Qualitative And Quantitative Relationship Between Common Aquatic Animals And Their Environmental DNA

The biodiversity of freshwater ecosystems on Earth, being under various threats, need comprehensive protection. It is a necessary step for us to get a thorough understands about the biodiversity status quo of a freshwater ecosystem before we can work out an efficient biodiversity protection countermeasure for it. Traditional species-centered biodiversity surveying method, being too costly, laborious and time-consuming, is far from satisfying the growing demand of biodiversity protection. In order to explore new biodiversity-surveying theory and technology for more efficiently surveying the biodiversity of freshwater ecosystems, here we try to use environmental DNA (eDNA) as the detecting target to survey freshwater ecosystems’ biodiversity. The main research contents include:1) collecting all possible literatures about most common aquatic animals in freshwater ecosystems in east China, searching and collecting their DNA barcoding sequences, designing specific primers of their COI gene and verificating their efficancy by experiment; 2) specifical detection of Brachionus calyciflorus Pallas, Cyprinus carpio, Misgurnus anguillicaudatus and Macrobrachium nipponense, the four kinds of common aquatic animals, under the condition of indoor breeding by using their eDNA; 3) analysis of quantitative relationship between individual density of Misgurnus anguillicaudatus and Macrobrachium nipponense and their eDNA abundance under the condition of indoor breeding. The main results were as follows:Find the common species of aquatic animals by literatures and search DNA barcoding library to get the DNA barcoding sequences. Then design primers by software and through the experiment to verify the specificity of the primers. We compared five kinds of eDNA extraction methods from water samples in the experiment, like Vacuum concentration meter enrichment, PEG2000 concentration respectively, rotary evaporation concentration, freeze drier concentration, alcohol precipitation concentrated many times. The results show that using the method of alcohol sink water samples to extract eDNA is the best effect and convenient to operate.According to certain density gradient raised four kinds of common aquatic animals, such as Brachionus calyciflorus Pallas, Cyprinus carpio, Misgurnus anguillicaudatus and Macrobrachium nipponense in laboratory simulation qualitative experiments. Applied the method of preliminary experiment, choose the alcohol sinking method to extract its eDNA in water samples, then through ordinary PCR, PCR products detection by agarose gel electrophoresis and sequencing. Results show that the four species are able to take advantage of specific primers amplification segments of purpose. Experiments prove that can use the eDNA for the qualitative detection of target species.Selected aquatic animals such as Misgurnus anguillicaudatus and Macrobrachium nipponense for quantitative analysis between breeding different density with the abundance of eDNA, using qPCR method to quantitative the eDNA from water samples in different breeding density. The results of two specises showed that the amount of their eDNA increased with the increase of the breeding density. The quantitative results show that with the increase of species density, the amount of eDNA is also increasing. Simulation curves between two species breeding density and the eDNA are based on binomial curve and according to density of animals and the eDNA there is nonlinear relationship.The above results show that:eDNA and corresponding aquatic animals does exist between the close inner link, we can use the eDNA to qualitative and quantitative detection of aquatic animals, detect abundance of eDNA can be used to test the species richness.