粤西镇海湾近江牡蛎线粒体16S rRNA基因序列变异分析

采用聚合酶链式反应(PCR)技术对粤西镇海湾水域的近江牡蛎Crassostrea rivularis (Gould)群体27个个体的线粒体DNA16S rRNA基因序列片段进行扩增，获得大约500bp的扩增产物。PCR产物经纯化后进行序列测定，经Clustal X同源排序，除去引物及部分端部序列，得到414bp的核苷酸片段。27个个体共检测到2个变异位点，均为颠换位点，没发现碱基位点插入、缺失及转换位点，共3种单倍型，每个单倍型只有一个碱基的差异。运用DNASP软件计算得该群体的核苷酸多样性和平均核苷酸差异数分别为0．00036和0．14815。此结果提示镇海湾近江牡蛎群体遗传多样性已很低，很有必要从其他分布区引进近江牡蛎亲贝来扩大该种群的遗传多样性。     

近江牡蛎两个野生种群的遗传多样性分析

采用RAPD分子标记和rDNA-ITS1序列分析了近江牡蛎Crassostrea rivularis湛江官渡和阳江程村野生种群的遗传多样性。12个RAPD引物共扩增出8838条片段,157个位点,平均每个引物可产生13个位点,片段长度在200～2200bp之间。湛江种群和阳江种群的多态位点比例分别为89.62%和89.57%,遗传多样性指数分别为0.4170和0.4334。种群间平均遗传距离为0.0327,平均遗传相似性为0.9681,平均遗传分化系数为0.0437。得到近江牡蛎18S、5.8S部分序列和ITS1全部序列,其中ITS1序列片段长度为478～485bp,共有11个变异位点,两个为转换(A/G),其他为插入/缺失(A/-、T/-)。湛江和阳江种群各获得8个单倍型,其中有一个单倍型为两个种群共享。两个种群的CG碱基平均含量较高,分别为58.29%和58.41%。种群间的遗传分化系数0.0254。结果说明,近江牡蛎湛江种群和阳江种群间具有较高的遗传多样性和较低的遗传分化。     

基于18S-ITS1-5.8S序列的吉富罗非鱼群体遗传多样性分析

运用PCR技术扩增吉富罗非鱼选育群体第20和21世代18S-ITS1-5.8S序列,分析二序列的遗传变异。结果表明,18S-ITS1-5.8S序列中,18S、内转录间隔区(ITS)1和5.8S序列分别为156、483~540、64 bp,主要变异位点位于ITS1中;基于ITS1序列的第20代吉富罗非鱼(17尾)群体内遗传距离为0.001,保守位点530个,变异位点10个,单倍型4个,单倍型多样性为0.331±0.143,核苷酸多样性为0.002,平均核苷酸差异为1.279;基于ITS1的第21代吉富罗非鱼(42尾)群体内遗传距离为0.015,6个个体存在严重碱基缺失现象,保守位点492个,变异位点49个,单倍型12个,单倍型多样性为0.638±0.083,核苷酸多样性为0.014,平均核苷酸差异为6.945。ITS序列在该两吉富罗非鱼群体中变异较小,基于ITS1序列分析的两代群体遗传多样性水平较低。     

钦州湾牡蛎线粒体16SrRNA和COI基因片段的序列变异分析

利用线粒体16SrRNA基因和线粒体细胞色素氧化酶亚基Ⅰ（cvtocllrome oxidase Ⅰ，COI）基因片段初步研究钦州湾牡蛎（Ostxea）的物种组成。以特异引物进行PCR扩增，对扩增产物进行纯化、测序，分析表明，16SrRNA基因部分长度为413bp，COI基因部分长度为535bp，2种牡蛎序列的碱基组成均显示出较高的A＋T比例：16SrRNA基因598％；COI基因605％。对位排序比较表明，16SrRNA片段种内个体间变异较小，存在7个变异位点，4种单倍型，其中包括5个转换位点突变，2个颠换位点突变；COI片段有30个碱基存在变异，7种单倍型，其中包括16个转换位点突变，14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离，并构建了NJ和UPGMA系统树。香港牡蛎（Crassostrea hongkongensis）16SrRNA和COI序列与白肉牡蛎的遗传距离均为0000，有明巨牡蛎（Crassostrea ariakensis）16SrRNA和COI序列和红肉牡蛎（redmeatostxea）的遗传距离分别为0000和0011，白肉牡蛎16SrRNA和COI序列和红肉牡蛎之间的遗传距离分别为0035和0146。结果表明，钦州湾牡蛎分为2个不同的种，线粒体16SrRNA和COI基因在种间存在明显的多态性，证实了16SrRNA和COI基因序列适用于牡蛎的系统学分析。     

