利用EST—SSR座位对鲤鱼4种生长性状的单标记回归分析

获得与经济性状连锁的分子标记或基因是开展该性状分子育种的工作基础。本研究以大头鲤／荷包红鲤抗寒品系的雌核发育群体为试验材料,利用Windows Map Manager2．0的标记回归法进行QTL单标记定位分析,从70个微卫星座位对吻长、头长、尾长、眼间距性状的标记回归研究中,共得到5个标记与性状相关。其中HLJE190、HLJE217、HLJE268与吻长相关,HLJE1与头长、尾长、眼间距相关;C88433与头长、眼间距相关。将C88433通过BLAST比对,结果显示与斑马鱼上控制V型离子泵（ATPase）的基因相关。     

利用EST-SSR座位对鲤鱼4种生长性状的单标记回归分析

获得与经济性状连锁的分子标记或基因是开展该性状分子育种的工作基础.本研究以大头鲤/荷包红鲤抗寒品系的雌核发育群体为试验材料,利用Windows Map Manager 2.0的标记回归法进行QTL单标记定位分析,从70个微卫星座位对吻长、头长、尾长、眼间距性状的标记回归研究中,共得到5个标记与性状相关.其中HLJE190、HLJE217、HLJE268与吻长相关,HLJE1与头长、尾长、眼间距相关;C88433与头长、眼间距相关.将C88433通过BLAST比对,结果显示与斑马鱼上控制V型离子泵(ATPase)的基因相关.     

鲤与生长性状相关的EST-SSRs标记筛选

以大头鲤(Cyprinus pellegrini Tchang)(母本)与荷包红鲤(Cyprinus carpio wuyuanensis)抗寒品系(父本)杂交产生的F1作为亲本,以其自交F2作为分离群体,结合"拟测交"策略,用EST-SSRs标记对其中92个个体进行基因型检测.利用Windows Map Manager 2.0的标记回归法对符合拟测交分离的70个EST-SSRs标记进行单标记回归分析,检测出8个与鲤体长、体高、体厚性状相关的标记.对同一标记不同基因型个体间生长性状进行显著性比较,通过t检验,找到与生长性状相关的基因型.将上述8个EST序列与GenBank数据库进行BLAST比对,有3条EST序列与蛋白库中已知功能的序列高度同源.其中HLJE225与编码斑马鱼(J9enio rerio)冷诱导基因RNA结合蛋白的基因同源性水平高达97%,HLJE222与编码斑马鱼DAZ相关蛋白1基因同源性水平也高达97%,HLJE599与编码斑点叉尾鲴(Ictalurus punctatus)核糖体蛋白L6基因的同源水平为87%.本研究鉴定的与鲤生长性状相关的功能基因及有利用价值的基因型,为鲤重要生长性状的QTL(数量性状基闪座)定位和分子标记辅助育种提供了一定的依据.[中国水产科学,2009,16(1):15-22]     

北极茴鱼形态性状对体重影响效果分析

为探明北极茴鱼(Thymallus arcticus)形态性状与体重的关系, 以 1⁺ ~3⁺ 龄北极茴鱼为研究对象, 测定了体重(Y) 和体厚(X₁)、眼间距(X₂)、体长(X₃)、体高(X₄)、头长(X₅)、眼径(X₆)、吻长(X₇)、尾柄长(X₈)、尾柄高(X₉)等 9 个形态性状, 利用相关分析、通径分析和多元回归分析等方法, 筛选出影响北极茴鱼体重的主要形态性状, 并建立回归方程。结果显示: (1) 不同年龄阶段, 与北极茴鱼体重显著相关(P＜0.05)的形态性状种类不同, 且数量也随着年龄的增加而降低。(2) 通径分析在 1⁺ ~3⁺ 龄北极茴鱼中分别保留了 4、4 和 2 个形态性状, 1⁺ 龄北极茴鱼中体长(X3)的直接作用最大(0.345), 尾柄高(X9)的间接作用最大(0.745); 2⁺ 龄北极茴鱼中体高(X4)的直接作用最大(0.473), 体厚(X1)的间接作用最大(0.378); 3⁺ 龄北极茴鱼中体厚(X₁)的直接作用最大(0.635), 尾柄高(X₉)的间接作用最大(0.344)。(3) 通径分析保留的形态性状对 1⁺ ~3⁺ 龄北极茴鱼体重的总决定系数较高, 分别为 0.943、0.778 和 0.997。(4) 多元回归分析拟合出 1+ 龄北极茴鱼形态性状(Xi)与体重(Y)回归方程为 Y=?90.510+15.345X₁+3.638X₃+10.473X₄+16.884X₉, 2+ 龄北极茴鱼形态性状(Xi)与体重(Y)回归方程为 Y=?142.449+29.023X₁+81.082X₂+27.126X₄?47.376X₇, 3⁺ 龄北极茴鱼形态性状(Xi)与体重(Y)回归方程为 Y=?228.922+75.063X₁+107.864X₉。本研究丰富了北极茴鱼基础生物学数据, 同时为将来人工养殖利用过程中北极茴鱼的选择育种提供待选形态性状。     

