Discrimination of species and strains of basidiomycete genusCoprinus by random amplified polymorphic DNA (RAPD) analysis

All five examined strains ofCoprinus cinereus could be clearly discriminated from the strains of five otherCoprinus species by RAPD patterns with 12 of 13 primers. Also one specimen of unknownCoprinus strain was identified to beC. cinereus by this method. The RAPD patterns were similar among the strains in the same species; many common DNA fragments were recognized as well as some strain-specific DNA fragments. Thus all seven strains ofC. cinereus and all four strains ofC. angulatus examined could be distinguished individually. Diakryotic strains showed the combined RAPD patterns of the two monokaryotic strains constituting the dikaryon. The combined RAPD markers observed in the dikaryons were segregated in their basidiospore progeny. All 18 randomly picked progeny showed different combinations of RAPD markers from the parental strains.     

Genome relationships among Lotus species based on random amplified polymorphic DNA (RAPD)

The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lotus was evaluated for several geographically dispersed accessions of four diploid Lotus species, L. tennis Waldst. et Kit, L. alpinus Schleich., L. japonicus (Regel) Larsen, and L. uliginosus Schkuhr and for the tetraploid L. corniculatus L., in order to ascertain whether RAPD data could offer additional evidence concerning the origin of the tetraploid L. corniculatus. Clear bands and several polymorphisms were obtained for 20 primers used for each species/accession. The evolutionary pathways among the species/accessions presented in a cladogram were expressed in terms of treelengths giving the most parsimonious reconstructions. Accessions within the same species grouped closely together. It is considered that L. uliginosus which is most distantly related to L. corniculatus, may be excluded as a direct progenitor of L. corniculatus, confirming previous results from isoenzyme studies. Lotus alpinus is grouped with accessions of L. corniculatus, which differs from previous studies. With this exception, these findings are in agreement with previous experimental studies in the L. corniculatus group. The value of the RAPD data to theories on the origin of L. corniculatus is discussed.     

Randomly amplified polymorphic DNA (RAPD) for rapid typing of Lactobacillus plantarum strains

Randomly amplified polymorphic DNA (RAPD) has been used for rapid typing of Lactobacillus plantarum strains. RAPD was used with either purified chromosomal DNA serving as template in the polymerase chain reaction, or with crude cell extracts, and using a 9-mer primer with 80% G + C content. Amplified DNA was visualized by ethidium bromide staining after separation on agarose gels. Patterns from 20 Lact. plantarum strains and two Lact. pentosus strains were analysed using the Pearson products moment correlation coefficient ( r ) and the unweighted pair group method with arithmetic averages (UPGMA). With some exceptions, the two sources of template DNA gave the same clusters and subclusters of strains at the similarity level of 50%. About 50% of the strains could be individually separated from all the other tested strains. The buffer brand, the amount of primer and crude cell extract used in the PCR-step were crucial for the final pattern.     

Assessment of random amplified polymorphic DNA (RAPD) for determining the origin ofYoungia koidzumiana Kitamura (compositae)

We determined the parental species ofYoungia koidzumiana (a natural interspecific hybrid) using PCR and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. These markers, generated by three primers, were sufficient to distinguishYoungia sonchifolia, Youngia denticulata, Youngia chelidoniifolia, andY. koidzumiana. The electrophoresis profiles of the amplified products from each of the four species were then compared. Three primers produced a total of 42 scorable markers; nine were specific markers forY. denticulata andY. chelidoni-ifolia. The length of the amplified DNA fragments ranged from 370 to 2500 b p. The three primers revealed polymorphic bands, which were indicators of the parental species ofY. koidzumiana. These bands showed a combination of specific profiles forY. denticulata andY. chelidoniifolia. Our results also were comparable to the data obtained for flowering times, floret numbers, and chromosome numbers of the four species. Therefore, we suggest thatY. koidzumiana is a hybrid betweenY. denticulata andY. chelidoniifolia}, and that RAPD markers are well suited for assessing the origins of plant species.     

