大菱鲆选育家系雌、雄群体的生长发育差异

采用全长/体长、体高/体长、全长/体高和体质量为评价指标,分别对9~33月龄大菱鲆雌、雄群体在生长发育过程中体型的动态变化及生长性能差异进行了比较。大菱鲆雌、雄群体形态比较结果显示,雌、雄群体的全长/体长、体高/体长、全长/体高在不同发育阶段呈现特有的变化,但在雌、雄群体间,除33月龄的体高/体长和全长/体高差异达到显著水平外,3项指标在不同阶段没有差异或差异不显著,即可以认为,对于统计初始体质量相同或相近的大菱鲆雌、雄群体,全长/体长、体高/体长和全长/体高3个形态比例指标是随发育时间序列同步变化。基于Logistic模型对大菱鲆雌、雄群体生长性能的比较结果显示,雌性群体的拐点月龄、拐点体质量和最大月增重均大于雄性群体;对于雌性和雄性群体的瞬时增长率,除在9~13月龄雌性群体稍低于雄性群体外,13~33月龄的雌性群体均高于雄性群体,且随着生长发育和体质量差异的增大,雌、雄瞬时增长率的差异也呈增大趋势;雄性群体进入快速生长期的始速点和进入缓慢生长期的终速点均比雌性群体提前,雄性群体的快速生长期时间区间长度小于雌性群体,其对应的体质量区间长度也小于雌性群体,雌性群体在快速生长期的月增重显著高于雄性群体。研究表明,大菱鲆雌、雄群体在生长发育过程中存在着显著的生长差异,而形态差异不显著。     

不同壳色杂色鲍F1家系的早期生长及遗传结构比较

在"东优1号"杂色鲍养殖群体中,实验发现了少量的黄壳色变异个体,为得到具有优良经济性状的新品种,实验通过自交和杂交的方法分别建立了正常壳色♀×正常壳色♂(BB)、黄壳色♀×黄壳色♂(YY)、黄壳色♀×正常壳色♂(YB)和正常壳色♀×黄壳色♂(BY)4组家系,对各家系的卵黄径、受精率、孵化率、变态率、存活率和生长性状等生物学参数进行了比较,分析了杂交家系的杂种优势,并利用16个微卫星标记比较了4个家系的遗传差异。结果发现,黄壳色家系的卵黄径显著大于正常壳色家系的卵黄径(P<0.05),各家系的受精率、变态率和存活率没有显著的差异(P>0.05),幼体壳长和壳宽在不同的发育时间里,表现的杂种优势不稳定,在90日龄时,4个家系生长差异不显著。家系BB的遗传多样性最低,遗传距离(0.198 6～0.457 3)和遗传相似性系数(0.632 9～0.897 2)分析表明,BB和YY家系间的遗传距离最大(0.457 3),遗传相似性系数最低(0.632 9),杂色鲍黄壳色的自交家系与正常壳色的自交家系产生了遗传分化。     

大黄鱼♀与黄姑鱼♂杂交F1家系初孵仔鱼的AFLP分析

为了评估大黄鱼♀与黄姑鱼♂杂交F₁在良种培育与遗传学研究中的应用潜力,利用AFLP标记研究了双亲基因在2个杂交F₁家系(HF1和HF2)初孵仔鱼中的传递和分离方式。8对AFLP选择性扩增引物在两对亲本中分别检出478和446个片段。在HF1中,检出片段包括215条母本特异条带(FSB)、165条父本特异条带(MSB)和98条双亲共有条带(MuB),其中,121条(56.3%)FSB、115条(69.7%)MSB和93条(94.9%)MuB传递给了全部后代,其余片段在后代中发生分离。FSB和MSB分离位点的平均显性表型频率之间没有显著性差异。AFLP标记在HF2的传递与分离和HF1相似,只是分离位点的比例较HF1低,而且检出了2个非亲位点。此外,在HF1和HF2中都出现了较高比例的偏离孟德尔定律的分离位点。这些结果表明,大黄鱼♀×黄姑鱼♂杂交F₁初孵仔鱼中同时含有来自双亲的基因;虽然计算结果显示杂交F₁在遗传上略偏向于母本,但是父源基因和母源基因没有明显的选择性丢失;母本特异位点多态比例与大黄鱼种内杂交F₁相当。研究结果为大黄鱼♀×黄姑鱼♂杂交F₁的开发与管理提供了科学依据。     

