Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species of Saprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except for S. hypogyna, S. delica, S. lapponica, and S. mixta. The rDNA data indicate that S. lapponica and S. lapponica and S. mixta are conspecific with S. ferax, whereas there is no support for the proposed synonymies of S. diclina with S. delica and of S. mixta with S. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in the S. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains of S. parasitica originating from the U.K. and Japan. HhaI and BsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonuclease BstUI was diagnostic for S. parasitica, generating identical fingerprints for all stains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved in S. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed that S. diclina and S. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification of Saprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.

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A ribosomal DNA restriction analysis of 17 strains belonging to the Succhromyces cerevkiue complex revealed two major groups, one of which corresponded to Succharomyces buyunus. Our results generally correlate with previously reported genetic and molecular data and support the conclusion that S. buyunus should be reinstated as a separate taxon.The schemes currently used for classifying yeasts in the genus Saccharomyces are based largely on physiological features, such as fermentation reactions and resistance to cycloheximide (3,29). The use of these characteristics is necessitated by the paucity of morphological criteria. However, mounting evidence suggests that such characteristics may not always be adequate for species delimitation, and interstrain variability has been observed (3,7,18,21,29). The instability of distinguishing characteristics (22, 23) and the apparent absence of reproductive isolation (13) , most Saccharomyces species can be differentiated only by using DNA-DNA hybridization data. On the basis of DNA reassociation results, these authors recognized S. bayanus and S. pastorianus as species that are distinct from S. cerevisiae, a view shared by Banno and Kaneko (2).Because of the presence of both highly conserved and variable regions, rRNA sequences have been used as a tool for analyzing phylogenetic relationships (10, 11). In this study, we examined restriction fragment length polymorphisms in the gene that codes for the small-subunit ribosomal DNA (rDNA), and we obtained further evidence of genetic heterogeneity in S. cerevisiae. We also found that restriction patterns could be used to assign most of the strains which we examined to either S. cerevisiae or S. bayanus. The original specific epithets are used below.We studied 17 strains ( Table 1) of organisms that were considered to be either synonyms or physiological races of S. cerevisiae (3,29,30). S. kluyveri, a species that is thought to be an outlying member of the genus (lo), was included for comparison. The procedure used for DNA isolation and the conditions used for the polymerase chain reaction (PCR) have been described previously (12). The primers which we used were those described by White et al. (28) Sau3AI, ScrfI, and TaqaI. The restriction fragments were electrophoresed on 2.6% NuSieve 3:l agarose (FMC Bioproducts) containing PGEM markers (ProMega) as external standards, stained with ethidium bromide, and photographed. The numbers of nucleotide substitutions per site (genetic distances) were estimated by using the method of Nei and Tajima (16). The values were arrayed in a matrix, and a cluster analysis was performed by using the SAHN feature of the NTSYS-pc program (19). A phenogram was then generated by using unweighted pair-group arithmetic average clustering (15). In order to test the goodness of fit of the clustering to the data set, a cophenetic value matrix was constructed (20), and the MXCOMP program was used to compare this matrix with the original matrix.The PCR products from all of the strains were approximately 1,8...

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Polymorphisms in enzymatically amplified ribosomal DNA (rDNA) were examined in 18 strains of Saccharomyces. Restriction patterns generated from the region spanning the internal transcribed spacers (ITS) and the 5.8S rDNA produced two clusters corresponding to S. bayanus and S. cerevisiae. The type culture of S. carlsbergensis (ATCC 76529), which could not be separated from the S. cerevisiae group by small subunit (SSU) rDNA patterns, showed a ScrfI profile that was distinct from all the other strains. The type culture of S. intermedius var. turicensis (ATCC 76519) is assigned to S. bayanus on the basis of the combined results of SSU and ITS restriction analyses. S. kluyveri occurred at a separate branch of the distance tree and is unrelated to any of the strains. Results were in general agreement with reported DNA homologies and are discussed in relation to other molecular and genetic data.

Molecular evidence supports the separation of Lentinula edodes from Lentinus and related genera. Can. J. Bot. 70: 2446-2452.Restriction polymorphisms in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 18 strains of Lentinus, Neolentinus, Pleurotus, and the shiitake mushroom Lentinula edodes. The polymerase chain reaction was used to separately amplify the 18s rDNA and the region spanning the two internal transcribed spacers and the 5.8s ribosomal RNA gene. Amplified products were digested with a battery of 10 restriction endonucleases and the two data sets were subjected to cluster analysis. All strains of Lentinula edodes consistently exhibited identical restriction profiles that were distinct from those of the genera Lentinus, Neolentinus, and Pleurotus. The internal transcribed spacer region exhibited more variability than the 18s rDNA, giving distinctive profiles for two strains of Lentinus tigrinus and for one strain of Neolerltin~is lepide~cs. Similarity coefficients were clustered with the unweighted pair group method with arithmetic average, single-linkage method, and complete-linkage method. Results from cluster analysis of the two data sets were highly congruent and tree topologies were consistent irrespective of the clustering method used. The distinctiveness of the groups was further confirmed by computing for consensus trees and cophenetic correlation coefficients. Ribosomal DNA restriction polymorphisms support the placement of the strains examined in separate taxa.

The gene coding for the small-subunit rRNA of 11 type strains belonging to the genus Dekkera and its anamorph, Brettanomyces, was amplified by using the polymerase chain reaction and subjected to digestion with a series of restriction endonucleases. Similarity coefficients were calculated from the number of shared and unique fragments, and a cluster analysis yielded four distinct groups with the following ascosporogenous states: Dekkera anomah, Dekkera bruxellensis, Dekkera custersiana, and Dekkera naardenensis. Results correlate with evidence from isoenzyme electrophoresis and DNA homology analysis. They also confirm previously reported anamorph-teleomorph connections and recently proposed synonymies within the genus.Most classification schemes for the genus Dekkera, the ascosporogenous genus established by van der Walt in 1964 (20) to accommodate the sexual state of the genus Brettanoinyces, are based on morphological and physiological characteristics (2,20,21). In their recent account of the genus, Barnett and coworkers (2) recognized four species: Dekkera iinomala (syn. Dekkera claussenii), Dekkera bruxellensis (syn. Dekkera abstinens, Dekkera intermedia, and Dekkera lambica), Dekkera custersiana , and Dekkera naardenensis .