关于大黄鱼Pseudosciaena crocea(Richardson)网箱育种技术的探讨

大黄鱼Pseudosciaena crocea(Richardson)的人工育苗和养成技术在“七五”期间福建省首次获得成功,推动我国海水养殖业的发展。1996年育苗量达6000万尾,网箱养殖大黄鱼2万箱,取得很好的成绩。作者就大黄鱼网箱育种技术作扼要的探讨。     

鲑鳟通用型低通量单核苷酸多态性芯片的开发

为开发常见鲑鳟养殖物种通用的遗传分析工具,本研究利用Affymetrix虹鳟(Oncorhynchus mykiss) 57K高通量单核苷酸多态性(Single nucleotide polymorphism,SNP)芯片,对国内代表性鲑鳟养殖群体开展了分型检测,包括山女鳟(Oncorhynchus masou masou)、银鲑(Oncorhynchus kisutch)、美洲红点鲑(Salvelinus fontinalis)、白斑红点鲑(Salvelinus leucomaenis) 4个物种,从57,501个SNP标记中筛选出96个共享多态性标记,应用Fluigidm 96.96动态芯片平台,构建了大麻哈鱼属(Oncorhynchus)和红点鲑属(Salvelinus)通用型低通量SNP芯片。该芯片分型结果准确性较高,与Affymetrix高通量芯片分型一致性达到96.55%。使用该芯片对来自6个家系的48尾银鲑个体及其候选亲本进行检测,应用Cervus 3.0.7软件进行亲权鉴定,结果能够准确重现复杂家系的真实系谱。在用于单亲本亲权鉴定时,第一亲本非排除率(Non-exclusion probability for first parent, NE-1P)为4.120×10~(–4);用于双亲本亲权鉴定时,双亲非排除率(Non-exclusion probability for parent pair, NE-PP)低至6.219×10~(–12),表明该芯片在鲑鳟养殖群体系谱鉴定应用中具有较高的准确性。使用该芯片开展4个鲑鳟养殖群体遗传结构分析,样本分群聚类结果与其所属的分类阶元相符,能够准确反映群体遗传组分构成和遗传关系。本研究构建的低通量SNP芯片在常见鲑鳟养殖物种中具有良好的通用性,将其应用于养殖群体遗传分析,能够为鲑鳟制种、育种和引种等科学决策提供基因组信息参考。     

大黄鱼“闽优1号”

大黄鱼“闽优1号”（品种登记号：GS-01--005—2010）是以在福建宁德市官井洋附近海域捕获的野生鱼种为起始亲本来源．通过常规的群体选择结合雌核发育技术,以生长速度、体型和成活率为主要指标,经5代选育获得的新品种。     

大黄鱼性别特异SNP标记的开发与验证

大黄鱼是我国养殖量最大的海水经济鱼类,其雌鱼生长显著快于雄鱼,但两性的外部形态差异不明显,也没有异形性染色体,依靠传统方法无法对其活体准确进行生理性别和遗传性别的判别与鉴定,需要开发性别特异的分子标记。本研究从2尾雌鱼和2尾雄鱼、以及分别由50雌鱼与50尾雄鱼组成的2个混合样品的基因组重测序数据比较中筛选与性别显著关联的SNP位点,对其中11个位点分别设计引物在15尾雌鱼和15尾雄鱼中扩增出PCR产物进行Sanger测序验证,鉴定出1个与性别完全连锁的位点(SNP6,15尾雌鱼均为纯合、15尾雄鱼均为杂合)。然后,设计等位基因特异性PCR引物,其中包括2条雌性与雄性通用引物和1条雄性特异引物,在闽—粤东族与岱衢族大黄鱼合计近2 200个个体中进行扩增,结果在全部雌鱼中都只扩增出1个348 bp的条带,而在全部雄鱼中还扩增出1个194 bp的Y染色体特异条带,检出率达到100%。研究表明,大黄鱼属于XX♀-XY♂类型的性别决定。本研究鉴定出一个雄性特异SNP标记,并建立了一种新的大黄鱼遗传性别鉴定技术,为大黄鱼单性育种、基因组选择育种和性别决定分子机制研究提供了重要的技术手段。     

大黄鱼血液基因组DNA的提取及其效果

应用Sangon血液基因组DNA小量抽提试剂盒,提取大黄鱼血液组织的基因组DNA,并以所提取的DNA为模板进行RAPD扩增效果分析。结果显示,血液基因组DNA稳定性好、质量高,分子量大小在20kb左右;RAPD扩增条带清晰,能满足RAPD等一般分子实验对于模板DNA的质量要求。为大黄鱼的隔代连续研究和濒危鱼类分子实验所要求的活体基因组DNA的获得提供了一个可行的样本组织采集方法。     

