Isolation Of Microsatellite Markers And Paternity Test Of Emei Moustache Toad (Leptobrachium Boringii)

Paternity testing is an important means of studying mating systems in animals.It enables us to get insights into the kinship between the adults and their offspring,which can not only help us understand the preference of mate selection,mating frequency,the nurturing behavior,and the adaptive sex ratio,but also can guide the conservation of endangered animals.At present,there are abundant studies on paternity testing in humans,mammals,fish,insects and reptiles,but relatively few in amphibians.This is mainly due to the fact that,compared to higher animals,amphibians receive less attention both at home and abroad with relatively few developments based on their molecular markers.In this thesis,Leptobrachium boringii was selected as the research object.By screening microsatellites with its transcriptome,11 polymorphic loci were found.What's more,paternity testing and data analysis were carried out on the adult and progeny samples collected from the Emei breeding ground in the Badagong Mountain National Nature Reserve in Hunan Province in 2017.With the SSRIT(simple sequence repeat identification tool),117 four-base Simple Sequence Repeats were found.According to the temperature gradient experiment,primer optimization and stability amplification were performed,and finally,34 pairs of primers stably amplified were obtained.Its genotyping was read in Genemarker Version 1.3.Then GenePop Version 1.2 was used to calculate whether it conforms to the Hardy-Weinberg expectation and detect whether there is a linkage between pairs of loci.The presence of invalid alleles and large allele dropout was analyzed with MicroChecker Version 2.2.3 and the polymorphism information was finally calculated using the program Cervus Version 3.0,and 11 pairs of microsatellite loci were finally obtained.According to the screened 11 polymorphic loci,DNA extraction,PCR amplification,and STR scanning were performed on the collected 305 adult toe(148 female,157 male)and 3,190 progeny embryos.The results were genotyped and their genetic diversity was calculated using Cervus Version 3.0.The diversities are as follows.The numbers of the alleles at 11 loci varied from 2 to 10.The average number of alleles per loci was 4.82.The average expected heterozygosity was 0.5243,and the average polymorphic information content was 0.4635.The analysis by using the parental calculating software Colony 2.0.6.5,the probability of paternity exclusion was 98.2%.Among the 95 egg masses,77.3%of the males had two or more egg masses which are their offspring and 33.3%of the egg masses had multiple patriarchal phenomena.This showed that the males of L.boringii had participated in the breeding activities of 2 times or more,so we speculated that the L.boringii adopted a polygamous mating system,and the multiple patriarchal phenomena of the same egg mass also provides the satellite strategy of the male L.boringii with some molecular evidence.With 11 microsatellite loci,the paternity identification was conducted on 95 egg masses of L.boringii.We concluded that the L.boringii had a polygamous mating system and males can rely on the satellite mating strategy to improve their fitness.