Fungal ITS amplicons for DNA extracted from root material of Cypripedium acaule.
Three roots were collected from a Cypripedium acaule individual. Root samples were flash frozen in liquid nitrogen and stored at -70? until processing. The presence of pelotons (i.e., intercellular coils of hyphae characteristic of mycorrhizal infection in orchid roots) was verified through systematic inspection of 3 cm intervals along the root using light microscopy. Mycorrhizal root segments were surface sterilized with a 10% bleach wash, followed by two washes with sterile water. Total genomic DNA was extracted from the crushed sample using a modified CTAB protocol (Doyle, 1991). The fungal ITS region (fITS) was PCR amplified from DNA extracts using a dual indexed version of the ITS1-F_KYO2 (GCATC-16 nucleotide barcode-TAGAGGAAGTAAAAGTCGTAA) and ITS4_Rev (GCATC-16 nucleotide barcode-TCCTCCGCTTATTGATATGC) primers with a 5? phosphorus modification (Toju et al., 2012; White et al., 1990). Sequencing libraries were constructed from resulting amplicons by the Georgia Genomics and Bioinformatics Core (GGBC) at the University of Georgia by circularizing amplicons and incorporating a PacBio sequencing adapter. Libraries were sequenced on a PacBio Sequel II housed at the GGBC facility using the consensus circle sequencing (CCS) approach.