4种养殖扇贝的群体遗传多样性及特异性AFLP标记研究

应用AFLP技术对我国4种主要养殖扇贝进行了遗传分析.虾夷扇贝、海湾扇贝、华贵栉孔扇贝、栉孔扇贝4种养殖扇贝群体中,种内遗传相似度依次分别为0.841 5,0.786 3,0.719 0和0.673 1,香侬氏指数分别为43.52,58.87,80.16和92.83,栉孔扇贝和华贵栉孔扇贝的遗传多样性水平明显高于海湾扇贝和虾夷扇贝;AFLP标记共享位点分布表明,在4种扇贝中栉孔扇贝与华贵栉孔扇贝之间的共享位点最多,同时4种扇贝的种间遗传相似度也以栉孔扇贝和华贵栉孔扇贝之间为最高(0.769 5),海湾扇贝和虾夷扇贝之间为最低(0.662 6),揭示所研究的几种扇贝中栉孔扇贝和华贵栉孔扇贝的遗传关系最近(遗传距离为0.262 1);找到了21个种内特异性AFLP标记和种间共享标记,可用于种质鉴定和分子辅助分类.     

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.     

高效非损伤性扇贝DN A提取方法的建立及效果评价

本研究建立了一种高效、经济的非损伤性扇贝DNA提取方法,在不影响扇贝个体生存状态的前提下,采用擦拭法和室温裂解相结合的方式在20 min内即可获得个体基因组DNA。以虾夷扇贝(Patinopecten yessoensis)、栉孔扇贝(Chlamys farreri)和海湾扇贝(Argopecten irradians)为实验对象,研究了不同擦拭材料(棉签、滤纸)和不同擦拭部位(外套膜、鳃丝、内脏团)的核酸提取效果,评估了本方法在贝类基因分型领域应用的可行性。研究表明,用棉签、滤纸2种材料擦拭扇贝的鳃丝、内脏团或外套膜组织均能获得主带清晰完整的基因组DNA,且不影响扇贝的存活状态。在3种扇贝中采用棉签擦拭法的DNA得率均高于滤纸法,以这2种方式获得的DNA为模板进行SSR和SNP标记分析,能获得均一稳定的扩增条带,SSR标记分型结果准确可靠。本研究建立了简便、快速进行扇贝基因型分析的非损伤性DNA提取方法,为贝类全基因组选育和珍稀贝类资源的种质保护开发提供了新的技术手段。     

Molecular identification of scallop planktonic larvae using species-specific microsatellites

The identification of scallop larvae is essential to understand the population structure and community dynamics and to assess the potential environmental impacts caused by scallop larvae released or escaped. However, the larvae identification by morphological characteristics is notoriously difficult, mainly due to the small size （usually being less than 150 μm） and vague morphological characteristics among different scallop species. A simple and accurate molecular method was developed to identify four economically farmed scallop species, the Zhikong scallop Chlamys farreri, the noble scallop C. nobilis, the bay scallop Argopecten irradians and the Yesso scallop Mizuhopecten yessoensis. The tests used the high degree of species-specific microsatellite markers, which was specified by transferability analyses, assessed by reference individuals and evaluated by BLAST searches. The sensitivity test indicated that the species-specific microsatellites were sensitive enough for the detection of 1% -2% larvae in mixed plankton samples. Larvae collected from scallop hatcheries and their effluents and from the artificially controlled crosses were well identified to the species/hybrid level. The results demonstrated that the one-step PCR-based assay was technically simple, inexpensive and robust in identification analyses, and also less sensitive to initial quality of template DNA extracted from the ethanol-preserved samples for several years.     