利用SSR﹑EST-SSR、SNP标记对鲤食物转化率、体厚、体质量3种性状的分析

利用Windows Map Manager2.0的标记回归法对鲤(Cyprinus carpio L.)F2群体进行单标记定位分析.用91个SSR标记、33个EST标记、364个SNP标记对鲤进行全基因组扫描,结果得到与食物转化率、体厚、体质量3个性状显著相关(P＜0.05)的标记,分别为27、35、27个,其中与食物转化率相关的标记SNP0041、SNP0044,其相关性达到极显著水平(P＜0.001),贡献率分别达到15%、16%.这些标记可作为分子标记辅助育种的参考标记.将得到的与食物转化率显著相关的EST座位HLJEl4、HLJE253和与体质量显著相关的HLJE253座位在NCBI上进行BLAST比对,结果显示HLJE253与斑马鱼上编码ORF2结构蛋白的基因相关,相关程度达61%.将得到的与3种性状相关的SNP标记在NCBI上进行序列查找,通过Blast比对找到相关的基因,并对可能的功能作了注释.     

大口黑鲈形态性状对体重的影响效果分析

对大口黑鲈全长、体长、体高、体宽、眼间距、头长、吻长、尾柄长、尾柄高和体重共 10个性状进行测定,运用相关分析、通径分析和多元回归分析,剔除与体长存在显著共线性的全长、体高、头长,尾柄高及回归方程中不显著的吻长和尾柄长,计算以体宽、体长、眼间距 3个形态性状为自变量,体重为依变量的相关系数、通径系数、决定系数及相关指数,定量分析大口黑鲈形态性状对体重的影响效果。结果显示,3个形态性状与体重的相关系数（0.942,0.979,0.928）均达到极显著水平（P＜0.01）;通径分析中,3个形态性状对体重的通径系数亦达到极显著水平（P＜0.01）,它们是直接影响体重的重要指标,其中体宽（P⁴=0.599）对体重的直接影响最大。所选形态性状与体重的相关指数R²＝0.980 ,说明所选性状是影响体重的主要形态性状。应用逐步多元回归分析建立了以体重为依变量(Y),体宽(X⁴)、体长(X²)和眼间距(X⁵)为自变量的回归方程：LgY=1.065 + 0.765 LgX²＋1.441 LgX⁴+0.543 LgX⁵。以上形态性状对体重影响效果相关数据的获得为大口黑鲈选育测量指标的确定提供了理论依据。     

盐度驯化对史氏鲟鳃Na+/K+ ATP酶活力、血清渗透压及离子浓度的影响

对史氏鲟在盐度驯化过程中鳃Na⁺/K⁺ATP酶活力、血清渗透压及血清离子（Na⁺、K⁺、Cl^-）浓度进行了检测和分析，探讨了史氏鲟驯化过程中血清渗透压调节机制。研究表明：史氏鲟在不同盐度（10、20、25）下经过驯化，鳃Na⁺/K⁺ATP酶活力显著高于对照组鳃Na⁺/K⁺ATP酶活力(P<0.05），其活力是对照组的2～2.5倍。驯化过程中，3种不同盐度阶段下鳃Na⁺/K⁺ATP酶活力首先表现为下降，随着驯化时间的延长，活力逐渐增加，最后下降并趋于平稳。血清渗透压也随盐度的增加而上升，盐度10时最高，达到(328.77±26.78) mmol·kg^-1，此后逐渐下降并稳定在290 mmol·kg^-1左右，略高于淡水中血清渗透压。不同盐度下，血清渗透压和鳃Na⁺/K⁺ATP酶活力的变化趋势相同。3种不同盐度下史氏鲟血清K⁺浓度平均值保持在3.00～3.30 mmol·L^-1之间，与对照组相比无显著差异（P>0.05）。3种盐度下血清Na⁺和Cl^-浓度变化趋势基本一致，随着盐度的增高而增高，盐度20时达到最高。盐度20以下血清Na⁺和Cl^-含量没有显著差异（P>0.05）。史氏鲟血清渗透压调节可以分为3个阶段：一是应激反应阶段，主要表现为鳃Na⁺/K⁺ATP酶活力受到抑制，陡然下降；二是主动调节阶段，鳃Na⁺/K⁺ATP酶被重新激活，且活力逐渐上升；三是适应阶段，鳃Na⁺/K⁺ ATP酶趋于平稳。     

