OPA1, OPA1-800, OPA1- 900, and OPA1-1200, are analyzed in this study. The study revealed that each of these products is composed of one Leishmania DNA band, taxonomically conserved among the different Old World species studied. Subsequently, only the electrophoretic position of the RAPD products can be considered species-specific. In addition, sequence data, genomic organization, and chromosomal location have proved that these fragments are different and physically independent. However, they possess common features related to the presence of different kinds of short DNA repeats, more particularly microsatellites and a CCCTTC motive, corresponding to the 3' half of the OPA1 primer. These results suggest that the OPA1 primer has initiated amplification from different priming sites, having a species-specific location. This corresponds to sequence micro-heterogeneity of DNA fragments present within the different species and leading eventually to a selective amplification of different RAPD products. This characteristic has been used to develop an original selective PCR test based on the sequence of the OPA1-800 product, in which only DNAs from the L. donovani species complex are amplified. Restriction site polymorphisms and sequence variations are identified within the PCR fragment amplified from these parasite DNAs. In fact, the OPA1-800 fragment proved to be a useful DNA marker either as a DNA probe or as a target for PCR-based assays. This tool can therefore be recommended for the control of Old World Leishmania parasites, such as species discrimination, molecular tracking of isolates, or study of polymorphisms within the L. donovani species complex. Moreover, the molecular bases underlying the amplification of the RAPD fragments studied correspond to mechanisms already described. Although they do not account for the amplification of all Leishmania RAPD products, such mechanisms stress some of the pitfalls of the technique, which need to be taken into consideration. We have identified at least misleading observations of DNA bands amplified in a species-specific manner, in spite of their presence in the genome of the other taxa, and relatedness between bands within the amplification profiles. Therefore, recommendations for careful interpretation of RAPD data in population genetics or phylogenetic analyses are reiterated. Molecular analyses are essential to validate conclusions.     

The use of random amplified polymorphic DNA markers in wheat

Summary An evaluation was made of the use of random amplified polymorphic DNA (RAPD) as a genetic marker system in wheat. Reproducible amplification products were obtained from varietal, homozygous single chromosome recombinant line and wheat/alien addition line genomic DNA with selected primers and rigorously optimized reaction conditions. Factors influencing the RAPD patterns are DNA concentration, Mg²⁺ concentration, polymerase concentration and denaturing temperature. In wheat, the non-homoeologous, non-dose responsive and dominant behaviour of RAPD products devalues their use as genetic markers for the construction of linkage maps, and the high probability that the amplified fragments derive from repetitive DNA limits their use as a source of conventional RFLP probes. However, RAPD markers will most certainly find many applications in the analysis of genotypes where single chromosomes or chromosome segments are to be manipulated.     

Recombinant bovine interferon alpha causes only a temporary prolongation of the lifespan of the corpus luteum

Abstract

A method is described for developing a sheep‐ vs. goat‐specific DNA marker using sequence characterized amplified regions (SCARs) derived from a random amplified polymorphic DNA (RAPD) marker from sheep DNA samples. A sheep 645 bp DNA fragment that was absent in goat DNA was identified by analyzing pools of sheep and goat DNA with RAPD primers. This fragment was cloned and partially sequenced to design extended, strand‐specific 24‐mer oligonucleotide primers. Each primer contained the original 10 bases of the RAPD primer and the following 14 internal bases. The pair of primers resulted in the amplification of a single band of 645 bp when used to amplify sheep DNA, and in no amplification when used to amplify goat DNA. These SCAR primers successfully amplified the equivalent of DNA from one nucleated sheep cell in a sample of 5000 nucleated goat cells. This level of sensitivity is especially desirable for research involving the detection of interspecific chimerism.     

