中国沿海三疣梭子蟹群体形态、生化与分子遗传多样性研究
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摘要
本研究在表型水平上应用多元分析（聚类分析、主成分分析、判别分析）对中国沿海三疣梭子蟹群体的形态学、脂肪酸与微量元素进行了比较研究，并在群体遗传水平上应用多种分子标记技术（ITS、D-Loop、AFLP、SSR）对其群体的遗传多样性和遗传分化进行了系统研究，以期为三疣梭子蟹种质资源合理开发利用提供基础性数据和参考。具体内容如下：
（1）运用三种多元分析方法，即聚类分析、主成分分析和判别分析，通过测量三疣梭子蟹23个形态学性状参数，对中国海大连海区、东营海区、连云港海区、舟山海区和湛江海区5个种群三疣梭子蟹的形态差异进行了比较研究。聚类分析结果表明，连云港种群和东营种群形态最为相似（形态距离0.07），大连种群与东营种群、大连种群与舟山种群的趋异程度逐渐增加，形态距离分别为0.08和0.09，而湛江种群的趋异程度最大，与其余4种群的形态距离达0.15以上。主成分分析构建了3个主成分，其贡献率：主成分1为27.39%，主成分2为15.23%，主成分3为10.27%，累积贡献率为52.89%。在第1主成分中，比例性状ICM/CL、BCW/CL、PWC/CL和SW/CL的影响比较大。判别分析结果表明，5个海区种群之间的形态差异极显著（P<0.01），通过建立5个地理种群的判别函数，结果判别准确率P1为63.64%～94.44%， P2为67.74%～92.86%，综合判别率为82.24%，因此，该判别方程具有较高的判别效果。
（2）为探明分布于中国四大海区的天然三疣梭子蟹群体脂肪酸组成状况及群体差异，并建立群体判定的指纹标记以应用于种质鉴定和资源保护。本文应用脂肪酸指纹标记结合多元分析方法在表型水平上研究中国四大海区中湛江、大连、连云港、东营、舟山和漳州海区6个天然种群三疣梭子蟹的脂肪酸差异和系统发生关系，并建立了群体判定的脂肪酸指纹标记。结果表明，四大海区6种群体三疣梭子蟹脂肪酸存在显著差异（P<0.01），均含有27种脂肪酸。其中，反式油酸、棕榈酸、DHA、EPA和芥酸这5种脂肪酸的总含量在6群体中均高达82%以上。油酸、二十碳一烯酸、芥酸、ARA和EPA这5种脂肪酸作为脂肪酸指纹标记可以有效的对中国四大海区的6群体三疣梭子蟹进行种质鉴定，其综合判别准确率达88.46%。应用所含的27种脂肪酸进行聚类分析显示四大海区6种群三疣梭子蟹系统聚类结果与地理距离不具有显著相关性，这种脂肪酸组成的群体差异可能与遗传和栖息地食物组成差异有关。本研究结果对于中国海三疣梭子蟹的种质鉴定和原产地资源保护具有重要的应用价值。
（3）运用聚类分析、主成分分析和判别分析，通过CAP6300等离子体测定三疣梭子蟹背甲11种微量元素含量参数，对中国海大连海区、东营海区、连云港海区、漳州海区4个种群三疣梭子蟹背甲的微量元素差异进行了比较研究。聚类分析结果表明连云港种群(LYG)与东营种群(DY)欧氏距离最小为0.55，最为接近，大连种群(DL)与东营种群(DY)和连云港种群（LYG）群这两种群的趋异程度逐渐增加，欧氏形态距离为0.59，而漳州种群(ZZ)与上述四个种群的形态距离最大，均在1.36以上，特别是漳州种群(ZZ)和连云港（LYG）种群形态距离达到1.76，趋异程度最大。运用主成分分析产生了3个主成分，它们贡献率分别为：第1种主成分为33.56%，第二种主成分为23.20%，第三种主成分为16.02%，总贡献率为72.78%，在第1主成分中，微量元素Co、Mn占主导作用。根据判别分析结果得出：4个海区种群之间背甲的微量元素显著（P<0.01），通过建立一个可以判别4个地理种群的函数，准确率P1为100%。因此，该判别具有较高的判别效果。
