基于转录组数据的大菱鲆(Scophthalmus maximus)SNP标记开发及多态性分析

大菱鲆(Scophthalmus maximus)是最具商业价值和养殖前途的海水鱼类之一,雌雄间生长有一定的差异。其高通量雌雄转录组测序的完成为大规模鉴定和开发SNP标记提供了参考序列。本研究基于大菱鲆雌雄转录组测序数据,选择其中45个SNP位点,设计引物63组,其中21个位点(46.7%)应用小片段高分辨率熔解曲线(HRM)技术分型成功。对其进行多态性检测,21个位点均具有二个单倍型。观测杂合度Ho的分布范围为0.256—1.000,期望杂合度He的范围为0.276—0.518,19个位点符合Hardy-Weinberg平衡。14个SNP位点位于基因编码区,其中3个属于非同义突变。含有这些SNP位点的基因大多与信号转导和转录翻译相关。本研究结果表明,高通量转录组测序和小片段HRM适合大规模SNP标记开发,为大菱鲆的分子遗传育种提供了候选标记资源。     

转录组数据的大菱鲆(Scophthalmus maximus) SNP标记开发及多态性分析

大菱鲆(Scophthalmus maximus)是最具商业价值和养殖前途的海水鱼类之一, 雌雄间生长有一定的差异。其高通量雌雄转录组测序的完成为大规模鉴定和开发SNP标记提供了参考序列。本研究基于大菱鲆雌雄转录组测序数据, 选择其中45个SNP位点, 设计引物63组, 其中21个位点(46.7%)应用小片段高分辨率熔解曲线(HRM)技术分型成功。对其进行多态性检测, 21个位点均具有二个单倍型。观测杂合度Ho的分布范围为0.256—1.000, 期望杂合度He的范围为0.276—0.518, 19个位点符合Hardy-Weinberg平衡。14个SNP位点位于基因编码区, 其中3个属于非同义突变。含有这些SNP位点的基因大多与信号转导和转录翻译相关。本研究结果表明, 高通量转录组测序和小片段HRM适合大规模SNP标记开发, 为大菱鲆的分子遗传育种提供了候选标记资源。     

大菱鲆生长性状相关单核苷酸多态位点的多家系验证

为在多家系群体中进一步验证与大菱鲆生长性状紧密关联的SNP位点,本研究以QTL定位作图筛选获得的100个与大菱鲆(Scophthalmus maximus L.)生长性状相关的候选单核苷酸多态(SNP)位点为基础,利用飞行质谱法,在4个大菱鲆家系群体中进行基因分型,并与体重、体长2个生长性状数据进行关联分析。对关联显著的位点,统计位点在家系群体中的多态性,并进行连锁不平衡分析和单倍型分析;根据显著位点所在基因序列信息,在NCBI数据库中进行比对分析,对匹配到的基因功能进行初步探讨。关联分析发现,有9个SNP位点与生长性状关联显著(P0.05)。9个位点中,4个位点与体重关联显著(P0.05),7个位点与体长关联显著(P0.05),其中,SNP51和SNP111与体重和体长2个性状均关联显著(P0.05)。SNP位点的多态性分析发现,观测杂合度在0.126~0.719之间(平均为0.387),期望杂合度在0.118~0.501之间(平均为0.338);9个位点的平均多态信息含量为0.267,属于中度多态性位点;哈迪温伯格平衡检验结果显示,9个位点中有5个位点偏离平衡。连锁不平衡和单倍型分析发现,标记间存在显著的连锁不平衡;单倍型CTTGT和CTTGA是2个主要的单倍型,单倍型频率分别为26.8%和25.9%。根据关联显著SNP位点所在的序列信息,在NCBI数据库比对分析后发现,COL11A1和Notch1基因可能是与大菱鲆生长性状相关的重要功能基因。本研究结果将推进大菱鲆生长相关分子标记的研究,为下一步基因定位和分子标记辅助育种提供更多的标记基础。     

