Complete nuclear ribosomal DNA sequence analyses of the black-footed abalone Haliotis iris

The black-footed abalone Haliotis iris is an economically important shellfish species in New Zealand. We successfully amplified, sequenced and analysed the complete nuclear ribosomal DNA (nrDNA) of H. iris. The length of the nrDNA was determined to be around 9.6?kb and included, in order, small subunit ribosomal RNA (nrSSU, 1858bp), internal transcribed spacer (ITS, 749?bp), large subunit ribosomal RNA (nrLSU, 3412bp) and an intergenic spacer (IGS, 3560–3662?bp). The nrLSU genes were identical in two individuals, whereas the nrSSU and ITS regions existed at three and four base differences, respectively. The IGS was more variable than the other nrDNA regions. A phylogenetic tree was constructed based on the ITS sequence datasets, which revealed that Haliotidae has two major subclades, mainly distributed in the North Pacific, Europe and Australia. The complete nrDNA sequence will be useful for the classification, phylogeny and breeding of this shellfish.     

Phylogenetic analysis of Pectinidae(Bivalvia) based on the ribosomal DNA internal transcribed spacer region

The ribosomal DNA internal transcribed spacer (ITS) region is a useful genomic region for understanding evolutionary and genetic relationships. In the current study, the molecular phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) was performed using the nucleotide sequences of the nuclear ITS region in nine species of this family. The sequences were obtained from the scallop species Argopecten irradians, Mizuhopecten yessoensis, Amusium pleuronectes and Mimachlamys nobilis, and compared with the published sequences of Aequipecten opercularis, Chlamys farreri, C. distorta, M. varia, Pecten maximus, and an outgroup species Perna viridis. The molecular phylogenetic tree was constructed by the neighbor-joining and maximum parsimony methods. Phylogenetic analysis based on ITS1, ITS2, or their combination always yielded trees of similar topology. The results support the morphological classifications of bivalve and are nearly consistent with classification of two subfamilies (Chlamydinae and Pectininae) formulated by Waller. However, A. irradians, together with A. opercularis made up of genera Amusium, evidences that they may belong to the subfamily Pectinidae. The data are incompatible with the conclusion of Waller who placed them in Chlamydinae by morphological characteristics. These results provide new insights into the evolutionary relationships among scallop species and contribute to the improvement of existing classification systems.     

杂色鲍繁殖亲本体型小型化原因初探

针对目前杂色鲍性腺发育提前的现象展开研究,以产卵量为指标分析了杂色鲍可量性状与产卵量之间的相关关系.结果表明,以体长的立方除以体重作为体型系数（F）,它与产卵量（G）之间存在着显著的负相关（p〈0.05）,相关系数为-0.9480.曲线回归方程为：G=-7.5009 ln（F）＋18.22（R2=0.9095）.进一步的分析结果表明,杂色鲍雌性亲本的产卵量和体长亦呈负相关,相关系数为-0.7182.结果提示,性腺发育较早的小个体的杂色鲍母本更多地参与了繁殖活动,而且产出的卵子的绝对重量大于大个体的杂色鲍亲鲍.这可能是造成人工育种中杂色鲍体型减小、性腺发育提前的潜在原因.     

基于皱纹盘鲍(Haliotis discus hannai)EST数据库的串联重复序列特征分析

皱纹盘鲍大规模EST的分析研究较少,为了研究其基因组转录本的基本特征并为皱纹盘鲍中开发EST-SSR功能性标记奠定基础,我们利用生物信息学手段对NCBI公共数据库中的5784条皱纹盘鲍EST序列进行EST-SSR特征分析。分析结果表明:(1)皱纹盘鲍EST中串联重复序列类型丰富;(2)皱纹盘鲍EST Gene Ontology注释序列SSR类型单一;(3)皱纹盘鲍EST-SSR分布广泛富,是EST-SSR标记开发的优良资源。     

