利用RAPD技术对三种绒螯蟹亲缘关系的研究

采用ＲＡＰＤ技术结合一定的形态学研究手段,对中华绒螯蟹,日本绒螯蟹及其合浦亚种进行亲缘关系的研究,为三种绒螯蟹的种及亚种分析重要资料,三种绒螯蟹的第四步足前节长宽比值,与其分类标本吻合或基本吻合,用一组引物,对每种各１０个个体的基因组ＤＮＡ进行扩增,得到一批特异,可重复的扩增图谱,扩增区带的相似率分析表明,中华绒螯蟹与日本绒螯蟹的亲缘关系远,而日本绒螯蟹合浦亚种与日本绒螯蟹近,最后,对文中ＲＡＰＤ     

四种绒螯蟹分子分类与系统发育

利用PCR技术,扩增了中华绒螯解、狭额绒螯蟹线粒体16SrDNA片段,经测序,与GenBank数据库中的日本绒螯解16SrDNA同源序列进行比较。结果显示,在长为376bp的16SrDNA同源序列中有33个多态性核革酸位点（8．78％）,其中种间多态性核苷酸位点28个（7．45％）,种间差异远大于种内差异。引入方蟹科其它近缘种类厚纹蟹、相手蟹和张口蟹的16SrDNA同源序列与上述4种绒螯蟹比较分析,MP法和NJ法构建的分子系统树表明：中华绒螯蟹与日本绒螯蟹亲缘关系最近,首先聚在一起,然后与台湾绒螯蟹聚为一支,狭额绒螯蟹则为相对独立的一支,且进化速度大于前3种绒螯蟹,但最后与前者仍聚在同一组,狭额绒螯蟹只是绒螯蟹属系统进化中的一个侧支,故本研究结果不支持Sakai（1983）和Guo等（1997）把狭额绒螯蟹和台湾绒螯蟹各自立为新属的观点。     

关于三种绒螯蟹种名的厘正

原称为绒螯蟹的种类 ,现隶属于甲壳纲、十足目、方蟹科绒螯蟹Ｅｒｉｏｃｈｅｉｒ、新绒螯蟹Ｎｅｏｅｒｉｏｃｈｅｉｒ和平绒螯蟹属Ｐｌａｔｙｅｒｉｏｃｈｅｉｒ 3个属。其中新绒螯蟹属由Ｓａｋａｉ于 1983年以绒螯蟹属中的狭颚绒螯蟹为模式种而建立 ,平绒螯蟹属是Ｎｇ等 (1999)建立 ,所用模式种为台湾绒螯蟹Ｅｒｉｏｃｈｅｉｒｆｏｒｍｏｓａ。有关平绒螯蟹属的有效性 ,作者持否定观点 ,另文详细讨论。到目前为止 ,所有绒螯蟹中有 3个种的学名拼写一直存在混乱 ,应予订正。1　日本绒螯蟹日本绒螯蟹 1835年被ｄｅＨａａｎ命名时归于方蟹属 ,…     

RAPD技术在系统与进化植物学中的应用

随着ＲＡＰＤ技术的发展,这一在ＤＮＡ分子水平上检测遗传多样性的方法被广泛用于植物系统与进化的研究之中,利用ＲＡＰＤ的分析结果,不仅可以探讨 物种间的亲缘演化关系,检测种内的遗传分化,而且还可以为属、种的分类提供强有力的证据。事实证明,ＲＡＰＤ在系统与进化植物学的研究中具有重要的参考价值,并已取得了可喜的成果。     

从线粒体16S rDNA序列探讨绒螯蟹类的系统发生关系

测定了绒螯蟹类各物种的线粒体16SrDNA部分片段的序列,构建了NJ树、ML树和MP树。序列歧异数据比较和各系统发生树都支持新绒螯蟹属(Neoeriocheir)为一个独立的属。在3种系统发生树中,直额绒螯蟹(Eriocheir recta)都是绒螯蟹属(Eriocheir)所有其它成员的姐妹群,并且广东珠江1只直额绒螯蟹标本的16SrDNA部分序列与台湾产台湾绒螯蟹(Eriocheir formasa)的相应序列相同。这些结果不支持平绒螯蟹属(Platyeriocheir)是一个有效的属,并表明E．formosa是E．recta的同物异名。绒螯蟹属(Eriocheir)所有其它成员聚为一个单系的分支,支持中华绒螯蟹、合浦绒螯蟹与日本绒螯蟹属于同一个物种Eriocheir japonica。16SrDNA部分序列的比对表明,产于台湾的日本绒螯蟹的此段序列与合浦绒螯蟹的相同,产于崇明岛的和产于美国旧金山海湾的中华绒螯蟹的此段序列与中华绒螯蟹单元型B的序列相同。     

