Reproductive Capacity Research Of The Artificially Cultured Yellow Pond Turtle(Mauremys Mutica)

There is a long history about the utilization of turtles,such as the sacrifice,food,medicine or ornamental animal.The need of market results in the huge turtle trade and many illegal trades.Because of environmental pollution,hunting,habitat loss and invasion of alien species,many wild turtles are facing the threat of extinction.Because of the high value and large demand,the wild population has largely declined in China.To protect and meet the market demand of turtles,the turtle farming is widespread and become an important industry in China now.Successive generations of farm-raised turtles show an obvious decrease in reproductive capability,farmers still need many wild individuals to meet the industry demand of offspring.In order to reduce the dependence and the damage to the wild resource,the reproductive research of turtles is important and impendency now.The method to keep or improve the fecundity of farm-raised turtle is the key point for a sustainable development.Because the similar physiology structure and behavior characteristics,the yellow pond turtles(Mauremys mutica)were chosen as the experimental subjects.In this study,we filtered out some female turtles with high fecundity,researched the effect of multiple paternity and repeated paternity on the breeding results of the experimental group and individual,described the ovarian histology and characterized the oogenesis of adult female turtle,cloned and screened reproduction related the gene and the SNPs of the female turtles.1.Asessment and screening of female fecundity in the artificial cultured Asian yellow pond turtle based on parentage assignmentThe present study designed to quantify the fecundity of the M.mutica and tried to screen good reproductive performance females.The population genetic variability and its genetic structure were analysed too.The parent-child relationships of all offspring(1263)in four successive years were confirmed using sixteen microsatellite loci.The genetic variability between the 129 parents and offspring were small and the offspring of different years existed little variability.We summarized the reproductive results of all females and counted annual offspring number and offspring number variation.Then the females were divided into three types(stable,undulate and leveling off type)according to the continuity of the reproductive performance.We screened seven females having good reproductive ability that could provide 16.94%annual contributions,while there were two females having no offspring in four years.Moreover,the possible reasons causing this difference and the importance of family survey was analyzed.These researches could provide the foundation and materials for the construction of good reproductive group and the studying of reproductive biology of M.mutica.2.Histopathological charateristics of oogenesis and vitellogenesis in yellow pond turtleThe oogenesis and vitellogenesis of adult females of M.mutica were characterized.The dynamic structural changes of ovarian and ovular were observed with the application of histology methods.The ovary of M.mutica has an irregular cortex and a well-developed reticular stroma.All stages of follicular cells,atretic follicle and corpus luteum were distributed in the cortex.The process of the oogenesis was divided into ten consecutive phases according to the morphological characteristics of the yolk granules,follicular cells and the pellucids zone.The structure of yolk granules gradually changed throughout the vitellogenesis.Several features of M.mutica ovarian were similar to Pelodiseus Sinensis.Nevertheless,those structures were different from lizards and snakes in reptiles and more different from mammals.These studies were important to research the follicular development and reproductive biology.It is also useful to help the breeding,protection and utilization of M.mutica.3.Multiple paternity and the reproductive capacity analysis of the male in cultured yellow pond turtlesAll studied freshwater turtles have polyandrous mating systems with multiple paternity.In order to survey the mating strategies of M.mutica,one year’s genetic data of parents and all offspring in an artificially cultured population were analyzed.Two groups of multiplex PCR containing 16 microsatellite loci were used to analyze the paternity of 302 hatchlings from 132 parents and from 138 clutches.The genetic data indicated that multiple paternity is rare in M.mutica,occurring in only seven of 138 clutches.Although the frequency of multiple paternity was 5.07%at least,our results indicate that M.mutica has a polyandrous mating system.In the breeding season,the successive clutches of 34 females each had the same paternity as the previous clutches.We observed that four males(f85,f58,f87,and f76)had 99 offspring that account for 32.78%of all offspring,more than 20 offspring