钦州湾牡蛎线粒体16S rRNA和COⅠ基因片段的序列变异分析

利用线粒体16S rRNA基因和线粒体细胞色素氧化酶亚基Ⅰ(cytochrome oxidaseⅠ,COⅠ)基因片段初步研究钦州湾牡蛎(Ostrea)的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16S rRNA基因部分长度为413bp,COⅠ基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A+T比例:16S rRNA基因59.8%;COⅠ基因60.5%。对位排序比较表明,16S rRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COⅠ片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎(Crassostrea hongkongensis)16S rRNA和COⅠ序列与白肉牡蛎的遗传距离均为0.000,有明巨牡蛎(Crassostrea ariakensis)16S rRNA和COⅠ序列和红肉牡蛎(red meat ostrea)的遗传距离分别为0.000和0.011,白肉牡蛎16S rRNA和COⅠ序列和红肉牡蛎之间的遗传距离分别为0.035和0.146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16S rRNA和COⅠ基因在种间存在明显的多态性,证实了16S rRNA和COⅠ基因序列适用于牡蛎的系统学分析。     

奥尼罗非鱼杂交子代及亲本mtDNA D-loop基因序列的多态性

利用PCR技术扩增奥利亚罗非鱼(Oreochromis aureus)、尼罗罗非鱼(O.niloticus)及其杂交子代(O.aureus♂×O.niloticus♀)3个群体mtDNA控制区全序列,分析其变异和遗传结构。结果显示:3个群体的mtDNA控制区序列长度多态性并不十分明显,全长921～933bp;3个群体间存在丰富的DNA序列多态性,共检测到138个变异位点(14.9%),44个个体具有44种单倍型(haplotype);3群体间的序列差异平均为4%,Tamura-Nei遗传距离平均为5.1%;采用NJ法和ME法构建的分子系统树中,奥利亚罗非鱼群体内所有的单倍型聚成一支,尼罗罗非鱼与其杂交子代的单倍型混杂在一起,聚成一支,表明杂交后代mtDNAD-loop表现为母系遗传。     

皱纹盘鲍和九孔鲍遗传差异nad2-cox1分析及鲍属系统发育

基于nad2-cox1核苷酸序列分析了皱纹盘鲍和九孔鲍3个养殖群体的遗传差异,基于cox1分析了鲍属19种鲍的系统发育关系。共获得625 bp的DNA片段,33个片段共检测到17种单倍型,其中皱纹盘鲍2个群体26个个体15种单倍型,九孔鲍7个个体2种单倍型。皱纹盘鲍大连群体与厦门群体有6种交叉共享单倍型,皱纹盘鲍与九孔鲍之间无共享单倍型。皱纹盘鲍群体内个体间的遗传距离(D)为0.002~0.015,九孔鲍个体间D值为0.003,两种鲍间的遗传距离为0.300~0.320。鲍属系统发育分析显示,皱纹盘鲍与日本鲍、盘鲍聚为一支(D值为0.000~0.004),而后与大鲍聚在一起(支持率为100﹪)(D值为0.018),九孔鲍、细纹九孔鲍、杂色鲍聚为一支(D值为0)。九孔鲍与马蹄螺科的Diloma bicanaliculata聚为一支,白鲍、黑鲍、红鲍、北国鲍、堪察加鲍聚为一支(支持率87﹪)。     

基于cox1和D-loop区序列雷州半岛红树林海区2种野生弹涂鱼的遗传变异

设计双层洞穴式弹涂鱼捕捉器,于雷州半岛红树林海区捕获弹涂鱼类野生群体,筛选出弹涂鱼(Periophthalmus modestus)、大弹涂鱼(Boleophthalmus pectinirostris)2个优势种群,并获取其线粒体控制区(D-loop)和cox 1基因部分序列,进行序列变异分析及遗传分析。结果表明,cox 1基因和D-loop区碱基组成A+T含量均大于C+G。cox 1数据揭示,大弹涂鱼变异位点占总长度2.1%,弹涂鱼占2.4%,种内平均遗传距离分别为0.003、0.006,种间遗传距离为0.158,37个个体共定义22个单倍型,单倍型多样性为0.950 45。而D-loop区数据指明,大弹涂鱼变异位点占总长度4.9%,弹涂鱼占7.5%,种内平均遗传距离分别为0.012、0.019,种间距离为0.409,37个个体定义32个单倍型,单倍型多样性水平极高(0.989 49)。中性检验结果显示,2种弹涂鱼进化历程符合中性进化假设,且存在群体扩张的可能。综合cox 1基因和控制区数据显示:弹涂鱼种群遗传多样性高于大弹涂鱼。     