七种方蟹科蟹类的遗传关系

利用水平电泳方法对方蟹科方蟹亚科粗腿厚纹蟹,弓蟹亚科日本绒螯蟹、中华绒螯蟹、肉球近方蟹、绒螯近方蟹、平背蜞及斜纹蟹亚科齿突斜纹蟹进行遗传分析,计检测了９种同工酶,１２个位点。结果表明,七种蟹类的多态座位比例及平均杂合度分别为：０．１６７和０．００３,０．２５０和０．０３６,０．３３３和０．０６２,０．２５０和０．０１２,０．０８３和０．００２,０．０８３和０．０５９,０．０８３和０．００２。最小遗     

中国特有种四合木种群遗传多样性的ＲＡＰＤ

采用随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术检测了西鄂尔多斯高原特有种四合木种群５个斑块的遗传多样性。２２个扩增引物产生ｌｌ６条带,Ｓｈａｎｎｏｎ信息指数和Ｎｅｉ指数对ＲＡＰＤ数据的分析表明：四合木种群存在较高的遗传多样氓,其中千里山斑块的遗传多样性和多态位点比例较高,石嘴山斑块的最低。遗传多样性的８６．５％存在于斑块内,斑块间的遗传变异为１３．５％。遗传距离与地理距离无直接相关关系。这些结果说明,遗传多样性反映了四合木种群基因组ＤＮＡ存在较高的变异性;同时各斑块间存在一定的基因流,四合木各斑块可看成是处于同一种群的半隔离状态（ｍｅｔａ—种群）,对四合木应注意保护遗传多样性丰富的ｍｅｔａ—种群。     

毛乌素沙地锦鸡儿（Caragana）种群种子蛋白多样性及其生物学意义

应用多种聚丙烯酰胺凝胶电泳方法对纯化的锦鸡儿种子球蛋白和清蛋白进行分离和分子量测定。占总蛋白７０％左右的球蛋白包括３个不同分子量的蛋白,它们由相同的２１个亚基组成,说明是不同的聚合态。清蛋白在ＰＡＧＥ和ＳＤＳＰＡＧＥ中部分出约２０种成分。全蛋白ＰＡＧＥ结果表明最主要的球蛋白和若干清蛋白在种群内和种群间有高水平的多态性,至少有４个可能的位点编码６个蛋白。ＳＤＳＰＡＧＥ结果表明全蛋白有约５０个不同分子量的单体,其中大多数是变异的。记录了其中１７个单体的变异,初步推测由１３个位点编码。从全部１０个种群３３７份样品的ＰＡＧＥ和３３８份样品的ＳＤＳＰＡＧＥ结果计算出２３个蛋白或单体的分布频率,进一步统计出各项种群遗传结构参数。数据表明毛乌素沙地锦鸡儿的全部遗传多样性中９０％以上存在于群体内,群体间只占７．６％,说明种群间存在强大的基因而。同一植株不同种子存在不同种子蛋白和单体成分,更进一步证明锦鸡儿的异交性。蛋白质的高水平多样性及其空间分布格局证实了所报道的形态变异的结果,说明毛乌素地区的锦鸡儿的确构成一个杂种带。     

闽江水系绒螯蟹的表型性状差异与分子遗传差异的比较与分析

表型性状差异(differentiation in phenotypictraits,PST)和分子遗传差异(differentiation at neutral molecular markers,FST)是近期进化生物学的研究热点之一。闽江水系是我国中华绒螯蟹与合浦绒螯蟹的主要混杂地域,是研究绒螯蟹遗传与进化的理想地之一。为探讨闽江水系绒螯蟹的PST和FST,以2009和2010年度闽江水系的133个绒螯蟹样本为材料,进行了14个表型数量性状差异分析和6个微卫星标记的遗传差异分析。结果发现:除3个表型性状不存在显著差异外,其他表型性状在不同年份间均存在极显著差异(P<0.01);2009年绒螯蟹的平均期望杂合度极显著高于2010年绒螯蟹(P=0.008),而平均等位基因丰富度、观测杂合度和近交系数均不存在显著差异(P=0.136～0.675);年份间的平均FST为0.1429;通过对PST与FST的比较发现,除第二步足掌节长度(F2)性状外,其他表型数量性状的PST值均高于FST值,表明这些性状均受到了较明显的选择压力。该文研究结果为绒螯蟹的分子进化研究积累了资料,也为其他水产生物的PST和FST比较研究提供了参考。     