（4）利用AFLP标记分析了我国六个地理种群三疣梭子蟹(Portunustrituberculatus)遗传多样性和遗传结构。8对引物组合在85个个体生成894个位点,结果显示,在这些群体具有很高的多态性。多态位点比例(PPB)从57.41%(ZS群体)-76.86%(DL群体)，据PPL、I和h,6种群遗传多样性有显著差异(p <0.05), ZZ,DY和DL的遗传变异最大，遗传多样性最高。为探讨群体间的分化,中国海整个三疣梭子蟹群体FST有适度的遗传分化，(p <0.01)，基因流Nm and GST分别为1.9354和0.2053。6群体除LYG和DL、ZS和ZJ、ZZ和DY的分化不明显FST <0.05、Nem>5，而其他组合均存在较明显分化，特别是ZS与其它4群体产生了高度的遗传分化，FST>0.25、Nem为0.5-0.7，基因流极低。ZJ分别与LYG、DL、DY3群体发生了中度分化，FST为0.18-0.23，Nem为0.63-0.93。总之,在6群体ZS和ZJ具有高遗传变异和高度的遗传分化，ZS、ZJ和LYG这三个群体面临相当大的选择性压力。
（5）以大连（DL）、东营(DY)、连云港(LYG)、舟山(ZS)、湛江(ZJ)和漳州(ZZ)6个三疣梭子蟹地理种群为研究对象，采用线粒体控制区D-loop全基因序列为分子标记，对中国海三疣梭子蟹野生群体的遗传多样性及群体遗传结构进行了分析。结果发现，在用于分析的1141bp的D-loop全基因序列中共有185个变异位点，129个简约信息位点。60个个体中共计48个单倍型，基因多样性和核苷酸多样性指数显示中国沿海三疣梭子蟹群体具有较高的遗传多样性，而且三疣梭子蟹在过去没有出现很强的选择效应，群体大小稳定。6种群三疣梭子蟹遗传分化指数（FST）为0.1897（P<0.05），将中国沿海三疣梭子蟹作为一个大群体来讲已产生了一定的分化。LYG分别和DY，ZJ、ZZ，以及ZJ和ZZ这4组之间无明显分化，基因流较大（Nem>5），而其他11个群组间已存在一定程度的分化。遗传距离与地理距离不存在显著的相关性，群体发生与扩散可能有更复杂的原因。
（6）利用ITS分子标记技术来探讨我国三疣梭子蟹的种质资源，目前应用的比较少。采用分子克隆方法对中国海6种群的三疣梭子蟹进行ITS1研究，使用DNASTAR、MEGA、Arlequin311等软件对测序结果进行处理，进而研究不同海区三疣梭子蟹的遗传多样性与遗传分化。结果显示：A、T、C、G四种碱基的含量在6种群内的变化不大；在ITS1序列中发现两个（TAC）n微卫星；6种群的三疣梭子蟹的转换/颠换比值平均为1.213。根据遗传距离，可以得出漳州和湛江群体之间最小为0.00651，而舟山和大连之间的最大为0.01111。在三疣梭子蟹内，湛江和漳州群体分为一小支，与大连、连云港聚为一支，东营和舟山群体则分为另一支。群体FST显示三疣梭子蟹6种群的遗传分化处于中度分化水平。
