Is It Feasible To Develop Environmental DNA Molecular Markers Based On Transposons?

Environmental DNA(eDNA)refers to DNA extracts that can be obtained from environmental samples such as soil,air,water,etc,and has been widely used in species detection,abundance assessment,and biodiversity survey.Most recently,eDNA has also been used to obtain population genetic information of aquatic species.At present,all eDNA population genetic studies use maternal mitochondrial markers,and thus paternal genetic variation cannot be included in the analysis,leading to a one-sided assessment of the genetic structure and lineage relationship of species.Therefore,it is imperative to develop novel environmental DNA molecular markers based on nuclear DNA.Transposons are an important part of the nuclear genome of organisms,and their high copy number makes them an alternative to mtDNA for eDNA population study.Here,we use medaka as a model to set up a platform for eDNA-based population genetic analysis using mitochondrial markers and verify the feasibility of developing environmental DNA molecular markers based on transposons.At last,the degradation kinetics of different eDNA fragments was compared.The main results are as follows:1.Population genetic analysis of the Chinese medaka(Oryzias sinensis)based on environmental DNA.Firstly,13 different medaka populations were adopted in Huai’an Guhuai Wetland Park(HA),Ji’nan Xiaomenya Scenic Area(JN),Liaocheng Xiaomeihe Wetland Park(LC),Lianyungang Qingkou Park(LYG),Nanjing Longshan Reservoir(NJ),Qingzhou Mihe(QZ),Rizhao Futuanhe(RZ),Shanghai Dianshan Lake(SH),Suzhou Tianping Mountain(SZ),Weifang Bailang Oasis Park(WF),Yangzhou(YZ)Shuangdun Reservoir,Yangzhou Sanwan Wetland Park(YZs),and Zhejiang Jinhua(ZJ).The collected medaka samples were cultured in the lab for one week before the culture water was filtered for eDNA extraction.Primers were designed to amplify Cytb,COI,and D-loop fragments of about 400 bp in length.After library construction,sequencing,quality control,and haplotype analysis by amplicon sequence variation(ASV)method,a total of 49,46,34,46,11,and 12 haplotypes were obtained for Cytb-1,Cytb-2,COI-1,COI-2,D-loop-1,and D-loop-2,respectively,among which 2,2,2,1,3,and 1 haplotype were shared among the 13 populations.Besides,there were more shared haplotypes between NJ and ZJ populations,and between NJ and SH populations.Population structure analysis based on the haplotype data showed that the 13 medaka populations could be divided into two large groups,and the genetic kinship of the population was consistent with the geographical distribution of each population.In other words,we successfully constructed a platform for eDNA-based population genetic analysis.2.The copy number and sequence polymorphism of transposon fragments in environmental DNA.To compare the copy numbers of transposons and mitochondrial genes,Japanese medaka(Oryzias latipes)eDNA was obtained by vacuum filtration of Japanese medaka culture water using mixed cellulose ester membranes with different pore sizes(0.45 μm,1 μm,5 μm,and 10 μm).At the same time,tissue genomic DNA was also extracted from the tail fin of a male Japanese medaka.Primers targeting single-copy nuclear genes(DMY,SHH),mitochondrial genes(D-loop,Cytb),and multi-copy nuclear genes(Rex6,18sRNA)were designed to amplify fragments around 100 bp in length.In addition,fluorescence-labeled probes targeting these genes were also designed.Then,the copy number of the aforementioned genes was determined by digital PCR.Here we show that the copy number of Rex6,a high-copy transposon in the genome of medaka,was much higher than the other gene in the genomic DNA.Moreover,the copy number ratio of DMY to SHH,and D-loop to Cytb was consistent with expectations.Also,Rex6 exhibited the highest copy number in the eDNA samples,regardless of the pore size of the membranes.As for the ratio of copy number between different gene fragments,however,a huge variation was observed between different pore sizes.Secondly,the cultured water eDNA of the 13 Chinese medaka populations and the laboratory Japanese medaka was used as a template to amplify both the mitochondrial fragments(COI,Cytb,and D-loop)and transposon fragments(Rex6,RTE,and TC1),two fragments per gene,for library construction and paired-end 250 high-throughput sequencing.Data analysis showed that the aforementioned mitochondrial and transposon fragments of the 14 samples of medaka produced 36,38,51,48,10,and 16,and 6581,8856,2514,3655,8807,and 2171 haplotypes,respectively.It was obvious that transposon fragments not only generated more haplotypes but also obtained more shared haplotypes between different populations.The above results showed that transposons not only own higher copy numbers but also recover more haplotypes from eDNA,indicating that transposons are potential alternatives to mtDNA for eDNA-based population analysis.3.Degradation kinetics of different eDNA fragments.In the second part of the study,we observed that the relative copy numbers of different gene fragments varied compared with tissue genomic DNA.It was speculated that there was a different degradation rate for different gene fragments after they were released into the environment.To test this hypothesis,we further conducted two experiments to study the copy number relationship of genes in aquaculture water samples at different time points.For the culture water experiment,water samples were collected after a one-week culture at 0h,6h,12h,24h,and 48h for eDNA extraction;while for the tissue homogenization experiment,water samples were collected at 0h,8h,24h,3d,7d,and 11d for eDNA extraction.Then,qPCR was adopted to analyze the relative abundance of 18sRNA versus Rex6,and Cytb versus D-loop over time.Here we show that 18srRNA and CytB degraded much faster than Rex6 and D-loop,respectively,in both experiments.The results of digital PCR analysis were consistent with qPCR results.It means that the selection of genes with lower degradation rates is beneficial to improve the sensitivity of eDNA detection.Analysis of degradation rates of environmental DNA.