大黄鱼“东海1号”

近日,第五届全国水产原种和良种审定委员会第一次会议审定通过了大黄鱼"东海1号"等15个水产新品种。其中大黄鱼"东海1号"、中国对虾"黄海3号"、三疣梭子蟹"科甬1号"、中华绒螯蟹"长江2号"、长牡蛎"海大1号"、栉孔扇贝"蓬莱红2号"、文蛤"科浙1号"、条斑紫菜"苏通1号"、坛紫菜"申福2号"、裙带菜"海宝1号"、龙须菜"2007"等11个品种为适宜推广的选育品种;北鲆2号、津新乌鲫、斑点叉尾鮰"江丰1号"、海带"东方6号"等4个品种为适宜推广的杂交品种。为推广优良品种,提高养殖效率,本刊将分期刊登这些品种的繁殖及养殖技术。     

SNP的研究进展及其在家畜育种中的应用

单核苷酸多态性(SNP)作为新的遗传标记,已广泛应用于动物遗传育种、基因定位、克隆和遗传多样性等方面的研究。文章对SNP的概念、检测方法进行了详细阐述,并综述了SNP在动物遗传育种和家畜繁殖技术中的应用。     

鲤低氧适应性状的全基因组关联分析

低氧适应是水产养殖物种的重要性状,筛选用于改良低氧适应性状的分子标记及候选基因有助于鱼类耐低氧品种选育.在数量性状和质量性状的遗传研究中,全基因组关联分析(GWAS)广泛应用于性状相关标记和基因的发掘.本研究对鲤(Cyprinus carpio)养殖群体开展了低氧胁迫实验,选取了低氧敏感和低氧耐受的极端性状个体作为研究...     

大黄鱼肌纤维性状的全基因组关联分析

大黄鱼是我国近海重要经济鱼类,因养殖条件限制,其肉质难以满足消费者的需求。肌纤维组织学特征是鱼类肉质评价中的常用指标,肌纤维平均直径和密度对鱼类肉质有重要影响。为定位肉质相关遗传位点,解析大黄鱼肉质性状的遗传调控机制,并为养殖大黄鱼肉质选育提供基础,本研究首次对大黄鱼肌纤维性状进行全基因组关联分析。采集一个大黄鱼养殖群体的背部肌肉样本,通过组织切片获得了483尾大黄鱼的肌纤维直径及253尾的肌纤维密度数据。随后用双消化限制性酶切位点相关的DNA测序（ddRAD-seq）法进行基因分型,获得了25 520和25 309个高质量单核苷酸多态性（SNPs）位点用于全基因组关联分析（GWAS）。最终,在5条染色体上定位到了6个显著位点,并注释到了lpin2、adcyap1、btg1、tcp11l2、lifr、thbs4、gmeb1、slc2a1、myom1、myom2共10个候选基因。对这些候选基因的分析表明,大黄鱼的肉质与肌纤维组成和生长,以及能量代谢有关。lpin2、adcyap1、btg1等基因可能在决定大黄鱼肌肉的物理特性中发挥作用。本研究首次对大黄鱼肌纤维相关性状进行基因组定位,研究结果为大黄鱼分子标记辅助选育提供了重要的理论参考,并为后续开展针对养殖群体大黄鱼肉质性状的遗传改良提供了分子工具。     

水产群体基因组重测序数据分析软件包的开发

为了帮助水生动物学研究者解决群体遗传学基础分析的困难,本实验在调研现有的水生动物重测序数据分析研究和成果的基础上,基于水生动物群体遗传学的常用方法和通用分析软件,构建可于本地运行的、能够完成大部分基因组重测序数据基础计算的软件包。软件包首先将质控过滤后的重测序数据与参考基因组序列进行比对,利用比对结果检测基因组的遗传变异,对群体进行系统发育分析、群体结构分析、主成分分析、遗传多样性重要量化指标的计算和选择性消除分析等,并通过R或Python语言工具包对分析结果进行可视化。根据该软件包对来自3个群体、共约30尾大黄鱼个体的简化基因组测序数据进行分析测试的结果,完成了软件包携带的测序数据比对、单核苷酸多态性(SNP)鉴定、系统进化树构建、群体结构预测、连锁不平衡检测和多样性指标等计算功能,并且较好地图形可视化了分析结果。该群体基因组重测序分析的简易软件包可用于野生和自然群体的群体遗传学分析的大部分基础统计、计算和绘图,适合包括水生生物学在内的相关领域的生物学研究者进行群体基因组学研究。本研究为水生动物重测序数据分析提供便利,节约科研时间,减少人力物力成本。相关源码和使用说明文档已公开上传至...     

