Population Genetic Structure Of Nemipterus Bathybius In South China Sea And Optimization Of DNA Extraction From Formalin-fixed Fish Specimens

There are two parts in this thesis. The first part is population genetic structure ofNemipterus bathybius in South China Sea, and the second part is optimization of DNAextraction from formalin-fixed fish specimens.Part one: population genetic structure of Nemipterus bathybius in South China Sea.Nemipterus bathybius is one of the widely distributed key species in South ChinaSea with high commercial value. The information of population genetic characteristicssuch as population composition in N. bathybius is unclear, which provides scientificreference for the assessment and management of this fishery resources. Therefore, wecollected the N. bathybius samples from the South China Sea and analyzed thepopulation genetic structure of the species using microsatellite markers.We isolated and characterized22polymorphic microsatellite loci in N.bathybiususing a (GT)13enriched genomic library. The numbers of alleles per locus ranged from4to13with an average of7.86. The observed and expected heterozygosities rangedfrom0.167to0.889and from0.278to0.904, with averages of0.590and0.690,respectively. Deviation from Hardy-Weinberg equilibrium was detected in three loci(the adjusted P≤0.0023). Two loci showed evidence for null alleles (null allelefrequency≥5%). No significant linkage disequilibrium was detected between locipairs.Subsequently, we analyzed population genetic diversity and genetic structure using9microsatellite loci among4samples of N. bathybius in South China Sea. A tatal of100alleles were detected in4samples. The average number of alleles per loci is11.11,ranged from6(J135A) to14(4-32). The average number of alleles per colony is7.9,ranged from6.2(Shantou) to8.8(Spratly Islands). The averages of observed andexpected heterozygosities per sample ranged from0.561(Shantou) to0.652 (Zhujiangkou) and from0.730(Shantou) to0.780(Spratly Islands), respectively. Theresults demonstrated that high genetic diversity was present in all4samples. PairwiseFSTvalues showed four pairs have significant genetic differences. Pairwise FSTvalueswere generally low, no exceeding0.0149. The analysis of molecular variance(AMOVA) showed that most of the variances were found within samples (97.94%),and few of the variance was found among samples (2.06%). Structure analysis showsthat all samples belong to the same population. MDS analysis showed that the geneticdistance arrangement has no corresponding relationand with geographic distancearrangement in2d space. The above results revealed that there were very weak geneticdifferences among N.bathybius samples, the N. bathybius pupulation in South ChinaSea was a near panmixia.Part two: optimization of DNA extraction from formalin-fixed fish specimens.In recent decades, population structure of many marine fish has dramatically changedunder the influence of anthropogenic activities and climate change. For betterunderstand the change mechanism, we need DNA extracted from formalin-fixed marinefish specimens. However, obtaining high quality DNA from formalin-fixed specimenswas difficult. In ordor to improve the quantity of DNA extracted from marine fishspecimens,10Nemipterus virgatus specimens fixed in1962were used as samples, andan orthogonal experiment was designed following the L8(4×24) table. The DNAconcentration was taken as the evaluation index. The sampling parts, anti-formaldehydetreatments, digestive times and extraction methods were taken as seletion factors. Thebest group was that muscle samples with long-time wash in buffer GTE and Alcoholgradient,90℃annealing for30min and drying in a vacuum centrifuge,3h55℃w aterboth digestion with Proteinase K, DNA extraction by kit. Anti-formaldehydetreatments could significantly improve the production of DNA. Digestion after90℃water bath was efficient. Quality of Specimen DNA was assessed by PCR amplificationand sequencing. Sequence compared between Specimens and fresh ones, which provedthat the methods were reliable.