安徽某淡水龙虾养殖群体分类的分子标记筛选

为筛选可用于淡水龙虾养殖群体物种分类的分子标记,对安徽省某水产养殖有限公司采集的淡水龙虾样本进行了线粒体COⅠ、16S rDNA、D-loop、Cyt b及12S rDNA基因的测序分析,并与GenBank中30个滑螯虾属(Cherax)物种的相应基因序列比对,同时,进行上述5种分子标记在种内个体间、属内种间、科内属间和目内科间的同源性变异范围比对分析。结果显示,本研究所采集淡水龙虾样本与四脊滑螯虾(C. quadricarinatus)的相似性最高,其中,1 113 bp的Cyt b相似性100%,COⅠ、16S r DNA及12S rDNA相似性都在99%以上;在以克氏原螯虾(Procambarus clarkii)为外类群构建的邻接系统发育树中,本研究样本与四脊滑螯虾聚为一支,然后与其他物种聚合构成系统树,确定本研究采集的淡水龙虾样本为四脊滑螯虾;通过同源性变异范围比对分析,在淡水龙虾养殖群体的分子分类工作中,16S rDNA、Cyt b及12S rDNA比COⅠ和D-loop这2种分子标记具有较强优势。本研究结果可为今后淡水龙虾养殖场中物种的分类鉴定工作提供参考。     

Molecular systematics of the Asian mitten crabs,genus Eriocheir (Crustacea: Brachyura)

To help resolve phylogenetic relationships among the mitten crabs, complete sequences of the nuclear DNA internal transcribed spacer (ITS) and portions of the mitochondrial genome corresponding to the cytochrome oxidase I (COI), were sequenced for all Asian mitten crabs of the genus Eriocheir and seven species of the Grapsoidea. The resulting phylogeny supports the establishment of a separate genus Neoeriocheir, but does not provide justification for the recognition of Platyeriocheir. A female mitten crab specimen from the Zhujiang River, China, was considered to be Eriocheir recta (), a species previously synonymized with Eriocheir japonica (de Haan, 1835). In the ITS analysis, a sequence from Eriocheir formosa (from Taiwan) falls within a well-supported E. recta group, which indicates that E. formosa may have to be synonymized with E. recta. Three previously recognized members of the genus, E. japonica, Eriocheir sinensis, and Eriocheir hepuensis constitute a monophyletic sister group to E. recta in all phylogenetic trees. We provide evidence for the conspecific status of these taxa. Phylogenetic trees based on COI and combined COI and ITS sequences indicate that E. japonica consists of three subgroups. Since the name E. japonica (de Haan, 1835) takes precedence over E. sinensis (H. Milne Edwards, 1853) and E. hepuensis, we suggest that these three subgroups correspond to three subspecies of E. japonica: E. j. japonica, E. j. sinensis, and E. j. hepuensis.     

中华绒螯蟹与合浦绒螯蟹两地理亚种的线粒体DNA序列变异

根据线粒体16SrDNA的PCR／RFLP鉴定和对Cyt b基因全序列的分析对中国大陆7水系绒螯蟹地理种群的遗传分化进行探讨。结果表明：中华绒螯蟹与合浦绒螯蟹两亚种在17条Cyt b全序列上以40个固定的碱基变异位点相区别。前者分布在瓯江及其以北的水系;后者主要分布在瓯江及其以南的水系。发现了4种在鸭绿江以南的北方水系中生活的合浦亚种单元型,对这些单元型的形成提出了3种可能的解释[动物学报51（5）：862—866,2005]。     

Mitochondrial DNA-inferred population structure and demographic history of the mitten crab (Eriocheir sensu stricto) found along the coast of mainland China

The mitten crab, Eriocheir sensu stricto , is economically important in East Asia, although it is an invasive species in Europe and North America. Little is known about its population structure and historical demography in its native range, especially along the Pacific coast of China. We collected mitochondrial cytochrome oxidase subunit II and cytochrome  b sequences from 154 individuals distributed in the rivers along the Chinese coast and 15 individuals from Japan. Phylogenetic analysis resulted in three major monophyletic groups: northern China, southern China and Japan. Negligible migration was detected among those groups by coalescent analysis. Hence, we support the recognition of three species: Eriocheir hepuensis in southern China, Eriocheir sinensis in northern China and Eriocheir japonica in Japan. The populations in the middle (the Oujiang and Minjiang Rivers) possess a mixture of haplotypes similar to either the northern or the southern haplotypes. We believe that secondary intergradation as the most likely cause of the clinal variation based on examining the genetic variation in the latitudinal space. The estimated divergence time between E. sinensis and E. hepuensis is 2.24 million years ago (Ma), while the divergence time between E. japonica and E. sinensis is 1.83 Ma. Both are in the late Pliocene, suggesting that land bridges associated with low sea level during that time might have severed as vicariant barriers for speciation. The divergence of the northern population and the 'northern haplotypes' in the middle population was estimated at 0.12 Ma, while the time separating the southern population and the 'southern haplotypes' in the middle populations was estimated as 0.16 Ma, implicating possible secondary contact in the late Pleistocene.     