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。     

Assessment of genetic diversity in 31 species of mangroves and their associates through RAPD and AFLP markers

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 31 species of mangroves and mangrove associates. Four AFLP primer combinations resulted in the amplification of 840 bands with an average of 210 bands per primer combination and 11 RAPD primers yielded 319 bands with an average of 29 bands per primer. The percentage of polymorphism detected was too high indicating the high degree of genetic variability in mangrove taxa both at inter- and intra-generic levels. In the dendrogram, species belonging to a particular family/ genus, taxa inhabiting similar habitats or having similar adaptations tended to be together. There were exceptions too; as many unrelated species of mangroves form clusters. The intrafamilial classification and inter-relationships of genera in the family Rhizophoraceae could be confirmed by molecular analysis. Both the markers RAPD and AFLP were found equally informative and useful for a better understanding of the genetic variability and genome relationships among mangroves and their associated species.     

Problems in the Use of RAPD to the Study of Genetic Diversity and Systematics

Rapid, simple and highly sensitive are the great advantages of RAPD (random amplified polymorphic DNA) method. However, its optimum condition of amplification varies greatly among different studies and, in turn, causes much debates in terms of its reproducibility and validation of its application to the study of systemetics. For this reason the authors tried to probe into some essential factors that might affect the result of RAPD assay. The results from the authors' genetic diversity research in Cathaya argyrophylla confirmed that (a) DNA templates, whether undergoing multiple steps of purification or not, showed the same RAPD profiles, hence, most of the steps would be unnecessary: (b) RNA, no matter deprived or not from the DNA templates, had no influence on amplification; (c) DNA templates with a wide range of concentration produced the same band patterns constantly; (d) DNA templates extracted from either fresh or dry leaves produced the same RAPD results. Therefore, it was concluded that RAPD exhibited very good reproducibility. Furthermore, the authors had also discussed some problems in product analysis and data analysis of RAPD. The use of RAPD method in taxonomic study was evaluated in the study on the relationships of congeneric species of Cimicifuga, Actaea asatica and Aconitum sungpanense, and the applicability of RAPD to systematic studies of species and closely related genera, although with some limitations, was highly appreciated.     

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.     

Linear nicking endonuclease-mediated strand-displacement DNA amplification

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly.     

Peracetic acid stress-induced genetic rearrangements in Escherichia coli H10407 detected by RAPD and RFLP analyses

The discriminatory powers of random amplified polymorphic DNA (RAPD) analysis and restriction fragment length polymorphism (RFLP) were assessed for the detection and comparison of DNA modifications caused by an oxidative stress. DNA extracted from peracetic acid (PAA)-treated Escherichia coli H10407 was randomly amplified with the 10-mer primer OPZ14, which generated one stress-induced fragment. RFLP and RAPD profiles were hybridized by Southern blotting with the digoxigenin-labelled RAPD product. Untreated and PAA-treated cells had difference band profiles. The results indicate that RAPD analysis could be used as a discriminatory tool for investigating genetic rearrangements in E. coli caused by oxidative stress and that RFLP analysis could be used to confirm the rearrangements.     

RAPD-marking of genomes of representative Hordeum species

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum. High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgare sensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between the Hordeum species, between the H. spontaneum populations, and between regional H. vulgare cultivars and a dendrogram was constructed.     

PCR-based molecular markers for assessment of somaclonal variation in <Emphasis Type="Italic">Pinus pinea</Emphasis> clones micropropagated <Emphasis Type="Italic">in vitro</Emphasis>

Four different markers [random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and selective amplified microsatellite polymorphism length (SAMPL)] were applied for evaluating somaclonal variation of micropropagated genotypes of stone pine (Pinus pinea L.). The total number of primers tested was 130, with 223 combinations assayed. A high number of them amplified successfully (178), representing 79.82 % of the total, and the average number of amplified fragments ranged from 2.47 (ISSR) to 65.76 (SAMPL). Based on internal controls, no problem of reproducibility was detected. Almost no somaclonal variation was detected within the clones. Of the tested markers, ISSR, AFLP, and SAMPL showed monomorphic amplification profiles, with only RAPD markers showing some interclonal variation.     

