尼罗罗非鱼微卫星标记与主要生长性状的相关性分析

为了筛选到与尼罗罗非鱼生长相关的分子标记,并对这些标记进行准确性鉴定,运用65个微卫星标记对鹭业和番禺2个尼罗罗非鱼群体进行了PCR扩增,再利用SPSS软件一般线性模型(GLM)对这些微卫星位点与尼罗罗非鱼体重等主要生长性状进行了标记性状连锁关联分析。结果表明,鹭业群体中有8个微卫星标记(UNH130,UNH183,UNH911,GM558,UNH211,UNH176,UNH914和UNH974)与主要生长性状显著或极显著相关(P<0.05或P<0.01),其中有2个微卫星标记(UNH914和UNH974)与番禺群体的主要生长性状显著或极显著相关(P<0.05或P<0.01),1个微卫星标记(UNH176)只与体重显著相关(P<0.05)。通过对与生长性状相关标记的基因型和表型值进行多重比较,得到了对体重、体长和体高3种性状有利的基因型或等位基因。发现了对罗非鱼生长性状有显著效应的微卫星位点,为开展罗非鱼的分子标记辅助育种提供了有价值的遗传标记。     

罗非鱼性别相关微卫星标记的初步筛选

为分析罗非鱼群体的遗传多样性以及筛选与罗非鱼性别相关的微卫星标记。应用24对微卫星引物,使用常规PCR及聚丙烯酰胺凝胶电泳的方法在两个尼罗罗非鱼(Oreochromis niloticus)群体和两个奥利亚罗非鱼(O.aureus)群体中初步筛选到UNH931、GM128、GM201、GM258、GM597以及UNH898共6个与性别相关的微卫星标记。然后使用降落PCR以及毛细管电泳的方法在两个尼罗罗非鱼群体、两个奥利亚罗非鱼群体以及ZY 1、WY 1、YY 1和YY 2型罗非鱼群体中进一步扩增这6个微卫星标记,统计各群体的遗传多样性参数:6个微卫星标记在上述群体共297个样本中检测到95个等位基因,其大小在97~302 bp之间,各位点在各群体等位基因数在1~13个之间,各群体平均观测等位基因数为2.167~9.333,平均有效等位基因数为1.624~4.966,平均观测杂合度为0.324~0.983,平均期望杂合度为0.329~0.782,平均多态信息含量为0.275~0.753;两个尼罗罗非鱼群体、WY 1和YY 2群体达到高度多态(PIC>0.5),两个奥利亚罗非鱼群体、ZY 1和YY 1群体为中度多态(0.25相似文献   

橙色莫桑比克罗非鱼微卫星遗传多样性分析及其与尼罗罗非鱼差异位点的筛选

用33对在尼罗罗非鱼（Oreochromis niloticus）中能有效扩增的微卫星引物,对橙色莫桑比克罗非鱼（Oreochromis mossambicus）进行PCR扩增,结果有32对引物能获得稳定的特异性条带,占总数的97．0％,其中15个微卫星位点具多态性,表明大部分尼罗罗非鱼的微卫星位点存在于橙色莫桑比克罗非鱼中。用具多态性的15个微卫星位点,对橙色莫桑比克罗非鱼30尾个体进行扩增分析,结果共检测到44个等位基因,大小在113～232bp之间,平均多态信息含量（PIC）为0．4308,平均观测杂合度（鼠）为0．5489,平均期望杂合度（垃）为0．5248,个体间平均遗传距离为0.3132,表明所选橙色莫桑比克罗非鱼群体遗传多样性较丰富,种群结构比较合理。本研究还对尼罗罗非鱼和橙色莫桑比克罗非鱼间特异位点进行了分析,发现有7个位点（UNH899、UNH208、UNH853、UNH876、UNH222、UNH933、UNH773）可有效区分莫桑比克罗非鱼和尼罗罗非鱼,可作为罗非鱼种质鉴定的分子标记。[中国水产科学,2008,15（3）：400-406]     

