Application Of MtDNA And Microsatellite Markers In Japanese Flounder Stock Enhancement

Japanese flounder belongs to offshore bottom fish,which is an important fishery resources and main sea culture species in china.It is very popular in the market because of its strong adaptability、fast growth、individual large、the succulent delicious.Recent year, the coastal environment is continuously serious in china, with the addition of over-exploitation, Japanese flounder resources are facing great threat, and the wild fish population decreases continuously.In order to augment natural population of Japanese flounder,from 1990 s,people released mass hatchery-reared juveniles to Huanghai and Bohai sea for many years.Mass hatchery-reared juveniles release to nature water,it is widely concerned its survival rate、growth rate and capture rate,in order to solve this problem, we should mainly distinguish “the released fishes”and “the unreleased fishes”.So far,people mainly use microsatellite markers in paternity test and genetic diversity of Japanese flounder.However they few use mt DNA the characteristics of maternal inheritance in paternity test of Japanese flounder stock enhancement. To this end, the author carried out the research about application of mt DNA and microsatellite markers together in research of Japanese flounder stock enhancement.The results are as follows:1.Paternity test of Japanese flounder from recaptured population inferred from mt DNA and microsatellite markersFirst,mt DNA were used for paternity test to parental、released and recaptured flounder.The results showed that fifty-five parents had twenty-six kinds of haplotypes;and the one hundred and twenty-nine released fishes had five kinds of haplotypes, allcorresponding to the parents’ haplotypes database, verified the accuracy of mt DNA markers in the anaylsis of paretal allocation for the released Japanese flounder.Four hundred and thirty-five recaptured fishes with seventy kinds of haplotypes were identified, among them, three hundred and thirty fishes with seventeen kinds of haplotypes were corresponded to the parents’ haplotypes database. These fish had the possibility to be the released fish. Another one hundred and five fishes with fifty-three kinds of haplotypes, could not be corresponded to the parents’ haplotypes database, and we categorized them into non-released fish of Beidaihe. We performed further paternity tests by using four high polymorphic microsatellite markers for the three hundred and thirty might be released fishes. The results indicated that three hundred and ten fishes’ alleles were all corresponded to candidate parents’ alleles, and we determined these fish as released fish; another ten fishes’ alleles were not corresponded to candidate parents’ alleles, and were determined as non-released fish. The results showed that,use mt DNA the characteristics of maternal inheritance,can categorize non-released fish from recaptured fish quickly;performed further paternity tests by using high polymorphic microsatellite markers for the might be released fishes can determine released fish. In conclusion, we could effectively determine the released Japanese flounder from the recaptured ones by using mt DNA and polymorphic microsatellite markers together,improve the efficiency and accuracy of paternity tests,it is a kind of valuable method.2.Parental contribution of Japanese flounder inferred from mt DNA and microsatellite markersApplication of mt DNA and microsatellite markers can analyze different parents have different contribution, the highest is 61.31%, the lowest is 0%.There is one couple detect have one hundred and thirty six offsprings,its contribution is 49.64%;another one female and one male have no contribution.In conclusion,there is big difference in Japanese flounder parental contribution.However, the reason of different parental contributions among parents needs further research.3.Genetic diversity and genetic structure of Japanese flounder broodstock populations analysis based on mt DNA makerApplication of mt DNA marker analyze Beidaihe、Weihai and Yantai three broodstock populations the average A, T, C and G contents were 38.4%, 28.1%, 17.9%and 15.6%, respectively, and the contents of A + T were 66.5%, higher than those of G +C(33.5%),it performs a significant A + T composition bias. The total variable sites in276 Japanese flounder individuals are 357,si/sv is 1.0,observed 54 mt DNA haplotypes.Haplotype diversity、nucleotide diversity and mean pairwise nucleotide differences of Beidaihe population were all higher than Weihai and Yantai populations.The Kimura and analysis based on D-loop showed that the significant genetic distance and genetic divergence existed in Beidaihe population and other two populations.In conclusion,there has very rich variation and genetic diversity in three Japanese flounder broodstock populations based on mitochondrial control region,Beidaihe population is richer than Weihai and Yantai populations,and it has better resources quality.The result of the experiment indicates that the method which based on paternity test could be used to distinguish released population and unreleased population,it has an important application prospect for evaluating the effect of stock enhancement; the different parents had different contribution to reproduction, so shen we match reproduct fishes we should consider not only the number of parents but also the difference contribution of different parents,in order to avoid change genetic structure of released population;the genetic diversity of three Japanese flounder broodstock populations in stock enhancement inferred from mitochondrial control region is very rich,Beidaihe population is much richer than Weihai and Yantai populations.The result of the research provide basic information for the analyzing of genetic diversity and artificial stock enhancement effect.