一种随机引物联合多特异性DNA片段检测结核分支杆菌的新方法

目的:针对目前结核性疾病实验室诊断的局限性,探索一种更为敏感和特异的结核分枝杆菌DNA检测新方法。方法:选取10株江苏地区流行的结核分支杆菌(MTB)菌株,选取临床其他常见菌株及分枝杆菌菌株作为对照组,分别提取DNA作为随机引物的模板。参考国内、外文献设计12条随机引物,并分别对MTB及对照菌株进行单个引物随机扩增,2%的琼脂糖凝胶电泳对扩增产物进行分离并切胶纯化,通过TA克隆将纯化片段连接到质粒pEASYTM-T5 Zero并进行测序,通过BLAST-nr比对验证是否为MTB DNA片段。按照所确定的MTB片段序列,在其内部设计、合成一对特异性引物。用此特异性引物扩增对应的随机引物扩增产物,获得MTB特异性条带图谱。并将该方法检测的敏感性和特异性与临床上常用的real-time PCR进行比较。结果:经BLAST-nr比对,随机引物IS986F,S535及IS986R扩增的条带与MTB DNA有高度同源性(均为99%)。随机引物IS986F、S535和IS986R分别联合其特异性引物可以检测稀释105倍、105倍和103倍的MTB DNA,其特异性分别为100%、90%和80%。常规real-time PCR可检测出稀释104倍的MTB DNA。结论:随机引物IS986F联合其特异性引物检测结核分枝杆菌的灵敏度和特异性优于S535、IS986R两组,特异性为100%,且灵敏度优于常规real-time PCR法。     

Advances in molecular marker techniques and their applications in plant sciences

Detection and analysis of genetic variation can help us to understand the molecular basis of various biological phenomena in plants. Since the entire plant kingdom cannot be covered under sequencing projects, molecular markers and their correlation to phenotypes provide us with requisite landmarks for elucidation of genetic variation. Genetic or DNA based marker techniques such as RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), SSR (simple sequence repeats) and AFLP (amplified fragment length polymorphism) are routinely being used in ecological, evolutionary, taxonomical, phylogenic and genetic studies of plant sciences. These techniques are well established and their advantages as well as limitations have been realized. In recent years, a new class of advanced techniques has emerged, primarily derived from combination of earlier basic techniques. Advanced marker techniques tend to amalgamate advantageous features of several basic techniques. The newer methods also incorporate modifications in the methodology of basic techniques to increase the sensitivity and resolution to detect genetic discontinuity and distinctiveness. The advanced marker techniques also utilize newer class of DNA elements such as retrotransposons, mitochondrial and chloroplast based microsatellites, thereby revealing genetic variation through increased genome coverage. Techniques such as RAPD and AFLP are also being applied to cDNA-based templates to study patterns of gene expression and uncover the genetic basis of biological responses. The review details account of techniques used in identification of markers and their applicability in plant sciences.     

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.     

Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.     

Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA

Twenty nine isolates of Fusarium spp. (twenty four of them belonging to the Gibberella fujikuroi complex) isolated from banana and corn from different geographical regions were analyzed for their ability to produce fumonisins B1 and B2 and for genetic relatedness using random amplified polymorphic DNA (RAPD) and restriction analysis of PCR amplification products of the 5.8s ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II). For RAPD analysis, six of twenty oligonucleotide primers were selected after testing with five Fusarium spp. isolates and used to characterize 24 additional isolates. DNA fragments from the 29 isolates of Fusarium spp., which were approximately 560 bp, were amplified with the universal primers ITS1 and ITS4. The restriction enzymes HaeIII, MboI, HpaII and MspI were useful for distinguishing the isolates. The RAPD analysis permitted to find interspecific differences among the isolates of Fusarium spp., between isolates with low and high capacity of fumonisin production and among isolates from different hosts. The restriction fragment length polymorphism (RFLP-PCR) analysis permitted to distinguish among different species of Fusarium. In combination with morphological analysis, the results of this research may find an application for the diagnosis of unknown Fusarium spp. and, particularly, for the characterization of fumonisin-producing isolates, which may be very useful in the food technology field.