ITS1-5.Captisol ic50 8S-ITS2 was PCR amplified as mentioned elsewhere. Amplification of D1/D2 region was carried out using primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′)

as previously described [43]. The amplified products were purified using NucleoSpin® Extract II gel extraction kit (Machery-Nagel, Düren, Germany) following manufacturer’s instructions. The PCR products were sequenced using ABI 3100 Genetic Analyser (Merck, Bangalore, India) with the same primers used for the amplification. The sequence reads were validated by analysing the electropherogram data using ChromasLITE software, version 2.01 (http://​technelysium.​com.​au/​). To identify the closest known relatives, the sequences were queried with NCBI and CBS yeast nucleotide databases. The sequences obtained from both sequencing and nucleotide databases were aligned using Clustal X algorithm and a neighbour joining tree was constructed by Kimura’s evolutionary TPCA-1 in vitro distance matrix obtained from the multiple sequence alignment using MEGA4 phylogenetic software. Bootstrap values selleck for 1000 replicates were shown at the node of cluster branch. The sequences were deposited to NCBI GenBank under the following accession numbers: JF439366 − JF439369 and KF268351 − KF268354. Results In silico selection of differentiating restriction enzymes The full length ITS1-5.8S-ITS2 sequences of M. guilliermondii and M. caribbica were retrieved

from NCBI and CBS yeast nucleotide databases and subjected to multiple sequence alignment followed by in silico restriction digestion. Three variable regions differentiating M. guilliermondii from M. caribbica were identified. Seven restriction enzymes (ArsI, BfaI, BsrI, Hpy188I, HpyCH4III, MmeI and TaqI) which cut the variable regions differently were identified (Figure 1A and Additional file 2: Figure S2). Considering the length and the number of polymorphic fragments with sizes greater than 100 bp (for easy analysis in normal agarose gel), BfaI, MmeI and

TaqI were found appropriate. Notably, commonly available TaqI gave distinct species-specific differentiation between the two species (Additional file 1: Table S4 and Additional file 2: Figure S3). We also tested the selected three restriction enzymes (BfaI, MmeI and Tau-protein kinase TaqI) for differentiating M. guilliermondii and M. caribbica from other closely related members of M. guilliermondii complex (Additional file 1: Table S4). Except C. carpophila and M. caribbica, all other members of M. guilliermondii complex were distinctly differentiated during the analysis. Figure 1 Differentiation of M. guilliermondii and M. caribbica by Taq I digestion of ITS1-5.8S-ITS2. A: Multiple sequence alignment of representative ITS1-5.8S-ITS2 sequences of the two species obtained from NCBI GenBank and CBS yeast database showing position of TaqI recognition site (highlighted) which distinctly differentiated the two species. B: TaqI restriction digestion profile of ITS1-5.