Analysis Of Genetic Diversity Of Obscure Puffer(Takifugu Obscurus)Populations

Obscure puffer(Takifugu obscurus) belongs to the genus Takifugu, is a commercially valuable anadramous in our country. Obscure puffer is a popular fish in China for its high-quality meat. However, the resource of wild populations has sharply declined under the effect of overfishing and environmental pollution. Therefore, in order to know the genetic structure of obscure puffer, microsatellite markers and SRAP markers were used to study the genetic structure and genetic diversity of four obscure puffer populations. The four obscure puffer populations were consisted of a captured population in Yangtze Rive, a released population and two cultured populations. The major results are as following:1. The transferability of Takifugu rubripes microsatellite primers to Takifugu obscurus was explored. Therefore,16microsatellite loci of Takifugu rubripes were analyzed for Takifugu obscurus. The conditions of polymerase chain reaction (PCR) were optimized for the fidelity of DNA synthesis during PCR amplification. It was found that6loci failed to be amplified and10loci (about62.5%) amplified specific products successfully. These10microsatellite primers can be used for Takifugu obscurus genetic diversity analysis.2.10microsatellite markers of Takifugu obscurus and10microsatellite Primers of Takifugu rubripes were used to analyze genetic diversity of four Takifugu obscurus populations. Four Takifugu obscurus populations were consisted of a captured population in Yangtze Rive, a released population and two cultured populations. It was found that20loci can be amplified successfully and the total of113alleles was detected. In each population, the average allele value was from4.95to5.50. The mean observed heterozygosity value was from0.6867to0.7617, while the average expected heterozygosity value was from0.6530to0.7000and the average polymorphic information content was from0.6013to0.6384. These results indicated a richer genetic diversity in four populations. But same loci were found deviating from Hardy-Weinberg Equilibrium in each population which indicated that the deficiency of heterozygote existed in same extent in four populations. The genetic differentiation coefficient among the four populations was0.0406.There was a little genetic differentiation among these populations. The gene flow was5.9056. It indicated that a little gene exchange among them. The genetic distances among populations ranged from0.0880to0.1519. Clustering analysis showed that shanghai population formed an independent cluster, the other three populations were another cluster.3. The orthogonal design was used to optimize Sequence-related amplified polymorphism (SRAP) PCR amplification system on Takifugu obscurus in four leveles of five factors (Mg2+, TaqDNApolymerase, dNTPs, DNA template, primer). The results showed that in20μL SRAP-PCR reaetion:Mg2+1.5mmol/L, TaqDNA polymerase0.5U, dNTPs0.15mmol/L, template DNA75ng, prime0.2μmol/L.The optimized SRAP-PCR system was tested on thirty Takifugu obscurus germplasms and proved to be steady and reliable.This optimized SRAP-PCR reaction system would play an important role in Takifugu obscurus genetic diversity analysis, linkage map construction, germplasm identification and so on.4. With the SRAP reaction system,18pairs of primer combinations, which were selected from49primer pairs, applied to four populations. Sequence-related amplified polymorphism (SRAP) marker was firstly used to analyze the genetic diversity of four populations of Takifugu obscurus. A total of231loci were detected, among them156were polymorphic. The proportion of polymorphic loci was ranged from54.98%to58.87%in four populations. The Nei’s genetic diversity of the four populations was between0.1992and0.2005while the Shannon information index was between0.2953and0.3016. The gene flow was4.1291among populations. Of which, the proportion of polymorphic loci, the Nei’s genetic diversity and Shannon information index of the captured population were a little higher than the other three populations. Individual clustering showed that the population of shanghai formed an independent cluster and the other three populations were another cluster. The analysis of molecular variance (AMOVA) showed that87.40%of variance was within populations. These genetic statistics indicated there is a richer genetic diversity in four populations and a certain gene flow between them.