Application Of Microsatellite Markers To Evaluate The Effect Of Paralichthys Olivaceus Stock Enhancement

Japanese flounder Paralichthys olivaceus is an important species for commercialfishing and aquaculture in China. It is not only an important fishing specie in ourcountry, but also the major cultured fish. Recently, Japanese flounder around China arefacing great challenges from habitat degradation, climate change and over-exploitation.Attempts to augment natural population of flounder, people released hatchery-rearedjuveniles, but the effectiveness of stock enhancement is difficult to evaluate. To solvethis problem, the key point is to distinguish―released ones‖and―wild ones‖.In the first half of this thesis, polymorphic microsatellite markers were used toidentify the parentage of cultured flounder. I analyzed the relation between probabilityof exclusion and paternity test accuracy, and then I compared the difference of parentalcontributions in the reproduction. In the rest part, I examined parental and recapturedflounder. With the paternity test method, released flounder and wild flounder weredistinguished from recaptured ones. The genetic diversity of the three populations wasanalyzed with8microsatellite markers. The results are as follows:(1) Thirteen parents (five, eight) and one hundred and eighty eight offspringof Japanese flounder (Paralichthys olivaceus), eighteen microsatellite markers wereused to study the Probability of Exclusion (PE), paternity test accuracy and the parentalcontributions in the reproduction. The average PE of18microsatellite markers was0.783, loci POLI-003-HFS had a highest value of0.916, while locus Newpao202andPoli1617were lowest, it’s0.604. When using4microsatellite markers, PE was greaterthan or equal to0.9999and accuracy was92.02%; Using6microsatellite markers, thePE was greater than or equal to0.999999and accuracy was96.81%; When using8microsatellite markers, the two values were0.999999999and97.87%; Using10microsatellite markers, the accuracy was99.47%, while12～18microsatellite markers,the accuracy could reach100%. The accuracy was increased with the number of microsatellite markers increasing. The results indicated that SSR markers could be usedfor the parentage determination and evaluating the effect of enhancement release, thepaternity test accuracy could be reached100%by using the method based on PE. In thisexperiment, although all the thirteen parents had contribution in the reproduction,different individuals showed large variance. The highest and the lowest parentalcontribution were47.34%and0.53%, respectively, however, the difference of parentalcontributions among parents needs further research.(2) Paternity test of three hundred and twenty-one recaptured flounder was carriedout with eight microsatellite markers which were already proved to be applicable informer experiment, and the genetic diversity of8microsatellite loci was analyzed inparental population, stock-enhancement population and wild population. The resultsindicated that ninety-one were released flounder which account for28.35%and theremaining were wild which account for71.65%. The allele number of three populationsat8microsatellite loci was103, the mean allelic number of per locus was12.88,indicating a rich gene pool. The mean observed heterozygosity of three populationswere0.8115、0.7756and0.7827, the expected heterozygosity were0.8257、0.7858and0.8154, and the former is smaller than the latter, which indicates heterozygotedeficiency in three populations. The mean PIC were0.7998、0.7550and0.7932, theShannon’s index were2.0076、1.8499and2.0028, which showed higher geneticdiversity. In this study, the genetic diversity of three populations was high, although thegenetic diversity of stock-enhancement population is lower than the others, there was noobvious difference among the three populations. Chi-square test was used to analyze thegenotypes based on Hardy-Weinberg equilibrium; the P value denoted that most locitested were not accorded with Hardy-Weinberg equilibrium. For the three populations,the genetic similarity index of0.9693between parental population and releasedpopulation is maximum, proving the phylogenetic relationship between the twopopulations are relatively nearer. Cluster’s analysis showed the three populations weredivided into two groups, parental population and released population were in the firstgroup, and then wild population was in the second group.The result of the experiment indicates that the difference of parents contributing toreproduction varied wildly, we should avoid cultured fish as broodstock during seedling.The method which based on paternity test could be used to distinguishstock-enhancement population and wild population, it has an important application prospect for evaluating the effect of stock enhancement. To use the wild flounder asparent and enlarge the parent population, stock-enhancement population would notaffect the genetic diversity of wild population.