吉富罗非鱼微卫星标记与体质量、体形性状相关性分析

根据罗非鱼微卫星遗传图谱,在不同的连锁群上随机选择30个微卫星位点,研究它们在192尾不同家系吉富罗非鱼(Genetically Improved Fanned Tilapia,GIFT)中的遗传多样性及这些位点与体质量、体形性状的相关性.结果表明,30个位点共检测出126个等位基因,片段长度124～310 bp.各位点等位基因数为1～7个,平均等位基因数4.2个;多态信息含量(P1C)为0.259 4～0.716 7,平均0.580 8;杂合度(H)为0.306 4～0.753 1,平均0.632 2,表明该吉富罗非鱼群体遗传多样性比较丰富.最小二乘方差分析结果显示,位点GM041、GM542和GM304与吉富罗非鱼雌鱼体质量相关;位点GM041、UNH860、GM519和GM304与雄鱼体质量相关,位点UNH952与雄鱼体形相关.位点GM041、UNH860、GM304的AA基因型和位点GM519的DE基因型都有可能作为以体质量为选育目标的辅助标记,其中UNH860和GM304的AA基因型有望成为作为以体质量为选育目标的首选辅助标记.实验结果说明,吉富罗非鱼具有与体质量相关的标记越多,体质量也越大.     

吉富品系尼罗罗非鱼抗病性状分子标记筛选及遗传多样性分析

文章采用微卫星标记技术,对吉富品系尼罗罗非鱼(GIFT strain Oreochromis niloticus)抗病群体和易感病群体进行了遗传差异分析。结果显示:在30个微卫星位点中20个能扩增出多态性高、重复性好和扩增条带清晰;抗病群体和易感病群体的平均等位基因数分别为2.5和2.4个;观测杂合度和期望杂合度分别为0.476和0.453,0.44和0.457;多态信息含量PIC分别为0.359和0.379;UNH845和UNH189位点在抗病群体中偏离Hardy-Weinberg平衡(P0.05),GM180位点在易感病群体偏离Hardy-Weinberg平衡,两个群体间的遗传距离和遗传分化指数0.055和0.054。此外GM251和GM462位点的差异等位基因片段(135bp和198bp)与抗病性状存在一定的连锁关系。以上结果表明,吉富品系尼罗罗非鱼抗病群体与易感病群体之间的遗传多样性差异不明显,筛选出2个与抗病性状相关的微卫星标记,为开展吉富品系尼罗罗非鱼抗病品种分子标记辅助选育奠定了基础。     

荧光原位杂交技术在鱼类基因定位研究中的应用

荧光原位杂交fluorescence in situ hybridization,FISH）技术是一种简单而有效的染色体上物理定位DNA序列的技术。本文介绍了荧光原位杂交技术的基本原理以及实验流程,综述了近年来FISH技术在鱼类基因定位方面的应用,如来源于细菌人工染色体（bacterial artificial chromosome,BAC）文库的单拷贝基因的定位、核糖体基因和组蛋白基因等中度重复序列的定位、着丝粒特异序列和性别特异序列等高度重复序列的定位以及其他重复序列的定位等,用于研究特定基因定位、性染色体鉴定及种间杂交等遗传学问题,并展望了此技术在定位鱼类经济性状相关标记或基因、性别相关标记或基因及种特异染色体标记中的应用前景。     

WY♀-YY♂型罗非鱼新品种选育和生物学研究

为获得罗非鱼新品种和创建尼罗型WY♀-YY♂繁育体系,采用不断回交的技术路线进行选育种。试验中罗非鱼新品种由奥利亚罗非鱼(Oreochromis aurea)与尼罗(Oreochromis Niloticus)超雄鱼杂交和回交选育而来。它由尼罗鱼的常染色体(N)、性染色体(Y)和奥利亚罗非鱼的性染色体(W)构成遗传基础,并形成NNWY♀-NNYY♂繁育体系。本实验主要介绍新品种的选育过程,同时对新品种鱼进行生物学研究,以期得到其一系列相关特性。     