长江下游鳊鱼遗传多样性的RAPD分析

应用RAPD技术对长江下游鳊鱼群体的遗传多样性进行分析,从40个随机引物中筛选出28个引物对鳊鱼24个个体的基因组DNA进行扩增,共检测到178个位点,分子片段在0.2～2.0kb之间,其中多态位点83个,占46.63%;个体间最大的遗传距离为0.1326,最小的遗传距离为0.0476,平均遗传距离为0.0885;群体的Nei基因多样性指数为0.2593,Shannon多样性指值为0.0986。与其他鱼类的遗传多样性研究结果相比,长江下游鳊鱼群体的遗传多样性处于中等水平。     

蛤蜊科3种贝类16S rRNA基因片段及ITS2核苷酸序列分析

利用PCR技术分别扩增连云港及启东沿海蛤蜊科的西施舌(Coelomactra antiquata)、中国蛤蜊(Mactrachinensis)和四角蛤蜊(Mactra veneriformis)3种双壳贝的16S rRNA基因片段和ITS2核苷酸序列,测序后用DNA star软件分析了核苷酸差异。结果显示:三种贝类16S rRNA基因片段长度相同,均为306bp(去除引物),核苷酸存在多态性,共有45个变异位点,54个核苷酸发生了变异,全部为碱基置换。西施舌与中国蛤蜊此片段核苷酸的同源性为88.9%,与四角蛤蜊的同源性为88.6%,中国蛤蜊与四角蛤蜊的同源性为90.6%。三种蛤蜊ITS2序列分别为390 bp(西施舌)4、41 bp(四角蛤蜊)和466 bp(中国蛤蜊),存在长度多态性,ITS2核苷酸差异分析结果显示,西施舌与中国蛤蜊的同源性为70.9%-71.1%,西施舌与四角蛤蜊的为70.5%-71.0%,中国蛤蜊与四角蛤蜊的同源性为88.1%-88.8%。ITS2序列分析结果与16S rRNA基因片段分析结果一致,2种分子分析法均显示中国蛤蜊与四角蛤蜊的亲缘关系近。     

2种亚洲龙鱼的D-Loop序列结构分析

采用PCR技术对2种亚洲龙鱼的mtDNAD_Loop全序列进行扩增和测序,序列结构分析和序列同源性比对结果表明,2种亚洲龙鱼的mtDNAD_Loop在靠近5’端有3个终止相关序列TAS(Ⅰ、Ⅱ、Ⅲ),靠近D_Loop的3’端有4个保守区域CSB1、CSB2、CSB3、CSB-D。在终止相关序列和保守区域之间是连续重复区域。经DNASP4.0软件分析,全序列中检测出多态位点数(S)为26,其中有17个转换,核苷酸多样性(Pi)为0.013,平均核苷酸差异数(K)为17.333。     

3种星虫线粒体16S rRNA、COI和Cyt b基因片段的序列比较

对裸体方格星虫(Sipunculus nudus)、可口革囊星虫(Phascolosoma esculenta)和澳洲管体星虫(Siphonosoma australe)的线粒体16S rRNA、COI和细胞色素b(Cytb)基因片段序列进行比较,并对其系统发生进行了初步探讨。采用PCR方法得到总长度分别为531～544bp(16S)、652～675bp(COI)和406～453bp(Cytb)的线粒体片段。片段碱基A+T比例较高(16S rRNA基因58.3%,COI基因56.9%,Cytb基因59.5%)。16S rRNA片段存在169个碱基变异位点(其中包括167个简约信息位点)和44个碱基插入/缺失,种内个体间变异较小;COI片段有512个碱基(333个简约信息位点)存在变异,79个碱基插入/缺失;Cytb片段存在347个碱基(318个简约信息位点)变异位点,16个碱基插入/缺失。数据分析结果支持3种星虫和环节动物的分类地位较近,与软体动物较远的分类观点。此外,裸体方格星虫与澳洲管体星虫之间亲缘关系较近(D=0.3159、0.3156、0.2361)。认为3种星虫线粒体16S rRNA、COI和Cytb基因在种间存在明显的多态性,证实了三种基因序列均普遍适用于星虫种及以上阶元的系统学分析。     

Bacterial diversity, composition and temporal-spatial variation in the sediment of Jiaozhou Bay, China

Studies on the diversity and distribution of bacterial populations will improve the overall understanding of the global patterns of marine bacteria and help to comprehend local biochemical processes and environments. We evaluated the composition and the dynamics of bacterial communities in the sediment of Jiaozhou Bay (China) using PCR-denaturing gradient gel electrophoresis (DGGE). Sediment samples were collected from 10 different sites in May, August, and November 2008 and in February 2009. There was significant temporal variation in bacterial community composition at all sites. However, the spatial variation was very small. The DGGE analyses of bacterial communities were used to divide the 10 stations into three types. Canonical correspondence analysis (CCA) revealed that the changes in bacterial communities were driven by sediment properties. Sequence analysis of DGGE band-derived 16S rRNA gene fragments revealed that the dominant bacterial groups in the sediment were of the classes γ-proteobacteria and δ-proteobacteria and phyla Bacteroidetes and Nitrospirae. Our results provide considerable insight into the bacterial community structure in Jiaozhou Bay, China.     

