罗非鱼微卫星DNA指纹图谱的构建

采用微卫星DNA指纹图谱技术对奥利亚罗非鱼、尼罗罗非鱼和橙色莫桑比克罗非鱼进行品种鉴定和遗传多样性分析。从103对罗非鱼微卫星引物中筛选出82对多态性高、带型清晰稳定的引物,用这 82对引物检测3种罗非鱼的遗传结构,共检测到605个等位基因,平均等位基因数7.37个,数据经Popgen32计算和EXCEL工具作图,构建3种罗非鱼的DNA指纹数据库,并绘制了DNA指纹图谱模式图。结果显示,奥利亚罗非鱼、尼罗罗非鱼和橙色莫桑比克罗非鱼的平均观测杂合度分别为0.179 6、0.530 1和0.312 2,平均期望杂合度分别为0.203 2、0.678 6和0.291 3,平均多态信息含量分别为0.075 4、0.608 3和0.152 8,由此可见,尼罗罗非鱼群体的遗传多样性水平最高,奥利亚罗非鱼群体遗传多样性最低。同时筛选得到16对具特异性条带的核心引物组合可将3种罗非鱼完全区分,每个位点可在其中一种罗非鱼中扩增出独有的条带,从而将这种罗非鱼与其它两种区分开来,将16对特异引物的图谱数据转化成计算机可以识别的数码指纹,可以方便地应用于罗非鱼及其杂交种的鉴定研究。为解决罗非鱼品种混杂遗传渐渗问题和保护罗非鱼种质资源提供技术支撑。     

尼罗罗非鱼与萨罗罗非鱼及其正反杂交后代的微卫星分析

微卫星（microsatellite）技术是近年来广泛应用的DNA标记,具有重复性好、较易操作和共显性的特点。在硬骨鱼类中表现出高度多态性,已在家系鉴定、遗传作图、群体遗传分析、育种以及系统发生研究等多个领域得到应用。尼罗罗非鱼（Oreochromis niloticus）是我国主要淡水养殖鱼类,生长快,耐盐能力一般;萨罗罗非鱼（Sarotherodon melanotheron）耐盐能力强,适合咸淡水养殖,但生长较慢。[第一段]     

中华鳖群体DNA指纹分析中的RAPD技术优化

以中华鳖(Pelodiscus Sinensis)4种不同区域代表性群体黄河鳖、太湖鳖、台湾鳖和日本鳖为研究材料,采用不同反应条件(如退火温度和循环次数等)对RAPD扩增技术进行了优化研究,结果表明,在条件优化后(退火温度为38℃,循环次数为40次)有20个RAPD引物在4个群体中均有扩增条带,其中随机引物S105能将黄河鳖群体和太湖鳖、台湾鳖、日本鳖群体区分开;随机引物S327能将日本鳖群体鉴别出来;而随机引物S474可将台湾鳖群体与其他群体分开.由此表明,退火温度和循环次数的优化有利于中华鳖RAPD鉴定技术的建立.本研究旨在为中华鳖的种质鉴定与保护提供技术支撑.     

微卫星标记在坛紫菜丝状体品系DNA指纹构建中的应用

用从条斑紫菜EST数据库中筛选合成的微卫星引物对8个坛紫菜丝状体品系进行微卫星DNA指纹扩增。5个微卫星引物共扩增出32条带,其中3对引物所扩增出的5个条带(AU192094—127、AU187410—335、AU187410—190、AU194267—203和AU194267—328)被用来构建8个坛紫菜丝状体品系的DNA指纹。在这个图谱中,每个丝状体品系都有独一的指纹模式,彼此很容易被区分开。所获得的DNA指纹图谱,可用来进行孟德尔分离研究,及为坛紫菜纯系鉴定提供分子基础。     

