中国近海铜藻ITS与coxI序列相似性分析

本研究分别对2016和2017年中国近海10个铜藻(Sargassum horneri)漂浮地理种群以及 3个定生种群的51个采集样本进行了ITS和coxI序列分析及相似性比对。结果显示,51个样本的coxI序列完全一致,ITS序列存在2个变异位点,按基因型的异同可分为4个类型,其中大连龙王塘、烟台大钦岛和南隍城岛的漂浮铜藻基因型相同,青岛金沙滩漂浮型、烟台大钦岛和大连獐子岛的定生铜藻基因型相同,威海俚岛和温州洞头的漂浮铜藻基因型相同,青岛雕塑园、王哥庄、大珠山、威海乳山的漂浮型和枸杞岛定生铜藻基因型相同,而同一种群内部即便是不同年份的个体间基因型并没有差异。基于ITS序列构建的系统树显示,来自中国的所有铜藻样本聚为一支,与来自韩国的铜藻样本有一定的遗传距离。以上结果说明,我国近海漂浮和定生铜藻的不同地理株间ITS和coxI的遗传变异水平较低,漂浮铜藻可能具有不同的来源,为进一步探明中国近海海域铜藻的分子遗传背景提供依据。     

中国近海铜藻ITS与coxⅠ序列相似性分析

本研究分别对2016和2017年中国近海10个铜藻(Sargassumhorneri)漂浮地理种群以及3个定生种群的51个采集样本进行了ITS和coxⅠ序列分析及相似性比对。结果显示,51个样本的coxⅠ序列完全一致,ITS序列存在2个变异位点,按基因型的异同可分为4个类型,其中大连龙王塘、烟台大钦岛和南隍城岛的漂浮铜藻基因型相同,青岛金沙滩漂浮型、烟台大钦岛和大连獐子岛的定生铜藻基因型相同,威海俚岛和温州洞头的漂浮铜藻基因型相同,青岛雕塑园、王哥庄、大珠山、威海乳山的漂浮型和枸杞岛定生铜藻基因型相同,而同一种群内部即便是不同年份的个体间基因型并没有差异。基于ITS序列构建的系统树显示,来自中国的所有铜藻样本聚为一支,与来自韩国的铜藻样本有一定的遗传距离。以上结果说明,我国近海漂浮和定生铜藻的不同地理株间ITS和coxⅠ的遗传变异水平较低,漂浮铜藻可能具有不同的来源,为进一步探明中国近海海域铜藻的分子遗传背景提供依据。     

马尾藻属新变种——拟披针叶马尾藻潭门变种的形态鉴定及系统进化分析

在海南省琼海市潭门镇发现了一种未见报道的马尾藻。形态鉴定结果显示,该马尾藻与拟披针叶马尾藻的形态特征十分相似,但气囊结构有明显差异,故将其定名为拟披针叶马尾藻潭门变种。对该新变种的内转录间隔区序列特征及其与马尾藻属其他物种的亲缘关系进行了分析,结果显示,拟披针叶马尾藻潭门变种隶属于真马尾藻亚属,并与瓦氏马尾藻和亨氏马尾藻具有较近的亲缘关系。     

我国南黄海海域漂浮铜藻的分子生物学鉴定

为探究南黄海海域漂浮马尾藻属(Sargassum)的种类,以2013年5月采自南黄海海域漂浮马尾藻属藻类为研究对象。采用PCR技术扩增出其ITS2序列,并用Clustal X和Mega 4.0软件对其进行序列分析和N-J系统发育树的构建。结果表明,南黄海海域漂浮的马尾藻属藻类为铜藻(Sargassum horneri),其ITS2序列大小为700 bp左右。     

华南沿海长鳍篮子鱼不同地理群体的遗传多样性分析

对南海北部沿岸及东海南部9个不同地理群体的长鳍篮子鱼(Siganus canaliculatus)进行遗传多样性及遗传结构分析。484条DNA D-loop 序列分析结果显示：序列长度约 828bp,包含57个变异位点,92个单倍型；所有群体总的单倍型多样性指数(Hd)和核苷酸多样性指数(π)分别为0.80798和0.00405；群体内的遗传距离 0.00386～0.00532,群体间的遗传距离 0.00383～0.00482,群体间的分化指数为-0.01935～0.00759,各种分组的AMOVA分析均显示群体内变异比例超过100%；Fu"s Fs检验值为显著负值(-25.53678,p<0.01),核苷酸错配分布图呈单峰,吻合度检验数值较小且不显著,这些结果均提示长鳍篮子鱼可能经历过种群扩张事件,推测扩张时间约在4.74万年～1.18万年前。综上,南海北部沿岸及东海南部9个长鳍篮子鱼群体间不存在显著的地理结构和谱系结构,可划归一个管理单元进行种质资源保护。     

