Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Data Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

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).

Sequence reads from each sample were mapped to the Potex-5/Potex-2RC amplicon region (with forward and reverse primer sequences removed) in the TVX genome (NCBI accession: NC_040644:van der Vlugt (2002)) using the “Map Sanger Reads to Reference” function implemented in Unipro UGENE v48.1 (Okonechnikov, 2012), with a trimming quality threshold of 20 and mapping minimum similarity of 70%. Electropherograms of mapped reads were manually inspected to remove primer sequences and low-quality bases and to resolve ambiguous bases. 

- Imported original file "Potexvirus_NCBI_accessions.xlsx" into the BCO-DMO system.
- Created "Aphia_id" field for Aphia reference identifier.
- Convert date from %m-%d-%y to %Y-%m-%d format

NSF Award Abstract:
Seagrasses are marine flowering plants (or angiosperms) that create expansive underwater meadows that form the basis of highly productive and valuable ecosystems in coastal oceans. Unlike terrestrial systems where angiosperms dominate plant diversity, seagrasses are the only flowering plants in marine environments. Based on the profound impacts of viral infections on terrestrial plants, viruses are expected to influence seagrass ecology. However, no prior work has investigated viral infection dynamics in seagrasses or the impact of viruses on seagrass health. This project provides fundamental knowledge about seagrass-virus interactions through field and laboratory studies of Thalassia testudinum (i.e., turtlegrass, a climax species and key ecosystem engineer), and turtlegrass virus X (TVX), the only seagrass virus currently reported from experimental research. The lack of a seagrass-virus study system has kept the scientific community from learning which factors drive viral infection in marine angiosperms. By establishing the first seagrass-virus study system, a novel virus-host pathosystem for which virtually nothing is known, this project contributes to a more comprehensive understanding of seagrass ecology and serves as a model for investigating the growing number of seagrass viruses discovered through sequencing efforts. This multifaceted project trains one postdoctoral researcher, two graduate students, and six undergraduate students. Dissemination of results and data through open access channels informs the broader community and provides scientists with data for their own research to propel the field of seagrass virology. This project also engages educators and students participating in programs that strive to increase participation from underrepresented groups in STEM fields. Teachers from the Jacksonville Teacher Residency Program are getting involved through development of lessons that dive into seagrass biology. Students from Girls Incorporated, Girl Scouts, and the University of South Florida’s Oceanography Camp for Girls are participating as citizen scientists by photographing Tampa Bay’s seagrass ecosystems and contributing their observations to the Seagrass Spotter website. This project also increases awareness of seagrass ecosystems and challenges the public perception that all viruses are pathogenic through hands-on activities at the annual St. Petersburg Science Festival.

Seagrass-virus interactions are being investigated through a two-tiered approach involving field studies in Tampa Bay, Florida and microcosm experiments. Field surveys focus on elucidating the nature of turtlegrass-TVX interactions (positive, neutral or negative) and the relationship between turtlegrass genotypic diversity and virus distribution in a natural population where TVX has persisted for at least five years. TVX load is monitored bimonthly over two years to assess how viral load relates to turtlegrass genotype and performance (growth, health, reproductive effort), and abiotic parameters. The investigated turtlegrass meadow contains TVX-positive and negative specimens, thus providing a perfect natural laboratory with homogenous environmental characteristics that allow exploration of the drivers of viral infection. Given that environmental changes may alter host-microbe interactions, complementary microcosm experiments are evaluating turtlegrass responses to TVX infection at the physiological (survival, photochemical capacity, cellular responses) and molecular (transcriptomic) levels in a controlled environment under normal conditions and in the context of salinity changes, an important seagrass stressor. Microcosm experiments also provide the first profiles of seagrass gene expression and measurement of cellular metabolites in response to viral infection. Expected results have direct implications for understanding seagrass production and resilience in the face of global climate change and anthropogenic stress.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.