respectively.This finding implies that paternity is competitive in this artificially cultured population and may bias the genetic diversity of the offspring.4.Repeated paternity affect male reproductive success and the offspring number in yellow pond turtleMating number is considered as the important factor affecting male reproductive success in polygynous systems,but differences of female reproductive qualities may also result in the variation of male reproductive success.We recorded the mating pairs and the offspring number viriation of the experimental group in four years.The results showed that all male turtles had offspring in 4 years and there were some preponderance male turtles in the experimental group.Moreover,the analysis of mating combination showed that some genotype combinations had the advantages in the groups and their offspring were the main part of the progeny in the female turtles.Repeated paternity was observed in the 45 female turtles with breeding in many years,excluding the female turtles spawned only in one year.Forty individuals(88.89%)repeated in the adjacent years and five individuals repeated in the overyear.The use of stored sperm to fertilize sequential clutches within and potentially among years could substantially increase the male’s reproductive success,particularly when females or males could make mating decisions on phenotypic characteristics correlated with reproductive quality.5.The gene clones of FSH β DNA,RNA of M.mutica and the polymorphic analyses of FSHβIn order to choose the female turtles quickly and accurately,the candidate gene method was used to screen the molecular markers.The vertebrate reproduction is regulated by the hypothalamic-pituitary-gonadal axis.The follicle-stimulating hormone could promote the follicular growth and development,and the follicle-stimulating hormone β(FSH β)is the key part to decide the function of FSH.The clone of FSH/3 gene and the SNPs analysis associated with the fecundity were researched in this study.Using the technique of RACE,the cDNA sequence of FSH β gene was firstly acquired.The complete gene was 745 bp and encoded 131 amino acids.Two tag sequences of Glycoprotein hormone beta family were found in the cDNA sequence.The protein conserved domain includes 26 dimer interfaces,7 receptor binding sites,6 cysteine knot motifs.Then the complete DNA sequence of FSH β gene was acquired with the Genome Walking technique.Two pairs of primers were designed to cover the gene and 83 female turtles were used to screen the SNPs.The SNPs associated with the average offspring number of 83 female turtles in four years(2013-2016).The F727 SNP with C→T mutation had a significant difference in the offspring number.6.Screening the SNPs associated with the females’ offspring in M.muticaIn this study,the 90 SNPs that were good supporting and associated with female reproductive activity were chosen from the previous transcriptome analysis of brain-gonad of M.mutica.The fragments containing the SNP were amplified with iPLEX Gold reagents and genetic typing with the MALDI-TOF mass spectrometry.The polymorphism of 64 SNPs was verified with 83 females in the early-experimental population.The genotypes of the selected SNPs were associated with the annual average offspring number of each female.The genotypes of RS 24 show significant difference in the offspring number(p<0.05),the offspring number of AA type(7.75±0.00)higher than GG type(3.59±2.36)and GA type(3.64±2.27).The heterozygous GG type(7.75±0)is higher than GA(3.60±2.42)and AA types(3.60±2.32),but AA and GG have no significantly difference.The genotypes of RS 5 only have two types,CG(5.88±2.61)and CC(3.53±2.30)show significant difference.The genotypes of RS 80 AA type(4.63±2.3 8)shows a significantly difference with AG type(3.32±2.45),but AA type has no significantly difference with GG(3.50±1.87).The analysis showed that SNPs of RS24、RS5、RS17 are closed with the reproductive activity and The AA of RS24,GG of RS17 and CG of RS5 may indicate the high fecundity.The SNPs need further verification with another population or the wild individuals according their offspring.In summary,M.mutica was chosen as the main representative to research the breeding activity of turtles in this study.The fecundity of all parents was determined according the paternity test of all individuals.The parent-child relationships of all offspring in four successive years were confirmed,so the female and male turtles having good fecundity were collected.The SNPs of transcriptome analysis were screened to find the marks associated with the reproductive activity of female turtles.The structure and function analysis of FSHβ genes was identified.The polymorphism and SNPs associated with the reproductive activity were screened and confirmed with 83 females.We found the male turtles also has great potential,because the multiple paternity and repeated paternity observed in the experimental population.