南海北部海域多齿蛇鲻的种群分析

对南海北部海域5个多齿蛇鲻地理群体(北海、湛江、茂名、东莞、汕尾)共78个个体的线粒体控制区基因序列进行了扩增测序。在78个个体的568bp序列中共检测到48个单倍型,39个变异位点。经分子变异等级分析(AMOVA)得出5个地理群体内遗传变异度为98.82%,群体间遗传变异度仅为1.18%,各群体间成对遗传变异固定指数FST值在0.00024～0.03008;78个个体间的遗传距离在0～0.025。通过对基于序列片段构建的NJ树和基于序列单倍型利用中介网络法构建的群体间关系网的分析发现,5个群体间有明显的基因交流,遗传分化差异不大;再结合种群、种及属的界定标准,判定5个多齿蛇鲻群体归属于同一种群;而且,在经历快速增长、变异和瓶颈期之后,多齿蛇鲻种群就以小群体模式继续快速增长,形成地理群体。     

基于mtDNA控制区序列的拟平鳅遗传变异和种群分化

采用DNA序列分析方法,分析拟平鳅不同地理种群间的遗传变异和亲缘关系。共检测了12条水系87尾拟平鳅的mtDNA控制区序列,在477 bp片段中有95个核苷酸变异位点,其中39个单个多态位点,56个简约信息位点。系统发育分析(NJ)结果显示,华南西部沿海地区拟平鳅20个单倍型分成3支;分支I由广东西部独立入海水系和珠江水系的种群组成,分支II由广西东部独立入海水系和海南岛昌化江的种群组成,而海南岛的万泉河和南渡江种群聚合成分支III。表明海南岛不同水系的拟平鳅已经发生了遗传分化,一部分与广西的种群相似,另一部分是海南特有的。3个分支内的变异率（0.583%～1.775%）都明显小于各分支间的变异率（4.205%～5.715%）,也远小于各分支与外类群间的变异率（11.825%～13.73%）。说明华南西部沿海和海南岛的拟平鳅已分化成3个在遗传上有明显差异的群。基于现在的地理格局,研究曾经假设华南西部沿海独立水系种群与珠江水系种群的关系较密切,而与海南岛种群的关系较疏远。分子变异分析（AMOVA）表明,大部分的变异（65.04%）分布于地理区内种群间,而地理区间的变异仅占2.24%,与假设不符。表明12条水系拟平鳅mtDNA控制区序列的遗传分化可能主要是由于第三纪水系的隔离,而不是第四纪冰后期海南岛与大陆的分离所致。而第四纪盛冰期低海平面所造成的陆连,使相互分离的水系有机会连接,因此,海南岛昌化江种群与广西沿海独立水系种群关系密切,珠江水系种群保持与广东西部沿海独立水系种群的密切关系。而琼中海峡这个地理屏障形成的时间还不够长久,并没有成为有效的生殖屏蔽。AMOVA分析结果与上述NJ分析中各个分支之内的分子变异非常小的结果一致。     

小球藻核rDNA ITS与叶绿体rbcL基因序列分析及应用

用核基因组rDNA的内转录间隔区(ITS)和叶绿体rbcL基因对小球藻属((Jhlorella)6株小球藻的种间和种内关系进行了分析.克隆序列与GenBank数据库检索到的相关小球藻序列(ITS和rbcL序列各为15条)一起进行比较分析.结果显示:ITS序列长度在小球藻种内高度保守,在种间变异较大;而rbcL序列长度在种间和种内水平都高度保守.15株小球藻ITS序列间遗传距离在0.000～0.663.之间;而15株小球藻rbcL序列间距离在0.000～0.216之间.6株实验藻中原始小球藻F-2与蛋白核小球藻F-5、F-9近似种内关系;蛋白核小球藻820与普通小球藻Cvq亲缘关系极近;椭圆小球藻Ce与其它5株小球藻亲缘关系最远.研究表明将变异程度较高的核ITS序列与相对保守的叶绿体rbcL基因相结合可以用于小球藻系统发生分析和分子鉴定.     