九孔鲍MSTN基因cSNP多态性及与生长性状关联分析

九孔鲍(Haliotis diversicolor supertexta)是中国南方具有较高经济价值的养殖贝类。肌肉生长抑制素(Myostatin, MSTN)是转化生长因子β超家族(TGF-β)中参与动物肌肉生长的重要调控因子, 因此MSTN基因是动物遗传改良的重要候选基因。为探究九孔鲍MSTN基因的多态性及其生长相关性, 实验采用PCR产物直接测序方法对九孔鲍MSTN基因进行单核苷酸多态性(SNP)筛选, 对MSTN基因SNP与体质量、壳长、壳宽进行关联性分析, 运用实时荧光定量PCR (qRT-PCR)技术检测九孔鲍MSTN基因不同基因型的表达水平。结果显示: 在九孔鲍MSTN基因中共筛选出9个SNP位点。SNP位点与生长性状关联分析发现, 九孔鲍MSTN基因编码区SNP位点g909C>T发生C/T同义突变与九孔鲍体质量、壳长、壳宽显著相关。TT基因型九孔鲍的体质量显著高于CC基因型和TC基因型个体; TT基因型的壳长、壳宽显著高于CC基因型(p<0.05)。通过qRT-PCR检测显示, 在九孔鲍MSTN基因SNP位点g909C>T的3种基因型中, TC和CC基因型的基因表达量显著高于TT基因型(p<0.05)。研究结果表明MSTN基因SNP位点g909C>T可作为九孔鲍标记辅助选择育种重要候选标记。     

刺参SNP标记与生长性状的关联分析

为探究SNP标记与刺参(Apostichopus japonicus)性状之间的相关性,本研究运用高分辨率溶解曲线(HRM)分析方法,采用46个SNP标记对刺参家系的遗传多样性进行分析,运用一般线性模型对SNP标记与刺参体长、体宽、体重、体壁重和出肉率性状的关联性进行检测。结果表明,平均多态信息含量(PIC)的范围从0.0302到0.3750,观测杂合度(Ho)的范围是从0.0312到0.4688,18个位点显著偏离哈迪-温伯格平衡(P0.05)。相关性分析显示,46个SNP位点中有10个位点与生长性状呈显著相关(P0.05),其中位点SNP183的基因型AB与出肉率显著相关(P0.05);位点SNP189的基因型AA与体长呈显著相关(P0.05);8个SNP位点(SNP4、SNP146、SNP154、SNP163、SNP164、SNP175、SNP178和SNP216)的基因型BB与体长、体宽、体重、体壁重性状中的部分或全部呈显著相关,推断基因型BB可能是这些位点的优势基因型。     

长牡蛎转化生长因子受体基因多态性与生长性状和糖原含量的相关性分析

本研究采用PCR-SSCP技术,对长牡蛎(Grassostrea gigas)转化生长因子受体基因(Cg-TGF-βRⅠ)编码区单核苷酸多态性(SNP)与来自5个家系316个长牡蛎个体的生长性状(壳高、壳长、壳宽、总体量和软体部量)和糖原含量进行了关联分析。研究表明,在Cg-TGF-βRⅠ基因编码区扩增出的671bp基因片段中检测到4个SNP,其中3个SNP位点(T726C,A741G,A786G)与经济性状相关;A741G和A786G与生长性状和糖原含量均存在显著相关性(P0.05),T726C仅与壳长和软体部重有显著相关性(P0.05);3个SNP位点构建得到5个单倍型,H1(TAG)和H4(CAG)单倍型的个体在总体重和软体部重上均显著高于其它3种单倍型的个体。研究结果表明,Cg-TGF-βRⅠ基因多态性影响长牡蛎的生长性状和糖原含量,可用于以后的长牡蛎的分子标记辅助育种和遗传性状的改良。     

栉孔扇贝EST-SNP标记开发及多态性分析

EST(表达序列标签)数据库发掘是单核苷酸多态性标记(SNP)开发的重要途径。本研究利用栉孔扇贝转录组测序数据进行SNP筛选,在含有4条EST以上的18 780条contig(拼接序列)中,共获得21 813个候选SNP位点。利用高分辨率熔解曲线结合非标记探针技术,对其中90个位点在4个野生群体中进行了位点多态性检测。得到33个(36.7%)具有二等位基因位点,并对其中26个位点进行了功能注释。进一步的标记验证显示,33个多态位点在青岛野生群体的48个个体中均成功分型,其观测杂合度Ho和期望杂合度He的分布范围分别为0.062 5～0.744 7和0.099 8～0.504 6,其中5个位点偏离Hardy-Weinberg平衡,各位点间没有检测到连锁不平衡。研究为栉孔扇贝群体遗传学和遗传育种分析提供了候选标记。     