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.     

Use of random amplified polymorphic DNA (RAPD) markers in the study of the parasitic weed Orobanche

Despite the tremendous economic impact of broomrapes (Orobanche spp.) on agriculture in many countries little is known of the pattern of genetic variation within this group of parasitic weeds. The present paper describes the use of RAPD markers for the study of five Orobanche species in agricultural fields in Israel. Pronounced genetic differentiation was found between the species, and RAPD markers were raised for the identification of each of them. Southern-hybridization patterns of RAPD products of the various species were used to confirm the interpretation. The same markers were valid both for broomrapes collected in agricultural fields and for those collected in natural habitats. The validity of the markers found for O. cumana and O. crenata was confirmed on plants of the same species that were collected in Spain. Parsimony analysis of 86 RAPD characters produced a tree that clearly distinguishes between the five studied Orobanche species, separates the two Orobanche species belonging to sect. Trionychon from those belonging to sect. Osproleon, and supports the separation of O. cumana from O. cernua and of O. aegyptiaca from O. ramosa.     

Evaluation of random amplified polymorphic DNA for species identification and phylogenetic analysis inStylosanthes (Fabaceae)

Random amplified polymorphic DNA (RAPD) was assessed for its suitability as a tool to be used in the identification of taxa from the genusStylosanthes (Fabaceae, Papilionoideae, Aeschynomeneae). Five random primers were used to fingerprint accessions from seven species in the genus, and generated RAPD profiles that were species-specific. Data were used to examine evolutionary relationships between taxa, employing both clustering and ordination techniques, and the results were compared with those from a previous cladistic analysis of chloroplast DNA (cpDNA) restriction fragments. Both multivariate approaches indicated relationships that were generally similar to those obtained by RFLP analysis of cpDNA. However, while cluster analysis grouped together all accessions within species, ordination placed certain accessions ofS. humilis, S. macrocephala andS. capitata into separate groups. Experiments to test the assumed homology of comigrating RAPDs estimated 85.7% homology for accessions within species, and 53.8% homology for accessions between species. The value of RAPD data in systematics is discussed.     

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA (RAPD) analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China. A captive population has been established in the Beijing Zoo since 1999. In order to determine the kinship of the offsprings in 2001, randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo. To amplify the genomic DNA of each individual, 53 arbitrary primers were selected. The results of amplifications showed that 14 primers had clear and distinct RAPD patterns. Totally, 226 amplified fragments were generated by RAPD in this study. Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male). This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds. __________ Translated from Journal of Beijing Normal University (Natural Science), 2005, 41(5): 507–509 [译自: 北京师范大学学报 (自然科学版), 2005, 41(5): 507–509]     

Use of random amplified polymorphic DNA (RAPD) markers in the discrimination and verification of genotypes in Eucalyptus

We carried out four separate studies using random amplified polymorphic DNA (RAPD) markers to analyse samples of Eucalyptus supplied by several different organisations. The objective was to examine the reproducibility of the RAPD technique and its ability to discriminate between individual genotypes for verification of clonal identities. We found that RAPD profiles that are unique to a genotype can be generated reliably and simply and that even closely related genotypes can be distinguished. In addition, in each of the four studies, we detected cases where the plant material studied had been mis-sampled or mis-labelled (i.e. the RAPD profiles were not consistent with the identification numbers): (1) ramets of a Eucalyptus grandis clone were found to be derived from 2 different clones; (2) ramets labelled as 2 different Eucalyptus hybrid clones were found to be the same clone, owing to a mis-planted clonal hedge; (3) samples supplied as a single progeny of a controlled E. nitens cross were derived from two crosses involving different pairs of parents; (4) mis-labelling was detected for ramets of 4 of a set of 10 clones of E. grandis and E. camaldulensis. For three of the four studies, the detection of genotype mis-identifications was unexpected, suggesting that labelling or sampling errors during the handling of plant material are a frequent occurrence, with potentially serious economic consequences.     

