Fig. 4.—

Polymorphism of centromere orientation among Komagataella phaffii strains. Genomic DNAs from four haploid laboratory strains and five natural isolates of K. phaffii from the USDA Agricultural Research Service NRRL collection (Kurtzman 2009) were amplified using orientation-specific PCR assays. For each centromere, two primers (B and C) bind to the mid region and two (A and D) bind to the chromosome arms outside the IR regions. The 16 primer sequences are listed in supplementary table S1, Supplementary Material online. Amplification with the AB and CD primer pairs for a centromere indicates that its mid region is in the same orientation as the reference genome sequence (Kuberl et al. 2011), whereas amplification with the AC and BD pairs indicates inversion of the mid region relative to the chromosome arms. PCR products are the size of the IRs (2.0–2.7 kb; table 1). PCR amplifications used GoTaq (Promega) polymerase for 30 cycles with an annealing temperature of 55 °C.