基于16S rRNA 部分序列探讨部分鳚亚目鱼类的分子系统进化关系

通过PCR扩增获得了3科5属6种中国黄渤海海域的鳚亚目(Blennioidei)鱼类的线粒体16S rRNA基因序列片段约669bp碱基,结合来自 GenBank的5种鳚亚目其他科鱼类的相应基因片段,并以眼斑雪冰鱼(Chionodraco rastrospinosus)为外群,生成供系统发育分析的序列矩阵,利用MEGA 4.0软件分析序列的碱基组成、差异百分比、转换/颠换值等,应用最大简约法(MP)和邻接法(NJ)构建系统发育树.结果显示:在鳚亚目鱼类的16S rRNA 片段生成的序列矩阵中发现有碱基的插入缺失现象,共有207 bp变异位点,转换/颠换值为0.8,碱基平均差异为3.36;支持绵鳚(Enchelyopus elongates)归于鳚亚目绵鳚科(Zoarcidae),鳚(Azuma emmnion)归于鳚亚目线鳚科(Stichaeidae);方氏云鳚(Enedrias fangi)和云鳚(Enedrias nebulosus)种间遗传距离只有0.01,亲缘关系最近.     

Genetic variation of Chinese and Japanese wild Pacific abalone(Haliotis discus hannai) measured by microsatellite DNA markers

1Introduction ThePacificabaloneHaliotisdiscushannaiisan importantspeciesinthecoastalfisheriesofnorthern China,andisofhighcommercialvalue.Thepro ductionofcultureseedsfortheabalonebeganin Chinaintheearly1980s.Duringthelasttwodec ades,productionofabalonehasi…     

Thermal constraints on glycolytic metabolism in the New Zealand abalone,Haliotis iris: the role of tauropine dehydrogenase

Black‐foot abalone, Haliotis iris, were sampled from two populations in warm northern waters, and from two in colder southern waters. Abalone muscle is characterised by high activity of the glycolytic pyruvate reductase enzyme, tauropine dehydrogenase (TDH). Adductor muscle TDH was profiled for thermostability and activity to test the hypothesis that the enzyme may show adaptation in titre or kinetic characteristics reflecting thermal habitat. Temperature dependency of the apparent Michaelis‐Menten constant of TDH for pyruvate (_appKm_pyr) suggested eurythermal enzyme behaviour below 20°C, and compromised function at the higher temperatures of northern populations occurring in the summer months. Thermostability profiles and enzyme activities suggest TDH expression does not differ significantly among populations (P > 0.05), indicating that this locus shows no compensation for temperature. The optimal temperature for efficient TDH function, estimated from V_max./_appKm_pyr, is close to 20°C. The possible thermal constraints on glycolytic metabolism in H. iris are discussed.     

6—DMAP抑制九孔鲍受精卵第二极体的释放诱导三倍体

当50％九孔鲍受精卵出现第一极体后，用6－DMAP不同的浓度进行不同的持续时间诱导三倍体。结果表明，6－DMAP的诱导浓度为300μmol/dm^3、诱导持续时间为10min时，其诱导的三倍体率为90％以上，并且胚胎的孵化率高达85％，幼体的畸形率较低，为50％－55％。用该方法多次进行了九孔鲍三倍体生产诱导和苗种培育试验。本文还对6－DMAP的作用机理以及九孔鲍三倍体诱导中的诱导因子等问题进行了讨论。     

绿鲍（Haliotis fulgens）线粒体基因组全序列测定及分析

为高效鉴别鲍属物种和更好地管理和保护鲍种质资源,本研究通过高通量测序获得了养殖绿鲍（Haliotis fulgens）稚贝的线粒体基因组全序列,并对其序列和结构特征进行分析。结果表明,绿鲍线粒体基因组全长17 041 bp,含有37个编码基因,其中蛋白质编码基因13个、tRNA基因22个、rRNA基因2个。13个蛋白质编码基因均以AUG为起始密码子,以UAG或UAA为终止密码子。除tRNA-Ser^(AGN)外的21个tRNA基因可折叠成典型三叶草结构。分析发现tRNA-Glu和COX3间存在富含A+T的非编码区,其内含有2个带回文序列的发卡结构。基于已报道的10个鲍属线粒体基因组全序列构建系统发育树,发现绿鲍与皱纹盘鲍（Haliotis discus hannai）、红鲍（Haliotis rufescens）、黑鲍（Haliotis cracherodii）聚为一支。将绿鲍与皱纹盘鲍13个线粒体编码蛋白的结构域比较,发现二者ND2、ND4的跨膜结构域数量存在差异,这是否与二者的高温耐受性差异有关,有待进一步研究。总之,绿鲍线粒体基因组全序列的首次获取和分析,丰富了鲍类细胞遗传信息,为分类、种质鉴定与种质资源保护提供了基础数据和参考。     

