Ribosomal DNA Restriction Analysis Reveals Genetic Heterogeneity in Saccharomyces cerevisiae Meyen ex Hansen

Abstract

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...

Rapid Characterization of Four Species of the Saccharomyces Sensu Stricto Complex According to Mitochondrial DNA Patterns

“…Furthermore, the 900-base sequences from S. pastonanus and S. bayanus were identical, and those from S. cerevisiae and S. paradoxus differed in only five nucleotides. rDNA restriction analysis from PCR-amplified fragments (13,(23)(24)(25) has also been used recently to differentiate between strains belonging to S. cerevisiae, S. bayanus, and S. pastorianus.…”

“…These include DNA fingerprinting (5,(31)(32)(33)(34)42), chromosomal DNA profiles (7,9,14,15,21,27), mitochondrial DNA (mtDNA) restriction analysis (22,48), and rRNA and ribosomal DNA (rDNA) sequencing (18) as well as restriction analysis (13,(23)(24)(25). Until now, only electrophoretic karyotyping, rRNA sequencing and rDNA restriction analysis have proved useful for differentiating Saccharomyces species.…”

Phylogenetic analysis of the Saccharomyces cerevisiae group based on polymorphisms of rDNA spacer sequences

“…However well-separated the ' cerevisiae' and 'bayanus' clusters may be, they consist of very closely related yeasts when the whole genus Saccharomyces is considered (7,11,12,13). Thus, a phylogenetic tree based on the 18s gene sequences of species belonging to Saccharomyces and related genera showed that the genus Saccharomyces is very heterogeneous, whereas the species of the Saccharomyces sensu stricto complex form a natural group quite separated from other Saccharornyces and non-Saccharomyces species examined (7).…”

Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers

“…Because ribosomal regions evolve in a concerted fashion, they show a low intraspecific polymorphism, and a high interspecific variability (Li, 1997) has been proved very useful for the classification of Saccharomyces species (Huffman et al, 1992;Molina et al, 1992;Valente et al, 1996;Wyder & Puhan, 1997), Kluyveromyces species (Belloch et al, 1998) and, recently, for the identification of a small collection of wine yeast species (Guillamon et al, 1998).…”