海湾扇贝4次引种后代的表型特征和遗传分化

分析了不同时期引入到我国的4个繁育历史清晰、未发生种质混杂的海湾扇贝养殖群体成体的壳形态指标和群体遗传结构。壳表型参数分析表明,海湾扇贝北部亚种(Northernbay scallop,Argopecten irradians irradians)的浙江养殖群体(ZJ)、加拿大新布朗斯威克群体(NB)、马萨诸塞-弗吉尼亚混合群体(M-V)及海湾扇贝南部亚种(Southern bay scallop,Arg-opecten irradians concentricus)的北卡罗来那群体(NC)等不同群体间在壳形态方面存在显著差异南部亚种的NC群体壳高与壳长基本相等[壳高与壳长的比值(H/L)达0.99,壳型宽壳宽和壳长的比值(W/L)达到0.59];而北部亚种3个群体的壳长均大于壳高,其中ZJ和NB群体的壳形态尤其偏长,其H/L分别是0.92和0.90,NB群体的壳宽显著小于其他群体,W/L仅为0.36。RAPD分析结果表明,ZJ、NB、M-V、NC群体的多态位点比例分别为31.82%、29.55%、28.79%和31.82%,平均杂合度分别为0.1078、0.1134、0.0966和0.1197,表明在我国独立繁育了19代的ZJ养殖群体仍然保持了与其他群体相近的遗传多样性水平。4个群体间的遗传分化系数(Gst)为0.1892,表明其中18.92%的变异来自不同群体间的遗传差异,说明4个群体在遗传上存在着较大的分化。从遗传距离分析,ZJ与NB较近,两者间的遗传距离为0.0319,而两者与M-V间的遗传距离相对较远,分别为0.0442和0.0524;NC与M-V间的遗传距离为0.0368。这些结果表明,从原产地引种到国外时间较早的ZJ、NB等2个群体与较晚引进我国的M-V群体之间的遗传距离已超过了M-V群体(北部亚种)与NC群体(南部亚种)之间的遗传距离,说明独立繁育了近20代后,ZJ群体和NB群体均已产生了较大的遗传分化,这种分化可能有利于海湾扇贝的品种培育。     

A prokaryotic mycoplasma-like organism infection in the scallop Argopecten irradians

Intracytoplasmic infection with a prokaryotic mycoplasma-like organism (MLO) were characterized in the scallop Argopecten irradians, and the first report of such infection in this scallop was represented. Ultrastructurally these microorganisms are usually pleomorphic and variable in morphology and size, and appear in several cell types such as small spherical body, rod-shaped bodies and longer filament-shaped body. They lack a cell wall. These observations revealed that these types of the prokaryote showed some characteristics of a mycoplasma-like organism (MLO). MLO reproduced in two ways: binary fission and budding. The results of isolation and purification showed that a large number of MLOs existed in the tissues of diseased scallops. The results of experimental infection revealed that the MLO is pathogenic to the scallop Argopecten irradians.     

海湾扇贝养殖过程中的流行病学调查研究

2001~2002年,对莱州湾养殖海湾扇贝进行了较系统地流行病学调查研究.以海湾扇贝养殖集中海区莱州市金城镇扇贝养殖场为定点调查和样品采集点.调查内容主要包括海湾扇贝各养殖期的死亡率和可疑病原生物检测,同时对pH,溶解氧,温度,盐度等理化环境因子进行连续监测.调查结果显示:海湾扇贝在0.5~1.3cm的幼贝阶段死亡率较高,达50%;随后死亡率降低,1.3~2.2cm的幼贝阶段死亡率约为20%,以后各阶段死亡率平稳,均在7%以下,累积死亡率约为60%.对环境理化因子的调查显示,pH,溶解氧、盐度的变化幅度不大,与扇贝死亡率无明显相关性;温度变化与死亡率有一定的相关性,即低温(20℃以下)可能是养殖海湾扇贝幼贝重要的环境胁迫因子,而高温(27℃左右)对海湾扇贝成贝生长无不良影响.通过对可疑致病性生物因子的调查研究及病理学观察表明,海湾扇贝在养殖过程中可感染多种微生物,其中原核微生物类立克次体(RLO)在养殖过程各生长阶段均可检出,即0.5,1.5,3.0,3.6和5.0cm的海湾扇贝RLO的感染率分别为15%,35%,80%,50%和20%;RLO的感染强度分别为1.0,1.5,16.0,11.5和5.0.在病原生物检查过程中,除RLO以外还发现有类支原体原核生物(MLO)寄生.结合感染强度和组织病理学的相关性分析,初步认为类立克次体(RLO)可能是海湾扇贝在海上吊养的幼贝阶段发病死亡的致病因子.     

