Microsatellite Markers Screening And The Application On Paternity Identification In Hynobius Chinensis

Identifying paternity for animals is the main issue of behavioral ecology.The studies of paternity identification in invertebrates and vertebrates,such as fishes,reptiles,birds and mammals have been comparatively sufficient.However,the studies of paternity identification in amphibians,especially for Caudata are scarce,which is largely limited by the development of polymorphic microsatellite markers.Identifying paternity for amphibians has a great significance for further studying the evolutionary mechanisms of animal mating systems and sexual selection.Therefore,we selected the Chinese Hynobiid(Hynobius chinensis),an endangered Caudata species endemic to China as our research object.We screened polymorphic microsatellite markers in H.chinensis transcriptome and applied them to identify paternity.According to the study results,we have found that:1)By using the online Simple Sequence Repeat Identification Tool,we identified 4,297 Simple Sequence Repeats among the 77,111 unigene sequences in H.chinensis transcriptome.According to the standard design of primer pairs,we designed 119 primer pairs for 74 unigene sequences by using the online Primer 3.0 software.After primers optimization and primers stability testing,we finally screened 13 polymorphic microsatellite loci.By using GenePop 1.2 software on the Web,Hardy-Weinberg Equilibrium and linkage disequilibrium were assessed for the 13 polymorphic microsatellite loci in 22 individuals of H.chinensis.We found that the 13 polymorphic microsatellite loci showed Hardy-Weinberg equilibrium,and no microsatellite loci showed linkage disequilibrium.The results of the Micro-Checker 2.2.3 software testing showed that neither evidence of stutter errors nor evidence of large allele dropout was found at the 13 polymorphic microsatellite loci.The 13 polymorphic microsatellite loci might be applied to identify paternity for H.chinensis.2)In the experiment of paternity identification in H.chinensis,we used 118 adults toe samples(78 males toe samples,40 females toe samples)and 2360 embryo samples(39 pairs of egg sacs)of H.chinensis as our experimental samples.By using the Cervus 3.0 program,we calculated the genetic diversity indices of the 13 polymorphic microsatellite loci in 2478 individuals of H.chinensis.The number of alleles per loci ranged from 2 to 6 and the mean number of alleles per loci was 3.15.The mean expected heterozygosity per loci was 0.402.The polymorphic information content per loci ranged from 0.019 to 0.550.By using the Cervus 3.0 program,we calculated that the cumulative paternity exclusion probability for the 13 polymorphic microsatellite loci was 92.6%.The results of paternity identification in H.chinensis showed that 35 out of 39 clutches of H.chinensis came from two or more genetic fathers,and the incidence of multiple paterniy was 89.74%.3)We preliminarily explored the formation reason of multiple paterniy in H.chinensis.For females,mating with several males simultaneously could increase the offspring genetic diversity.We deduced that the genetic diversity hypothesis could be used to explain the multiple paterniy in H.chinensis.