A revised classification of Glossopetalon (Crossosomataceae) based on restriction site-associated DNA sequencing
Allen, Maya; Ayers, Tina (2021), A revised classification of Glossopetalon (Crossosomataceae) based on restriction site-associated DNA sequencing, Dryad, Dataset, https://doi.org/10.5061/dryad.zkh18937v
Genomic DNA was isolated from silica dried leaf material and herbarium vouchers using an amended Sorbitol protocol (Štorchová et al. 2000) with the exception of G. texense, which was extracted using a CTAB protocol with the addition of pvp-40 (Doyle and Doyle 1987). Preliminary DNA quality was assessed with 1% agarose gel electrophoresis. DNA quantifications and purity determinations were conducted via a Nanodrop 1000 Spectrophotometer (Thermo Scientific, Carlsbad, California) and PicoGreen quantification was conducted with a Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments, Winooski, Virginia). All samples were normalized to 10ng/uL using 10mM Tris-Cl pH 8.0 before library preparation.
The libraries were prepared using an amended protocol of Peterson et al. (2012). Template DNA was digested with restriction enzymes MspI and EcoRI (NEB, Inc.). Adapter ligation was simultaneously conducted during the same reaction. Preparation of the adapters (Eurofins Genomics, USA) were as follows: the P1.1 EcoRI Adapter 5’-CCTATGTGGAGAGCCAGTAAGCGATGCTATGGT-3’ was annealed to P1.2 EcoRI Adapter 5’-[PHO]AATTACCATAGCATCGCTTACTGGCTCTCCACATAGG-3’ using a PTC-100 Programmable Thermal Cycler heated to 95°C for 5 minutes followed by a cool down to 25°C. Afterwards the EcoRI adapter was diluted to a concentration of 0.05μM with sterile water. The P2.1-MspI Adapter 5’-GTCAACGCTCACTACTGCGATTACCCAAGTCAG-3’ was likewise annealed to P2.2 Adapter 5’-[PHO]GCCCTGACTTGGGTAAGATAGCAC-3,’ but subsequently diluted to a concentration of 0.5μM using sterile water. The differences in concentration of the adapters were to account for the higher frequency of EcoR1 restriction enzyme sites. T4 DNA Ligase (NEB, Inc) was employed to ligate the adapters to digested DNA fragments. Furthermore, the reagents utilized for this reaction were: BSA (100x), EcoR1 10x Buffer, T4 DNA Ligase 10x Buffer with 10μM ATP, and sterile water. The reaction underwent 6 cycles of 37°C for 20 mins followed by 25°C for 20 mins and remained in the thermal cycler overnight at 10°C.
A 1:1 bead cleanup was performed with 25% PEG before the PCR indexing reaction. This amplification reaction consisted of Phusion HS II (Thermal Scientific), MgCl2, custom primers, template DNA, and sterile water. Each sample was double indexed using distinctive forward and reverse indices. Indexing was performed over 25 cycles of 95°C for 1 min, 35°C for 15 secs, 55°C for 15 secs, 72°C for 30 secs, and 72°C for 7 mins. Now that samples were indexed, all samples were pooled and underwent a 1:1 bead clean up with 18% PEG. Samples were subsequently quantified using a Nanodrop 1000 Spectrophotometer and analyzed on an Advanced Analytical Fragment Analyzer (Advanced Analytical Technologies GmbH, Heidelberg, Germany). Based on the high presence of the fragments from 200-550 base pairs, a size selection at that range was conducted using the Pippen Prep (Sage Science, Beverly, Massachusetts) for the ddRAD library. These libraries were sequenced on a single lane on a HiSeq 4000 instrument (Illumina, San Diego, California) at the University of Oregon’s Genomics and Cell Characterization Core Facility using custom primers to produce single-end 150 base pair reads.
Sequence Data Preparation— Demultiplexing of raw data was conducted in accordance with akutils RADseq utility protocol using the module fastq-multx from EA-UTILS (Aronesty 2011; Andrews 2018). The demultiplexed data has been uploaded to Dryad.