Advances in Food Mycology

72 Ai Lin Beh et al.
2000; Scorzetti et al., 2002) yeast species. Sequencing of the D1/D2
domain of the 26S ribosomal DNA is now widely used for the routine
identification of yeasts and the construction of phylogenetic taxonomy.
Sequence comparisons have also been done for the small subunit,
18S ribosomal DNA but, so far, the databases are not extensive and
sequence differences may not be sufficient to allow the discrimination
of closely related species (James et al., 1997; Naumov et al., 2000;
Daniel and Meyer, 2003). The ribosomal spacer regions (ITS) show
higher rates of sequence divergence than the D1/D2 domain of the
26S subunit and have proven useful for species differentiation (James
et al., 1996; Naumov et al., 2000; Cadez et al., 2003). For example, the
Hanseniaspora uvaurm-guillermondii cluster is poorly resolved, and
species of Saccharomyces pastorianus/Saccharomyces bayanus, and
Cryptococcus magnus/Filobasidium floriforme/Filobasidium elegans are
indistinguishable using D1/D2 sequences. Sequencing of the ITS
region can provide the required level of differentiation.
Sequencing of mitochondrial and protein encoding genes are also
being used to determine phylogenetic relationships among yeasts.
These genes include the translation elongation factor 1α, actin-1,
RNA polymerase II, pyruvate decarboxylase, beta tubulin gene, small
subunit rDNA and cytochrome oxidase II (Daniel et al., 2001;
Kurtzman and Robnett 2003; Daniel and Meyer 2003).
The basic protocol for sequencing ribosomal DNA segments is: (i)
prepare a pure culture of the yeast isolate, (ii) extract and purify the
DNA, (iii) perform PCR amplification of the region to be sequenced,
(iv) verify the amplified product by gel eletrophoresis, and (v)
sequence the product using internal or external primers. Procedures
for conducting these operations are well established but are not standardised,
and may vary from one laboratory to another. Primer
sequences used to amplify the different segments of the rDNA have
been tabulated in White et al. (1990), Valente et al. (1999), Sipiczki
(2002) and Kurtzman and Robnett (2003). Table 1 lists some key publications
on the identification of yeasts by ribosomal DNA sequencing.
Some yeasts are not reliably identified by sequencing single gene
segments and it is suggested that sequences be obtained for multiple
genes or gene segments for more reliable data (Kurtzman, 2003).
2.2. Restriction Fragment Length Polymorphism
(RFLP)
RFLP analysis of the ribosomal DNA segments is emerging as one
of the most useful methods for rapidly identifying food and beverage