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。     

雌性虾夷扇贝与雄性栉孔扇贝杂交子代及其亲本的核型研究

采用滴片法制片-吉姆萨染色对雌性虾夷扇贝(Patinopecten yessoensis)与雄性栉孔扇贝(Chla-mysfarreri)杂交子代及其双亲的核型进行了研究,报道了在扇贝杂交过程中产生的特殊的细胞遗传学现象,结果表明,栉孔扇贝染色体核型2n=38=6m+10sm+22st;虾夷扇贝染色体核型2n=38=6m+10sm+16st+6t;两者杂交子代染色体数目2n=38,但其染色体组型不符合预期型,不是简单地由两个亲本各自一套染色体组型组合而成,呈现出复杂的形态多态性,端部着丝点染色体明显增多.     

紫扇贝与海湾扇贝种间杂交的研究

为推动中国扇贝养殖业的进一步发展,作者于2008年从秘鲁引进了紫扇贝(Argopecten purpuratus),尝试将紫扇贝与海湾扇贝(Argopecten purpuratus)进行杂交,以期培育出具有优势性状的杂交子代.利用引进的紫扇贝与海湾扇贝进行种间杂交,培育出紫扇贝(雌)×海湾扇贝(雄)杂交扇贝.实验证明紫扇贝的卵子与海湾扇贝的精子可正常受精,受精卵发育正常.杂交扇贝的生长速度在幼虫阶段和成体阶段均快于海湾扇贝.至收获时,杂交贝比海湾扇贝平均壳高、壳长和壳宽分别大27.3%,25.2%和19.3%,体质量增加107.0%,杂种优势明显.     

栉孔扇贝(♀)×虾夷扇贝(♂)子一代杂种优势的SRAP分析

采用新型的分子标记技术(Sequence-related amplified polymorphism,简称SRAP)对栉孔扇贝(Chlamys farreri)(♀)×虾夷扇贝(Patinopecten yessoensis)(♂)子一代及其双亲的遗传变异进行分析.从不同的引物组合中筛选出9对条带清晰、多态性丰富的组合,9对引物共产生了121条扩增带,其中多态性条带107条,平均每对引物组合产生11.89条多态标记.Shannon信息指数I分别为0.299 9、0.218 9、0.334 3,Nei's基因多样性指数h分别是0.196 4、0.141 1、0.218 9,栉孔扇贝与杂交后代的遗传距离是0.107 4,虾夷扇贝与杂交后代的遗传距离是0.228 7,聚类分析也显示了这一特点.从变异贡献率来看,3个群体的总变异中,有37.31%的变异来源于群体间,62.69%的变异来源于个体间.     

Selective feeding of bay scallop Argopecten irradians on phytoplankton community revealed by HPLC analysis of phytopigments in Bohai Sea,China

