Genetic Diversity And Population Structure Of The Tiger Frog (Hoplobatrachus Rugulosus) In Zhejiang Area

The Tiger frog (Hoplobatrachus rugulosus) is widely distributed in the Southern part of China. This species has been listed in Appendix II of CITES and as one of the Class II national protected species in China. Also this species has been classified as Vulnerable on IUCN Red List and Species Red List of China. But the species population decreased 30 percent in last 3 decades, because of the habitat fragment and destruction, over-hunting by human. AS an important distributed area, Zhejiang province has experienced drastic decline at population size in recent years. In this research, we started from the nondestructive sampling in endangered Anura, and used affinitive species to cross-species amplification and enriched genomic library to select polymorphic microsatellite loci in tiger frog respectively. Finally, genetic diversity and population structure of tiger frog have been researched in Zhejiang area by these microsatellite loci. And hope to provide theories for this species protecting in Zhejiang province. The results as follow:1. Used nondestructive sampling to extract DNA from blood and toes of tiger frog respectively, and compared with the DNA which was extracted from the muscle. The results showed that the DNA from blood and toes samples was equal to its muscle sample. Then PCR analysis was applied to mitochondrion 16S rRNA and the nucleus DNA simple sequence repeats of the three types of samples respectively. The results also showed that the amplification products in three types of sampling were equivalent, and all these can be appropriate for conservation genetics research. So, the nondestructive sampling strategy can completely instead of the traditional muscle sampling method.2. Only 33.3% loci can be amplification and 46.7% loci had no amplification products using 30 pairs of microsatellite loci belong to other affinitive species. The kinship between cited species and need to researched species, and genomic DNA size of need to researched species will affect amplification efficiency using cross-species amplification. 3. 9 pairs of polymorphic microsatellite loci of tiger frog was isolated from CA-enriched genomic library. The allelic number per locus varied from 6 to 11 (average 8.56). The observed heterozygosity and expected heterozygosity varied from 0.571 to 0.935 (average 0.796) and from 0.765 to 0.888 (average 0.837) respectively. Hrug07 showed significant deviation from Hardy-Weinberg equilibrium (P<0.01) as null allele existence. Linkage disequilibrium tests showed significant linkage between loci Hrug05 and Hrug09.4. Genetic diversity was detected in 7populations of Zhejiang area (n=87) used 9 pairs of microsatellite loci. The number of alleles per locus ranged from 5 to 13 with an average of 7 and the expect heterozygosity of all populations were at higher level (>0.5). So, the genetic diversity of these 7 populations are high.5. Only Lishui population was not experienced bottleneck in recent years, other 6 populations experienced bottleneck under IAM, TPM, SMM mutation models.6. 61.9% samples in 21 pairs showed significant genetic differentiation (P<0.001) after detecting between all pairs of sampled sites. According to Nei's unbiased genetic distance constructed UPGMA dendrogram and Neighbour-Joining tree, the results showed there were two clumps about centre of Changshan and Tangxi, including Changhua, and centre of Linan and Yongkang, contains Lishui and Qingtian.7. The gene flow between all pairs of sampled sites showed there must existed gene flow between all pairs of sampled sites (N_m > 1). At the mean time, most of F_is > 0 in all sampled sites, so there must be more inbreeding between individuals. There are probabilities occurring genetic differentiation in future.