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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.


Absorbance spectrum of DNA prepared from mucus samples (grey lines), blank (i.e., card only, black dotted) and a tissue sample with DNA extraction kit (black solid) measured by Nanodrop™.
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fig-4: Absorbance spectrum of DNA prepared from mucus samples (grey lines), blank (i.e., card only, black dotted) and a tissue sample with DNA extraction kit (black solid) measured by Nanodrop™.

Mentions: Time between sampling and lab analyses ranged from 81 to 343 days. DNA concentration estimated by spectrophotometric measurements of the concentration of DNA templates ranged from 12.18 to 29.00 ng/µl (23.16 ± 4.05 ng/µl, mean ± s.d., n = 18). Estimate for blank sample (i.e., card only) was 11.7 ng/µl. Absorbance spectra lacked the typical peak at wavelength 260 nm preceded by a dip at 230 nm, which was observable in DNA templates prepared from tissue samples using a commercial DNA extraction kit (e.g., Qiagen DNeasy Kit) (Fig. 4). Instead, spectra showed high absorbance around wavelength 230–240 nm that was also present in blank sample. Fluorometric measurements ranged from 0.0743 to 2.16 ng/µl (0.589 ± 0.536 ng/µl, mean ± s.d., n = 18). Measurement for blank sample (i.e., card only) was lower than the detection limit of 0.05 ng/µl. There was no visible band or smear with gel electrophoresis loaded with 10 µl of samples. Samples concentrated approximately ten times by both standard ethanol precipitation and vacuum drying also failed to show a band or smear.


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)

Absorbance spectrum of DNA prepared from mucus samples (grey lines), blank (i.e., card only, black dotted) and a tissue sample with DNA extraction kit (black solid) measured by Nanodrop™.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-4: Absorbance spectrum of DNA prepared from mucus samples (grey lines), blank (i.e., card only, black dotted) and a tissue sample with DNA extraction kit (black solid) measured by Nanodrop™.
Mentions: Time between sampling and lab analyses ranged from 81 to 343 days. DNA concentration estimated by spectrophotometric measurements of the concentration of DNA templates ranged from 12.18 to 29.00 ng/µl (23.16 ± 4.05 ng/µl, mean ± s.d., n = 18). Estimate for blank sample (i.e., card only) was 11.7 ng/µl. Absorbance spectra lacked the typical peak at wavelength 260 nm preceded by a dip at 230 nm, which was observable in DNA templates prepared from tissue samples using a commercial DNA extraction kit (e.g., Qiagen DNeasy Kit) (Fig. 4). Instead, spectra showed high absorbance around wavelength 230–240 nm that was also present in blank sample. Fluorometric measurements ranged from 0.0743 to 2.16 ng/µl (0.589 ± 0.536 ng/µl, mean ± s.d., n = 18). Measurement for blank sample (i.e., card only) was lower than the detection limit of 0.05 ng/µl. There was no visible band or smear with gel electrophoresis loaded with 10 µl of samples. Samples concentrated approximately ten times by both standard ethanol precipitation and vacuum drying also failed to show a band or smear.

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.