单条固定线虫基因组DNA提取及18S rRNA基因PCR扩增

根据线虫18S核糖体RM基因PCB扩增效果比较了丙酮、乙醇、乙醇 0．05mol／L FDTA(pH8．0)和5％海水福尔马林4种固定剂,碱裂解和蛋白酶K处理2种单条线虫基因组DNA提取方法的优劣。用乙醇固定的样品最适合制备RR模板DNA,而5％海水福尔马林固定的样品能最完整地保持样品形态。蛋白酶K处理获得的DNA较碱裂解获得的更适合PCR扩增。结果有助于分子生物学方法在海洋线虫分类、多样性和生态学研究中的应用。     

广东沿海杂色鲍养殖群体遗传多样性的微卫星分析

实验选取的7个杂色鲍(Haliotis diversicolor)养殖群体分别来自广东汕头、汕尾、惠东、湛江和徐闻,深圳野生驯养,利用7对微卫星引物对上述群体进行了遗传多样性和群体遗传结构的分析。结果显示:7个位点产生的等位基因数从7～13个不等,共检测到68个等位基因,平均每个位点的等位基因数为9.71个。有效等位基因数的范围为3.8～8.1,平均观测杂合度范围为0.422～0.906,平均期望杂合度范围为0.748～0.917。7个群体的多态信息含量变化范围为0.657～0.883,PIC值均在0.500以上,表现为高度多态性。与两个野生驯养的养殖群体相比,其他5个养殖群体均存在不同程度的遗传多样性降低。AMOVA分析显示,4.79%的遗传变异来自于群体之间,95.21%的变异来自于群体内的个体之间。研究表明,广东沿岸的杂色鲍具有较高的遗传多样性水平,7个群体之间呈现一定的遗传分化。     

The complete DNA sequence of the mitochondrial genome of Sinonovacula constricta (Bivalvia: Solecurtidae)

The nucleotide sequence of the mitochondrial genome of the solecurtidae Bivalvia mollusca Sinonovacula constricta (GenBank accession number EU880278) has been determined and is reported here. We determined the complete mitochondrial genome sequence using long-PCR and Shot Gun Sequencing. Contained within the 17 225 base pairs (bp) are the two ribosomal RNA genes and 12 protein coding genes typical of metazoan mitochondrial genomes. The S. constricta mitochondrial DNA (mtDNA) did not contain a gene for atp8, similar to the mtDNA of Crassostrea virginica, Crassostrea giga and Mytilus edulis. The S. constricta mtDNA is 67.0% A+T (A 25.9%, C 10.5%, G 22.5%, and T 41.1%). This value is higher than that for many invertebrate mitochondrial genomes. Only 19 putative tRNA genes are present in S. constricta and 27 noncoding regions, of which two are large in size. The trnE and trnW genes as well as a second trnS were absent in S. constricta. The gene arrangement of S. constricta is different from the other Bivalvia genomes.     

A spatial- and age-structured assessment model to estimate the impact of illegal fishing and ecosystem change on the South African abalone Haliotis midae resource

The management of abalone stocks worldwide is complicated by factors such as illegal fishing combined with the difficulties of assessing a sedentary (but not immobile) resource that is often patchily distributed. The South African abalone Haliotis midae fishery is faced with an additional problem in the form of a relatively recent movement of rock lobsters Jasus lalandii into much of the range of the abalone. The lobsters have heavily reduced sea urchin Parechinus angulosus populations, thereby indirectly negatively impacting juvenile abalone which rely on the urchins for shelter. A model is developed for abalone that is an extension of more standard age-structured assessment models because it explicitly takes spatial effects into account, incorporates the ecosystem change effect described above and estimates the magnitude of substantial illegal (‘poached’) catches. The model is simultaneously fitted to catch per unit effort and Fishery-Independent Abalone Survey abundance data, as well as to several years of catch-at-age (cohort-sliced from catch-at-size) data for the various components of the fishery and different spatial strata. It constitutes the first quantitative approach applied to the management of this commercially valuable resource in South Africa and has provided a basis for management advice over recent years by projecting abundance trends under alternative future catch levels.     

