Dataset: Sanger Sequencing Analysis of Potexvirus PCR Products (Potex-5 and Potex-2RC)

Sample collection

A systematic seagrass survey was conducted on 133 defined sites ~10m apart within a ~9000 m2 seagrass meadow in Terra Ceia Aquatic Preserve. These sites are routinely monitored for turtlegrass Virus X (TVX). This dataset includes sequence references for 40 Thalassia testudinum samples from multiple Florida sites, including Terra Ceia Aquatic Preserve, Tampa Bay seagrass sites S1T5 and S3T8 (Lassing Park), Panacea located in the Florida Panhandle, and Florida Keys sites including Bush Key, Garden Key, Marquesas Key, and Key West.

The survey examined the host species Thalassia testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, and Zostera marina. Potexvirus PCR products were successfully generated only from Thalassia testudinum samples. Thalassia testudinum specimens successfully amplified with the Potex-5/Potex-2RC primer pair were collected from:

1. A systematic seagrass survey at Terra Ceia Aquatic Preserve on August 1st, 2022
2. Tampa Bay seagrass site S3T8 (Lassing Park) on October 3rd, 2023
3. Panacea located in the Florida Panhandle on May 10th, 2023
4. Florida Keys and Dry Tortugas National Park, Florida, USA collected between May 16th-20th, 2022

RNA extraction and cDNA synthesis

Total RNA extraction was performed on 30-100 mg of leaves from multiple shoots pooled by seagrass species and collection site using Zymo Research’s (ZR) Quick-RNA™ Plant Miniprep kit. Each pooled seagrass sample was homogenized in a BashingBead Lysis Tube containing 2 mm ceramic beads and 800 μL RNA lysis buffer (provided in the kit) for 5 minutes at maximum speed using a FisherbrandTM Bead Mill 4 Homogenizer (Fisher Scientific, Waltham, MA, USA). Tissue homogenates were centrifuged at maximum speed (21,130 g) for 1 minute and RNA was extracted from the total volume (~800 μL) of the supernatant. To ensure successful RNA extraction, after each round of extraction, a random subset of RNA samples was quantified using the QubitTM RNA high sensitivity (HS) assay (Invitrogen™). From each sample, cDNA was synthesized from 8 μL RNA using the SuperScriptTM IV First-Strand Synthesis System (Invitrogen™) and following manufacturer’s instructions for random hexamers. 

PCR amplification and sequencing 

PCR was performed under the following conditions: initial denaturation at 95°C for 10 minutes, 40 cycles of denaturation at 95°C for 30 seconds, annealing at 51.5°C (as published in van der Vlugt and Berendsen, 2002) for 30 seconds, extension at 72°C for 1 minute, followed by elongation at 72°C for 10 minutes and cooling at 11°C. The PCR product was visualized following gel electrophoresis on a 1% (wt/vol) agarose gel stained with ethidium bromide. All PCR reactions, except for the no template control, yielded visible bands. All PCR products were purified using the Zymoclean Gel DNA Recovery Kit (Irvine, CA, USA), quantified using the QubitTM DNA high sensitivity (HS) assay (Invitrogen™, Waltham, MA, USA), and Sanger sequenced bidirectionally by Eurofins Genomics (Louisville, KY, USA).