羊栖菜养殖技术

羊栖菜(sargassum,fusiforme Harv setch)为北太平洋西部特有种类,分布于朝鲜、日本和我国沿岸。有较高的药疗和经济价值。 一、羊栖菜的生物学特征 羊栖菜隶属褐藻门,圆子纲,墨角藻目,马尾藻科,马尾藻属。藻体呈黄褐色,株高一般为30—50cm,高的可达200cm左右。采用生殖细胞人工育苗经养殖后高的可达330cm。藻体分为假根、茎、叶片和气囊4部份。藻体外形由于南北地理环境的不同,出现较大的差异,北方种群株技密集,叶、气囊扁宽多锯齿,南方株枝稀长,叶、气囊线形或棒状。 羊栖菜的生长方式是由顶端细胞进行立体型分裂。随着藻体生长,叶片由下向上逐渐脱落。 羊栖菜的生活史中只有孢子体阶段,而无明显的     

基于线粒体COⅠ基因的中国沿海和泰国普吉岛里氏拟石磺群体遗传结构分析

通过对COⅠ序列的分析,研究了中国沿海及泰国普吉岛8个里氏拟石磺群体的种群遗传结构,171个样本中共检测出单倍型101个,117个多态性位点,里氏拟石磺具有高的单倍型多样性(0.974±0.005)和核苷酸多样性(0.032 2±0.001 3)。分子方差分析(AMOVA)表明,50.99%的变异存在于种群间,49.01%的变异发生在种群内。群体间遗传分化固定指数(F st)、基因流(N m)及遗传距离分析表明,里氏拟石磺已明显分化为显著的遗传结构。遗传距离模式(IBD)检测显示,里氏拟石磺群体的遗传距离与地理距离之间不存在明显的线性关系。历史动态检验推断,湛江(ZJ)、苍南(CN)、东寨港(HN)、文昌(WC)及普吉岛(TH)种群可能经历过历史上的种群扩张事件。中国沿海群体扩张时间推测大约为0.781~0.725 Ma BP,泰国普吉岛群体约为0.035 Ma BP,两者可能伴随更新世冰期的气候变暖、冰川消融和海平面上升等现象发生。     

11种鲈形目鱼类ITS2特征及系统应用

ITS2 (Internal transcribed spacer 2)是位于核糖体5.8S和28S基因之间的非编码序列。为了探讨该片段的多样性特征以及进化模式,本研究选取了鲈形目(Perciformes) 5科11种鱼类为研究对象,共获得了444条ITS2克隆序列,其长度范围为332～515 bp。比较种内不同序列的长度发现,金带细鲹(Selaroides leptolepis)在种内存在24 bp的差异,剑鱼(Xiphias gladius)在种内存在32 bp的差异,这2种鱼类的差异较为明显;其余9种鱼类的长度相对比较保守,长度差异小于14 bp。依据11种鱼类的保守位点数、变异位点数、简约信息位点数、单倍型数、保守位点比例、单倍型多样性指数、核苷酸多样性等特征分析发现,种内存在着不同程度差异,特别是金带细鲹的ITS2序列存在着Type A、Type B和Type C 3种类型,各类型间差异较大。根据序列的多样性特征推断,金带细鲹和剑鱼的进化方式为非协同进化;蓝圆鲹(Decapterus maruadsi)、大甲鲹(Megalaspis cordyla)、吉打副叶鲹(Alepes djedaba)和日本竹筴鱼(Trachurus japonicas)的长度和变异位点均存在着一定程度的差异,但差异并不明显,视为不严格的协同进化;泰拉鰆鲹(Scomberoides tala)、布氏鲳鲹(Trachinotus blochii)、尖吻鲈(Lates calcarifer)、射水鱼(Toxotes chatareus)和军曹鱼(Rachycentron canadum) 5种鱼类为协同进化;另外,协同和非协同进化状态与分类系统没有相关性。序列比对发现,大甲鲹种内存在着由协同进化方式演变为非严格的协同进化方式的过度序列;在金带细鲹的3个不同个体中,序列间存在着从协同进化、非严格的协同进化演变为非协同进化的3种进化方式。基于ITS2序列构建的11种鱼类的邻接系统树显示,每种鱼类的克隆都分别按种单独聚为一支,鲹科7属鱼类各属也是单独聚支,表明ITS2不仅可以用在种类的分子鉴定,同时也可以作为分子标记应用于鲹科和属级水平的系统关系研究。     