饲料中添加硫酸锌对奥尼罗非鱼幼鱼生长合机体抗氧化功能的影响

试验以硫酸锌为锌源,评价不同锌水平对奥尼罗非鱼幼鱼（Oreochromis aureus♂×Oreochromis niloticus♀）生长和抗氧化能力的影响。体质量为(4.13±0.32)g罗非鱼随机分配在18个水族箱中,每箱20尾,每3个箱为1个处理组,分别以添加锌为0、20、40、80、160和320 mg•kg^-1的6种饲料投喂,日投喂率为鱼体重5%～9%。8周的试验结果表明：20mg•kg^-1锌饲料组的增重率和鱼体蛋白含量显著高于其它各组(P＜0.05),蛋白质效率、饲料转化率也明显高于其它各组;全鱼脂肪含量随着饲料锌水平的升高而升高,但320mg•kg^-1锌饲料组降低;20mg•kg^-1锌饲料组,脊椎骨中锌离子浓度达到最大值且显著高于0mg•kg^-1锌饲料组(P＜0.05);20mg•kg^-1与40mg•kg^-1锌饲料组的血液中红细胞数量显著高于其它各添加组(P＜0.05),20mg•kg^-1锌饲料组的血液中血比容显著高于0mg•kg^-1组(P＜0.05),与其它各组没有显著差异;肌肉中,硫代巴比妥酸反应产物(TBARS)(Thiobarbituricacid reactive substances)的量,锌为0mg•kg^-1饲料组显著高于其它各组(P＜0.05)。综上所述,饲料中锌添加量为20mg•kg-1饲料促进了罗非鱼生长,使鱼体抗氧化功能增强。     

西沙群岛珊瑚礁海域鱼类分类学多样性

为评价南中国海西沙群岛珊瑚礁海域鱼类分类学多样性和更好地保护其鱼类,根据西沙群岛珊瑚礁海域鱼类物种组成的历史资料对西沙群岛及其16座岛礁的珊瑚礁水域鱼类物种数目、平均分类差异指数和16座岛礁的G-F多样性指数作了分析。结果显示,西沙共记录鱼类661种,隶属于25目、93科、305属。其中,永兴岛(351种),华光礁(173种),东岛(160种)记录鱼类物种数目较多。西沙鱼类平均分类差异指数(Δ⁺)约为60.2,各岛礁的Δ⁺与物种数目无关。中建岛、晋卿岛、玉琢礁等岛礁鱼类平均Δ⁺显著低于西沙鱼类Δ⁺。西沙鱼类Δ⁺小于陆架和河口区鱼类Δ⁺,说明珊瑚礁海域鱼类亲缘关系比陆架和河口区域的都要接近,此外大尺度上,一般纬度越低,中国海洋鱼类Δ⁺越小。但G-F多样性测度各指数由物种数目的大小决定,一般物种数目较大的岛礁G-F多样性各指数也相对较高,而R_G-F指数更适合该海域鱼类多样性的测度。以石岛为例初步探讨了G-F指数为负的原因,一是物种数目较少,二是多样性测度的关键函数Y=-XLnX并不是单调函数所致;物种数目较大的岛礁如永兴岛、华光礁、东岛等应该成为西沙珊瑚礁海域鱼类保护的重点区域。     

鲤饲料转化率性状的关联分析及优异等位变异挖掘

饲料转化率是鲤养殖重要的亟待改良的经济性状之一。本研究利用分布于鲤全基因组的1 536个SNP标记,分析68尾全同胞家系镜鲤Cyprinus carpio的基因分型,采用TASSEL软件的GLM和MLM模型检测与饲料转化率性状紧密关联的标记位点,发掘相关位点内的优异等位变异。本研究共获得17个与饲料转化率性状显著关联(P0.05)的SNP标记,贡献率在6.4%~16.6%之间,分布于鲤基因组第2、6、11、16、20、32、33、41,和42染色体以及Scaffold1032、1078和2323上,注释了一批饲料转化率相关的候选基因。从显著关联的标记位点内发掘了16种优异等位变异,其平均表型效应值较群体均值高6.2%,较非优势等位变异高12.5%。其中,SNP0919、SNP0167和SNP1016 3个标记的优异等位变异增减效差异最大,暗示这些标记对性状的选择效果明显。本研究发掘的饲料转化率相关的标记及优异等位变异可为鲤饲料转化率性状的分子标记辅助选择及分子设计育种提供依据。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.