基于DNA条形码技术的珠江口春季鱼卵和仔稚鱼种类组成和分布特征的研究

采用线粒体COⅠ和12S rRNA基因片段作为DNA条形码,分析珠江口春季鱼卵和仔稚鱼种类组成和分布特征,并探究两种条形码在鱼卵和仔稚鱼种类鉴定中的适用性。研究共扩增样本391个,成功鉴定的鱼卵和仔稚鱼共7目25科42属60种（2种未鉴定到种）。其中,以鲈形目（Perciformes）种类和数量最多,种类数占比为51.6%,数量占比为47.91%;其次为鲱形目（Clupeiformes）,种类数占比为25%,数量占比为34.56%。优势种10种,其中凤鲚（Coilia mystus）优势度最高,为0.071;棘头梅童鱼（Collichthys lucidus）最低,为0.014。COⅠ和12S rRNA基因片段扩增结果显示,鱼卵和仔稚鱼12S rRNA基因片段扩增成功率（95.60%）明显高于COⅠ基因（43.22%）。遗传距离和ABGD分析显示,COⅠ基因种内遗传距离为0～0.005（平均0.003）,种间遗传距离为0.061～0.376（平均0.253）,两者间存在明显的“条形码间隙”,ABGD划分结果与数据库比对结果一致;12S r RNA基因种内遗传距离为0～0.011（平均0...     

16S rRNA is a better choice than COI for DNA barcoding hydrozoans in the coastal waters of China

Identification of hydrozoan species is challenging, even for taxonomic experts, due to the scarcity of distinct morphological characters and phenotypic plasticity. DNA barcoding provides an efficient method for species identification, however, the choice between mitochondrial cytochrome c oxidase subunit I（COI） and large subunit ribosomal RNA gene（16S） as a standard barcode for hydrozoans is subject to debate. Herein, we directly compared the barcode potential of COI and 16S in hydrozoans using 339 sequences from 47 pelagic hydrozoan species. Analysis of Kimura 2-parameter genetic distances（K2P） documented the mean intraspecific/interspecific variation for COI and 16S to be 0.004/0.204 and 0.003/0.223, respectively. An obvious ＂barcoding gap＂ was detected for all species in both markers and all individuals of a species clustered together in both the COI and 16S trees. These results suggested that the species within the studied taxa can be efficiently and accurately identified by COI and 16S. Furthermore, our results confirmed that 16S was a better phylogenetic marker for hydrozoans at the genus level, and in some cases at the family level. Considering the resolution and effectiveness for barcoding and phylogenetic analyses of Hydrozoa, we strongly recommend 16S as the standard barcode for hydrozoans.     

10种蟹类18S rDNA 和COⅠ基因序列分析及其分子系统发育研究

应用分子系统发育学的方法,以蟹类18S rDNA和线粒体细胞色素C氧化酶亚单位Ⅰ(COⅠ)基因序列片段为分子标记,结合形态学特征对10种蟹类的分类地位进行探讨。实验共获得10 条18S rDNA序列,长度为1780~1787 bp,其中A、T, G,C平均含量分别为23.72%, 24.58%,24.52%和27.17%;通过序列对比,发现18S rDNA序列相对保守,只含有1个从88 bp 到130 bp 约 50 bp的高可变区;物种间碱基距离比较小,从0.001到0.017。18S rDNA系统发育树为方蟹总科、沙蟹总科和梭子蟹总科起源于同-海洋蟹类祖先提供了分于生物q证惦,开上火亚N内尔量分别为26.88%.弓蟹科。获得的5条CO Ⅰ基因序列,长度均为709 bp,A、T,G、C平均含量分别为26.88%,37.62%,17.50%和18.00%;COⅠ基因片段变异性高,物种间碱基距离从0.016到0.138。COⅠ基因系统发育树真实地反映了住属各物种之间的进化关系,和传统分类非常吻合,为形态特征相似的姆类鉴定提供了-种快速准确的方法。     

流式细胞术比较研究4种双壳贝类血细胞的分群

应用流式细胞术对太平洋牡蛎(Crassostrea gigas)、海湾扇贝(Argopecten irradians)、虾夷扇贝(Patinopecten yessoensis)和毛蚶(Scapharca subcrenata)4种双壳贝类的血细胞分类进行了比较。根据其血细胞的前向角散射光(FSC)和侧向角散射光(SSC)特性的不同将血细胞分群,FSC和SSC二维图分析发现太平洋牡蛎、海湾扇贝和虾夷扇贝的血细胞都可分为3个亚群,即透明细胞、小颗粒细胞和大颗粒细胞,而毛蚶的血细胞只可区分为透明细胞和颗粒细胞2个亚群。同时还对这4种双壳贝类各种血细胞所占比例进行了比较研究,发现各亚群细胞所占比例差异很大。     