凡纳滨对虾CTSL基因与生长相关的SNP位点的特征

对3个凡纳滨对虾品系的CTSL基因进行单核苷酸多态性（single nucleotide polymorphism,SNP）筛查,发现在生长指标差异的对虾中存在特征较为显著的7个SNP位点,其中3个属于外显子上的突变,4个属于内含子上的突变．B210对虾品系体重月平均生长量约为7．58g,体长月平均生长量约为2．31cm,B310对虾品系体重月平均生长量约为3．96g,体长月平均生长量约为1．48cm,HNIT对虾品系体重月平均生长量约为3．04g,体长月平均生长量约为2．18em．从市场价值来看,B210和B310品系比HNTr品系生长相对好些．进一步的PCR产物直接测序比较分析表明在生长指标较好的对虾品系,这7个SNP位点表现为纯合子;而在生长指标较差的对虾品系,在这7个SNP位点上31．6％表现为杂合子．虽然国内外有不少关于CTSL基因SNP位点的报道,但是与生长快慢的相关分析还鲜有报道,且这些SNP位点与前人发现的位点有所不同．本研究发现的CTSL基因的SNP位点与对虾生长快慢相关,将为凡纳滨对虾生长研究提供有用信息,从而有助于为对虾选育提供可靠的分子标记．     

基于线粒体D-loop区分析黄颡鱼(Pelteobagrus fulvidraco)五个淡水湖泊群体的遗传多样性

采用线粒体D-loop区测序技术,对巢湖、滆湖、洪泽湖、鄱阳湖和太湖5个地理群体的黄颡鱼(Pelteobagrus fulvidraco)遗传多样性及种群遗传结构进行了研究。结果表明,890个位点中变异位点有151个,简约信息位点24个。143个个体共检测到72个单倍型,平均单倍型多样性指数为0.951±0.011,核苷酸多样性为指数为0.00614±0.00087。鄱阳湖群体和其它4个湖泊群体的遗传距离相对比较大,滆湖和巢湖的遗传距离最小,总体遗传分化指数Fst为0.0896(P<0.01)。群体间基因流系数Nm=2.22,表明5个黄颡鱼种群间存在着一定的基因流动。     

ARTP诱变法分析深海沉积微杆菌Microbacterium sediminis YLB-01的耐压耐冷机理

深海微生物由于长期生活在高压、低温、黑暗、寡营养的极端环境中,必然会发展出与之相应的分子基础。近年来,对深海微生物极端环境适应性机理的研究是深海微生物研究的前沿课题。通过对一株耐冷、耐压的深海沉积微杆菌Microbacterium sediminis YLB-01进行ARTP诱变,成功获得了对低温和高压敏感的突变子9-3E5,突变子9-3E5与野生型菌株的基因组比较分析结果表明,SNP和In Del在突变子9-3E5全基因组上分布均匀,838个SNP位点位于编码区且为非同义突变,占总SNP数量的77.45%; 27个In Del中11个位于编码区且发生了移码突变;突变子9-3E5共有11条SV序列,长度为300～400 bp的序列占比为81.82%。以OR值 2、p 0.05为标准对突变基因进行富集,共富集到3个相关的功能集。在与细胞分裂相关的蛋白中发现了11个SNP突变位点,主要涉及细胞分裂蛋白Fts I、Fts W、Fts X和细胞分裂起始蛋白;在与DNA修复、合成相关的6个基因中发现了10个SNP突变位点;在与胞内运输和能量合成调控相关的2个基因中发现了3个SNP突变位点。我们推测细胞分裂、DNA损伤修复及合成、胞内运输对能量合成的影响这3类功能基因发生改变可能是突变子9-3E5相对于野生株YLB-01低温、高压耐受性改变的原因,M.sediminis YLB-01通过调节这些胞内的生理生化过程来应对外界低温和高压环境。     

Microbial Biomass and Organic Nutrients in the Deep-Sea Sediments of the Central Indian Ocean Basin

In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance.     