团头鲂耐低氧F3的建立及其在低氧环境下的生长差异

为了构建团头鲂耐低氧新品种,实验从鄱阳湖引进和挑选野生优良亲本为奠基群体F0,2009年–2011年通过群体选育获得F1,2011年再通过群体选育实现了F1到F2的传代。2012年,以鄱阳湖选育F2亲本和团头鲂"浦江1号"F9亲本为基础,经夏、秋季2次低氧胁迫,筛选出536尾耐低氧能力强的F2亲本,构成团头鲂耐低氧F2。2013年,挑选个体大、体形好的F2亲本(雌鱼50尾、雄鱼48尾)建立了24个F3家系群体(2♀×2♂群体22个、3♀×2♂群体2个),共100个F3家系。对上述F3家系群体进行1龄阶段的低氧胁迫养殖,通过测定相应生长指标和耐低氧性状,共筛选出5个快速生长家系群体(A2、A3、A18、A19和A20)和6个生长较快的家系群体(A4、A6、A17、A25、A27、A28),微卫星分析其分别归属于20个和28个家系。结果显示,团头鲂的生长性能指标与耐低氧性状呈正相关。在1龄阶段长时间低氧胁迫养殖条件下,团头鲂耐低氧F3中耐低氧能力强的家系的体质量显著大于耐低氧能力弱的家系,选育出的团头鲂耐低氧F3家系在2龄阶段同样保持了快速生长特征。本研究旨在建立团头鲂耐低氧F3新品系,以供后续团头鲂耐低氧新品种的选育。     

红鲫(♀)×湘江野鲤(♂)F2和F3的染色体研究

采用血细胞培养制片法和肾脏细胞直接制片法对红鲫(♀)×湘江野鲤(♂)杂交种F2和F3代的染色体数目和核型进行了比较分析，得出F2代染色体数目为100条，染色体组型为22m＋34sm＋22st＋22t，NF＝156；F3代染色体数目为4n＝200，染色体组型为44m＋68sm＋44st＋44t，NF＝312。为证明红鲫×湘江野鲤杂交后代由异源二倍体转变为异源四倍体发生于F2与F3代之间提供了直接证据。     

大菱鲆(Scophthalmus maximus)PRL基因、Na+-K+-ATPase a1基因对盐度胁迫的响应

采用荧光定量PCR技术对不同盐度胁迫下各时间点大菱鲆(Scophthalmus maximus)幼鱼肠、鳃中催乳素(PRL)基因和Na+-K+-ATPase a1两种基因的表达量进行检测。以盐度30为对照组,盐度5、10、40和50为实验组进行数据分析。结果显示,两种基因在两种组织中均有表达,且基因的表达量具有组织和时间特异性。肠组织PRL、Na+-K+-ATPase a1基因的表达量,在盐度50和5条件下,随胁迫时间的延长呈先升高后降低的变化趋势;鳃组织PRL基因表达量,在盐度50和盐度5条件下,随胁迫时间延长先升高后降低,而Na+-K+-ATPase a1基因表达量在低盐条件下(盐度5)没有显著变化,在高盐条件下(盐度50)随时间延长呈现先降低后升高的变化趋势。在肠组织中,两种基因存在极显著的协同作用,随着盐度的升高,两种基因的表达量都呈现先升高后降低的趋势,且相关系数均接近于1;在鳃组织中,在10–40盐度范围内,两种基因的表达存在明显的拮抗作用,当PRL基因的表达量呈现升高(或下降)趋势时,Na+-K+-ATPase a1基因的表达量呈现下降(或升高)趋势,且两种基因的相关系数均为负值。研究表明,PRL具有抑制Na+/K+-ATP酶活性的作用,为今后盐度胁迫分子调控机理研究提供理论依据。     