赤潮异弯藻和海洋原甲藻LSU Rdna扩增及序列分析

利用引物D1R和D2C扩增并测定了赤潮异弯藻(Heterosigma akashiwo Hada)和海洋原甲藻(Prorocentrum micans Ehrenberg)的LSU rDNA D1与D2序列,并与GenBank中相关序列进行比较分析.结果表明:在种内水平,所测的H.akashiwo 6个株系之间共有5个变异位点,序列H.k与H.k-2,H.k-4均存在碱基替换;原甲藻属不同种内各株系之间的遗传距离为0.002～0.023之间,所测序列P.mi与P.micans其他株系之间均存在碱基替换.在种间水平上,原甲藻属不同种之间的遗传距离在0.045～0.139之间,大于种内遗传距离,每个种都具有特定的保守序列.根据序列间的遗传变异,可设计特异性的探针对不同株系和物种进行检测和计数.     

中国沿海常见蜑螺科贝类的DNA条形码

DNA条形码不仅为物种鉴定提供了有效方法,而且也有助于分类学和生物多样性研究。本研究旨在探讨将COI和16S rRNA基因序列应用于中国沿海蜑螺科贝类物种鉴定的可行性,获得了该科3属7种贝类61个个体的COI和16S rRNA基因序列。基于COI基因序列的种内遗传距离为0.00—1.29%,平均为0.67%;属内种间遗传距离为4.62%—19.25%,平均为13.02%;基于16S rRNA基因序列的种内遗传距离为0.00%—0.48%,平均为0.23%;属内种间遗传距离为2.47%—8.48%,平均为6.37%。两种基因序列在所研究的蜑螺中,种内遗传差异均小于种间遗传差异,存在明显的条形码间隙,所有物种在系统发生树上都表现为独立的单系群。结果表明,线粒体COI和16S rRNA基因序列可以作为DNA条形码标准基因对蜑螺科贝类进行有效地物种鉴定。     

南海长棘海星物种有效性探讨

南海长棘海星暴发已严重威胁到该海域珊瑚礁生态系统健康,乃至整个南海生物多样性。针对南海长棘海星拉丁学名混用、中文名不统一的现状,我们采集了中沙群岛济猛暗沙海域长棘海星样品,结合长棘海星此前物种分类和分布的研究结果,对南海长棘海星物种有效性进行探讨。结果表明：所有长棘海星序列明显分为4个类群,各类群间遗传距离范围为0.087 5~0.104 7,达到了种间差异水平。南海长棘海星与长棘海星的太平洋种聚类到一起,实为太阳长棘海星(Acanthaster solaris),与其余3个种存在明显的种间差异。太阳长棘海星中2个支系间的遗传距离为0.005 3,在COⅠ基因层面属于种内差异。南海长棘海星物种有效性的研究结果为后续开展其遗传特征与适应性机制、种群分布与扩散机制、种群暴发机制等内容的研究提供科学依据。     

The mitochondrial genome of Chaeturichthys stigmatias provides novel insight into the interspecific difference with Amblychaeturichthys hexanema

Chaeturichthys stigmatias and Amblychaeturichthys hexanema belong to the family Gobiidae, which are offshore warm fish species and widely distribute in the western Pacific Ocean. In this study, the mitochondrial cytochrome c oxidase subunit I(COI) sequences and 12 S ribosomal RNA(12 S rRNA) sequences were used to analyze the interspecific differences between the two species. The phylogenetic analysis showed that the interspecific distance was significantly higher than the intraspecific genetic distance. The Neighbor-Joining tree showed two separate clusters, without sharing haplotype. The mitochondrial genome sequence of C. stigmatias was also reported. This genome was 17 134 bp in size, with a high A+T content of 55.9%. The phylogenetic analysis based on the tandem 13 coding protein genes nucleotide sequences indicated that C. stigmatias showed a close relationship with A. hexanema. This study can provide the basic genetic data for two species and will help for constructing the phylogeny of the Gobiiade.     

