Nanodrop one instrument

Leaf samples were taken using clean tweezers and scalpel, and flash frozen in liquid nitrogen and stored at −80°C until use. Approximately 50 mg of frozen tissue was ground in a 1.5 ml reaction tube using a disposable tube pestle. Genomic DNA from the ground leaves of all species grown was isolated using the E.Z.N.A. Plant DNA Kit (OMEGA Bio-Tek, Norcross, GA) following the manufacturer’s recommendations. The gDNA was eluted using 100 μl nuclease-free water and the concentration was estimated using a NanoDrop™ One instrument (ThermoFisher Scientific, Waltham, MA). The 1:10 diluted DNA was used to amplify a region of the maturase K gene using Q5® High-Fidelity DNA Polymerase (NEB, Ipswich, MA) following the manufacturer’s recommendations with following primer combinations: matK_F1 (5′-ATA CTT TAT TCG ATACAA ACT CCT TTT TTT-3′) and matK_R1 (5′-AGT ATT GCA ATT TGA ATA GTT TCA TTA C-3′) using 53°C as annealing temperature or matK_F2 (5′-ATA CTT TAT TCG ATA CAA ACT CCT TTT TTT GGA AG-3′) and matK_R2 (5′-AGT ATT GCA ATT TGA ATA GTT TCA TTA CTC GAA A-3′) using 56°C as annealing temperature. Both primer pairs amplify the same target while matK_F2 and matK_R2 bind approximately 40 bp upstream of their respective F1/R1 counterparts. PCR products were directly sequenced using Sanger sequencing at the Cornell Institute of Biotechnology using the above primers.