西伯利亚鲟(F2)胚后发育的形态观察

对人工养殖西伯利亚鲟(F₂)仔鱼的胚后发育形态进行了观察,从仔鱼刚出膜［0日龄,(9.16±0.21) mm］开始一直观察到早期稚鱼阶段［53日龄,(87.12±1.92) mm］。从形态发育来看,西伯利亚鲟仔鱼的胚后发育可以分为2个时期：早期仔鱼,即从刚出膜（0日龄）到初次开口（9日龄）摄食;晚期仔鱼,从开口摄食至器官发育基本完全（37日龄）;以后进入早期稚鱼期。早期仔鱼期的形态建成与分化明显地比晚期仔鱼和早期稚鱼快,其中,早期仔鱼的感觉、摄食、呼吸、游泳等器官快速分化;晚期仔鱼,主要表现为各骨板的分化和完善,当仔鱼在形态上完成向成鱼转变时,表示进入了早期稚鱼期。西伯利亚鲟胚后发育的观察结果显示,发育的早期阶段各器官协调并快速发育,感觉、摄食、呼吸、游泳等器官的出现和完善,使仔鱼在短时间内具备了躲避敌害和摄食的能力,其生存能力大大提高。     

转Hu-IFN-α基因草鱼雌核发育F1的遗传配型分析

The Hu-IFN-α gene, which was transducted into downstream promoter of β-actin gene of common carp (Cyprinus carpio), was recombined by DNA recombination technology. These recombined genes were injected into 1-2 cell fertilized eggs of grass carp (Ctenophatyngodon idellus) by microinjection technology, we gained transgenetic fish by molecular detection methods. In order to analyse the genetic expression of tranHu-IFN-α gene gynogensis F₁, which male individualization were gained by raising methyltestosterone, molecular genetic marker technology was used. In our research, 30 random primers were picked out from 48 and were used into RAPD-PCR, the result indicated that 1 169 clear, steady and repeated DNA finger printing bands were achieved. On the basis of gentic distance matrix among tranHu-IFN-α gene gynogenesis F₁ group, the genetic relationship of gynogenesis F₁ were analysed by UPGMA, the results showed the genetic patterns are close between the 3# male gynogenesis F₁ and the the 23# female of gynogenesis F₁, 5# and 27#, 2# and 28#, 2# and 30#. The data indicated that these group could be served as parent of tranHu-IFN-α grass carp (Ctenophatyngodon idellus) pure line.     

大黄鱼22个微卫星标记在F1家系中的分离方式及与生长性状的相关分析

研究了1个大黄鱼F1家系150个个体22个微卫星位点的基因型分布,并分析了标记位点与生长性状的相关性。结果显示,22个位点共检测到60个等位基因,平均等位基因数为2.73,平均有效等位基因数为2.37;平均观测杂合度与期望杂合度分别为0.75和0.73,部分位点基因型分布严重偏离孟德尔定律,暗示其可能与适应性基因相连锁,其中LYC0446位点附近可能存在隐性纯合致死基因。LYC0077位点与体质量、体长和体高均呈显著相关(P<0.05),其中等位基因A(165 bp)对应的生长性状表型值最大,可以作为选育快长性状的有效分子标记;LYC0015和LYC0243与体高呈显著相关(P<0.05),与体长、体质量的相关不显著(P>0.05),LYC0015位点的等位基因C(110 bp)和LYC0243位点的等位基因A(160 bp)为有利的等位基因。对LYC0015、LYC0077和LYC0243进行不同基因型个体表型值的多重比较,找到3种对生长性状有利的基因型,分别为BC、AA和AB。同时,以体质量性状为参照,对3个位点不同基因型组合进行比较,找到一个最优基因型组合(BC/AA/AB),与3个位点单独分析对应的最优基因型完全一致,符合加性作用模型。     