两种相手蟹线粒体 COI 基因序列的比较研究

测定了褶痕相手蟹(Sesarma plicata)和红螯相手蟹(S.haematocheir)线粒体细胞色素氧化酶I亚基(COI)基因片段的序列,其长度均为658bp,A,T,G,C的平均含量分别为28.6%,37.5%,17.0%,16.9%和29.5%,39.0%,16.2%,15.3%。2种蟹共出现5种单倍型,褶痕相手蟹2种,红螯相手蟹3种。两种间有75个(11.4%)多态性核苷酸位点,其中转换42个,颠换33个;种间差异远大于种内个体间差异。2种相手蟹的AT含量明显高于GC含量,这与其它海产甲壳动物COI基因研究结果相似。     

Species delimitation in the green algal genus Codium (Bryopsidales) from Korea using DNA barcoding

Codium, one of the largest marine green algal genera, is difficult to delimit species boundary accurately based on morphological identification only. DNA barcoding is a powerful tool for discriminating species of seaweeds. The plastid elongation factor TU(tuf A) is considered as maker to perform DNA barcoding of green algal species than rbc L gene due to universality and rapid evolution rate. We conducted DNA barcoding application to Codium specimens from the Jeju Island, Korea to overcome the limit of morphological identification and to confirm the species diversity. As a result of applying tuf A marker, we newly generated fifty-five tuf A barcodes to resolve eight species. Tuf A marker exhibited 6.1%–21.8% interspecific divergences, wider than the gap of rbc L exon 1,3.5%–11.5%. Molecular analysis of rbc L exon 1 sequences of Codium revealed eight distinct species like tuf A analysis separated in five phylogenetic groups. DNA barcoding of the genus Codium using tuf A marker is more helpful to overcome the limit of morphological identification, and this is more potential to reveal cryptic species and to resolve the relationships among subspecies than rbc L analysis alone. The complement of tuf A barcoding and rbc L analyses including morphology for the genus Codium in the northwestern Pacific will give much more reliable achievement for discovering species diversity and resolving the phylogenetic relationships.     

Cytogenetic analysis in two scallops (Bivalvia: Pectinidae) by PRINS and PI banding

Cytogenetic analysis was performed for the bay scallop(Argopecten irradians Lamarck 1819) and the Japanese scallop(Patinopecten yessoensis Jay 1857) by primed in situ labeling(PRINS) and propidium iodide(PI) banding techniques.The PRINS analysis revealed that major rRNA genes were clustered in two loci on the telomeric regions of the short arms on two acrocentric chromosome pairs in A.irradians and on two submetacentric pairs in P.yessoensis.The histone H3 gene sites differed in number and location between these two species.The C-band-like patterns revealed by PI staining varied considerably between these two species.A.irradians displayed terminal bands at long arms on all chromosomes,centromeric bands on some pairs and interstitial bands on five pairs.P.yessoensis exhibited only centromeric bands on all chromosomes.These results would contribute to the better understanding of karyotype evolution in A.irradians and P.yessoensis.     

魁蚶线粒体16S rRNA和COI基因片段序列测定及其应用前景

魁蚶（Ｓｃａｐｈａｒｃａｂｒｏｕｇｈｔｏｎｉｉ）是蚶科贝类的一种大型经济种类，主要分布于我国、日本和朝鲜半岛及俄罗斯东南部沿海。在我国，主要分布于辽宁、河北和山东沿海，生活在３～５０ｍ（多为２０～３０ｍ）水深的软泥或泥沙质海底，是我国北方沿海重要的经济贝类之一。但但有有关关其其自自然然群群体体的的遗遗传传变变异异及及群群体体间间遗遗传传分分化化等等方方面面的的研研究究不不多多 ;;同同时时，，作作者者和和日日本本研研究究者者发发现现，，中中国国、、韩韩国国和和日日本本魁魁蚶蚶群群体体在在形形态态和…     

黄海西部细弱刚毛藻（刚毛藻目，绿藻门）的表型可塑性与分类——暗示基因序列作为DNA条形码的可行性

由于广泛的形态可塑性,导致刚毛藻属物种的分类仍存在不确定性。作为分布较广的物种之一,细弱刚毛藻已报道了许多变异类型,这为对其鉴定造成了困难。针对这个问题,本研究通过采集于黄海西部相似于细弱刚毛藻的9个样品,分析了它们的形态多样性。一些样品具有明显的可变鉴定特征,难于利用形态分类准确鉴定。因此,采用18S rDNA和 ITS序列对它们进行了分子鉴定。其中,样品的18S rDNA序列相似度为99.6%-100%,而ITS的为98.7% -100%。分子数据强烈地支持了形态可变的样品可准确定位为细弱刚毛藻。通过对样品分类特征的比较分析发现,作为分类标准的分枝方式和密度、藻体颜色、藻体高度和质地受环境影响和藻体成熟度而发生较大变化。此外,作为相对稳定的特征,细胞大小在种内水平上也常发生变化。18S rDNA和ITS序列在本种鉴定上的成功应用,表明以DNA条形码为基础的基因序列分析可作为传统形态分类学强有力的辅助方法。     

