Transcript Species detection using Environmental DNA from water samples

Species detection using Environmental
DNA from water samples
2012.10.25. 정다금
Position
Biodiversity studies
Species distribution
Ecology
Biogeography
Conservation biology
Problem:
- Difficulty to detect a species, particular time periods or developmental stages
Solution:
- Detect the presence of a species using the DNA in the environment
especially specific primers
Benefits
Extraction of DNA from environmental samples:
- Allows characterization of their micro-organisms
- Provide information on extinct communities of macro organisms
(eg: old sediments, permafrost and ice cores)
- Unexplored potential about highly concentrated organisms in present-day
Novel approach: based on the persistence of DNA in the environment
Purpose:
- To detect the presence of a species in fresh water,
- To examine shether DNA fragments can be used for a reliable assessment
of current species presence
1. Controlled environments
2. Natural field conditions
American bullfrog(황소개구리): Rana catesbeiana
(=Lithobates catesbeianus)
invasive amphibian
(high-quality census data)
=>Reliable field validation
American bullfrog:
- Native to western North America
- Introduced into ecosystems around the glove
- One of the world’s most harmful invasive species
Materials and methods
Controlled conditions
-
Tadpole in Aquarium with 3L of water
Natural alpine spring at 1000m sealevel,
80km from the nearest bullfrog record.
0, 1, 5, 10 tadpoles / aquarium
Each density: 6 replicates,
After 24 hrs, collected 15ml water sample from each aquarium
Natural populations: Ponds: 1000-10000m2
Low density: 3 ponds
1-2 adults, no reproduction
High density: 3 ponds
Dozens of adults, thousands of tadpoles
No detection: 3 ponds
Never been detected, 30km from the nearest bullfrog record
Immediately after collection
* 1.5 ml sodium acetate 3M + 33ml absolute ethanol
To recover precipitated DNA/cellular remains
-> Centrifuge/ discard supernatant
PCR
primers 5’-TGCCAACGGAGCATCATTC-3’ and 5’-ATAAAGGTAGGAGCCGTAGT-3’
:amplify a 79 bp segment of mitochondrial cyt-b, which is
monomorphic in all 397 individuals analysed by population genetic
studies covering the whole native and European range of the
species ( Ficetola et al. 2008).
-> Primers :
-Specificity confirm-> Genbank
-Try amplifying DNA of all other frog species living in France
(genus Rana) 2 ind. from different sites per each species.
-Each water sample: 3-5 amplification using the multi-tube
approach
PCR product of one pond was sequenced using the 454
pyrosequencing.
Generalized mixed models: assuming a binomial error
to compare the amplification rates among ponds with different
bullfrog densities, fitted using lme4 in R
Results
All 18 aquarium water samples:
using selective primers PCR was successful.
(0.3,1.7,3.3 tadpoles per L)
- All PCR products were sequenced and corresponded perfectly to
the published bullfrog cyt-b sequence.
-674 fragments from one PCR product were sequenced using 454
pyrosequencing technology.
-False negative: approximately 1.5%
Using a multi-tube approach and ancient DNA precautions,
which are suitable for analysing DNA that is degraded
and/or at low concentrations
Average
amplification
success
0.37+/- 0.1
0.79+/- 0.08
22%
89%
0
- To ensure that the positive amplification is not due to artefacts
-To ensure that the negative amplification is not due to chance
-Generalized linear mixed model: significant
Differences in amplification rates among ponds with differing densities
of target species were significant (Average amplification success)
Disucssion
The way to use environmental DNA and Strengths
- To ascertain species presence: discriminating between absence and presence
even low density
-To allow the reliable detection of secretive organisms in wetlands
w/o direct observation
-Answer to many situation where traditional census techniques
give low-quality results
-To quantify secretive harmful, invasive or threatened species
-Assessment of distribution of rare threatened species(target of conservation plans)
Several precautions
Influence factor: the amount of DNA in environmental samples
: volume of water, size and density of the organism and volume of secretions
Duration time: difficult to evaluate how long DNA fragments persist in water
(short DNA fragments can persist a long time under dry cold conditions… )
eg: 10000 year old dry cave sediments amplification(Willerslev et al. 2003)
400bp may persist up to 1 week at 18℃ in lake water(Matsui et al. 2001)
New avenues for the study of biodiversity:
DNA barcodes for identifying species from degraded DNA will be more applicable
to more and more plant and animal species
Massive sequencing techniques:
To analyse PCR products generated with universal primers working
on degraded substrates
-> To make possible the assessment of the current biodiversity of macro-organisms
from environmental samples