| Asian Yellow Pond Turtle(Mauremys mutica)is an fresh turtle that has high edible, medical and ornamental value. Due to the effects of habitat destruction and overfishing, the population size of wild Asian Yellow Pond Turtle has been decreased dramatically. Asian Yellow Pond Turtle, used to be vulnerable species, has been listed in AppendicesⅡ of Convention on International Trade in Endangered Species of Wild Fauna and Flora(CITES). With the consumer demand increasingly rising, more and more people choose to cultivate Asian Yellow Pond Turtle, which has become an most popular aquaculture species in recent years. Because of the long time, about five years, it takes for Asian Yellow Pond Turtle to reach sexual maturation and the relatively small amount of eggs, hatchlings is in short supply, which seriously restrict the development of cultivation scale. Therefore, to ease this situation, selective breeding some high prolific varieties is in need. Some members in our laboratory has been devoted to the breeding work of improving the fertility of Asian Yellow Pond Turtle for years, which include selection of high prolific varieties. In the process of selection, which need be based on a relatively large population, to estimate the reproductive performance of female turtle, the number of its offspring should be clear, which is difficult to obtain in a random mating group. Thus to solve this problem, it is necessary to develop a technique for parentage assignment. Recently, in the research of parentage assignment, the technology of microsatellite markers was widely used. In this experiment, regarding parents and two offspring group, born in 2013 and 2014, as experimental subjects, we conduct the research of genetic diversity and parentage assignment with the two multiplex PCR sets developed based on microsatellite markers, through which the number of female parent?s offspring can be worked out. All of our work will lay the foundation for selective breeding of high prolific varieties. The specific experiment contents include two aspects, which are as follows.1. Design and screening of the microsatellite primerWe conduct PCRs with 50 microsatellite primers on Asian Yellow Pond Turtle?s genomic DNA, 44 of which developed by our laboratory and 6 chosen from published literature. The amplification stripes of 34 primers are clear, stable and specific. 30 random individuals from parents group are detected to test the polymorphism of the 34 microsatellites, six of which has only one allele, the remaining 28 of which has 4 alleles at least. The analysis of genetic parameters shows: the number of alleles of the 28 microsatellite locus ranges from 4 to21, with an average of 10.1; the polymorphism information contents(PIC) of the 20 microsatellite varies from 0.337 to 0.992, with an average of 0.717, 22 of which show high polymorphism(PIC≥0.5) and 6 show intermediate polymorphism(0.25<PIC<0.5); the observed heterozygosity varies from 0.333 to 0.967 with an average of 0.678, while the expected heterozygosity varies from 0.396 to 0.942 with an average of 0.754; the frequency of invalid allele ranges from-13.63% to 40.31%.2. Development of microsatellite multiplex PCR set and analysis of genetic diversity in Asian Yellow Pond TurtleTwo multiplex PCR sets are established by optimizing primers ratio, annealing temperature, concentration of fluorescent joint and times of cycle, Each set made up with eight loci. The set 1 is made up with sg8, mmu13, mmu1, mmu7, mmu2, sg10, mmu3, mmu4, with their primers mix ratio at 5:8:8:10:2:6:2:11and the concentration of mixed fluorescent joint being 2μmol/L. Annealing temperature and times of cycle of PCR in set 1 are 57.5℃ and 22 respectively. The set 2 is made up with sg3, sg16, sg11, sg29, sg22, sg5, sg23, sg27, with their primers mix ratio at 10:10:4:5:5:7:15:10 and the concentration of mixed fluorescent joint being 1.2μmol/L. Annealing temperature and times of cycle of PCR in set 2 are 62.5℃ and 20 respectively. The genotyping of individuals in parent and offspring groups and genetic diversity analysis are performed with the multiplex PCR tools and ABI3130 genetic analyzer. The result shows: there is a relatively high genetic variability on the 16 microsatellite locus in both parent and offspring groups; most of the genetic information in parent is expressed in offspring, and the offspring?s performance of genetic diversity is close to the parent whose genetic diversity is richer.3. Parentage assignment of Asian Yellow Pond Turtle and its application in selective breeding of parent with high prolificacyWith the data of genotype and software Cervus 3.0, analysis of exclusion rate of single parent is conducted in the group of female parent and offspring(2013). The results indicate that the exclusion rate of a single parent of the sixteen microsatellite makers range from 9.2% to 74.7% and the accumulated exclusion rate is 99.9%, when two parental genotype is unknown. The results of parentage assignment in the group of female parent and offspring, born in 2013 and 2014 shows: both the simulate assignment rates approach 100%, and the real assignment rates are 87% and 94% respectively.According to the results of parentage assignment, the offspring number of the female parent is counted: on one hand, there is one female parent at least who didn?t give birth in 2014 and there is likely to be female parent who didn?t give birth in 2013; on the other hand, the offspring number of the female parent marked with m2、m16、m17、m23、m27、m38、m43、m73、m117、m124 is no less than 5 in both 2013 and 2014. These female parents show so good performance in fecundity, therefore they and their offspring can be used as objects in selective breeding of parent with high prolificacy.Microsatellite multiplex PCR set developed in this experiment can used as tool in parentage assignment for Asian Yellow Pond Turtle and parentage assignment for female parent and their offspring, born on 2013 and 2014, is the initial work of selective breeding of parent with high prolificacy, laying a foundation for the development of marker-assisted breeding. |
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