Testing use of mitochondrial COI sequences for the identification and phylogenetic analysis of New Zealand caddisflies (Trichoptera)

We tested the hypothesis that cytochrome c oxidase subunit 1 (COI) sequences would successfully discriminate recognised species of New Zealand caddisflies. We further examined whether phylogenetic analyses, based on the COI locus, could recover currently recognised superfamilies and suborders. COI sequences were obtained from 105 individuals representing 61 species and all 16 families of Trichoptera known from New Zealand. No sequence sharing was observed between members of different species, and congeneric species showed from 2.3 to 19.5% divergence. Sequence divergence among members of a species was typically low (mean = 0.7%; range 0.0–8.5%), but two species showed intraspecific divergences in excess of 2%. Phylogenetic reconstructions based on COI were largely congruent with previous conclusions based on morphology, although the sequence data did not support placement of the purse‐cased caddisflies (Hydroptilidae) within the uncased caddisflies, and, in particular, the Rhyacophiloidea. We conclude that sequence variation in the COI gene locus is an effective tool for the identification of New Zealand caddisfly species, and can provide preliminary phylogenetic inferences. Further research is needed to ascertain the significance of the few instances of high intra‐specific divergence and to determine if any instances of sequence sharing will be detected with larger sample sizes.     

中国近海大型底栖动物DNA条形码的研究进展

DNA条形码是利用标准的、变异率适度、易于扩增且相对较短的DNA片段对物种进行快速准确鉴定的技术。海洋大型底栖动物分布广泛,具有多样性、复杂性和趋同性等特点,DNA条形码技术能够在传统分类学基础上对物种进行快速、准确地鉴定。本文概述了DNA条形码技术在中国近海大型底栖动物研究中的发展现状,如多孔动物(Porifera)、刺胞动物(Coelenterata)、多毛动物(Polychaeta)、软体动物(Mollusca)、甲壳动物(Crustacea)、棘皮动物(Echinodermata)等,介绍了该技术在物种鉴定、隐存种发现、生物多样性评估等领域的研究进展,指出了目前条形码技术在应用中的局限性,并对未来的研究工作进行了展望。     

基于16S rDNA和看家基因序列分析技术的鲍鱼养殖水体弧菌种类的鉴定

采用TCBS选择性培养基从厦门某鲍鱼养殖场的养殖水体中分离到19株细菌.经16S rDNA序列分析,所有菌株均属于弧菌属（Vibrio）,且与最近似的弧菌模式种的同源性都在98.99%以上.由于弧菌种间16S rDNA序列相似度极高,无法仅靠16S rDNA序列比对来鉴定这些菌株到种的水平.16S rDNA序列的系统发育分析也显示,大多数菌株与弧菌模式种无法归为一簇,表明16S rDNA基因在弧菌种的分类鉴定上分辨率不高.进一步采用基于4种看家基因（rpo A、pyr H、gap A和top A）的多位点序列分析技术（MLSA）对分离到的弧菌菌株进行分类鉴定,结果显示19株海洋弧菌归于溶藻弧菌（Vibrio alginalyticus）与魔鬼弧菌（Vibrio diabolicus）2个种,表明这两种弧菌是该鲍鱼养殖场水体环境中的优势弧菌种,在鲍鱼养殖弧菌病害防治方面需要重点关注.此外,看家基因与16S rDNA基因的多态性分析表明,4种看家基因的多态位点比率均高于16S rDNA.4种看家基因串联后的多态位点比率高达41.1%,远高于16S r DNA基因的13.4%,表明看家基因相较于16S rDNA基因有着更高的分辨率,更适合于海洋弧菌的分类鉴定.     