三株赤潮硅藻5.8S rDNA及转录间隔区(ITS)的克隆及序列分析

对引发赤潮的3株硅藻——1株尖刺拟菱形藻(Pseudo-nitzshia pungens)和2株中肋骨条藻(Skeletonema costatum)的5.8SrDNA和ITS(internal transcribed spacers)序列进行了PCR扩增、克隆和序列测定,并分析了硅藻门10株赤潮藻(7株从GenBank获得)的系统进化关系.研究结果表明,尖刺拟菱形藻的ITS和5.8SrDNA的长度为693bp,SK-1(分离自东海赤潮暴发区)测序得到715bp,除ITS和5.8SrDNA外,还包含部分18SrDNA和28SrDNA;SK-2(分离自青岛养殖场)的ITS和5.8SrDNA的长度为331bp,尖刺拟菱形藻与从GenBank中获得的2株尖刺拟菱形藻相似程度最高,为100%,与该属的多列拟菱形藻相似程度稍低,为82.9%.SK-2的ITS序列与SK-1的相似程度很低,只有51%,但与拟中肋骨条藻的ITS序列相似程度高,为95.5%.SK-1的ITS序列与拟中肋骨条藻的相似程度也低,为56.7%.系统进化树反映的结果与相似性反映的结果一致.研究的该株尖刺拟菱形藻从根据ITS序列研究的结果与形态鉴定的结果看是一致的;SK-2可能属于拟中肋骨条藻;SK-1比较特殊,有待于用其他的方法进一步研究确定其分类地位.     

笛鲷鱼类的线粒体DNA控制区结构及其系统发育分析

采用PCR扩增直接测序的方法获得了11种笛鲷鱼类的线粒体DNA控制区全序列。结合从GenBank中下载的6种笛鲷鱼类的相应序列进行结构分析,可以将笛鲷鱼类的控制区分为终止序列区、中央保守区和保守序列区三个区域,找到了终止相关的序列ETAS以及一系列保守序列(CSB-F,CSB-E,CSB-D和CSB-1,CSB-2,CSB-3),并给出了它们的一般形式。以黄谐鱼为外群,采用NJ法和ME法构建笛鲷鱼类的分子系统树。分子系统发育分析表明,线粒体DNA控制区序列适合于笛鲷鱼类的系统发育分析,而且支持Allen关于笛鲷属的系统形态分类学观点。     

利用线粒体DNA 控制区部分序列分析不同地理群体大泷六线鱼遗传多样性

利用PCR技本扩增了6个野生地理群体大泷六线鱼(Hexagrammos otakii)线粒体DNA控制区的部分序列,共获得352bp碱基序列,31尾个体定义了22种单倍型.中性检验Fu's Fs值为-0.14881(P＜0.01).分子变异分析(AMOVA)结果显示:遗传分化指数Fst=0.7398(P＜0.01),73.98％的变异来自群体间,26.02％的变异来自群体内.根据不同个体间的遗传距离构建了NJ和MP系统树,两种方法获得了相似的进化树.其中青岛群体与旅顺群体的一部分个体单独成支,其它群体聚为1支.研究结果表明,大泷六线鱼群体遗传多样性较为丰富,群体间发生了较大的遗传分化,证明线粒体DNA控制区基因可用于大泷六线鱼群体内及群体间遗传多样性的分析.     

