Molecular Markers And Genetic Diversity Analysis In Populations Of Trachidermus Fasciatus

The adaptability, viability and evolutionary potential of a species are closelyrelated to its genetic diversity. Therefore, studying the genetic diversity ofTrachidermus fasciatus, an endangered species, which is very important for itsprotection and management strategy formulation. Researchers had discussed the levelof genetic diversity of T. fasciatus, but it was not objectively reflect the currentsituation. Consequently, RAPD-SCAR marker, microsatellite DNA and cytochrome bgene (Cyt b) sequences were conducted to analyze genetic diversity and variation of T.fasciatus in the Fuchun River population (FR), Yellow River population (YR), LuanRiver population (LR), and Yalu River population (YL). The study results willprovide basic information for utilization of germplasm resources, protection andidentification of T. fasciatus. The results are shown as follows:1． Analysis of genetic diversity in populations of Trachidermus fasciatus byRAPD and the transformation of two SCAR markersFirstly, RAPD was conducted to analyze genetic diversity of T. fasciatus in theFuchun River population (FR), Yellow River population (YR), Luan River population(LR), and Yalu River population (YL), with32polymorphic10-bp random primersselected from294ones. Thirty wild individuals were detected in each population. Theresults indicated that the genetic diversity of T. fasciatus was relatively rich. Themajor results were as the following:1) Altogether,591bands were detected and515of them were polymorphic, accounted for87.14%. The range of proportion ofpolymorphic loci (P) was: FR (89.17%)>YR (87.99%)>YL (86.63%)>LR (83.25%).2) The Shannon’s information index (I) and Nei’s genetic diversity (H) amongpopulations were0.3393~0.3566and0.2157~0.2279, respectively. Compare to other three populations, LR population had relative lower values. If took the populations asa whole, the total Nei’s genetic diversity (H) and Shannon’s information index (I) was0.2336±0.1643and0.3710±0.2153, respectively.3) The value of gene flow (Nm)(5.76103~19.84497) were high, indicating certain gene exchange existed among thefour populations. But the AMOVA results exhibited significantly differentiation(P<0.05or P<0.01) among the populations.4) In the UPGMA tree constructedaccording to genetic distance, YL and YR populations clustered firstly, then with FRpopulation, and finally they joined to LR population. Obviously, the YL, YR and FRpopulations had relatively close relationship according to their geographic distance,whereas LR population showed clear divergence to the other three populations.Secondly, out of the five special RAPD bands (S1225525bp, S1225605bp, S1225841bp, S1345695bpand S1345825bp), SCAR01560bpand SCAR02443bpwere successfully transformed fromS1255605bpand S1255841bp, respectively. After large samples examination of the twomarkers, we found the highest frequency (96.67%and93.33%) in the YL population,higher frequency (83.33%and90%) in the FR population, high frequency (56.67%and66.67%) in the YR population, and the lowest frequency (13.33%and20%) inthe LR population. Therefore, SCAR01560bpand SCAR02443bpcan be used as specialmolecular markers for the population identification between LR and other threepopulations.2. Isolation and characterization of microsatellite markers in Trachidermusfasciatus and analysis of genetic variation in populationsBiotinylated magnetic beads method were used to enrich microsatellite loci of T.fasciatus. By using (CA)15as probes,158positive were identified from222clones,and129microsatellites loci were isolated．Among the129microsatellites loci,59were perfect (45.74%),59were imperfect (45.74%) and only11were compound type(8.52%).60pairs of primers were designed using software Primer Premier3.0andwere tested on60individuals from YL population and YR population. Of the60microsatellite loci19were found to be polymorphic. The number of alleles in these19markers ranged from10to20. The observed (Ho) and expected (He) heterozygosityranged from0.8000to1.000in YL and from0.8638to0.9497in YR respectively. Thepolymorphism information content (PIC) varied from0.8230to0.9300(P>0.5) in thetwo populations. The results indicated that the19microsatellite markers can be usedas molecular markers to discuss genetic variation of T. fasciatus. The genetic variation of four populations (FR, YR, LR and YL) of T. fasciatuswas investigated using19microsatellite markers. Results showed that the PIC of19loci was relatively high, varied from0.7179to0.9300(P>0.5). The number ofaverages allele and effective allele of4populations were13.9474to14.8421and9.3877to9.9143, the observed (Ho) and expected (He) heterozygosity ranged from0.8668to0.9117and0.8966to0.9111. It turned out that the genetic diversity of T.fasciatus was relatively rich. The value of Nm(9.36412~15.94901) indicating certaingene flow existed among the four populations, but the AMOVA results exhibitedsignificantly differentiation (P<0.01) between the populations. There washeterozygote deficiency and excess in four populations. In the UPGMA treeconstructed according to genetic distance, YL and YR populations clustered firstly,then with FR population, and finally they joined to LR population. Obviously, the YL,YR and FR populations had relatively close relationship according to their geographicdistance, whereas LR population showed clear divergence to the other threepopulations.3. Genetic diversity in populations of Trachidermus fasciatus based onmitochondrial cytochrome b gene sequence analysisThe genetic diversity of four populations (FR, YR, LR and YL) of T. fasciatuswas based on cytochrome b gene (Cyt b) sequences analysis. Thirty individuals weredetected in each population and a total of fifty three haplotypes were identified. Fiftyeight variable sites were detected, of which thirty nine were single base mutation sitesand nineteen were parsimony site. The haplotype diversity (0.786~0.947) wasrelatively abundant, but the nucleotide diversity (0.001283~0.003177) was low.Genetic distance among four populations ranged from0.00120to0.00341, of whichthe genetic distance between YR and LR was minimum (0.00120), and the geneticdistance among YL and the other three populations were relatively far. Although thefifty three haplotypes were divided into two clusters in the NJ tree, they did not formdifferent monophyletic group according to the geographical area. The AMOVA resultsexhibited significantly differentiation (P<0.05or P<0.01) among the populations, butthe genetic variation mainly from individuals within population (78.02%). Tajima’s Dtest, Fu and Li’s test and mismatch distribution of the Cyt b sequences suggested thatthe four populations of T. fasciatus might not had experienced a large-scaleexpansion.