Understanding the feeding selectivity on phytoplankton by shellfi sh is currently a big challenge. In order to investigate the feeding behavior of bay scallop ( Argopecten irradians ) on phytoplankton, we compared its compositions of phytopigments in digestive glands with those in the surrounding seawater, and conducted fi ve consecutive investigations between July and November 2016 in a bay scallop culture area along coast of Qinghuangdao City, northwest of the Bohai Sea, China. Phytopigments in four-size fractionated phytoplankton of seawater (micro-(20-200 μm);nano(L)-[10-20 μm];nano(S)-[2.7-10 μm], and pico-[<2.7 μm]) and digestive glands of A . irradians were examined to investigate the selective feeding of A . irradians . Results show that fucoxanthin and peridinin constituted the major part of taxonomically diagnostic carotenoids (TDCs) in the micro- and nano(L)-phytoplankton in seawater. Compared with total phytoplankton biomass of seawater (TPB, sum of the four sizes), a substantial decrease of fucoxanthin proportion to total DCs in digestive glands was observed while that of peridinin, 19′-butanoyloxyfucoxanthin, alloxanthin and 19′-hexanoyloxy-fucoxanthin showed an obvious increase when those pigments were mainly confi ned to micro-sized phytoplankton (20-200 μm). However, zeaxanthin and prasinoxanthin were mainly confi ned to nano(s)- and pico-phytoplankton, of which the proportions in digestive glands were usually lower in TPB. The contribution of lutein to total DCs in digestive glands (with an average of 7.23%) increased compared with TPB of seawater (with an average of 0.63%) during all fi ve sampling times.     

控制虾夷扇贝闭壳肌积累类胡萝卜素相关基因的筛查

利用抑制性差减杂交技术(Suppression subtractive hybridization,SSH)对虾夷扇贝闭壳肌积累类胡萝卜素相关基因进行筛查和分析。以虾夷扇贝橘红色闭壳肌和白色闭壳肌cDNA构建正反向差减文库,结合454测序技术在正向差减文库中测序得到2 640条序列,拼接得到50个contigs(重叠群);反向差减文库中测序得到2 496条序列,拼接得到187个contigs。经过同源性比对分析,共获得150多个差异表达基因,从中选择3种清道夫受体SRB1、SRF2和SRCR作为候选基因进行研究,对其在虾夷扇贝各组织中的表达量水平进行检测,初步阐述了其在虾夷扇贝中的组织表达规律。根据结果推测在虾夷扇贝橘红色闭壳肌中,清道夫受体有可能是调节类胡萝卜素累积和转运的关键基因。本研究为虾夷扇贝闭壳肌类胡萝卜素累积相关通路的探索提供了初步研究的基础数据。     

Spatial difference in net growth rate of Yesso scallop Patinopecten yessoensis revealed by an aquaculture ecosystem model

Identifying the main factors on spatial differences in net growth rate of Yesso scallop ( Patinopecten yessoensis ) in culture system is the key to effective aq...     

海湾扇贝微卫星标记开发及其分离方式分析

利用尼龙膜吸附杂交法构建了海湾扇贝的(AC)_(15)、(AG)_(15)微卫星富集文库,随机挑取3 000个克隆,经菌落原位杂交二次筛选共获得阳性克隆1 268个(42.3%).经序列测定和生物信息学分析后得到521个独立的阳性克隆,其中微卫星506个,小卫星15个.微卫星中,完美型248个,占总数的49.0%;非完美型216个,占42.7%;复合型42个,占8.3%;其中AG/TC重复占70.4%(356个),AC/TG重复29.6%(150个).选择其中的55条序列设计引物,筛选出其中的20对引物检测它们在海湾扇贝CC10家系的双亲和94个子代个体中的分离情况,结果表明:(1) 其中6个位点在母本和子代中发生分离,10个位点在父本和子代中发生分离,4个为双亲共享位点;(2) 2个位点存在无效等位基因;(3) 16个位点符合孟德尔遗传定律,4个位点的子代个体基因型频率偏离孟德尔遗传定律.上述结果表明,这些微卫星引物可以用于构建海湾扇贝的遗传连锁图谱,并且所构建的富集文库适用于微卫星标记的大规模开发.     

