Study On Genetic Diversity And Skeletal System Of Artificial Preserved Asian Giant Soft-Shelled Turtles

The Asian giant soft-shelled turtles(Pelochelys cantorii)belong to Testudines,Trionychidae,and Pelochelys.As one of the largest aquatic turtles in China,it is also an important indicator species for the health of the river ecosystem in the Pearl River Basin and even the southern part of China.Because of the long history of P.cantorii,it is of great scientific value in the study of environmental changes and biological evolution.P.cantorii was widely distributed in Southeast Asia of China in history.However,due to unfair trade,excessive harvesting and killing of turtles and water conservancy projects,the population of P.cantorii has been greatly reduced.Currently,P.cantorii have become an extremely endangered species.There are only 13 P.cantorii in captivity in China.In this background,in order to strengthen the protection of endangered animals can work and management,Pearl River Fisheries Research Institute,Chinese Fishery Academy of Sciences bred more than 800 P.cantorii successfully as a conservation population from 2015 to 2020 on the basis of four sexually mature P.cantorii(2 females,2 males).Twenty healthy P.cantorii aged 4 to 5 years and weighing 1.5-2 kg were selected for a rewilding adaptive protection test in 2020.Nonetheless,the genetic structure and genetic diversity of the artificially conserved population are still unclear,so scientific management is urgently needed to obtain healthy second-generation P.cantorii with high genetic diversity,in addition to restoring the wild population through artificial propagation and release.This research use transcriptome data design trinucleotide repeat and repeat four nucleotides microsatellite primers 30000 on,the choice of 230 pairs of primer synthesis,received 10 after two screening amplification with high efficiency and high specificity of microsatellite marker,and build a multiple PCR system,comparative analysis and raise a generation of children can,and 2020 of 2019 generation can,genetic diversity.According to Nei’s genetic distance,182 Asian giant soft-shelled turtles were divided into two subgroups by constructing an evolutionary tree and Yebes algorithm.Two dead soft-shelled turtles were dissected,their skeletons were observed,described and illustrated by hand,and some of their skeletons were compared with those of Chinese soft-shelled turtles.The results of this study are useful for the genetic protection and morphological study of soft-shelled turtle F2.1.Development of microsatellites.More than 30,000 pairs of tri-nucleotide repeat and tetrinucleotide repeat microsatellite primers were designed based on soft-sheller soft-sheller transcriptome data.Among them,230 pairs were selected to synthesize the primers.A total of 10 excellent microsatellite markers were obtained after two screening.2.Establishment of microsatellite multiplex PCR and genetic diversity analysis of softshelled turtles.The genetic diversity of soft-shelled turtles born in 2019 and born in 2020 was compared and analyzed using a multiplicase polymerase chain reaction(PCR)system with selected microsatellite primers.The results showed that most of the 10 microsatellite loci had polymorphic information content(PIC)ranging from 0.313 to 0.674,with an average value of 0.401,and 2 loci had high polymorphic information content(PIC≥0.5).The number of alleles(NA)of the second generation soft-shelled turtles was 2～5.The effective number of alleles(NE)ranged from 1.6349-3.6142.The observed heterozygosity(HO)ranged from0.3407 to 0.8132,with an average of 0.5819.The average expected heterozygosity(HE)ranged from 0.3894 to 0.7253,and the average value was 0.4932.3.Islet child generation analysis.Based on the microsatellite data of 103 and 79 Asian giant soft-shelled turtles born in 2019 and 2020 respectively,a single sample was treated as a population by Popgene1.3.2 software,and the Nei’s standard genetic distance of each individual was calculated to obtain the pair difference degree clustering(evolutionary tree)constructed by MEGA5.0 software,and the classification relationship between each individual was obtained.Then,the Structure software was used to simulate the number of subgroups,and the clustering status and lineage composition of the child turtles were calculated based on the Bayesian algorithm,and clumps were drawn with the software.Cluster analysis was carried out on the progeny to lay the foundation for the breeding of F3 progeny in the later period.4.The anatomy of the skeletal system.The bone specimens of Asian giant soft-shelled turtles and Chinese soft-shell turtles were prepared by insect erosion.This paper describes and draws the morphological characteristics of the whole skeletal system of P.cantorii.Skeleton is composed of exoskeleton(dorsal and ventral)and endoskeleton(axial bone and appendage bone).Compared with Chinese soft-shell turtles,the results showed that there were obvious differences in the skull and cervical vertebrae,such as the length of the snoutand the structure of the cervical vertebrae.The results can not only enrich the basic ecological data of turtles,but also provide skeletal basis for the species identification of turtles,the evolution of turtles and the adaptability of turtles to the ecological environment.