Genetic variation of the small yellow croaker (Larimichthys polyactis) inferred from mitochondrial DNA provides novel insight into the fluctuation of resources

The small yellow croaker (Larimichthys polyactis) belongs to the family Sciaenidae, which is an offshore warm fish species and widely distributed in the western Pacific. In this study, the variation of genetic diversity and genetic differentiation among L. polyactis populations was analyzed by mitochondrial DNA control region. A total of 110 polymorphic sites were checked, which defined 134 haplotypes. High level of haplotype diversity (h=0.993±0.002) was detected in the examined range. Population genetic structure analyse (analysis of molecular variance, F_st) showed there were high gene flow among L. polyactis populations. The result showed that there were relatively high genetic diversity and low genetic differentiation among the Yellow Sea and the East China Sea populations, which can be attributed to diverse habitats, wide distribution range and high mutation rate of control region. Using phylogenetic methods, coalescent analyses (neutrality tests, mismatch distribution analysis, Bayesian skyline analyses) and molecular dating interpreted in conjunction with paleoclimatic and physiographic evidence, we inferred that the genetic make-up of extant populations of L. polyactis was shaped by Pleistocene environmental impacts on the historical demography of this species. Besides, relatively constant genetic diversity and larger effective population size were detected in recent L. polyactis population. The result showed that the fishing policy certainly, such as the summer closed fishing, played a role in protecting resources of L. polyactis. This study can offer a wealth of biological novelties which indicates genetic structure of L. polyactis population and provides the foundation for resources protection and policy setting.     

Molecular analysis of complete ssu to lsu rdna sequence in the harmful dinoflagellatealexandrium tamarense (korean isolate, HY970328M)

New PCR primers (N=18) were designed for the isolation of complete SSU to LSU rDNA sequences from the dinoflagellateAlexandrium tamarense. Standard PCR, employing each primer set selected for amplifications of less than 1.5 kb, successfully amplified the expected rDNA regions of A. tamarense (Korean isolate, HY970328M). Complete SSU, LSU rDNAs and ITS sequences, including 5.8S rDNA, were recorded at 1,800 bp, 520 bp and 3,393 bp, respectively. The LSU rDNA sequence was the first report inAlexandrium genus. No intron was found in the LSU rRNA coding region. Twelve D-domains within the LSU rDNA were put together into 1,879 bp (44.4% G+C), and cores into 1514 bp (42.8% G+C). The core sequence was significantly different (0.0867 of genetic distance, 91% sequence similarity) in comparison withProrocentrum micans (GenBank access. no. X16108). The D2 region was the longest in length (300 bp) and highly variable among the 12 D-domains. In a phylogenetic analysis using complete LSU rDNA sequences of a variety of phytoplankton,A tamarense was clearly separated with high resolution against other species. The result suggests that the sequence may resolve the taxonomic ambiguities ofAlexandrium genus, particularly of the tamarensis complex.     

厚蟹线粒体16S rRNA基因序列分析及系统发育研究

对我国沿海天津厚蟹6个群体、侧足厚蟹4个群体、伍氏仿厚蟹2个群体和日本仿厚蟹1个群体的线粒体16S rRNA基因片段进行了序列测定;结合从GenBank下载的其他厚蟹序列,分析了厚蟹分子系统发育关系.除天津厚蟹丹东群体的2个个体16S rRNA基因片段长度为526 bp外,其他天津厚蟹和侧足厚蟹均为525 bp;除日照...     

Molecular phylogeny of the genus Clibanarius(Decapoda: Anomura: Diogenidae) based on mitochondrial and nuclear DNA sequences

Clibanarius , a genus of hermit crabs, is one of the most diverse genera within Diogenidae. However, studies on the phylogeny within Clibanarius is very limited. We reconstruct the molecular phylogeny of the genus Clibanarius based on two mitochondrial (16S rRNA and COI) and two nuclear protein-coding (NaK and PEPCK) genes using a multigene phylogenetic approach. Eleven selected Clibanarius species from the Indo-West Pacifi c are analyzed and the Bayesian and maximum likelihood analyses produced identical results in topology. Results suggest that C . rutilus and C . infraspinatus are more closely related to each other than to C . snelliusi , and C . striolatus shows a close relationship to C . longitarsus . The close association of C . merguiensis with C . englaucus and C . humilis is strongly supported by the divergence of C . virescens from them. Our phylogenetic results contradict the morphological classifi cation scheme proposed for Clibanarius and indicate that the relative length of dactyls and propodi is not phylogenetically signifi cant in Clibanarius . In addition, we speculate that the morphological characteristic of the median cleft of the telson might be phylogenetically important for Clibanarius .