尼罗罗非鱼♀×萨罗罗非鱼♂杂交后代的遗传分离分析

应用微卫星标记对尼罗罗非鱼(Oreochromis niloticus)♀×萨罗罗非鱼(Sarotherodon melanotheron)♂杂交后代(F1、F2)的遗传分离情况进行了初步研究.在86对微卫星引物中,筛选出21对在尼罗罗非鱼和萨罗罗非鱼中存在差异等位基因的特异性引物,共检测出59个等位基因,包含32个尼罗罗非鱼和24个萨罗罗非鱼特异等位基因.F1观测杂合度(Ho=0.998)大于 F2(Ho=0.739), F2有效等位基因数(Ne=2.48)、平均多态信息含量(PIC=0.497)与 F1的(Ne=2.45、PIC=0.489)相似.F1中所有位点均表现为尼罗罗非鱼与萨罗罗非鱼杂合基因型.F2中位点等位基因分离情况各异,通过卡方检验,有18个位点符合孟德尔遗传(P>0.05),2个位点GM145、UNH003表现为偏离孟德尔遗传(P<0.05),1个位点GM276不分离.在18个孟德尔遗传位点中, F2中尼罗罗非鱼特异等位基因频率为48.3%,萨罗罗非鱼特异等位基因频率为47.7%;基因型出现F1型、尼罗罗非鱼型、萨罗罗非鱼型的比例分别为58.2%、19.9%、21.9%.结果表明, F2较好地继承了F1的遗传杂合性,但也存在一定程度的分离.     

ZY异配型超雄罗非鱼的人工产生

罗非鱼 (Ｏｒｅｏｃｈｒｏｍｉｓｓｐｐ )的雄鱼比雌鱼长得快、长得大 (叶冀雄 ,1983) [1] 。有两种方法可生产全雄或超雄罗非鱼用作商品鱼 :其一是激素法(马仲波 ,1984 ;胡逸农等 ,1979) [2 ,3] ,在鱼苗开始吃食时人工哺拌有甲基睾丸酮的饲料以促使雌鱼雄性化 ,但是对人体和生态环境的影响受到质疑 ;另一种是通过杂交法产生 (江山 ,1984 ) [4 ] ,如奥利亚雄鱼 (性染色体组成是ＺＺ)与尼罗罗非鱼雌鱼 (性染色体组成是ＸＸ)杂交 ,由于染色体Ｚ的雄性性别决定基因强于Ｘ染色体上的雌性基因 ,所以其子代鱼应该全是ＺＸ型雄鱼 ,但如果其它染色体…     

黄姑鱼染色体识别与重复序列定位

黄姑鱼是我国重要的海水经济鱼类。然而,由于细胞遗传标记匮乏,黄姑鱼染色体仍然难以辨识。为了提高黄姑鱼染色体的配对识别水平,本研究利用荧光原位杂交(fluorescence in situ hybridization,FISH)、吉姆萨染色和荧光染色技术分析了黄姑鱼染色体的特征。以总DNA为探针进行基因组DNA荧光原位杂交(genomic fluorescence in situ hybridization,GISH),从而获得黄姑鱼染色体图谱,可使每对染色体呈现特定的荧光信号。依据GISH荧光信号分布模式,可以辨识黄姑鱼的24对染色体。18S r DNA FISH结果显示,18S r DNA只有一对信号,分布于1号染色体臂间,并与吉姆萨染色呈现的次缢痕、DAPI阴性带和DPI染色高亮区域同位。5S r DNA有一强一弱两对信号,信号强的一对分布于1号染色体着丝粒端,信号弱的一对分布于4号染色体的远端。端粒信号在所有染色体的端部显示,但个别染色体一端信号微弱。本研究结果丰富了黄姑鱼的细胞遗传标记,为解决黄姑鱼染色体辨识问题提供参考依据,也为进一步研究石首鱼科染色体进化提供了资料。     

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).     

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.     

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.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.