Development And Preliminary Application Of Environmental DNA Technology In Asian Giant Softshell Turtle(Pelochelys Cantorii)

The Asian giant softshell turtle（Pelochelys cantorii）belongs to Trionychidae,Pelochelys,is one of the larger aquatic turtles in China.In China,it is mainly distributed in the middle and lower reaches of the Yangtze River and Guangdong,Zhejiang,Fujian,and other provinces.Abroad distribution in India,Myanmar,Vietnam,the Philippines,and other countries.Since the last century,due to human activities,the habitat has been severely damaged and the habitat has become less and less.Coupled with the wanton killing of humans and their longer age of sexual maturity,the Asian giant softshell turtles are extremely rare in the wild environment of our country.In 1989,the Asian giant softshell turtle was listed as a national level wild protected animal.Because the wild resources of the Asian giant softshell turtle are extremely scarce and have the habit of long-term submerged sand.Therefore,the traditional resource survey method has a low accuracy rate for the field resource survey of the turtle,and a scientific survey method is urgently needed as an auxiliary.In recent years,with the rapid development of molecular biology,environmental DNA（e DNA）technology has developed rapidly in the field of aquatic animal resources investigation,and has been widely used in species detection,biodiversity assessment,and biomass assessment.Compared with traditional resource survey methods,e DNA technology has higher sensitivity in detecting rare and endangered species,and will not cause harm to target animals,which greatly saves manpower and material resources.Therefore,in this study,specific primers and Taq Man probes were designed based on the mitochondrial genome sequence of the Asian giant softshell turtle.The optimal annealing temperature and primer probe concentration of real-time fluorescence quantitative PCR（q PCR）were explored by Taq Man probe q PCR experiment,and the e DNA detection technology of the Asian giant softshell turtle was established.The most suitable filter membrane pore size for water sample filtration,the stability time and degradation days of the Asian giant softshell turtle e DNA in indoor environment,and the influence of different water temperatures on Asian giant softshell turtle e DNA concentration were tested.To explore the relationship between the e DNA concentration and biomass of the Asian giant softshell turtle at the laboratory level,and to conduct a preliminary application in the Asian giant softshell turtle nature reserve of Guangning County.It provides new ideas and auxiliary methods for the field resource investigation and release effect evaluation of Asian giant softshell turtle,and provides important reference value for the protection of other endangered species.The main findings are as follows:（1）A set of specific primers and probe on the ND5 gene were designed and screened according to the mitochondrial genome sequence of Pelochelys cantorii in Foshan City,Guangdong Province in NCBI database.Using Primer-BLAST verification,the ND5 gene sequence of P.cantorii in NCBI database can be completely matched.The comparison of the amplified sequences of the ND5 gene in seven regions showed that the designed primers could match the ND5 gene sequences in the Asian giant softshell turtle of different regions.The Asian giant softshell turtle,the Chinese softshell turtle（Pelodiscus sinensis）,the spiny softshell turtle（Apalone spinifera）,the Burmese narrow-headed softshell turtle（Chitra vandijki）DNA were used for q PCR amplification.The results showed that the Asian giant softshell turtle and the Burmese narrow-headed softshell turtle were positive for amplification,but the Burmese narrow-headed softshell turtle had no wild distribution in China.Therefore,this group of primers probe has strong specificity for the amplification of the Asian giant softshell turtle DNA and can be used for subsequent experiments.The annealing temperature of q PCR and the concentration of primer and probe were tested.It was found that the annealing temperature at 60°C and the concentration of primer and probe at 2μM had the smallest CT value for the amplification of the Asian giant softshell turtle DNA.The standard curve was drawn by q PCR amplification using gradient diluted standard.The results showed that the q PCR experiment could amplify DNA in the range of 10²-10¹⁰ copies/μl.The correlation coefficient of the standard curve was R²=0.999,and the regression equation was y=-3.7389x+46.234.（2）By comparing the filtration time of different pore size filter membranes and the concentration of enriched Asian giant softshell turtle e DNA.It was found that the concentration of e DNA detected by 1μm pore size filter membrane was the highest,and the filtration time was shortened by one sixth compared with 0.45μm pore size filter membrane.Considering the filtration time and enrichment effect,the filtration efficiency of 1μm pore size filter membrane is the highest,and 1μm pore size filter membrane can be used to improve the filtration efficiency in subsequent experiments.The days required to explore the stability and degradation of e DNA concentration in the indoor environment were 1 d and 9 d,respectively.At different water temperatures,the e DNA concentration was higher at 28°C.This may be related to the increased feeding behavior of Asian giant softshell turtles,accelerated metabolism,and increased e DNA shedding into the water.（3）To explore the relationship between e DNA concentration and biomass of Asian giant softshell turtle under laboratory conditions.It was found that the e DNA concentration increased with the increase of the number of Asian giant softshell turtles.Through linear regression analysis,there was a good linear relationship between the biomass and the CT value obtained by e DNA amplification.The correlation coefficient R²=0.9683,the linear equation is y=-0.4401x+30.47.EDNA was successfully amplified in both indoor and outdoor Asian giant softshell turtle ponds.（4）In March 2023,water samples were collected from the Asian giant softshell turtle nature reserve in Guangning County to detect turtle e DNA.No Asian giant softshell turtle e DNA was detected at the set sampling points.Analysis of the reasons may have the following points:First is that there is no distribution of Asian giant softshell turtles in the sampling point or the biomass of turtles in the protected area is very small.The DNA released in the environment did not reach the minimum threshold for q PCR assay.Second,in the sampling season,the water temperature was low（21°C）,the activity of the Asian giant softshell turtle was not large,and the DNA released in the water was less.Third,there are many factors affecting the degradation rate of e DNA in wild rivers.Subsequent sampling surveys can be considered in the peak season of the Asian giant softshell turtle growth and spawning（June-July）.