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Evaluating manta ray mucus as an alternative DNA source for population genetics study: underwater-sampling, dry-storage and PCR success.

Kashiwagi T, Maxwell EA, Marshall AD, Christensen AB - PeerJ (2015)

Bottom Line: Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging.DNA stored on cards was found to be reliable for PCR-based population genetics studies.We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Fisheries Laboratory, University of Queensland , St. Lucia, QLD , Australia ; Marine Megafauna Foundation , Truckee, CA , USA ; Current affiliation: Center for Fisheries, Aquaculture and Aquatic Sciences, Southern Illinois University Carbondale , Carbondale, IL , USA.

ABSTRACT
Sharks and rays are increasingly being identified as high-risk species for extinction, prompting urgent assessments of their local or regional populations. Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging. DNA is typically amplified from tissue samples which are collected by hand spears with modified biopsy punch tips. This technique is not always popular due mainly to a perception that invasive sampling might harm the rays, change their behaviour, or have a negative impact on tourism. To explore alternative methods, we evaluated the yields and PCR success of DNA template prepared from the manta ray mucus collected underwater and captured and stored on a Whatman FTA™ Elute card. The pilot study demonstrated that mucus can be effectively collected underwater using toothbrush. DNA stored on cards was found to be reliable for PCR-based population genetics studies. We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species. As the yields of DNA with the tested method were low, further improvements are desirable for assays that may require larger amounts of DNA, such as population genomic studies using emerging next-gen sequencing.

No MeSH data available.


Mucus sampling with a toothbrush mounted on an extendable pole.
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fig-2: Mucus sampling with a toothbrush mounted on an extendable pole.

Mentions: Mucus from eighteen Manta birostris was collected on SCUBA from Isla de la Plata in Ecuador (1°15 29.62S, 81°4 25.96W) between 2 September and 20 September 2012. Samples were obtained using a small toothbrush held in the diver’s hand (Video S1) or mounted on an extendable pole (Fig. 2). For each sample, the dorsal surface of the ray was rubbed back and forth or in a circular motion ∼3–5 times, then the brush was placed into an individual 50 ml plastic tube to prevent cross contamination. On dry land, approximately 120 µl mucus was transferred from the brush with a clean sterile cotton bud and then onto FTA™ Elute Cards and/or Indicating FTA™ Elute Cards (GE Healthcare) using three side-to-side motions, 90° each way (Fig. 3), spreading mucus and cells evenly to an area of approximately 625 mm2. These cards, which are impregnated with a chemical formula that lyses cells and denatures proteins upon contact, are designed for room temperature storage and shipment of DNA from biological samples for PCR analysis. The applied volume of liquid samples is the recommended amount to avoid overloading the chemicals (GE Healthcare). Cards were then air dried and placed in separate resealable plastic bags. Samples were then transported via land and air as normal domestic and international postage and kept at room temperature with desiccants until further analysis in the lab.


Evaluating manta ray mucus as an alternative DNA source for population genetics study: underwater-sampling, dry-storage and PCR success.

Kashiwagi T, Maxwell EA, Marshall AD, Christensen AB - PeerJ (2015)

Mucus sampling with a toothbrush mounted on an extendable pole.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581770&req=5

fig-2: Mucus sampling with a toothbrush mounted on an extendable pole.
Mentions: Mucus from eighteen Manta birostris was collected on SCUBA from Isla de la Plata in Ecuador (1°15 29.62S, 81°4 25.96W) between 2 September and 20 September 2012. Samples were obtained using a small toothbrush held in the diver’s hand (Video S1) or mounted on an extendable pole (Fig. 2). For each sample, the dorsal surface of the ray was rubbed back and forth or in a circular motion ∼3–5 times, then the brush was placed into an individual 50 ml plastic tube to prevent cross contamination. On dry land, approximately 120 µl mucus was transferred from the brush with a clean sterile cotton bud and then onto FTA™ Elute Cards and/or Indicating FTA™ Elute Cards (GE Healthcare) using three side-to-side motions, 90° each way (Fig. 3), spreading mucus and cells evenly to an area of approximately 625 mm2. These cards, which are impregnated with a chemical formula that lyses cells and denatures proteins upon contact, are designed for room temperature storage and shipment of DNA from biological samples for PCR analysis. The applied volume of liquid samples is the recommended amount to avoid overloading the chemicals (GE Healthcare). Cards were then air dried and placed in separate resealable plastic bags. Samples were then transported via land and air as normal domestic and international postage and kept at room temperature with desiccants until further analysis in the lab.

Bottom Line: Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging.DNA stored on cards was found to be reliable for PCR-based population genetics studies.We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Fisheries Laboratory, University of Queensland , St. Lucia, QLD , Australia ; Marine Megafauna Foundation , Truckee, CA , USA ; Current affiliation: Center for Fisheries, Aquaculture and Aquatic Sciences, Southern Illinois University Carbondale , Carbondale, IL , USA.

ABSTRACT
Sharks and rays are increasingly being identified as high-risk species for extinction, prompting urgent assessments of their local or regional populations. Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging. DNA is typically amplified from tissue samples which are collected by hand spears with modified biopsy punch tips. This technique is not always popular due mainly to a perception that invasive sampling might harm the rays, change their behaviour, or have a negative impact on tourism. To explore alternative methods, we evaluated the yields and PCR success of DNA template prepared from the manta ray mucus collected underwater and captured and stored on a Whatman FTA™ Elute card. The pilot study demonstrated that mucus can be effectively collected underwater using toothbrush. DNA stored on cards was found to be reliable for PCR-based population genetics studies. We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species. As the yields of DNA with the tested method were low, further improvements are desirable for assays that may require larger amounts of DNA, such as population genomic studies using emerging next-gen sequencing.

No MeSH data available.