| In this study, mitochondrial molecular markers (Cytb gene and D-loop region)and nuclear genetic markers (SSR) were used to analyze phylogeography relationsand population genetic structure among six populations of the mudskipper(Boleophthalmus pectinirostris)[Lianyungang(LYG) in Jiangsu, Jiuduansha(JDS) inShanghai, Cixi(CX) and Zhoushan(ZS) in Zhejiang, Xiapu(XP) in Fujian, Beihai(BH)in Guangxi]. In addition, genetic variation and evolution history of the populations ofB.pectinirostris were discussed. The major results were as following:1. The characteristics of complete mitochondrial genome of the B.pectinirostrisIn this paper, the complete mitochondrial genome of B. pectinirostris was firstlydetermined, Long-PCR and the method of primer walking were applied and theprimers for long-PCR were designed on the basis of COI and Cytb sequencesinformation of this species. The length of mitochondrial DNA (mtDNA) was17,111bp,containing13protein-coding genes,2rRNA genes,22tRNA genes and2non-codingregions (OL region and D-loop control region). The DN6gene and8tRNA geneswere encoded on the light strand (L strand), and the rest were on the heavy strand (Hstrand). The total base composition of H strand showed two features, the negative biasfor G (15.4%) and AT bias. The phenomenon of gene overlap were detected on theclose genes, the largest overlap was7bp between ND4L and ND4gene. Allprotein-coding genes started with ATG as initiation codon except COI and ATPase6,which used GTG and ATA, respectively. The open reading frame of genes wereterminated using TAA (ND1, ND2, COI, ATPase6, ND4L and ND6), TAG (ATPase8and ND5), while the others used incomplete termination codons (T). All tRNA genescan be folded into typical cloverleaf secondary structures, whice had some nucleotidemispairings in every stem and major mispairings in the aminoacyl stem and TψC stem.A termination-associate sequence (TAS), a central conserved region (CSB-E), threeconserved sequence blocks (CSB-1, CSB-2and CSB-3) and unique tandem repeat sequence were found in the D-loop control region.130bp complete repeat unit wasfound in the tandem repeat sequence. Five, six, seven and eight complete repeat unitsfollowing an incomplete repeat unit (53bp in length) were found in139individuals.This four types were1453bp、1583bp、1713bp and1843bp in length, respectively, thisfeature was firstly found in gobies (Gobiidae).2. Analysis on population genetics and phylogeography of the B.pectinirostrisbased on the Cytb geneGenetic diversity and genetic structure six wild populations from Yellowsea(LYG), East sea(JDS, CX, ZS and XP) and South sea(BH) of B.pectinirostris wereanalyzed using complete sequences of Cytb gene of mtDNA, besides phylogeographyand evolution history of the populations of B.pectinirostris were also discussed. Theresults showed that:①138haplotypes were defined in242individuals, in which16haplotypes was shared among populations. And only haplotype H23was shared by allpopulations, suggesting possible for the original haplotype.②The haplotypediversity(h) and nucleotide diversity(π), ranged from0.903to0.981and from0.00325to0.00658, respectively in six populations of B.pectinirostris, showing that geneticdiversity was higher in the populations of China coastal B.pectinirostris.③Thepairwise Fst value between populations ranged from-0.0281to0.07236, except BHpopulation, no significant genetic differentiation among other populations(p>0.05).Gene flow (Nm) value between populations was higher, all greater than6.40943.Genetic distance between populations ranged from0.0033to0.0066. Analysis ofmolecular variance (AMOVA) showed that the genetic variation mainly came frombetween individuals of within population (96.3%, P=0.00465). The results indicatethat, gene exchange was frequent, and genetic differentiation was weaker in thepopulations of China coastal B.pectinirostris. But, there were genetic structurebetween BH population and other populations.④Neutrality test, nucleotidemismatch distribution analysis and minimum spanning networks analysis indicatedthe B.pectinirostris in China coastal Sea might have experienced a rapid populationexpansion, and this event may occurred in late Pleistocene, about0.077~0.031millionyears ago. And the trend of the expansion was from the south to north, South seapopulation before East sea population, East sea population before Yellow seapopulation.