The effect of the causative algae of large-scale HAB in the East China Sea on egg hatching of Argopecten irradians, and population growth of Brachionus plicatilis and Moina mongolica

The impacts of Prorocentrum donghaiense Lu and Alexandrium catenella Balech, causative species of the large-scale HAB in the East China Sea, were studied under laboratory conditions. According to bloom densities, the effects of monoculture and mixture of the two species were examined on the egg-hatching success of Argopecten irradians Lamarck, and the population growth of Brachionus plicatilis Müller and Moina mongolica Daday. The results showed that monoculture of A. catenella had a significant inhibition on the egg hatching success of A. irradians, and the population growth of B. plicatilis and M. mongolica. The median effective densities (ED_50) inhibiting the egg hatching success of A. irradians for 24 h and the population growth of B. plicatilis and M. mongolica for 96 h were 800, 630, and 2 400 cells/cm3, respectively. Monoculture of P. donghaiense has no such inhibitory effect on the egg hatching success of A. irradians; P. donghaiense at lower suitable densities could sustain the population growth of B. plicatilis (1×104~3×104cells/cm3) and M. mongolica (2×104~5×104cells/cm3); P. donghaiense at higher densities had significantly adverse effect on the population growth of B. plicatilis (4×104~10×104cells/cm3) and M. mongolica (10×104cells/cm3). When the two algae were mixed according to bloom densities, P. donghaiense at suitable densities to some extent could decrease the toxicity of A. catenella to B. plicatilis and M. mongolica. The results indicated that the large-scale HAB in the East China Sea could have adverse effect on zooplankton, and might further influence the marine ecosystem, especially when there was also Alexandrium bloom.     

中国北方海域扇贝重金属含量的比较与质量评价

于2005年10～11月在大连湾、秦皇岛、莱州湾、烟台、威海、胶州湾、胶南7个海域,进行了海湾扇贝(Argopecten irradians)和栉孔扇贝(Chlamys farreri)As,Hg,Pb,Cu,Zn5种重金属含量的调查。重金属含量的测定采用原子吸收法进行。结果表明:(1)重金属含量存在着组织差异性,贝边的含量高于贝柱;(2)取自同一海域的栉孔扇贝重金属含量高于海湾扇贝;(3)5种重金属的平均含量排序为:Zn>Cu>As>Pb>Hg;(4)胶州湾栉孔扇贝Zn有超标现象,贝柱和贝边中的含量分别是限量标准的1.24倍和1.59倍;Hg,Pb,As和Cu均未超标。     

扇贝糖胺聚糖提取和纯化方法研究──Ⅰ．提取方法研究

研究从海湾扇贝中提取扇贝糖胺聚糖的一种实用而有效的方法。提出用胃蛋白酶、胰蛋白酶和枯草杆菌中性蛋白酶结合的水解方法，并给出糖胺聚糖提取过程的工艺流程。     

Response of Yesso scallop Patinopecten yessoensis to acute temperature challenge: physiological and biochemical parameters

Water temperature is generally considered to be a major factor af fecting the physiological and biochemical activities of marine bivalves. Here, the physiological and biochemical responses of Yesso scallop, Patinopecten yessoensis, to acute water temperature changes in summer were studied. Scallops were transferred directly to a lower temperature( T dec treatment)(from 23°C to 15°C) or to a higher temperature( Ti nc treatment)(from 15°C to 23°C) for 72 h, respectively. Results showed that the oxygen consumption and ammonia-N excretion rates of P. yessoensis decreased signi?cantly in the T dec treatment but increased dramatically at 6 h in the Ti nc treatment( P <0.05). In the T dec treatment, hepatopancreas antioxidant enzyme activities, superoxide dismutase(SOD) and catalase(CAT) activities, increased substantially within 72 h( P <0.05). However, a signi?cant decrease in CAT activity was found at 12 h in the Ti nc treatment( P <0.01).A signi?cant enhancement of acid phosphatase(ACP) activity and malondialdehyde(MDA) content was detected when scallops were acutely exposed to a temperature of 15°C. The levels of Cu/Zn-SOD gene expression in their gills up-regulated signi?cantly in response to acute temperature changes( P <0.01). These data suggest that acute temperature change af fects physiological and biochemical functions, and improve our knowledge of P. yessoensis under conditions of thermal stress.