The Mating System And Its Influence On The Genetic Diversity Of The Chinese Alligators

The Chinese alligator,Alligator sinensis is a freshwater crocodilian species which is endemic in China and one of the most endangered species in the world.The Chinese government listed this species as a first-level state-protected species,while the IUCN Red List of Threatened Species listed as “Critically Endangered” as the wild population is rare.Captive breeding is an integral part of many species recovery plans.Lower genetic diversity means lower reproductive fitness and the increased risk of extinction for endangered species.Additionally,the preservation of population genetic diversity is strongly influenced by the mating system and ultimately determines how the variation is transmitted in generations.The mating system plays an important role in revealing the genetic diversity and destiny of their offspring,and enhancing profound understanding of sexual selection and the evolution of species.Genomic DNA was extracted from the umbilical cord and egg-shell membranes of the Chinese alligator,which can be used as a non-invasive sampling method to the conservation studies about the genetic resources of oviparous endangered animals.In addition,to study the mating system,microsatellite technology and whole-genome resequencing technology were used and to study the influence of mating system on the genetic characteristics of the offspring.The main results are as follows:1.Paternity analysis of the 50 nest Chinese alligators(n1=755)were using microsatellite genotyping technology,the result showed the evidence of multiple paternity in Chinese alligators with 40%(20/50)of clutches being of single paternal origin,52%(26/50)of clutches from two paternal origins,and 8%(4/50)of clutches with a minimum of three paternal origins.Meanwhile,the three paternal origins is a new discovery for the research of Chinese alligator mating system.However,single paternity was found in a nest of wild crocodiles(n2=17)from the Changle protection site.Additionally,multiple paternity rate would be influenced by multiple factors at the same time,including mate encounter rates(population density),sex ratios,age and the localities except the sample size.2.Microsatellite genotyping data showed multiple paternity couldn't increase the egg production significantly,and there is no significant relationship between litter size and males in one nest(P>0.05).Polyandry females choose to mate with males that are more distant in relatedness compared with monogamy females.Multiple paternity can decrease the inbreeding coefficient,furthermore,there was an increased allelic diversity(though not heterozygosity)in multiple paternity sired offspring compared with the single paternity sired offspring in F2 generations,as predicted by the genetic diversity hypothesis,which suggested that multiple paternity is a adaptive strategy of the Chinese alligator for maintaining the genetic diversity.3.Whole-genome resequencing was carried about a total of 49 samples including the Chinese alligator of F3 generation(multiple paternity group and single paternity group)which were used for mating system studying,and samples collected from Changle(CL,2015),F1 generation(Xuancheng,1999),and from Zhejiang(1999),and also one from wild collected in 2007.49 samples were got about 2222.16 Mb Clean Reads,665.35 Gbp the Clean Data,and Q30 reached an average of 89.96%.The average ratio of the samples to the reference genome was 98.35%,the average coverage depth was 5X,and the genome coverage was 97.54%.The results showed that the captive alligators are derived from wild alligators,the genetic variation of the whole alligator population was very small,interestingly,compared with other samples,alligators from Changle protection site showed a relatively large genetic variation.4.Whole-genome sequencing data also showed that the multiple paternity can increase the genetic diversity and genetic variation of the offspring,which is similar to the results of the microsatellites.In addition,the candidate differential genes were filtered out from the significant differential region of different mating systems by using selective sweep method combined Fst with ? ratio,and these genes including TLR3 which is a immune signal recognition receptor,and ARPC3 that can regulate salmonella in epithelial cell invasion,and HSPA4 L the gene mutation can cause early neonatal respiratory distress and increase the mortality rate,a total 15 gene which are related to the disease,immunity and important life activities of the Chinese alligator.Multiple paternity is a more favorable adaptation strategy that plays an important role in maintaining the genetic diversity of the population and may increase the ability of disease resistance of the offspring.The present study will provide basic data for the further understanding of the Chinese alligator reproduction breeding strategies and other reproductive biology and can also guide artificial breeding management,genetic conservation and reintroduction of endangered species.In addition,this is the first time to use whole-genome resequencing to study the mating system of animals,which provide a new way of studying animal mating systems.