（7）采用磁珠富集法成功构建了三疣梭子蟹基因组微卫星文库,采用限制性内切酶MseⅠ对三疣梭子蟹基因组DNA酶切,用MseⅠ接头连接;用生物素标记的寡核苷酸探针(GA)15进行筛选,磁珠富集含有微卫星的DNA单链序列;对DNA模板进行PCR扩增,连接pUCm-T载体,转入用氯化钙制备的感受态大肠杆菌中,得到微卫星序列文库;利用蓝白斑筛选获得113个阳性克隆,对其测序,得85个含微卫星序列,完美型、非完美型、复合型序列分别占总数的64.71%、22.35%、12.94%。最终选择设计出31个理想的微卫星引物，用一个人工养殖群体进行了检验，18个新的微卫星标记被验证具有多态，等位基因数2-4，群体平水上的Ho和He分别间于0.1481-0.8621和0.4898-0.7475,2个位点显著偏离Hardy–Weinbergequilibrium (P<0.01)。为三疣梭子蟹种质评价与遗传育种研究奠定了科学基础。
为开发更多有用的多态性标记，用（CA）15为探针，对连云港30个野生三疣梭子蟹进行了扫描，结果设计的19对引物共筛选出条带清晰稳定、多态性高的引物14对，见表2。14个标记中，等位基因数3-9个，群体水上的Ho和He分别间于0.5417-1和0.6164-0.8404,为三疣梭子蟹种质评价与遗传育种研究奠定了科学基础。
（8）为探明三疣梭子蟹养殖群体对野生资源的遗传影响，本文利用20对SSR引物对于来自海州湾三疣梭子蟹野生群体与两个养殖群体进行群体遗传结构和遗传分化的研究。结果表明，野生种群遗传多样性明显高于养殖群体，Ho=0.8509，而两养殖群体的杂合度低于野生群体，Ho分别为0.4525和0.5283。经单因子方差分析显示海州湾野生三疣梭子蟹的Ne、Ho、He、PIC均显著高于两养殖群体（P<0.05），但两养殖群体的Ne、Ho、He、PIC均无显著差异（P<0.05）。以上结果说明海州湾天然三疣梭子蟹群体的遗传多样性显著高于养殖群体。三群体的Fst间于0.1085-0.1448间，处于中度分化状态，Nm处于1.5-2.0间，野生群体与养殖群体的遗传分化要较养殖群体内部之间更大，基因流也较养殖群体内部之间要小，表明野生群体与养殖群体存在一定的分化，基因流处于中等程度。总之，当前海州湾三疣梭子蟹遗传状况良好，但要定期监测天然资源的遗传状况，防止养殖活动和增殖放流对现有遗传资源产生影响。
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.
（1）运用三种多元分析方法，即聚类分析、主成分分析和判别分析，通过测量三疣梭子蟹23个形态学性状参数，对中国海大连海区、东营海区、连云港海区、舟山海区和湛江海区5个种群三疣梭子蟹的形态差异进行了比较研究。聚类分析结果表明，连云港种群和东营种群形态最为相似（形态距离0.07），大连种群与东营种群、大连种群与舟山种群的趋异程度逐渐增加，形态距离分别为0.08和0.09，而湛江种群的趋异程度最大，与其余4种群的形态距离达0.15以上。主成分分析构建了3个主成分，其贡献率：主成分1为27.39%，主成分2为15.23%，主成分3为10.27%，累积贡献率为52.89%。在第1主成分中，比例性状ICM/CL、BCW/CL、PWC/CL和SW/CL的影响比较大。判别分析结果表明，5个海区种群之间的形态差异极显著（P<0.01），通过建立5个地理种群的判别函数，结果判别准确率P1为63.64%～94.44%， P2为67.74%～92.86%，综合判别率为82.24%，因此，该判别方程具有较高的判别效果。