Dietary threonine requirement of juvenile large yellow croaker,Larmichthys crocea

A feeding trial was conducted to determine the dietary threonine requirement of juvenile large yellow croaker (Larmichthys crocea). Six diets were formulated containing 45% crude protein with six graded levels of threonine (0.71–2.46% in about 0.35% increment). Each diet was randomly assigned to triplicate groups of 60 juvenile fish (initial body weight 6.00 ± 0.10 g). Fish were fed twice daily (05:00 and 16:30) to apparent satiation for 8 weeks. The result indicated that significant difference was observed in the weight gain among all treatments. Specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER) and nitrogen retention (NR) increased with increasing levels of threonine up to 1.75% diet (P < 0.05), and thereafter, declined. No significant differences in body dry matter, crude protein, crude lipid or ash content were found among dietary treatments. Theronine contents of fish muscle were significantly affected by dietary threonine levels (P < 0.05). Fish fed the diet with 0.71% threonine showed the lowest threonine content (2.94%) in fish muscle, while fish fed the diet with 1.75% threonine had the highest value (3.16%). Other essential amino acid contents of muscle were not significantly different among the dietary treatments. On the basis of SGR, FE or NR, the optimum dietary threonine requirements of juvenile L. crocea were estimated to be 1.86% of diet (4.13% of dietary protein), 1.90% of diet (4.22% of dietary protein) and 2.06% of diet (4.58% of dietary protein), respectively, using second‐order polynomial regression analysis.     

Artificial fertilization and generating families for a selective breeding programme of large yellow croaker ( Larimichthys crocea )

Large yellow croaker is an important marine aquaculture species in China. The aim was to determine an appropriate protocol of artificial fertilization for family construction in the breeding programme based on two trials. In trial 1, luteinizing hormone-releasing hormone A3 (LHRHA3) was injected once, with a dosage of 2 μg/kg for females and 1 μg/kg for males. The latency time was in the range of 30–34 h. The maturation stage was checked by extracting a few eggs with a Pasteur pipette. The fertilization rate and hatching rate were 27 and 52%, respectively. The percentage of females with spawning difficulties was 30%. In trial 2, the females were injected LHRHA3 twice: with a first dose of 0.8 μg/kg and a second dose of 2 μg/kg, at an interval of 10 h, whereas the males were still injected once. The latency time was in the range of 29.5–35 h, determined by only observing courtship behaviour of males. The females with spawning difficulties decreased to 10%, and the fertilization rate and hatching rate also improved to 41 and 62%, respectively.     

Nocardiosis in large yellow croaker, Larimichthys crocea (Richardson)

An epizootic in seawater-cage reared large yellow croaker, Larimichthys crocea, in China was caused by a Nocardia sp. from August to October 2003. The cumulative mortality rate was 15% and the diseased fish were 16 months old with individual length varying from 25 to 30 cm. Multiple, white nodules, 0.1-0.2 cm in diameter, were scattered on the heart, spleen and kidney. The morphology of isolated bacteria from Lowenstein-Jensen medium and tryptic soy agar was bead-like or long, slender, filamentous rods. Experimental infection indicated that the isolated bacterium was the pathogen responsible for the mortalities. A partial sequence of the 16S rRNA gene of the organism and the type strain of Nocardia seriolae JCM 3360T (Z36925) formed a monophyletic clade with a high sequence similarity of 99.9%. Based on the morphological, physiological, biological properties and the phylogenetic analysis, the pathogenic organism was identified as N. seriolae. This is the first report on N. seriolae-infected large yellow croaker in aquaculture.     

Optimal dietary lipid level for large yellow croaker (Pseudosciaena crocea) larvae