Assessing the Validity of Seismo-Acoustic Predictor Equations for Obtaining Seabed and Sub-Surface Sediment Physical Properties

The interdependence between the seismo-acoustic properties of a marine sediment and its geotechnical/physical parameters has been known for many years, and it has been postulated that this should allow the extraction of geotechnical information from seismic data. Though in the literature many correlations have been published for the surficial layer, there is a lack of information for greater sediment depths. In this article, a desktop study on a synthetic seafloor model illustrates how the application of published near-surface prediction equations to subsurface sediments (up to several tens of meters burial depth) can lead to spurious predictions. To test this further, acoustic and geotechnical properties were measured on a number of sediment core samples, some of which were subjected to loading in acoustically-equipped consolidation cells (oedometers) to simulate greater burial depth conditions. For low effective pressures (representing small burial depths extending to around 10 meters subsurface), the general applicability of established relationships was confirmed: the prediction of porosity, bulk density, and mean grain size from acoustic velocity and impedance appears generally possible for the investigated sedimentary environments. As effective pressure increases through, the observed relationships deviate more and more from the established ones for the near-surface area. For the samples tested in this study, in some instances increasing pressure even resulted in decreasing velocities. There are several possible explanations for this abnormal behavior, including the presence of gas, overconsolidation, or bimodal grain size distribution. The results indicate that an appropriate depth correction must be introduced into the published prediction equations in order to obtain reliable estimates of physical sediment properties for greater subsurface depths.     

A Review of Some Environmental Issues Affecting Marine Mining

This article reviews information recently available from existing marine and coastal mining for responses to environmental issues affecting marine mining at different depths. It is particularly but not exclusively concerned with those issues affecting seabed biodiversity impact and recovery. Much information has been gathered in the past 10 years from shallow mining operations for construction aggregate, diamonds, and gold, from coastal mines discharging tailings to shallow and deep water, and from experimental deep mining tests. The responses to issues identified are summarized in a series of eight tables intended to facilitate site-specific consideration. Since impacts can spread widely in the surface mixing layer SML, and can affect the biologically productive euphotic zone, the main issues considered arise from the depth of mining relative to the SML of the sea. Where mining is below the SML, the issue is whether it is environmentally better to bring the extraction products to the surface vessel for processing (and waste discharge), or to process the extraction products as much as possible on the seabed. Responses to the issues need to be sitespecific, and dependent on adequate preoperational environmental impact and recovery prediction. For deep tailings disposal from a surface vessel, there are four important environmental unknowns: (1) the possible growth of "marine snow" (bacterial flocs) utilizing the enormous quantities of fine tailings particles (hundreds or thousands of metric tons per day) as nuclei for growth, (2) the possibility that local keystone plankton and nekton species may migrate diurnally down to and beyond the depth of deep discharge and hence be subjected to tailings impact at depth, (3) the burrow-up capability of deep benthos and their ability to survive high rates of tailings deposition, and (4) the pattern and rate of dispersion of a tailings density current through the deep water column from discharge point to seabed. Actions to obtain relevant information in general and site-specifically are suggested.     

Increased Particle Fluxes at the INDEX Site Attributable to Simulated Benthic Disturbance

Particle fluxes were measured 7 m above the sea bottom during the predisturbance, disturbance, and postdisturbance periods by using time series sediment traps attached to seven deep-sea moorings deployed in the INDEX experiment site in the Central Indian Basin. The predisturbance particle fluxes varied between 22.3 to 55.1 mg m -2 day -1 . Increased and variable particle fluxes were recorded by the sediment traps during the disturbance period. The increase observed was 0.5 to 4 times more than the background predisturbance fluxes. The increases in particle fluxes (~4 times) recorded by the sediment trap located in the southwestern direction (DMS-1) were the greatest, which could be the result of preferential movement of resuspended particles generated during the deep-sea benthic disturbance along the general current direction prevailing in this area during the experimental period. Also, the traps located closer to the disturbance area recorded greater fluxes than did the traps far away, across the Deep Sea Sediment Resuspension System path. This variability in recorded particle fluxes by the traps around the disturbance area clearly indicates that physical characteristics such as grain size and density of the resuspended particles produced during the disturbance had an important effect on particle movement. The postdisturbance measurements during ~5 days showed a reduction in particle fluxes of ~50%, indicating rapid particle settlement.     