近江牡蛎耗氧率和排氨率的研究

采用室内实验生态学方法研究近江牡蛎的耗氧率和排氨率。结果表明,在实验温度13~33℃范围内,近江牡蛎的耗氧率(RO)和排氨率(RN)与体重(W)均呈负相关,可用Y=aW-b表示。耗氧率和温度的关系可表示为RO=-c+b1t-b2 t2,耗氧率为0.626~2.354 mg.g-1.h-1;在温度13~28℃范围内,耗氧率随温度的升高而增加,28℃时,耗氧率达最大值,温度升高至33℃时,耗氧率反而下降。排氨率与温度的关系可表示为RN=c1e d1t,排氨率为0.075~0.318 mg.g-1.h-1,且随温度的升高,排氨率呈持续升高趋势。近江牡蛎呼吸和排泄Q10值范围分别为0.631~3.399和1.046~2.288。在28℃,不同规格近江牡蛎呼吸氧原子数与排出氨氮原子数的比值(nO/nN)均达到最大值。方差分析表明,体重、温度及二者的交互作用对近江牡蛎的耗氧率和排氨率均有极显著的影响(P<0.01)。近江牡蛎的日常代谢明显高于标准代谢,耗氧率和排氨率平均值分别提高46.8%和67.7%。     

Molecular identification and population differentiation of Aurelia spp. ephyrae in sea cucumber aquaculture ponds of northern China

Aurelia spp. ephyrae have been reported to form blooms in sea cucumber aquaculture ponds in the Bohai and Yellow Seas. To identify the species, we carried out a genetic analysis of Aurelia spp. ephyrae and medusae based on mitochondrial 16 S rRNA gene. Samples offour Aurelia sp. ephyrae populations were collected in sea cucumber aquaculture ponds and samples offour Aurelia sp. medusae populations were collected in coastal waters. Using a BLASTn search, we found that both the ephyrae collected in the aquaculture ponds and medusae collected in coastal waters belong to Aurelia coerulea. Seventeen haplotypes were recovered from the 16 S rRNA gene. The overall haplotype diversity and nucleotide diversity of the 166 A. coerulea individuals were 0.686% and 0.329%, respectively, indicating high haplotype diversity and low nucleotide diversity. Moreover, the haplotype diversity of ephyrae populations were generally lower than that of medusae populations with close sampling points. The genetic differentiation between ephyrae populations collected in the sea cucumber aquaculture ponds and A. coerulea medusae collected in coastal waters was not significant, suggesting the ephyrae populations in the sea cucumber culture ponds were part of the same genetic group as the medusae populations in the coastal waters. Phylogeographic analysis of the 16 S rRNA region revealed that there was no significant correlation between the haplotypes and the geographic distribution of populations. Pairwise fixation index values showed significant genetic differentiation and limited gene flow between A. coerulea population of Weifang and other locations.     

Archaeal diversity and abundance within different layers of summer sea-ice and seawater from Prydz Bay,Antarctica

Fluorescence in-situ hybridization （FISH） and 16S rRNA gene clone library analyses were used to determine the abundance and diversity of archaea in Prydz Bay, Antarctica. Correlation analysis was also performed to assess links between physicochemical parameters and archaeal abundance and diversity within the sea-ice. Samples of sea-ice and seawater were collected during the 26th Chinese National Antarctic Research Expedition. The results of FISH showed that archaea were relatively abundant within the top layer of the sea-ice, and correlation analysis suggested that the concentration of 4NH＋ might be one of the main factors underlying this distribution pattern. However, using 16S rRNA gene libraries, archaea were not detected in the top and middle layers of the sea-ice. All archaeal clones obtained from the bottom layer of the sea-ice were grouped into the Marine Group I Crenarchaeota while the archaeal clones from seawater were assigned to Marine Group I Crenarchaeota, Marine Group II Euryarchaeota, and Marine Group III Euryarchaeota. Overall, the ifndings of this study showed that the diversity of archaea in the sea-ice in Prydz Bay was low.