Two Subgroups within the Saccharomyces bayanus Species Evidenced by PCR Amplification and Restriction Polymorphism of the Non-Transcribed Spacer 2 in the Ribosomal DNA Unit

Abstract

Summary A PCR-RFLP method has been improved for the rapid identification of the four species of Saccharomyces sensu stricto . We used the NTS2-ETS sequence of the rDNA as target and the amplification reaction was realized by chosing a pair of primers that could anneal to the end and the beginning of the 5S and 18S conserved sequences, respectively. PCR products obtained from S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus (synonym S. carlsbergensis ) type strains displayed a clear polymorphism when digested with several restriction enzymes. Using only three enzymes: Ban I, Alu I and Taq I we were able to differentiate S. cerevisiae, S. paradoxus and the S. bayanus/S. pastorianus complex which shared the same pattern. The S. bayanus taxon now regroups the former species S. abuliensis, S. globosus, S. heterogenicus, S. intermedius, S. inusitatus, S. uvarum . PCR-RFLP of these species showed that, except for S. abuliensis and S. uvarum , they shared the same patterns as S. bayanus type strain. That lead us to conclude that there are two subgroups in S. bayanus and that S. abuliensis and S. uvarum belong to a distinct subgroup. Remarkably, wine strains that were previously identified as S. bayanus by genetic hybridization techniques, as well as strains isolated from cider fermentation, belong to the S. uvarum subgroup.