（2）为探明分布于中国四大海区的天然三疣梭子蟹群体脂肪酸组成状况及群体差异，并建立群体判定的指纹标记以应用于种质鉴定和资源保护。本文应用脂肪酸指纹标记结合多元分析方法在表型水平上研究中国四大海区中湛江、大连、连云港、东营、舟山和漳州海区6个天然种群三疣梭子蟹的脂肪酸差异和系统发生关系，并建立了群体判定的脂肪酸指纹标记。结果表明，四大海区6种群体三疣梭子蟹脂肪酸存在显著差异（P<0.01），均含有27种脂肪酸。其中，反式油酸、棕榈酸、DHA、EPA和芥酸这5种脂肪酸的总含量在6群体中均高达82%以上。油酸、二十碳一烯酸、芥酸、ARA和EPA这5种脂肪酸作为脂肪酸指纹标记可以有效的对中国四大海区的6群体三疣梭子蟹进行种质鉴定，其综合判别准确率达88.46%。应用所含的27种脂肪酸进行聚类分析显示四大海区6种群三疣梭子蟹系统聚类结果与地理距离不具有显著相关性，这种脂肪酸组成的群体差异可能与遗传和栖息地食物组成差异有关。本研究结果对于中国海三疣梭子蟹的种质鉴定和原产地资源保护具有重要的应用价值。
（3）运用聚类分析、主成分分析和判别分析，通过CAP6300等离子体测定三疣梭子蟹背甲11种微量元素含量参数，对中国海大连海区、东营海区、连云港海区、漳州海区4个种群三疣梭子蟹背甲的微量元素差异进行了比较研究。聚类分析结果表明连云港种群(LYG)与东营种群(DY)欧氏距离最小为0.55，最为接近，大连种群(DL)与东营种群(DY)和连云港种群（LYG）群这两种群的趋异程度逐渐增加，欧氏形态距离为0.59，而漳州种群(ZZ)与上述四个种群的形态距离最大，均在1.36以上，特别是漳州种群(ZZ)和连云港（LYG）种群形态距离达到1.76，趋异程度最大。运用主成分分析产生了3个主成分，它们贡献率分别为：第1种主成分为33.56%，第二种主成分为23.20%，第三种主成分为16.02%，总贡献率为72.78%，在第1主成分中，微量元素Co、Mn占主导作用。根据判别分析结果得出：4个海区种群之间背甲的微量元素显著（P<0.01），通过建立一个可以判别4个地理种群的函数，准确率P1为100%。因此，该判别具有较高的判别效果。
（4）利用AFLP标记分析了我国六个地理种群三疣梭子蟹(Portunustrituberculatus)遗传多样性和遗传结构。8对引物组合在85个个体生成894个位点,结果显示,在这些群体具有很高的多态性。多态位点比例(PPB)从57.41%(ZS群体)-76.86%(DL群体)，据PPL、I和h,6种群遗传多样性有显著差异(p <0.05), ZZ,DY和DL的遗传变异最大，遗传多样性最高。为探讨群体间的分化,中国海整个三疣梭子蟹群体FST有适度的遗传分化，(p <0.01)，基因流Nm and GST分别为1.9354和0.2053。6群体除LYG和DL、ZS和ZJ、ZZ和DY的分化不明显FST <0.05、Nem>5，而其他组合均存在较明显分化，特别是ZS与其它4群体产生了高度的遗传分化，FST>0.25、Nem为0.5-0.7，基因流极低。ZJ分别与LYG、DL、DY3群体发生了中度分化，FST为0.18-0.23，Nem为0.63-0.93。总之,在6群体ZS和ZJ具有高遗传变异和高度的遗传分化，ZS、ZJ和LYG这三个群体面临相当大的选择性压力。
（5）以大连（DL）、东营(DY)、连云港(LYG)、舟山(ZS)、湛江(ZJ)和漳州(ZZ)6个三疣梭子蟹地理种群为研究对象，采用线粒体控制区D-loop全基因序列为分子标记，对中国海三疣梭子蟹野生群体的遗传多样性及群体遗传结构进行了分析。结果发现，在用于分析的1141bp的D-loop全基因序列中共有185个变异位点，129个简约信息位点。60个个体中共计48个单倍型，基因多样性和核苷酸多样性指数显示中国沿海三疣梭子蟹群体具有较高的遗传多样性，而且三疣梭子蟹在过去没有出现很强的选择效应，群体大小稳定。6种群三疣梭子蟹遗传分化指数（FST）为0.1897（P<0.05），将中国沿海三疣梭子蟹作为一个大群体来讲已产生了一定的分化。LYG分别和DY，ZJ、ZZ，以及ZJ和ZZ这4组之间无明显分化，基因流较大（Nem>5），而其他11个群组间已存在一定程度的分化。遗传距离与地理距离不存在显著的相关性，群体发生与扩散可能有更复杂的原因。