草鱼、赤眼鳟及其杂交F1遗传多样性的RAPD分析

采用RAPD-PCR分子标记技术分析了草鱼、赤眼鳟及其正、反交子一代的遗传多样性。从100条引物中筛选出13条扩增效率高、多态性高的有效引物,对4个群体共120尾个体样本进行扩增,获得115个基因位点,扩增产物大小集中在300~3 000 bp,草鱼、正交F_1、赤眼鳟和反交F_1多态位点率分别为85.22%、77.39%、80.87%和70.43%,Shannon信息指数分别为0.392 4、0.396 9、0.443 3和0.358 3。结果表明,赤眼鳟的遗传多样性最丰富,正交F_1的遗传多样性优于草鱼,反交F_1的多样性最差。遗传同源性和遗传距离聚类分析表明,正交F_1和反交F_1与赤眼鳟的遗传关系更近。种群平均遗传分化指数为0.302 6,表明4个群体之间的遗传分化程度较高,30.26%的变异来源于群体间。     

Dietary protein requirement for young turbot (Scophthalmus maximus L.)

This study was conducted to determine the optimum dietary protein level for young (an initial weight of 89 g) turbot, Scophthalmus maximus L. Duplicate groups of the fish were fed the five isoenergetic diets containing the various protein levels ranging from 290 to 570 g kg^?1 diet for 45 days. Survival was not affected by dietary protein level. Weight gain and feed efficiency were improved with dietary protein level up to 490 g kg^?1 diet. Dietary protein requirement of young turbot using the broken‐line model was estimated to be 494 g kg^?1 diet based on weight gain response. Protein efficiency ratio was not influenced by dietary protein level. The highest protein retention was obtained from the fish fed the 490 g protein kg^?1 diet. Proximate composition of the fish was not significantly affected by dietary protein level. In considering these results, it was concluded that the 494 g protein kg^?1 diet with 100 g lipid kg^?1 diet (15 MJ kg^?1 diet) provided optimal growth of young turbot under these experimental conditions.     

大菱鲆种质资源研究与开发

综合介绍了大菱鲆种质资源的分类、自然分布和生物学特性。从生化和分子生物学方面阐述了大菱鲆自然群体遗传结构及国内外研究现状。详述了大菱鲆野生资源的利用和养殖开发现状．特别是我国对引进的大菱鲆种质资源的养殖开发。指明了我国目前在引种及养殖方面存在的不足．提出了建立全国性大菱鲆良种引育中心,以保证我国大菱鲆养殖业的持续、稳定、健康发展的建议。     

Statistical properties and performance of pairwise relatedness estimators using turbot (Scophthalmus maximus L.) family data

The statistical properties and performance of four estimators of pairwise relatedness were evaluated in several scenarios using the microsatellite genotype data from a set of large known full‐sibships of turbot. All estimators showed a significant negative bias for the four kinships commonly used in these studies (unrelated: UR, half‐sibs, full‐sibs and parent–offspring), when allele frequencies of the reference population were estimated from the individuals analysed. When these frequencies were obtained from the base population from which all families proceeded, the bias was mostly corrected. The Wang (W) and Li (L) estimators were the least sensitive to this factor, while the Lynch and Ritland (L&R estimator) was the highest one. The error (mean around 0.130) was very similar in all scenarios for W, L and Queller and Goodnight (QG) estimators, while L&R was the highest error‐prone estimator. Parent–offspring kinship resulted in the lowest error, when using W, L and QG estimators, while UR resulted in the lowest error with the L&R estimator. Globally, W was the best‐performing estimator, although L&R could perform better in specific sampling scenarios. In summary, pairwise estimators represent useful tools for kinship classification in aquaculture broodstock management by applying appropriate thresholds depending on the goals of the analysis.     

