Segregation analysis of random amplified polymorphic DNA (RAPD) markers in Picea abies Karst.

The reliability of arbitrarily primed amplification products was tested. The segregation analysis of 266 amplification products obtained using 17 different 10-mer oligonucleotides in 34 megagametophytes from a single tree of Picea abies was carried out. Fifty-four out of the 165 variable bands fit the 1:1 segregation ratio expected for Mendelian traits. The segregation ratio of a subset of six RAPD markers in five other individuals from the same population confirmed their genetic nature. Our results strengthen the evidence previously reported that RAPDs markers can be considered Mendelian traits useful in the detection of genetic variability among both different individuals and populations.     

Strain typing of Lentinula edodes by random amplified polymorphic DNA assay

Abstract Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shiitake mushroom, Lentinula edodes . Seven primers produced polymorphisms in all 15 strains tested, producing 12–19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applications in mushroom breeding and strain improvement programs.     

Identification and genetic homogeneity of Trichophyton tonsurans isolated from several regions by random amplified polymorphic DNA

Trichophyton tonsurans is an anthropophilic dermatophyte mostly causing tinea capitis and tinea corporis. This study was carried out to identify T. tonsurans and to clarify whether it has any genetic differences depending on the phenotype or region of isolation by random amplified polymorphic DNA (RAPD) analysis with three random primers. The assay was performed in 11 Korean, 2 Japanese, 2 Taiwanese, 5 Brazilian and 1 American isolates of T. tonsurans together with the other 10 anamorphic species of dermatophytes and 3 Arthroderma spp. All tested species of dermatophytes showed distinct bands and T. tonsurans was differentiated from other dermatophytes. It was most clearly ifferentiated from T. mentagrophytes by using primer 5-GAAGGCTCCC-3 (OPAO-15). No difference was found in RAPD band patterns in all strains of T. tonsurans with these random primers. It was considered that T. tonsurans is a genetically homogeneous species regardless of its isolation regions, morphologic or physiologic characteristics.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.     

Genetic diversity study of Chromobacterium violaceum isolated from Kolli Hills by amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD)

Aim: Chromobacterium are saprophytes that cause highly fatal opportunistic infections. Identification and strain differentiation were performed to identify the strain variability among the environmental samples. We have evaluated the suitability of individual and combined methods to detect the strain variations of the samples collected in different seasons. Methods and Results: Amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD) profiles were obtained using four different restriction enzyme digestions (AluI, HaeIII, MspI and RsaI) and five random primers. A matrix of dice similarity coefficients was calculated and used to compare these restriction patterns. ARDRA showed rapid differentiation of strains based on 16S rDNA, but the combined RAPD and ARDRA gave a more reliable differentiation than when either of them was analysed individually. Conclusion: A high level of genetic diversity was observed, which indicates that the Kolli Hills’C. violaceum isolates would fall into at least three new clusters. Significance and Impact of the Study: Results showed a noteworthy bacterial variation and genetic diversity of C. violaceum in the unexplored, virgin forest area.     

Genetic relatedness among dioecious Ficus carica L. cultivars by random amplified polymorphic DNA analysis,and evaluation of agronomic and morphological characters

A collection of 64 fig (Ficus carica L.) accessions was characterized through the use of RAPD markers, and results were evaluated in conjunction with morphological and agronomical characters, in order to determine the genetic relatedness of genotypes with diverse geographic origin. The results indicate that fig cultivars have a rather narrow genetic base. Nevertheless, RAPD markers could detect enough polymorphism to differentiate even closely related genotypes (i.e., clones of the same cultivar) and a unique fingerprint for each of the genotypes studied was obtained. No wasteful duplications were found in the collection. Cluster analysis allowed the identification of groups in accordance with geographic origin, phenotypic data and pedigree. Taking into account the limited information concerning fig cultivar development, the results of this study, which provide information on the genetic relationships of genetically distinct material, dramatically increase the fundamental and practical value of the collection and represent an invaluable tool for fig germplasm management.     

Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants

Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoog's basal medium (MS) supplemented with dicamba, picloram (2 mg l⁻¹) or 1-naphthaleneacetic acid (NAA) (5 mg l⁻¹) in combination with benzyladenine (BA) (0.5 mg l⁻¹). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l⁻¹) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l⁻¹), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Occurrence of larval Pacific lamprey Entosphenus tridentatus from Japan, detected by random amplified polymorphic DNA (RAPD) analysis

Species identifications of the Pacific lamprey Entosphenus tridentatus from four other Japanese lampreys, Lethenteron japonicum, L. kessleri, and two undescribed Lethenteron species (L. sp. N and L. sp. S), were carried out on the basis of random amplified polymorphic DNA (RAPD) using the polymerase chain reaction (PCR) method. Of 65 RAPD loci, seven loci possessing species-specific fragments were obtained for E. tridentatus. Based on these RAPD loci, four larval individuals of E. tridentatus from the Naka River (eastern Honshu Island, Japan) were recognized in 2001 and 2002. The existence of larval individuals of E. tridentatus, as well as spawning adults previously reported from the same river, indicated the possibility of residence in that species.     

Application of random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis for high-resolution identification of <Emphasis Type="Italic">Monascus</Emphasis> strains

Monascus fungi are commonly used for a variety of food products in Asia, and are also known to produce some biologically active compounds. Since the use of Monascus is expected to increase in food industries, strain-level identification and management of Monascus will be needed in the near future. In the present study, random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis was applied for this purpose. Evaluations of the analysis stability revealed that reproducible results could be obtained, although template DNA fragmentation could influence the resulting RAPD pattern. RAPD analysis using 15 Monascus strains consisting of four species, M. ruber, M. pilosus, M. purpureus, and M. kaoliang showed that each strain generated a unique RAPD pattern, which allows strain-level identification of Monascus. In addition, the phylogenetic tree constructed from RAPD patterns reflected M. ruber–M. pilosus and M. purpureus–M. kaoliang clusters inferred from both ITS and β-tubulin gene sequences, which indicated that the RAPD pattern could reflect their phylogenetic traits to a certain extent. On the other hand, RAPD analysis did not support the monophyletic clustering of the four Monascus species used in this study, which suggests the necessity of reexamination of species boundaries in Monascus.