（6）利用ITS分子标记技术来探讨我国三疣梭子蟹的种质资源，目前应用的比较少。采用分子克隆方法对中国海6种群的三疣梭子蟹进行ITS1研究，使用DNASTAR、MEGA、Arlequin311等软件对测序结果进行处理，进而研究不同海区三疣梭子蟹的遗传多样性与遗传分化。结果显示：A、T、C、G四种碱基的含量在6种群内的变化不大；在ITS1序列中发现两个（TAC）n微卫星；6种群的三疣梭子蟹的转换/颠换比值平均为1.213。根据遗传距离，可以得出漳州和湛江群体之间最小为0.00651，而舟山和大连之间的最大为0.01111。在三疣梭子蟹内，湛江和漳州群体分为一小支，与大连、连云港聚为一支，东营和舟山群体则分为另一支。群体FST显示三疣梭子蟹6种群的遗传分化处于中度分化水平。
（7）采用磁珠富集法成功构建了三疣梭子蟹基因组微卫星文库,采用限制性内切酶MseⅠ对三疣梭子蟹基因组DNA酶切,用MseⅠ接头连接;用生物素标记的寡核苷酸探针(GA)15进行筛选,磁珠富集含有微卫星的DNA单链序列;对DNA模板进行PCR扩增,连接pUCm-T载体,转入用氯化钙制备的感受态大肠杆菌中,得到微卫星序列文库;利用蓝白斑筛选获得113个阳性克隆,对其测序,得85个含微卫星序列,完美型、非完美型、复合型序列分别占总数的64.71%、22.35%、12.94%。最终选择设计出31个理想的微卫星引物，用一个人工养殖群体进行了检验，18个新的微卫星标记被验证具有多态，等位基因数2-4，群体平水上的Ho和He分别间于0.1481-0.8621和0.4898-0.7475,2个位点显著偏离Hardy–Weinbergequilibrium (P<0.01)。为三疣梭子蟹种质评价与遗传育种研究奠定了科学基础。
为开发更多有用的多态性标记，用（CA）15为探针，对连云港30个野生三疣梭子蟹进行了扫描，结果设计的19对引物共筛选出条带清晰稳定、多态性高的引物14对，见表2。14个标记中，等位基因数3-9个，群体水上的Ho和He分别间于0.5417-1和0.6164-0.8404,为三疣梭子蟹种质评价与遗传育种研究奠定了科学基础。
（8）为探明三疣梭子蟹养殖群体对野生资源的遗传影响，本文利用20对SSR引物对于来自海州湾三疣梭子蟹野生群体与两个养殖群体进行群体遗传结构和遗传分化的研究。结果表明，野生种群遗传多样性明显高于养殖群体，Ho=0.8509，而两养殖群体的杂合度低于野生群体，Ho分别为0.4525和0.5283。经单因子方差分析显示海州湾野生三疣梭子蟹的Ne、Ho、He、PIC均显著高于两养殖群体（P<0.05），但两养殖群体的Ne、Ho、He、PIC均无显著差异（P<0.05）。以上结果说明海州湾天然三疣梭子蟹群体的遗传多样性显著高于养殖群体。三群体的Fst间于0.1085-0.1448间，处于中度分化状态，Nm处于1.5-2.0间，野生群体与养殖群体的遗传分化要较养殖群体内部之间更大，基因流也较养殖群体内部之间要小，表明野生群体与养殖群体存在一定的分化，基因流处于中等程度。总之，当前海州湾三疣梭子蟹遗传状况良好，但要定期监测天然资源的遗传状况，防止养殖活动和增殖放流对现有遗传资源产生影响。
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.
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