Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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