Randomly amplified polymorphic DNA (RAPD) profiling of Plasmodiophora brassicae

M. MÖLLER AND R. HARLING. 1996. A technique is described for the preparation of DNA suitable for use in RAPD analysis from pure, sterile resting spores of Plasmodiophora brassicae . Using this technique, random 10-base pair primers were applied to P. brassicae DNA from three single spore isolates. The resulting profiles were compared with the race classification based on inoculation of the European Clubroot Differential (ECD) series of Brassica hosts. Out of 40 primers tested, 23 gave amplification products, three gave isolate-specific profiles and one a profile which corresponded with the ECD race classification of the isolates. RAPD profiling can provide a faster means of race classification in P. brassicae .     

葡萄早熟芽变品种“早生高墨”的RAPD分析

以中国野生葡萄的部分株系、河岸葡萄、砧木品种S04和欧洲葡萄部分品种的幼叶为试材,采用改良CTAB法,提取葡萄基因组DNA的完整性好、纯度高。以所提葡萄基因组DNA为模板,共筛选随机寡核苷酸引物180个,对葡萄品种高墨及其早熟芽变品种早生高墨(紫玉)进行RAPD分析,结果发现引物OPW02和OPG06在两者之间扩增出了与葡萄早熟性状相关的多态性DNA片段OPW02—590、OPG06—1300和OPG06—400。这为进一步克隆、测序并分析它们的序列构成,了解葡萄早熟芽变的分子机理乃至其它果树植物芽变的分子机理提供了分子依据。     

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.     

Evaluation of genetic relationship in Typhonium species through random amplified polymorphic DNA markers

Studies were undertaken to identify genetic relationships in three species of Typhonium and to evaluate the genetic variance within populations of Typhonium trilobatum, Typhonium roxburghii and Typhonium flagelliforme by using random amplified polymorphic DNA (RAPD) markers. A total of 193 distinct DNA fragments ranging from 0.2 to 3.2 kb, were amplified using 22 selected random decamer primers. The cluster analysis indicated that the three species of Typhonium formed two clusters: the first one consisted of T. trilobatum and T. roxburghii, the second one was represented by T. flagelliforme. A maximum similarity of 63 % was observed in T. trilobatum and T. roxburghii. T. flagelliforme shared up to 43 % similarity with T. trilobatum and T. roxburghii. The closest genetic distance was obtained within populations of different Typhonium species.     

DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.     

Use of random amplified polymorphic DNA analysis to detect genetic variation inPyrus species

Pyrus communis L. is the most important pear species for European production. Very few cultivars satisfy standards for fruit quality and clonal fidelity; thus, accurate verification of cultivar identity for checking propagation material and patent protection is important. We evaluated the randomly amplified polymorphic DNA (RAPD) technique for its ability to identify genetic differences among standard pear (Pyrus communis L.) cultivars, William, Passa Crassana, and Conference, and three gamma-ray induced variants. To identify genotype-specific markers, we used thirty 10-mer and two 11-mer sequences, annealing temperatures from 36–45°C, 2Taq polymerases (AmpliTaq and Stoffel fragment, both from former Perkin Elmer Cetus), and 2–4 replicate amplifications. Of the 32 primers (30 from Operon Technologies, Alameda, CA, USA), very few distinguished William from Passa Crassana, and only 1 could clearly differentiate all 3 cultivars. Two primers that did not reveal polymorphisms when used singly, generated polymorphic patterns that distinguished standard from gamma-ray-treated material when used in combination. We show that RAPD analyses can discriminate pear genotypes and suggest this technique as a reliable and inexpensive method for marker-facilitated screening of propagation material and for patent protection.     

Molecular characterisation of Sargassum polycystum and S. siliquosum (Phaeophyta) by polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers

Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10) tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships between species within a genus and in developing individual fingerprints for individual samples.     