Cryopreservation of turbot Scophthalmus maximus (L.) sperm: fertilization and hatching rates

The present paper assesses the fertilization and hatching rates of an artificial fertilization series (n=1153) using fresh and cryopreserved sperm from 49 specimens of turbot Scophthalmus maximus (L.), carried out to confirm the results of a previous study, with the ultimate aim of transferring these cryo‐preservation techniques to commercial hatcheries. No significant differences were found between the fertilization rates of the two groups (fresh and cryopreserved sperm) when their respective fertility rates were >69.2%, which was the case in 75% of all fertilizations. Likewise, no significant differences in hatching rates were found. In order to use the cryopreserved sperm more efficiently, a key concern for commercial use of this technique, we also experimented with a lower sperm:diluent ratio (1:1) than used previously at our centre. We also compared the traditional 0.5‐mL straws with 2‐mL cryotubes able to contain a higher volume of sperm, finding no significant differences in the resulting fertilization and hatching rates. In conclusion, the use of cryopreservation for turbot sperm presents major advantages for broodstock management in commercial hatcheries.     

Pathology of Edwardsiella tarda infection in turbot, Scophthalmus maximus (L.)

Macroscopic and histopathological changes in cultured turbot, Scophthalmus maximus (L.), in Spain caused by infection with Edwardsiella tarda are described. Eye tumefaction, inflammation, haemorrhages, ascites and the presence of a purulent fluid were the main macroscopic lesions observed. Histopathological lesions were found in the kidney, spleen and liver. In the kidney and spleen these were characterized by a severe apostematous inflammatory reaction, with a large number of abscesses. The liver was affected to a lesser degree and only some phagocytes loaded with bacteria were observed. Ultrastructural observations indicated that macrophages were the main cell type implicated in the inflammatory response. Most of the bacteria observed within the phagocyte cytoplasm showed no degenerative changes and some were dividing. Degenerative changes observed in macrophages indicate their failure in preventing the infection.     

一种吻蛭类大鱼蛭在大菱鲆鱼体上的感染

在山东省威海崮山育苗场发现大菱鲆成鱼及幼鱼鱼体感染寄生虫，受感染的鱼由于处于感染初期，并经过及时的处理，所占比例较低。病鱼症状为鱼体体色变黑、瘦弱、离群、不摄食甚至死亡。寄生虫寄生于大菱鲆的体表及鳃部，虫体黑褐色，全长40～50mm。经鉴定，该寄生虫属于环节动物门(Annelida)，蛭纲(Hirudinea)，吻蛭目(Rhynochobdellida Blanchard)，鱼蛭科(Piscicolidae Johnson)，鱼蛭属(Piscicola Blainville)，大鱼蛭(Pisciola magna Yang)。     

大菱鲆AFLP分析体系的建立

本研究构建了大菱鲆扩增片段长度多态性(AFLP)分析体系,对DNA提取、双酶切反应、连接反应、预扩增反应、选择性扩增反应和银染等步骤进行了分析。结果表明,用EcoRI/MseI双酶切、核心序列加3个选择性碱基的选择性扩增引物、1∶6(w∶w)的EcoRI端与MseI端选择性扩增引物比和20μl选择性扩增体积,可得到十分稳定的结果。所得产物经电泳和银染后可获得条带清晰、背景干扰小的图像。该体系的构建为AFLP技术在大菱鲆相关研究中的应用奠定了基础。     

大菱鲆家系生长和耐热性状对比分析

利用40个大菱鲆(Scophthalmus maximus)家系进行耐热性实验,测定相应生长和耐热性状指标,对耐热性状进行方差分析,并分析生长与耐热性状的相关情况。结果表明:在各家系生长前期,快速生长家系6个,生长较快家系26个,生长速度一般家系8个。在生长后期阶段,快速生长家系9个,生长较快家系28个,生长速度一般家系3个,仅有8号家系一直为快速生长家系。在耐热性状方面,各家系耐热性平均值在1 367.30～836.30℃.h之间,总体平均值为1 113.00℃.h。高耐高温家系为8号、33号和37号家系,其中33号家系耐热性数值最高,其耐热性平均值为1 367.30℃.h,不耐高温家系为3号、9号、16号、38号和40号家系,其中38号家系耐热性数值最低,其耐热性平均值为836.30℃.h。方差分析结果显示,不同家系间耐热性存在极显著差异(P<0.01),相关分析结果表明,各家系不同生长阶段与耐热性呈现不同的相关性。旨在探明生长性状与耐热性状的相关性,为大菱鲆的耐热家系选育奠定研究基础。     

Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.)

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.