基于18S和ITS-5.8S rDNA基因序列的白色霞水母(Cyanea nozakii)的分子鉴定与检测

采用通用引物PCR扩增法,测定了辽东湾海域的白色霞水母(Cyanea nozakii)螅状体、碟状体及水母体的18S以及ITS-5.8S r DNA序列,同时利用Gene Bank数据库中已有同源序列对其进行序列分析及系统分析。结果显示,白色霞水母3个个体的18S和ITS-5.8S r DNA序列完全一致。白色霞水母样品的ITS-5.8S r DNA序列与Gen Bank中未知真核生物的序列高度相似(≥99%),推测该物种可能是早期发育阶段(卵、浮浪幼虫或碟状体)的白色霞水母样品。霞水母属不同种间18S r DNA序列经比对后同源序列长度为1709bp,多态位点数33个;比对后ITS1同源序列长度为368bp,其中变异位点203个,简约信息位点数178个,单变异位点21个。基于18S r DNA基因序列的霞水母属种内和种间平均遗传距离分别为0、0.008,而基于ITS1序列的霞水母属种内和种间平均遗传距离分别为0.019、0.284。基于ITS1的种间遗传距离是种内遗传距离的15倍,适合于进行物种鉴定。NJ系统树的结果也表明同种的不同个体各自聚枝,其聚类结果大致与形态分类吻合。研究表明,ITS基因片段在霞水母不同种间变异较大,更适于大型水母种类鉴定、检测及属内种间水平的系统进化研究。     

新月细柱藻的分离培养及形态和分子鉴定

自胶州湾分离培养2株微藻(MGB0401和MGB0402)。经形态学鉴定为新月细柱藻(Cylindrotheca closterium)。MGB0401和MGB0402的18S核糖体RNA基因(rDNA)扩增片段长度为1775 bp,存在10个碱基差异。MGB0401和MGB0402的rbcL基因扩增片段长度为1190 bp,有45个碱基差异,对应的氨基酸序列存在4个氨基酸残基差异。系统学分析表明,2株微藻与已知新月细柱藻相应序列亲缘关系最近。微藻常用系统学分析用基因的序列不仅是分类鉴定的补充指标,也是不同研究结果相互比较的重要基础。     

用16S rDNA序列初步探讨部分真虾类的系统发育关系

利用DNA序列测定技术测定了真虾下目7科8个种的线粒体16S rDNA部分序列,与从GenBank检索得到的12个相关种的16S rDNA部分序列进行同源性比对,探讨其系统发育关系及16S rDNA在真虾类系统发育研究中的应用。比对序列长353bp,其中变异位点221个、简约信息位点180个。碱基转换替代速度比颊换替代速度慢。以螯虾下目的Austropotamobius pallipes为外群构建了这20种真虾的系统发育关系分支图,结果展示：（1）长臂虾科隐虾亚科的Awhistus custos远离长臂虾亚科分支。而且它的16S rDNA序列在Genbank中,首先与六足纲（Hexapoda）的昆虫比对上。（2）长臂虾科长臂虾亚科的两个近缘属Palaemon和Palaemonetes问的4个种混合相聚,说明传统形态分类中以大颚有无触须作为这两个属的属级形态鉴定特征尚需进一步审定。（3）鼓虾总科藻虾科的Exhippolysmata ensirostris与褐虾总科褐虾科的Crangon affinis处在同一分支,而没与鼓虾科的种类形成姐妹群。研究结果表明,16S rDNA片段的序列很适合研究真虾类的属间系统发育关系,而在研究属上高级阶元或种下阶元间的系统发生关系时较不敏感,因其变异对于种级水平显得过于保守,而对于科级以上阶元又显得太快。     

Isolation and phylogenetic assignation of actinomycetes in the marine sediments from the Arctic Ocean

Actinomycetes in five marine sediments collected from the Arctic Ocean at depths of 43 to 3 050 m were cultivated using a variety of media. A total of 61 actinomycete colonies with substrate mycelia only were observed, and no colonies with aerial mycelia were observed under aerobic conditions at 15 ℃. From these colonies, 28 were selected to represent different morphological types.Denaturing gradient gel electrophoresis (DGGE) was used to check the purity of isolates and select representatives for subsequent sequencing. Phylogentic analyses based on nearly full-length 16S ribosomal RNA gene (rDNA) sequences indicated that the actinomycetes isolated were accommodated within genus Rhodococcus of family Nocardiaceae, genus Dietzia of family Dietziaceae,genera Janibacter and Terrabacter of family Instrasporangiaceae and genera Kocuria and Arthrobacter of family Micrococcaceae. One of the strains (P27-24) from the deep-sea sediment at depth of 3 050 m was found to be identical in 16S rDNA sequence(1474/1474)with the radiation-resistant Kocuria rosea ATCC 187T isolated from air. More than halfofthe isolates showed the similarities ranging from 99.5% to 99.9% in 16S rDNA sequence to dibenzofran-degrading, butyl 2-ethylhexanoate-hydrolysising and nitrile-metabolizing actinomycetes. All the strains isolated were psychrotolerant bacteria and grew better on the media prepared with natural seawater than on the media prepared with deionized water. Three of them (Dietzia sp. P27-10, Rhodococcus sp. S11-3 and Rhodococcus sp.P11-5)had an obligate growth requirement for salt, confirming that these strains are indigenous marine actinomycetes.