⑤According to the NJ tree based on the genetic distances among sixpopulations, Yellow sea population and East sea population beong to one clade, while South sea population have originated from one independent evolutionary branch.3. Analysis on population genetic structure of the B.pectinirostris based on thecontrol region of mtDNAGenetic structure of six populations of B.pectinirostris was analyzed usingcomplete sequences of Control region which were obtained by clone sequencing. Theresults were as following:①The haplotype diversity(h) and nucleotide diversity(π),ranged from0.991to1.000and from0.0112to0.0142, respectively in six populationsof B.pectinirostris, mean was0.998and0.0124, showing that genetic diversity washigher in the populations of China coastal B.pectinirostris.②The pairwise Fst valuebetween populations ranged from-0.0144to0.4887, except BH population, nosignificant genetic differentiation among other population(p>0.05). Gene flow (Nm)value between populations was higher, all greater than4.6036. Genetic distancebetween populations ranged from0.0112to0.0147. Analysis of molecular variance(AMOVA) showed that the genetic variation mainly came from between individualsof within population (95.72%, P=0.00535). The results indicate that, gene exchangewas frequent, and genetic differentiation was weaker between Yellow sea populationsand East sea populations. But, there were significantly genetic differentiation betweenBH population and other populations.③Neutrality test, nucleotide mismatchdistribution analysis and minimum spanning networks analysis indicated theB.pectinirostris in China coastal Sea might have experienced a population expansion,and this event may occurred in late Pleistocene, about0.102~0.026million years ago.④According to the UPGMA tree based on the genetic distances among sixpopulations, except XP population, the rest of the population were clusted from northto south the same as the geographical location, while South sea population haveoriginated from one independent evolutionary branch.4. Isolation polymorphic microsatellite markers and population genetics analysisof the B.pectinirostrisFirst, the microsatellite loci were isolated using the magnetic beads enrichmentmethods. The markers were tested on30individuals collected from Lianyungang(LYG) coast in yellow sea.16polymorphic microsatellite loci were isolated andcharaterized. Rusults were as following: The number of alleles (NA) in these markersranged from5to11. The observed (HO) and expected (HE) heterozygosity rangedfrom0.5333to0.8000and from0.6808to0.8729, respectively. No significantly linkage disequilibrium and deviates from Hardy–Weinberg equilibrium (HWE) weredetected at any pair of loci. The polymorphism information content (PIC) varied from0.6255to0.8434, belong highly polymorphic microsatellite loci. These microsatelliteloci have being employed in population genetics studies of B. pectinirostris.Secondly, genetic diversity and genetic structure of the populations of B.pectinirostris were studied using twenty-two microsatellite markers. The resultsshowed that:①For the six populations studied, the average number of alleles(NA)was8.864to9.727, average effective number of alleles(NE) was5.712to6.430, thevalue of average observed heterozygosity(Ho) was0.692to0.733, the value ofaverage expected heterozygosity(He) was0.802to0.832, the value of averagepolymorphism information content(PIC) was0.764to0.797. In general, geneticdiversity was very high.②The pairwise FSTvalue between populations ranged from0.0079to0.0469, and genetic differentiation was weaker. Gene flow (Nm) valuebetween populations was10.1587to40.3455, and gene exchange was frequent.Analysis of molecular variance (AMOVA) showed that95.72%of genetic variationwas tested between individuals of within population (P<0.05), there were nosignificantly genetic differentiation among three groups.③Nei’s genetic distancebetween populations ranged from0.0745to0.2553. The UPGMA tree based on thegenetic distances among six populations showed that, Yellow sea population and Eastsea population beong to one clade, while South sea population have originated fromone independent evolutionary branch on foot. This suggesting that, BH population andother population has more distant relationship. |
PDF Full Text Request