The Study On Morphology, Biochemistry, Molecular Phylogeography And Genetic Diversity Of The Swimming Crab Portunus Trituberculatus Along China Coast

Multivariate analysis, including cluster analysis, principal component analysis anddiscriminant analysis were use to analyze the phenotype variation in morphologyvariation,fatty acid composition and mirco-elements biochemistryf or the swimmingcrab Portunus trituberculatus along China coasts. Some molecular markers, includingITS, AFLP, SSR, D-loop also were used in the analysis of molecular phylogeographyand genetic variations of swimming crab along China coast. All the results gave thepowerful support for the following research.Based on23morphological characters of P. trituberculatus, three multivariationanalysis methods (cluster analysis, principal component analysis and discriminantanalysis) were used to comparably reveal the morphological variation among the fivegeographical populations from China sea (Dalian, Dongying, Lian yungang, Zhoushanand Zhanjiang). The results of cluster analysis and principal component analysisindicated that the morphological characters were similar between Dongying andLianyungang, but quite different from that of Zhanjiang. In the principal componentanalysis, three principal components were constructed, and the contributory ratio of thefirst principal component was27.39%, the second15.23%, and the third was10.27%.The cumulative contributory ratio was52.89%.The results of discriminantanalysis indicated that there were significant differences among the five populations(P<0.01). The identification accuracy was63.64%～94.44%(P1) and67.74%～92.86%(P2), The total discriminant accuracy was82.24%.A multivariate analysis was used to study fatty acid composition difference ofswimming crab P. trituberculatus along China coast, the six geographical populationsfrom China sea (Dalian, Dongying, Lianyungang, Zhangzhou, Zhoushan and Zhanjiang). A set of fatty acid as fingerprint technique was developed for identifying differentpopulations. The crabs from the six population all contained27kinds of fatty acidsamong which the total volume of five kinds of fatty acids C18:1n9t, C16:0, C22:1n9,DHA and EPA were over82%, and multivariate anova revealed that there weresignificant differences among the six populations (P<0.01). As fatty acid fingerprintmarkers, the five kinds of fatty acids C18:1n9c, C20:1, C22:1n9, ARA and EPA, wereeffective to identify the six-population crabs for the total discriminant accuracyamounted to88.46%. The results of cluster analysis indicated that there were nosignificant correlations between geographical distance and Eucidean distance based on27kinds of fatty acids. The reason population differences of fatty acids were probablyrelated with genetics or the feed composition in habitat of the swimming crabs.By using the clustering analysis,the principal component analysis and thediscriminant analysis, contents of the11kinds of trace elements were measured in P.trituberculatus through the CAP6300plasma.The differences were compared in fourpopulations from the ocean of Lianyungang, Zhanghzou, Dalian and Dongying.Through the the clustering analysis,we draw the conclusion as follows:the populationfrom the Lianyugang and the population form the Dongying were much more similar,the distance is the smallest(0.55); the Dalian population, the Dongying population andthe Lianyungang population were getting longer in the form distance(0.59); however theZhangzhou population was different with them all,the distance was more than1.36, andthe distance between the Zhangzhou population and the Liangyungan population wasthe longest(1.76).We set up three primary components by the principal componentanalysis.Contribution rate of them was33.56%,23.20%,16.02%,and the totalcontribution rate was72.78%.The results of the discriminant analysis show that thedifferences of the trace elements in four different populations were obvious. Because theresults come from the different discrimination function in four places, these results werereliable.Genetic diversity and structure of the six swimming crab (P.trituberculatus)geographical stocks from the coast of China was analyzed using AFLP markers to assessgenetic resource state. Eight primer combinations generated925loci among the85individuals, and revealed high polymorphism within these stocks. The percentage ofpolymorphic bands (PPB) varied from57.41%(ZS stock) to76.86%(DL stock). Therewere significant difference (P<0.05) for He among the six stocks except from between DY and DL, and between LYG and ZJ. The first three greatest He were ZZ,DY and DL,and the three lowest were ZJ and LYG and ZS. Therefore,ZZ,DY and DL have highergenetic variation than LYG, ZJ and ZS according to PPL,I and He together.To exploreinter-stock differentiation, the pairwise FSTresults revealed there were significantdifferentiation among the six stocks (P <0.01). Especially, genetic differentiation wasmoderate among the six stocks whose the pairwise FST value all surpassed0.1besidesto between DL and LYG(0.04418) and between ZJ and ZS(0.02633) and between ZZand DY(0.03218).Gene flow Nm and GSTacross all stocks as a whole was1.9354and0.2053, respectively.Generally, among the six stocks there existed high genetic variationand genetic differentiation.UPGMA Cluster found the six stocks belonged to threeclades which were ZS and ZJ consisting of fist clad, LYG and DL,second clade and thethird was DY and ZZ,and the geographical