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D1/D2 sequencing

The D1/D2 region of the 26S subunit was previously adopted as a convenient mechanism for characterizing yeast to the species level. By analysing this sequence, Kurtzman and Robnett (1998) were able to identify a large number of yeast species based on sequence divergence within the D1/D2 domain. These authors demonstrated that strains belonging to a species exhibited less than 1% difference within the D1/D2 sequence, while distinct species had a much greater variation. Analysis of the D1/D2 domain can be conducted using a

Identification of yeast species based on analysis of ribosomal RNA gene sequences

Figure 11.7 Identification of yeast species based on analysis of ribosomal RNA gene sequences. Several sections of the rRNA gene region are variable between species but conserved between strains, including the D1/D2 domain of the 26S subunit, and the ITS and NTS regions. The D1/D2 domain is typically analysed by PCR followed by sequencing of the =600 bp fragment. The ITS region (including the 5.8S region) can be amplified in its entirety by primers designed to target the ends of the 18S (small subunit) and 26S (large subunit) ribosomal RNA genes (1). The PCR product is then cut using restriction enzymes (2) to yield a number of DNA fragments that can be resolved using gel electrophoresis (3). The sizes of each resulting fragment (along with that of the original PCR product) are determined by comparison to a DNA ladder (molecular weight marker). For analysis of species using ITS or NTS RFLP, or D1/D2 sequencing, identification is based on comparison to the literature, or to an internal or external database.

Table 11.6 Primer sequences employed for the identification of yeast species

Primers

Sequence

Target

Reference

ITS1

ITS4

5' TCC GTA GGT GAA CCT GCG G 3' 5' TCC TCC GCT TAT TGA TAT GC 3'

ITS region

White et al. (1990), Kurtzman and Robnett (1991)

ITS4

ITS5

  • 5' TCC TCC GCT TAT TGA TAT GC 3'
  • 5' GGA AGT AAA AGT CGT AAC AAG G 3'

ITS region

White et al. (1990), Scoch et al. (2012)

r-1234

r-2516

5' AAC GGT GCT TTC TGG TAG 3' 5' TGT CTT CAA CTG CTT T 3'

NTS2 region

Nguyen and Gaillardin (1997)

NL1

5' GCA TAT CAA TAA GCG GAG GAA AA 3'

D1/D2

O’Donnell (1993), Kurtzman and Robnett

NL4

5' GGT GCG TGT TTC AAG ACG G 3'

Domain

(1998)

combination of PCR and DNA sequencing. PCR is required to amplify the region of interest, generating an amplicon of =600 bp based on targeted primers (Table 11.6). This region is then sequenced to allow for the generation of qualitative data that can be analysed using sequence alignment software or compared directly to a database. There are

several sources of reference that can be used, many of which are freely available online. This is most easily performed by conducting a BLAST (Basic Local Alignment Search Tool) analysis of deposited sequences that are maintained in databases such as GenBank or the YeastIP gene database for molecular taxonomy and phylogeny of yeasts (Weiss et al.,

2013). Either approach can be used to calculate phylogenetic relationships, as well as for the identification of unknown species.

 
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