利用ITS和AFLP标记探讨中国、日本和澳大利亚3种珠母贝的亲缘关系

我国合浦珠母贝 Pinctada fucata 的命名较混乱,与日本的 P.fucata martensii 和澳大利亚的 P.imbricata 的分类关系存在争议。用 ITSs 序列和 AFLP 标记对这3个种的遗传关系进行了分析。结果表明这3个地理种的遗传分化很低,种内和种间的遗传距离相互重叠,AFLP 数据的聚类分析表明澳大利亚种群大部分个体单独聚合成一支,主成分分析结果相似。种问的遗传距离与分化程度与其地理距离呈正相关。分子方差结果也表明种间的差异较小,低于6%。这些结果表明这3个种实际上是同一物种。由于大西洋的 P.imbricata 与澳大利亚的不同,因此根据分类命名的优先原则,正确的种名应为 P.fucata。     

Molecular systematics of bisexual Artemia populations

To help resolve phylogenetic relationships among bisexual Artemia populations, phylogenetic analysis was conducted using DNA sequences from the nuclear DNA internal transcribed spacer 1 (ITS‐1) and portions of the mitochondrial genome corresponding to the cytochrome oxidase I (COI). DNA sequences were generated for nine bisexual Artemia populations living in different regions of the world. Phylogenetic trees based on ITS‐1 and COI sequences indicated that bisexual Artemia populations consist of four groups. The bisexual Artemia populations from Tibet and Kazakstan always clustered with Artemia urmiana in the same group; there is small sequence divergence and genetic distance among them. Therefore, we deduced that bisexual Artemia populations from Tibet and Kazakstan may belong to the A. urmiana group. Our study did not support that bisexual Artemia populations from Tibet are a new, separate species A. tibetiana. We also found that A. sinica and A. urmiana have a small genetic distance. Based upon these findings, we conclude that A. urmiana may have played an important role in the evolution of A. sinica.     

Genetic variations within Symbiodinium clade C among zooxanthellate corals (Scleractinia) in the temperate zone of Japan

In a recent study most of the temperate-living corals were found to harbor stress-sensitive Symbiodinium clade C, which is common in the tropical zone. In this study, we investigated the genetic variations within Symbiodinium clade C among corals living in temperate waters of Japan (17 genera; 26 species) using rDNA internal transcribed spacers (ITSs) to clarify the genetic differences between climatic zones. We also focused on two widespread coral species, Acropora hyacinthus and Pocillopora damicornis, to compare the genetic differences of Symbiodinium clade C between subtropical and temperate regions. We found multiple haplotypes of Symbiodinium ITSs in clade C, which formed nine subclades. Our data indicate that five genera (Acropora, Cyphastrea, Stylocoeniella, Porites, Pocillopora) contained host-specific Symbiodinium, while members of the family Faviidae and others contained several Symbiodinium ITS haplotypes from two or three subclades. A comparative analysis using Acropora and Pocillopora also revealed that the temperate zone coral population formed a single subclade of Symbiodinium clade C that was genetically isolated from those of subtropical populations. In summary, the majority of corals living in temperate waters of Japan, such as faviid corals, have several ITS haplotypes which possibly are utilized as an opportunistic survival strategy under severe environmental conditions, whereas Acropora and Pocillopora at the very least harbor temperate zone-specific Symbiodinium.     

Denaturing Gradient Gel Electrophoresis (DGGE) as a tool for the characterisation of Brachionus sp. strains

Many zooplanktonic organisms, like the cyclic parthenogenetic rotifer Brachionus plicatilis (Rotifera: Monogononta), are actually a complex of species and biotypes with a high degree of morphological similarity (i.e. cryptic species). Various phylogenetic studies with molecular markers (e.g. ITS1 and COI) on wild Brachionus populations described the presence of at least nine genetically divergent Brachionus species and biotypes. Because different studies found evidence that these cryptic species and biotypes differ significantly in ecological preferences and thus probably behave differently in response to rearing conditions in the hatchery, questions rise on the actual identity of the rotifer strains used in aquaculture, where Brachionus discrimination is still based on morphology. This study is a part of an investigation of the genetic make-up of strains used in hatcheries, aquaculture research institutes and laboratories, and describes the rapid and sensitive PCR-DGGE method for the detection of Brachionus species and biotypes based on nucleotide sequence variation within the mitochondrial 16S rRNA gene. Considerable genetic diversity was found, albeit smaller within hatcheries than within laboratories and aquaculture research institutes. All 16S haplotypes produced an unambiguous DGGE fingerprint out of which a database was constructed.     

