Development Of Specific Primers For Environmental DNA Detection Of Rare Marine Animals In The South China Sea And A Complementary Method For Primer Development Of Barcode Sequences

The south china sea is within the scope of marine biodiversity in China even all over the world in key areas,the condition of the biodiversity and biological resources affected by climate change and human activity is bigger,so that timely and accurate grasp of these important information for understanding the south china sea marine ecosystems in human and global climate change under the background of the evolution of the law is of great significance.However,due to the limitation of traditional survey methods,the monitoring of the status of important and rare biological resources in the south china sea is still unable to be carried out quickly and effectively up to now.Environmental DNA(e DNA)refers to the DNA information left in the environment by organisms when they are moving in the environment,and environmental DNA samples can be used to monitor the biodiversity and abundance in the environment.Compared with some shortcomings of traditional survey methods,this technique has the advantages of high sensitivity,low invasion and good economy,and has the potential to become an important survey method for monitoring marine living resources.Even in this area,its application is still restricted by the lack of barcode sequence resources and other problems.In this study,nest primers were designed and tested for the rare fish of south china sea,Cromileptes altivelis and Cheilinus undulatus,so as to improve the development and evaluation process of specific detection primers.The primer evaluation results showed that the primer could meet the species specificity and the lower limit of detection was 1.11 copies/μL and 1.03 copies/μL,respectively.Then,the nest primers were tested by using the aquaculture seawater environment DNA,and the target species in aquaculture seawater environment DNA mixed with a variety of DNA could still be detected.In addition,in order to solve the problem of insufficient barcode sequence resources of rare marine organisms in the south china sea,it is proposed to select the genus conch as the representative and use high-throughput sequencing technology to establish an efficient and feasible mitochondrial genome sequence assembly and analysis process.The specific results are as follows: First,Charonia tritonis,a rare animal in the south china sea,was selected as the target species.After DNA extraction,high-throughput sequencing technology was used for shotgun sequencing.The filtered,high-quality sequencing raw data was assembled and annotated through soapdenovo-trans,resulting in the complete mitochondrial genome of farnavus.The mitochondria encoded a total of 37 genes,including 13 protein encoding genes,22 transport RNA(t RNA)genes and 2ribosomal RNA(r RNA)genes.The whole genome has obvious A+T bias.Among the13 protein-coding genes in the mitochondrial genome,the start codon of all of them is ATG,the stop codon of 11 of them is TAA,and the stop codon of the remaining two protein-coding genes is TAG.The results show that this process can quickly and economically supplement the barcode sequence resources applicable to environmental DNA studies.However,the target species were not detected in the Xisha coral reef area.In this paper,we designed and tested the nest primers for specific detection of Cromileptes altivelis and Cheilinus undulatus,and improved the development process of the primers.The method of obtaining barcode sequences was developed by using high-throughput sequencing technology,which provided reference resources for the development of specific primers in the future.It lays an important foundation for the application of environmental DNA in the dynamic detection of rare species and key species in the south china sea.