distribution and UPGMA Cluster tree wasnot exactly accordant.Neutrality test results revealed all have significant difference inthe number of selection effect locus (P<0.05). ZS, ZJ and LYG these three stocks faceda considerable selective pressure.The Population differentiation and genetic diversity of Portunus trituberculatusfrom six geographical populations of China Sea (Dalian DL, Dongying DY,Lianyungang LYG, Zhoushan ZS, Zhangzhou ZZ and Zhangjiang ZJ) were studiedbased on mitochondrial D-loop whole gene sequences. There were1141bp,185variation sites and129parsimony informative sites in D-loop whole gene sequences.And there were48haplotypes among60swimming crab individuals. Nucleotidediversity index revealed a high genetic diversity and low selective effect for swimmingcrab populations in China Sea. The genetic differential index FST of total sixpopulations was0.18972, which meant that the genetic differentiation amongpopulations was significant（P<0.05）. Low Gene flow indicated that there were geneticdifferentiation among the15groups,except from between LYG and DY，ZJ, ZZ,respectively, in addition to ZJ and ZZ（Nem>5）.The genetic distance had no significantcorrelation with geographical distance,so there may have more intricate reasons inpopulation occurrence and spread.A primary result using ITS as molecular marker to discuss the P.trituberculatusgermplasm resources of China are relatively rare. In this experiment, using themolecular cloning of P. trituberculatus from six populations of China Sea for ITS1research, DNAStar、MEGA、Arlequin311computer software dealt with the results of sequencing, and ITS1sequences handled well were built molecular evolutionary tree,then to study the genetic diversity of P. trituberculatus from different populations. Theresults indicated that A T C G base constitutions hardly varied in6populations; Therewere two loci（TAC）n SSR in ITS1sequence; Ts/Tv is1.213. According to Geneticdistance, the smallest distance was0.00651between ZZ and ZJ populations, howeverthe largest was0.01111between ZS and DL. The gene tree of the six populations of P.trituberculatus composed in first clade of ZJ and ZZ, second clade DL and LYG，the lastone DY and ZS. The ITS gene showed a moderate genetic differentiation of the sixpopulations along China coasts.Eighteen novel polymorphic microsatellite loci were isolated from the swimmingcrab, P. trituberculatus, and characterized in a cultured stock of swimming crab throuth(GA)15probe. The number of alleles varied between two and four, and the observedand expected heterozygosity at population level ranged from0.1481to0.8621and0.4898to0.7475, respectively. Two loci significantly deviated from Hardy–Weinbergequilibrium (P<0.01). These eighteen microsatellite markers will be useful for parentage,population genetics and genome mapping studies in this species.Additionally, to development more useful microsatellite loci, a new (CA)15probewas used to accumulate CA-motif SSRs, the result indicated that there were14SSRswere successfully identified,and these markers to be applied to next step research.In order to find out the genetic impact of swimming crab culuture on wildswimming crab genetic resources in Haizhou Bay,20SSR primers were used to assessthe genetic structure and genetic differentiation of swimming crab wild stocks andculture stocks in Haizhou bay.The results indicated that the genetic diversity is the highest that wild populations,Ho=0.8509, and the heterozygosity of two culture stocks was lower than the wildgroup, and Ho was0.4525and0.5283, respectively.The one-way ANOVA showed, thegenetic parameters of Ne Ho, He, PIC in wild crab were significantly higher thanculture stocks(P<0.05), but those genetic parameters in culture stocks were nosignificant difference (P>0.05). The results showed that genetic diversity of naturalresources of swimming crab in Haizhou bay higher than that in culture stocks.The Fst ofthese stocks was between0.1085and0.1448, in a moderately differentiated state, Nm in 1.5-2.0. between, genetic differentiation was more significant founed between thewild stocks and culture socks than among culture socks. The results indicated therehave a moderate genetic differentiation between the wild stocks and culture socks andgene flow was in a moderate state.In order to find out the genetic impact of swimming crab culture on wildswimming crab genetic resources in Haizhou Bay,20SSR primers were used to assessthe genetic structure and genetic differentiation of swimming crab wild stocks andculture stocks in Haizhou bay. The results indicated that the genetic diversity is thehighest that wild populations, Ho=0.8509, and the heterozygosity of two culture stockswas lower than the wild group, and Ho was0.4525and0.5283, respectively.Theone-way ANOVA showed, the genetic parameters of Ne Ho, He, PIC in wild crab weresignificantly higher than culture stocks(P<0.05), but those genetic parameters in culturestocks were no significant difference (P>0.05).The results showed that geneticdiversity of natural resources of swimming crab in Haizhou bay higher than that inculture stocks.The Fst of these stocks was between0.1085and0.1448, in amoderately differentiated state, Nm in1.5-2.0. between, genetic differentiation wasmore significant founed between the wild stocks and culture socks than amongculture socks. The results indicated there have a moderate genetic differentiationbetween the wild stocks and culture socks and gene flow was in a moderate state.