Molecular divergence and application of the ITS-5.8S rDNA and RUBISCO spacer in <Emphasis Type="Italic">Porphyra haitanensis</Emphasis> Chang et Zheng (Bangiales,Rhodophyta)

To select a reliable and sensitive method for discriminating strains of Porphyra haitanensis, the nucleotide sequence of the internal transcribed spacer 1 to internal transcribed spacer 2 regions (ITS-5.8S) of nuclear ribosomal DNA and the intergenic spacer region of RUBISCO were compared in five wild and five cultivated Porphyra haitanensis strains. Based on molecular analyses, sequences of ITS-5.8S (about 1,210 bp) could be divided into three regions: ITS1, 5.8S, and ITS2. The ITS1 and ITS2 sequences of each strain differed, even between individuals collected from the same site. In contrast, 5.8S rDNA and RUBISCO spacer sequences were identical among the ten P. haitanensis strains, although differences were found among different Porphyra species. Phylogenetic analysis also supported these conclusions. These sequence features of highly conserved regions and diversified regions that occurred repeatedly in ITS-5.8S could be useful in discriminating germplasm of P. haitanensis strains or Porphyra species. In contrast, the RUBISCO spacer is only suitable for identifying Porphyra species. New coupled primers were designed to amplify only the 5.8S rDNA and ITS2 region of Porphyra. The sequences of these amplified fragments can be readily used to identify germplasm or to perform phylogenetic analysis of Porphyra spp.     

中国东南沿海青蟹线粒体COI基因部分序列分析

对我国东南沿海5个地区的72个青蟹个体的线粒体细胞色素氧化酶亚基I (COI)部分序列序列进行了测定和分析。获得的72个细胞色素氧化酶亚基I (COI)序列可分为12个单倍型，与GenBank中已知的Scylla paramamosain COI序列的相似性达到98%以上，与其它3种 青蟹的差异为7.36%～15.54%。这些序列与S. paramamosain的遗传距离仅为0.00783，但是与S. serrata、S. olivacea和S. tranquebarica〗的遗传距离却分别达到0.11659、0.17812和0.08423。序列特征、遗传距离和系统进化等分析结果都表明本文研究的青蟹为S. paramamosain。结果提示，在进行青蟹属相关研究应当仔细鉴别采集样本的种类。     

5科11种鱼类ITS1特征分析及其在系统分类研究中的适用性

为了探讨ITS1作为分子标记用于鱼类系统演化的适用性,实验选取鲈形目5科11种鱼类为研究对象,包括尖吻鲈科、射水鱼科、军曹鱼科、剑鱼科和鲹科。通过克隆和测序等技术共获得了348条ITS1序列,长度范围为442~661 bp;通过对所有序列的长度、变异位点数量、GC含量、核苷酸多样性及单倍型多样性指数等遗传特征比较分析发现,11种鱼类ITS1序列无论是在种内还是在种间,长度和序列都表现出较为明显的多态性。特别是在军曹鱼中,70条克隆的长度范围为648~661 bp,但有一条序列存在55 bp缺失,结合该序列的GC含量,二级结构和最小自由能,推断该序列为假基因。以鮣为外类群,基于核糖体ITS1序列构建的邻接树显示在物种种类水平上,不同个体的克隆都按种类聚支,ITS1可以用于该类群物种的区分;在属级水平上,ITS1将11属鱼类完全区分开,能够用于属级水平的区分;在科级水平上,虽然鲹科分为2支的分子结果和形态分类存在差异,但ITS1构建的系统关系与线粒体分子标记构建的系统进化树相似。研究表明,核糖体ITS1可以作为一种有效的分子标记用于研究鱼类的系统分类研究,并且不同的分类阶元其解析能力不同,这将为鱼类核糖体的研究提供科学依据。     

Phylogenetic analysis and species identification of popular shrimp species in southeast China using the first internally transcribed spacer of ribosomal DNA

The ribosomal DNA internally transcribed spacer 1 (ITS1) was investigated in the search for an appropriate genetic marker that was suitable for phylogenetic study and species identification of eight major exported shrimp species in southeast China. Using the selected primers, the amplified ITS1 sequences exhibited a high degree of length polymorphisms, ranging from 448 bp in Metapenaeopsis dalei to 1491 bp in Macrobrachium nipponense . Many microsatellite loci were found at the 5' end and in the middle region of ITS1, which seemed to be associated with intragenomic sequence variation among samples of the same species. This variation might obscure the phylogenetic relationship between some shrimp populations, but the separation of five Penaeus species was well supported. In combination with polymerase chain reaction-restriction fragment length polymerism methods analysis, ITS1 sequences from shrimp species belonging to different families and genera could also be easily discernable. The results suggested that ITS1 was highly variable among different shrimp groups and could be an appropriate marker for species identification and molecular systematic studies.