A 30‐day feeding experiment was conducted in blue tanks (70 × 50 × 60 cm, water volume 180 L) to determine the effects of dietary lipid levels on the survival, growth and body composition of large yellow croaker (Pseudosciaena crocea) larvae (12 days after hatchery, with initial average weight 1.93 ± 0.11 mg). Five practical microdiets, containing 83 g kg^?1 (Diet 1), 126 g kg^?1 (Diet 2), 164 g kg^?1 (Diet 3), 204 g kg^?1 (Diet 4) and 248 g kg^?1 lipid (Diet 5), were formulated. Live feeds (Artemia sinicia nauplii and live copepods) were used as the control diet (Diet 6). Each diet was randomly assigned to triplicate groups of tanks, and each tank was stocked with 3500 larvae. During the experiment, water temperature was maintained at 23(±1) °C, pH 8.0 (±0.2) and salinity 25 (±2) g L^?1. The results showed that dietary lipid significantly influenced the survival and growth of large yellow croaker larvae. Survival increased with the increase of dietary lipid from 83 to 164 g kg^?1, and then decreased. The survival of larvae fed the diet with 83 g kg^?1 lipid (16.1%) was significantly lower than that of larvae fed other diets. However, the survival in larvae fed the diet with 16.4 g kg^?1 lipid was the highest compared with other artificial microdiets. Specific growth rate (SGR) significantly increased with increasing dietary lipid level from 83 to 164 g kg^?1 (P < 0.05), and then decreased. The SGR in larvae fed the diet with 164 g kg^?1 lipid (10.0% per day) was comparable with 204 g kg^?1 lipid (9.6% per day), but were significantly higher than other microdiets (P < 0.05). On the basis of survival and SGR, the optimum dietary lipid level was estimated to be 172 and 177 g kg^?1 of diet using second‐order polynomial regression analysis respectively.     

大黄鱼选育子二代生长性状研究

通过对选育和未选育2个群体大黄鱼（Larimichthys crocea）繁育得到的子代的卵径、油球径和口径进行测量,以及对其子代养殖过程中的生长性状进行测量比较,分析选育效果。结果显示,选育大黄鱼子代（XY-F2）和未经选育大黄鱼子代（HB-F1）平均卵径、平均油球径和平均口径之间差异不显著（P〉0.05）。拟合1~10月龄XY-F2和HB-F1的体长（x）、体质量（y）的生长曲线及体长（x）与体质量（y）的关系表明,两群体间体长（x）与体质量（y）的关系存在差异;XY-F2在体长（x）和体质量（y）的生长在后期高于HB-F1。分析其数量性状,选育子一代（XY-F1）2龄鱼体长、体质量现实遗传力分别为0.26、0.18,而XY-F2的体长、体质量现实遗传力分别为0.029、0.134。结果说明选育具有一定的作用,今后选育应该结合家系选择和家系内选择进行。     

大黄鱼网箱养殖水体的细菌群落结构

以16S rDNA为分子标记, 通过构建克隆文库、限制性片段长度多态性(Restriction Fragment Length Polymorphism, RFLP)分析等技术手段, 研究大黄鱼(Larimichthys crocea)网箱养殖水体中细菌的群落结构。样品采自福建省宁德市三都湾富发养殖基地。随机选取5个不同养殖网箱水样混合, 取3 L混合水样过滤富集细菌后提取总DNA, 用细菌通用引物27F和1 492R扩增其16S rRNA基因, 构建克隆文库。从文库中随机挑选154个克隆子进行分析, 得到137个阳性克隆, 92种RFLP带型。对部分代表性克隆子进行测序的结果表明, 大黄鱼养殖水体中细菌多样性非常丰富。序列分析结果显示, γ-变形菌纲的假交替单胞菌属(Pseudoalteromonas)细菌为最优势菌(占γ-变形菌纲克隆子数的31.2%), 海源菌属(Idiomarina)次之(占γ-变形菌纲克隆子数的15.1%)。此外还存在6.6%的厚壁菌门(Firmicutes)、5.9%的拟杆菌门(Bacteroidetes)、各0.7%的浮霉状菌门(Planctomycetes)、绿屈挠菌门(Chloroflexi)、绿菌门(Chlorobi)细菌和OP11类群。本研究结果阐明了网箱养殖大黄鱼水体细菌的群落结构, 为大黄鱼养殖区病害防治、环境监测以及探讨大黄鱼健康养殖与养殖水体细菌间的关系奠定了基础。     

Dietary lysine requirement of large yellow croaker (Pseudosciaena crocea,Richardson 1846) larvae

A 30‐day feeding experiment was conducted to estimate the lysine requirement of large yellow croaker larvae (2.75 ± 0.11 mg). Six isonitrogenous (509.5–519.7 g kg^?1 crude protein) and isoenergetic (22.3–22.5 kJ g^?1 energy) microdiets containing graded levels of lysine·HCl ranging from 24.8 to 41.0 g kg^?1 diet in placement of glycine and glutamic acid were formulated. Mixture of crystalline amino acids (MAA) was supplemented to simulate the amino acid (AA) profiles of whole body of this larva, except for lysine. The MAA and supplemented lysine for each diet were coated with tripalmitin. Triplicate groups of 3000 fish were fed to apparent satiation by hand eight times per day. The results showed that specific growth rate (SGR), survival, body composition and the specific activity of digestive enzymes were significantly affected by dietary lysine levels (P<0.05). The optimal dietary lysine requirements estimated by second‐order polynomial model based on SGR and survival were 33.7 (65.5 g kg^?1 dietary protein) and 33.4 (64.9 g kg^?1 dietary protein) g kg^?1 dry diet respectively. The estimated requirements for the other essential AAs were calculated by A/E ratios of whole body AA profile of this larva based on lysine requirement.     

