Characterization And Application Of Free Environment DNA In Freshwater Environment

Ecological environment and biodiversity research has been the focus in related field.Environmental DNA including the various species DNA from environmental samples,because of environment DNA can quickly to identify the environmental biological species caused the researchers a greatly growing interest.Freshwater ecosystems is one of the important resources to the survival of people.In recent years,ecological environment deterioration,biodiversity reduction made fresh water resources investigation and monitoring of aquatic species become the research hotspot in the field of ecology related.Environmental DNA have the advantage of fast and non-invasive compared with traditional species detection methods.But species DNA will be affected by various factors in the water environment and appear larger fluctuation.So characterize free DNA have an an important significance for species identification,diversity analysis,environmental protection.This thesis intends to focus on the various factors influencing the free environment DNA concentration under controlled condition and natural environment,and applying the latest high-throughput DNA sequencing technology to conduct a comprehensive analysis,and establish a system of analysis platform based on environmental DNA.Lay a foundation for the further application of environment DNA.This paper mainly completed with the following research contents:1?Characterization of free DNA in freshwater environment:The paper focus on the free DNA purification method and the factors influencing environment DNA concentration.First,we investigate the extracted method?sample volume.Found that environmental free DNA concentration with isopropyl alcohol precipitation is higher than ethanol precipitation and less reagent consumption;10 ml water sample volume can satisfy the investigation of the environmental factors affecting DNA concentration;We characterize free eDNA two temperature levels in controled condition?abuandance,different geographic location,sampling time under natural environment,eDNA existence time after removed species how affect eDNA concentration.Free DNA concentration was positively related to species abundance,the concentration of eDNA between 15? and 25? have no significant difference under controled condition;Free eDNA can be detected in a month after species removed.Further study found microvesicles including species DNA in water samples,this result provides a new direction in the future to study environment DNA.And the environment DNA concentration have differences in the different location and the different period.2?We explored microbial community diversity of XuanWu Lake in Nanjing based on environment DNA:We use environment metabarcoding with specific primer amplification of 18 s ribosomal DNA V4 regions,and combining with high throughput sequence on HiSeq 2500 PE250 sequence platform to explore microbial community diversity in the different location and the different period of XuanWu Lake.From which we found that at the genus level,Cyptomonas,Didinium,Ochromonas,Woloszynskia,Peridinipsis were most abundance in Jiefangmen samples in March;Platanus,Bicosoeca,Rhizophydium,Sphaeroeca were abundance in Jiefangmen samples in April;Dileptus,Strongylidium,Pseudotontonia were abundance in Jiefangmen samples in May;Cyrtohymena and Tintinnidium were abundance in Jiefangmen samples in June.This result suggest that different periods samples have different species abundance.Relative abundance species in different location environment samples in May.This result indicated that environment DNA can reflect species distribution and the dynamic change of species,environment DNA combining with high throughput sequence can provide a comprehensive,innovative means for studying aquatic biological species diversity.