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.     

Random amplified polymorphic DNA (RAPD) identification of genetic variation in three species ofPorphyra (Bangiales,Rhodophyta)

The random amplified polymorphic DNA (RAPD) technique was used to characterize three species ofPorphyra from the western North Atlantic and adjacent Gulf of Mexico. Twenty 10-mer primers were screened for DNA amplification usingPorphyra template DNA. Nine of these oligonucleotide primers, all (G+C)-rich, were positive or band-producing, but yielded poor or variable band resolution. Subsequent use of the universal 20-mer M 13 primer resulted in both clear band resolution with a minimum of secondary bands and a high degree of reproducibility. Amplification products for DNA from six regional isolates ofPorphyra carolinensis Coll et Cox,P. leucosticta Thuret in Le Jolis andP. rosengurttii Coll et Cox were compared to each other and toBangia atropurpurea (Roth) C. Agardh. Results provide evidence of both genetically hetero- and homogeneous populations. Use of the RAPD method with the M 13 primer yields amplification products which can be used to fingerprint specific genotypes. This procedure could be used to discriminate between hetero- and homokaryotic fusion products from previously characterized donor strains.     

Organellar DNA restriction fragment length polymorphism (RFLP) and nuclear random amplified polymorphic DNA (RAPD) analyses of morphotypes of Gracilaria (Gracilariales,Rhodophyta) from Chile

The extreme phenotypic variability recognized among the species of Gracilaria has highlighted the need for the application of refined methods to help solve taxa identifications. In Chile, there still exists uncertainty about the exact number of Gracilaria species. Our investigations are centered on DNA analyses of morphotypes collected from different geographical locations, namely Lenga and Isla Santa María, Region VIII (36°00 S to 38°00 S), and Maullín, Region X (39°30 S to 43°40 S). These two regions of Chile are considered as areas of confluence of G. chilensis, G. verrucosa, and a species of Gracilariopsis. In this study four morphotypes, from a natural bed located in Maullín, were analyzed for RFLP of plastid DNA and the results compared with data of four morphotypes from a bed in Lenga. The DNA banding patterns from each enzyme digest were identical irrespective of morphotypes and/or locations. In an attempt to unravel the nature of the morphological differences found among Lenga and Maullín morphotypes, RAPD analyses of nuclear DNA were also performed; however, no polymorphism has been found yet. Therefore, the data of this study, as well as concurrent data from preliminary interfertility tests, suggest that all morphotypes belong to a single taxon, Gracilaria chilensis.Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas     

Random amplified polymorphic DNA (RAPD) markers reveal genetic homogeneity in the endangered Himalayan species Meconopsis paniculata and M. simplicifolia

Random amplified polymorphic DNA (RAPD) marker-based analysis was carried out to study the extent of genetic polymorphism between populations of the two endangered Himalayan poppy species, Meconopsis paniculata and M. Simplicifolia. Of the 90 primers tested, 38 revealed marked inter-species genetic polymorphism between individuals of the two species from geographically isolated populations. However, intra-species genetic homogeneity was also evident with respect to a number of primers both within and between populations. A comprehensive analysis incorporating data from RAPDs, DNA fingerprinting and isozyme pattern was carried out and, based on the presence or absence of bands, three matrices of similarity indices were estimated. These matrices were subsequently utilized in cluster analysis. In order to compare the three clusters generated using these three different marker systems, a Mantel matrix-correspondence test was carried out on the basis of comparisons of co-phenetic values. The overall representation of relationships by cluster analysis was similar for all three marker systems and this was substantiated by high correlations among the three analyses revealed by the Mantel matrix-correspondence test. Our results point to very low or absence of, genetic polymorphism in M. paniculata and M. simplicifolia, and are in broad agreement with our previous observations on genetic diversity of Meconopsis species which point to a genetic basis for the possible extinction of this economically important genus.