The First Internal Transcribed Spacer (ITS-1) of Ribosomal DNA as a Molecular Marker for Phylogenetic and Population Analyses in Crustacea

The objective of the present study is to explore the feasibility of using the first internal transcribed spacer (ITS-1) of ribosomal DNA as a molecular marker for studying the interspecific and intraspecific genetic variations among crustaceans. We designed primers that could amplify ITS-1 from a majority of taxonomic groups of crustaceans. The gene was found to exhibit a high degree of length polymorphism among different groups, ranging from 182 bp in the barnacle Balanus amphitrite to approximately 820 bp in the spiny lobster Panulirus japonicus. With respect to differences between congeneric species, it was found that the ITS-1 sequences of 3 mitten crabs, Eriocheir sinensis, Eriocheir leptognathus, and Eriocheir formosa, exhibit 5.4% to 16.3% nucleotide divergence, suggesting that ITS-1 is informative for phylogenetic analysis at the species level. Yet there are extensive (0.9%–2.3%) variations within individual E. formosa, so that phylogenetic analyses could be obscured. ITS-1 was found to vary between 2 geographical populations of the shrimp Penaeus japonicus. The variations involved substitutions as well as insertions/deletions between shrimp from Australia and South China Sea. These results show that ITS-1 is highly divergent among different crustaceans and could be an appropriate marker for molecular systematic studies at the species and population levels, although the presence of intragenomic variation needs to be taken into consideration. Received August 15, 2000; accepted February 9, 2001     

Microsatellite analysis of genetic diversity and population structure of Chinese mitten crab （Eriocheir sinensis）

Chinese mitten crab (Eriocheir sinensis) has higher commercial value as food source than any other species of Eriocheir in China.To evaluate the germplasm resources and characterize the genetic diversity and population structure of the crabs in different water systems,two stocks and two farming populations were assessed with 25 polymorphic microsallite loci available in public GenBank.Basic statistics showed that the average observed heterozygosity (Ho) amongst populations ranged from 0.5789 to 0.6824.However,a remarkable presence of inbreeding and heterozygote deficiencies were observed.To analyze population structure,pairwise FST coefficients explained only ～10.3% variability from the subdivision of mitten crab populations,the remaining variability stems from the subdivision within subpopulations.Although the four populations had slight differentiation,different allelic frequencies resulted in distinct population structures.Two stocks and one farming population were clustered together to the phylogenetic branch of Yangtze crab,with an approximate membership of 95%.Whereas,another fanning population was clustered singly to the phylogenetic branch of the Liaohe crab,with a membership of 97.1%.The tests for individual admixture showed that Yangtze crab had probably been contaminated with individuals from other water systems.Genetic relationships between populations also supported the conclusion that Yangtze crab and Liaohe crab had different gene pools in spite of the origins of the same species.     

阳澄湖原产及“洗澡”中华绒螯蟹背甲的几何形态分析

为有效确认非阳澄湖产“洗澡蟹”与阳澄湖原产中华绒螯蟹(Eriocheir sinensis)背甲形态的差异, 研究运用几何形态分析法, 比较研究了前者在阳澄湖中人为“洗澡”式养殖1个月始末时, 其与阳澄湖原产蟹的背甲形态特征。背甲的形态分析基于所建立的35个地标点系统来进行, 即背甲经图像处理后提取地标点坐标, 开展相对扭曲主成分以及判别分析, 再利用薄板样条分析和网格变形将形态变异矢量可视化。相对扭曲得分的逐步判别分析结果显示: “洗澡”式养殖前后阳澄湖原产蟹与非阳澄湖产蟹背甲形态差异均极其显著, 判别准确率均为100%。网格变形结果进一步发现, 与非阳澄湖蟹情况不同, “洗澡”式养殖前后的阳澄湖产蟹背甲形态变化不显著。研究首次定量证实, 非阳澄湖产中华绒螯蟹即使在阳澄湖“洗澡”式养殖1个月, 背甲形态仍无法与原产蟹趋同。这种差异性具有区别阳澄湖原产和“洗澡”蟹的应用潜力。