海上大直径钢管桩打桩过程中桩周土强度弱化研究

海上大直径钢管桩打桩过程中,桩周土体受到强烈扰动而发生强度弱化,掌握桩周土体强度弱化规律对于准确预测打桩过程、保证工程安全具有重要意义。为研究土体强度弱化规律,开展了环剪试验模拟打桩对桩周土体的扰动,测试土体强度随剪切速率的变化规律,建立了描述土体强度弱化规律的拟合公式,引入到打桩分析软件中。研究结果表明：土体的强度折减程度不仅与土体本身的性质有关还受到土体的埋深和剪切速率的影响,埋深越深土体强度折减程度越低,剪切速率越高土体强度折减越高,在打桩分析中可采用这里推荐的线性折减方法来模拟不同深度处土体强度的折减规律。     

Geoacoustic Inversion via Genetic Algorithm and Its Application to Manganese Sediment Identification

An acoustic inversion method using a wide-band signal and two near field receivers is proposed and applied to multiple layered seabed models including a manganese sediment. The inversion problem can be formulated into a probabilistic model comprised of signals, a forward model, and additive noise. The forward model simulates wide-band signals, such as chirp signals, and is chosen to be the source-waveletconvolution plane wave modeling method. The wavelet matching technique, using weighted least-squares fitting, estimates the sediment sound-speed and thickness on which determination of the possible numerical ranges for a priori uniform distribution is based. The genetic algorithm is applied to a global optimization problem to find a maximum a posteriori solution for determined a priori search space. Here the object function is defined by an L 2 norm of the difference between measured and modeled signals. Not only the marginal pdf but also its statistics are calculated by numerical evaluation of integrals using the samples selected during importance sampling process of the genetic algorithm.     

Determination of In Situ Sediment Shear Strength from Advanced Piston Corer Pullout Forces

The advanced piston corer (APC) has been used by the Ocean Drilling Program since 1985 for recovering soft sediments from the ocean floor. The pullout force measured on extracting the core barrel from the sediment is shown to correlate with the average shear strength of the sediment core measured in the ship's laboratory. A simple rule of thumb is derived relating the shear strength of the sediment to the pullout force. Multiple APC holes at individual sites allow the consistency of the pullout measurements to be assessed. The effects of different operational procedures during APC coring are also explored. Although generally applicable, the correlation between pullout force and laboratory measurements of shear strength breaks down for some APC holes, possibly because of the disturbance of some sediment types during the APC coring process. A better understanding of the physical process of APC coring, and its effect on the properties of the sediment both inside and immediately outside the core barrel, would indicate what confidence can be put on the measurement of pullout force as a way of evaluating the in situ shear strength of deep sea sediments.     

Interannual Sea Level Variations and Annual Tides in the Northwestern Pacific

Specific properties of the interannual sea level variations and annual tides in the Northwestern Pacific were studied. Several tide stations were monitored. The monthly mean sea level for the year of 1995 was analyzed at each tide station. A seismic event in 1995, some tectonic activity around the subject area, and the Kuroshio (the oceanic western boundary current) may possibly contaminate results which would have occurred from the astronomical annual tide alone.     

Flow Cytometric Analysis of Coastal Lagoon Bacterioplankton and Picophytoplankton: Fixation and Storage Effects

The effect of different fixation and storage protocols on the flow cytometric (FCM) simultaneous analysis of bacterioplankton and phytoplankton in coastal seawater samples (Mediterranean coastal lagoons) was investigated. FCM measurements (cell number, fluorescence and scatter characteristics) were obtained through DAPI staining. Three fixatives [glutaraldehyde (GA), formaldehyde (FA) and paraformaldehyde (PFA)] and two storage (3 months duration) methods (5 °C and −196 °C) were tested. Two dominant populations were detected in studied samples: bacteria and eukaryotic picophytoplankton. Adding fixatives (2% final concentration) appears necessary to obtain FCM exhaustive counts of all the bacteria and phytoplanktonic cells. This was related to the permeation effect of fixatives which allowed a better DAPI staining of the cells. Maximum fluorescence, i.e. optimal staining of the cells was obtained with FA or PFA, and significant lower fluorescences with GA. Fixed samples stored at 5 °C induced rapid cell loss. Only storage in liquid nitrogen of samples fixed with FA or PFA, allows mid-term (≥4 months) preservation of bacteria or picophytoplankton cell numbers, and limited evolution of DAPI-induced fluorescence and scatter characteristics.