Abi 310 genetic analyzer

To investigate the population diversity of T. candicans in order to protect wild resources, we screened 11 cpDNA segments from noncoding regions and designed primers using Geneious 11.1.5 based on hyper-variable regions of the two cp genomes of T. candicans. More than 30 populations were sampled covering most distributions on record, and each population sampled one or two individuals. Total genomic DNA was extracted by an improved CTAB method (Doyle, 1987 ). PCR amplification was carried out in a 30 µL volume, and included15 µL ddH₂O, 9 µL mix (Tiangen, Beijing, China), 3 µL DNA solution, 1.5 µL Forward primer and 1.5 µL reverse primer solution (10 µmol/L⁻¹). The PCR procedure was as follows: pre-denaturation for 4 min at 94 °C, followed by 35 cycles of denaturation for 45 s at 94 °C, annealing at 50–55 °C for 1 min and extension at 72 °C for 1 min, and finally, extension at 72 °C for 7 min. The PCR products were sequenced (BGI, Beijing, China) by ABI 310 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). The paired-end sequences were assembled by Geneious 11.1.5. DnaSP5 was available for nucleotide diversity, haplotype diversity, and mismatching analyses.