操作胁迫对大黄鱼幼鱼的影响

研究操作胁迫(追赶惊扰)对大黄鱼(Pseudosciaena crocea Richardson)幼鱼的生长、行为、肝脏、脾脏、免疫功能等方面的影响.实验用大黄鱼幼鱼全长(5.85±0.45)cm,实验共设3个组对照组、实验组1(每日操作胁迫1次)、实验组2(每日操作胁迫2次),每组2个重复.分别于实验开始后第10天、20天、30天,取样测定其全长、体质量、脾脏重以及肝脏重,计算脾系数和肝系数;断尾取血做血涂片,在油镜下进行白细胞分类计数,并对各项指标进行统计分析.结果表明,长期的剧烈操作胁迫会显著抑制大黄鱼幼鱼的生长,同时也影响其肝脾脏功能和机体免疫机能,如增加大黄鱼幼鱼脾系数(即脾肿大),肝脏的合成代谢先受到抑制;各种白细胞比率受剧烈操作胁迫(每日2次胁迫)影响发生显著变化,嗜中性粒细胞和单核细胞在实验前期(10 d)或中期(20 d)显著增多,而淋巴细胞则明显减少,但在实验末期均又恢复正常水平,其原因可能是大黄鱼的免疫机能对长期操作胁迫表现一定的适应性.     

大黄鱼连续4代选育群体遗传多样性与遗传结构的微卫星分析

利用13个多态性微卫星位点分析了大黄鱼官井洋优快01品系F1到F44个选育世代的遗传结构与遗传多样性变化情况。结果显示,随着选育的进行,4个世代群体遗传多样性指标值渐次下降,F1到F413个微卫星位点的平均多态信息含量从0.638下降到0.524,平均等位基因数从5.462下降到4.308,平均观测杂合度从0.779下降到0.532,平均Shannon多样性指数从1.356下降为1.092。F1与其后各代遗传相似系数逐渐减小(从0.7194到0.5813),遗传距离逐渐增加,而相邻世代间的遗传相似性逐步升高,遗传分化指数(FST)渐次变小(F1～F2为0.0619,F2～F3为0.0511,F3～F4则为0.0475)。随着选育的进行,微卫星位点LYC0002和LYC0054等位基因频率有规律地发生变化,推测其可能与选育性状存在遗传上的相关。结果表明,经过连续4代的选育,部分不利基因遭到淘汰,选育群体的遗传基础逐步得到纯化,基因型逐渐趋向纯合、稳定,经进一步的选育可望获得较稳定的品系。     

Dietary phosphorus requirement of large yellow croaker,Pseudosciaena crocea R

A study was conducted to estimate the optimum requirement of dietary phosphorus for large yellow croaker in floating sea cages (1.0 × 1.0 × 1.5 m). Five practical diets were formulated to contain graded levels (0.30%, 0.55%, 0.69%, 0.91% and 1.16%) of available phosphorus from dietary ingredients and monocalcium phosphate (MCP). Each diet was randomly assigned to triplicate groups of 180 juvenile fish (initial body weight, 1.88 ± 0.02 g). Fish were fed twice daily (5:00 and 17:00) to satiation for 8 weeks. During the experimental period, the water temperature fluctuated from 26.5 to 32.5 °C, salinity from 32‰ to 36‰ and dissolved oxygen was more than 7 mg l^− 1. Specific growth rate (SGR) significantly increased with increasing available phosphorus from 0.30% to 0.69% of diet (P < 0.05), and then leveled off. The body composition analysis showed that the whole-body ash and lipid, as well as phosphorus content in the whole body, vertebrae and scales were significantly affected by dietary available phosphorus level (P < 0.05). Broken-line analysis based on SGR indicated the minimum available phosphorus requirement for the optimal growth of large yellow croaker was 0.70%. Based on the phosphorus content in either vertebrae or whole body, the requirements were 0.89% and 0.91%, respectively.