Isolation And Polymorphic Analysis Of Microsatellite Loci For The Chinese Alligator

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. Although the captive Chinese alligator populations have been greatly developed in recent years, there has been genetic depression in the filial generation because of the founder effect. As the startup of the reintroduction project for the captive Chinese alligator population, it is urgent to investigate the genetic status of this species and provide feasible strategies for its conservation.In this study, we isolated polymorphic microsatellite loci from the Chinese alligator based on magnetic beads enrichment protocol, and did comprehensively research on genetic diversity and population structure of this species. The major results are:1. Approximately 4,000 clones were screened and a total of 260 recombinants that potentially contained microsatellite sequence were obtained. 150 positive clones were chosen randomly for DNA sequencing, out of which 92 clones had sufficient flanking sequence for primer design. The result of PCR amplification revealed that 56 primer pairs could yield consistent specific products. Further experiments showed that 11 pairs of them were polymorphic and well amplificated for each sample.2. The 11 polymorphic microsatellites presented a total of 31 alleles among 57 individuals scored, yielding an average of 2.82 alleles per locus. One allele was unique to the American reintroduced population(Alsi05-2) but four private alleles(Alsi05-1; Alsi06-1; Alsi07-1; Alsi10-1) were detected in the Changxing indigenous population. The average PIC (polymorphic information content) of the 11 polymorphic loci obtained in this study is 0.384.3. The individual identity probability was evaluated for the 11 polymophic markers. Results suggested that when the 11 polymorphic loci were combined, the individual identity probability was 0.9998. Therefore, the microsatellite markers could be utilized to genetic pedigree construction for the captive Chinese alligator populations. In addition, it could be helpful in the selecting of proper individuals for the reintroduction program.4. In this study, we also used 8 polymorphic loci isolated by cross-species amplification to assess the genetic status of this species, of which 6 loci was amplificated effectively. Finally, the exclusion probabilities of paternity testing went up to 0.991 if 11 species-specific polymorphic loci calculated in combination with 6 previous cross-species markers, which achieved the desired results.5. A complete Bayesian method in BAPS (Bayesian Analysis of Population Structure) was chosen to perform individual clustering without prior population information. The results showed that the 57 individuals were grouped into 2 clusters (named as the Changxing indigenous population and the American reintroduced population). The further genetic admixture analysis revealed that although genetic difference was present between the two populations, there was certain admixture among them.6. Based on the microsatellite analysis results, we conclude that the Chinese alligator has very low genetic diversity, but the Changxing indigenous population and the American reintroduced population still has some rare genes of their own. Hence, we strongly suggest that individuals from different populations should be chosen as the founders in the reintroduction program to largely protect the genetic diversity of this critically endangered species.