The restriction fingerprints were analysed for the absence or presence of discriminating fragments using GelCompar II software, version 6.5 (Applied Maths, Sint-Martens-Latem, Belgium). mtDNA-RFLP A single colony of 24 − 48 h old culture from YEPD agar was inoculated to Cilengitide price 5 mL of YEPD broth supplemented with antibiotics, and incubated for 18 h at 30°C with shaking at 200 rpm. The grown culture was inoculated into 50 mL of

fresh YEPD broth (initial OD600 = 0.1) and incubated in the above conditions till mid-logarithmic growth phase (final OD600 = 0.4 − 0.8). Cells of 20 OD600 were harvested at 1,800 g for 5 min at 4°C (A-4-81, Centrifuge 5810R, Eppendorf). The mtDNA was extracted as previously described [42] with some modifications. The cells were resuspended and washed with 5 mL of yeast resuspension buffer (50 mM Tris-Cl, 20 mM EDTA, pH 8.0) and stored at −20°C for 10 min. Lyticase (50 U) (Sigma-Aldrich) was used to produce spheroplast and 15 μL of 1 mg/mL RNase A solution (Sigma-Aldrich) was added during cell lysis. The total DNA was precipitated at −20°C for 1 h. After quantifying the DNA Vactosertib content spectrophotometrically, the DNA was freeze dried, re-dissolved in sterile deionized water to a final

concentration of 1 μg/μL and stored at −20°C till further use. Restriction digestion was carried out on 10 μg of the DNA in a 20 μL reaction volume using 10 U each of HaeIII and HinfI (Promega) according to manufacturer’s instructions. The restriction patterns were generated

by 1.0% (w/v) agarose gel electrophoresis of the 20 μL reaction volume at 80 V in 0.5× TBE buffer for 4 h in parallel with 1 kb DNA ladder (Promega). After staining and documentation, the restriction of fingerprints were subjected to cluster analysis using unweighted pair group method with arithmetic mean (UPGMA) algorithm on Jaccard similarity coefficients using GelCompar II. Composite data set of the restriction digestion profiles was generated with 1.0% position tolerance to generate the clustering. Bootstrap analysis with 1,000 replicates was performed to indicate the branch quality. Electrophoretic karyotyping Intact chromosomal DNA for electrophoretic karyotyping using PFGE was prepared as previously described [32]. The electrophoresis was carried out in 1.0% (w/v) PFGE-grade agarose gel (Sigma-Aldrich) and 0.5× TBE buffer at 13 − 14°C and 150 V in contour-clamped homogeneous electric field electrophoresis apparatus (Gene Navigator, Amersham Biosciences, Uppsala, Sweden). The gel was run for 22 h with a switch interval of 90 s for 8 h followed by 105 s for 6 h and finally 120 s for 8 h in parallel with PFGE marker (225 − 22,000 kb) from Saccharomyces cerevisiae strain YPH80 (Sigma-Aldrich). Staining and documentation were performed as mentioned elsewhere. ITS and D1/D2 sequencing and sequence analysis The representative RAD001 in vitro isolates from each ITS-RFLP genotype group were randomly selected for sequencing ITS1-5.