动物亲权鉴定的几种方法

本文阐述了DNA指纹、STR、线粒体DNA技术在动物亲权鉴定中的应用原理。分析了这三种方法在动物亲权鉴定中的优点及不足。     

不同鳊鲂鱼类群体微卫星DNA指纹图谱的构建和遗传结构分析

为对不同鳊鲂鱼类进行群体鉴定和遗传多样性分析,从60对微卫星标记中筛选出18对多态性高的引物,构建了6个鳊鲂鱼类群体的微卫星DNA指纹图谱。结果显示,东江三角鲂、钱塘江三角鲂、厚颌鲂、广东鲂、团头鲂和长春鳊6群体的平均等位基因数(N a)分别为5.17、6.11、3.50、6.56、5.22、5.22,平均期望杂合度(H e)分别为0.634 2、0.720 4、0.546 2、0.681 2、0.675 2、0.559 7,平均多态信息含量(PIC)分别为0.575 6、0.666 9、0.472 0、0.630 6、0.606 4、0.517 0,表明钱塘江三角鲂的遗传多样性最高,厚颌鲂的遗传多样性最低;聚类分析表明,钱塘江三角鲂和团头鲂首先聚为一支,遗传距离较近,为0.560 6;厚颌鲂与长春鳊的遗传距离最远,为1.759 2。引物Mam03和EST37产生的特异条带可将鲂属和鳊属鱼类区分,鉴定出鳊属鱼类长春鳊;引物TTF3、EST37、TTF2/TTF10、EST66依次组合可区分出鲂属东江三角鲂、厚颌鲂和广东鲂这3个群体。研究结果为我国鳊鲂鱼类种质资源保存、种群鉴定和良种选育奠定了基础。     

牙鲆家系亲权鉴定的微卫星DNA标记分析

为准确进行不同家系的亲权鉴定,筛选具有高亲本排除概率的微卫星DNA标记,从牙鲆选育基础群体中挑选性腺发育良好的雌雄亲本各10尾建立全同胞家系10个,从独立饲养的每个家系中随机选取30尾个体组成系谱结构已知的家系群体,从混合培育的子代群体中随机选取200尾个体组成系谱结构未知的混合群体。48个微卫星DNA标记选自牙鲆第二代遗传连锁图谱,且均匀分布于24个连锁群上,每个连锁群2个标记。家系群体的遗传分析结果发现,10个拥有丰富遗传多态性的微卫星DNA标记表现出高的亲本排除概率,其Excl1和Excl2的范围分别为0.655~0.719和0.792~0.837。随鉴定所用微卫星DNA标记数目的增加,累计排除概率逐步升高。当使用8个微卫星DNA标记鉴定时,累计排除概率达到100%。利用这些标记开展混合群体的亲权鉴定,显示共有13个雌雄个体参与繁殖过程,不同亲本配组产生的后代数量存在差异,亲本与后代群体之间的各项遗传学统计指标不存在明显差异。研究表明,筛选出具有高亲本排除概率的微卫星DNA标记能够有效进行牙鲆家系的亲权鉴定,可以作为今后开展家系选育与DNA分子标记联合育种的候选标记。     

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]     

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ZY₁、WY₁、YY₁和YY₂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、W...     

14个尼罗罗非鱼家系遗传差异的微卫星标记分析

利用12个微卫星标记分析14个尼罗罗非鱼Oreochromis niloticus家系的遗传多样性,以P_0代为对照,研究P_4、P_5代家系的遗传多态性,指导尼罗罗非鱼的选育。研究结果表明:14个罗非鱼家系的平均有效等位基因数、平均杂合度、平均多态信息含量分别为3.1819、0.6276、0.5753,其遗传多样性丰富;家系N312与N314遗传距离最小(0.1214),而家系N304与N306遗传距离最大(0.3137),14个家系间的平均遗传距离为0.1973;UP_GMA进化树分析结果表明,14个尼罗罗非鱼家系聚为4个分支,第一分支为家系N303,第二分支由N301、N306和N311组成,第三分支由N302、N308和N310组成,其余家系组成第四分支;P_4代尼罗罗非鱼群体遗传多态性比P_0代小幅降低,而构建P_5代家系时参考了微卫星标记的分析结果,其遗传多态性明显高于P_0和P_4代。结果表明:参考微卫星标记计算的家系间的亲缘关系,人为控制选配提高后代稀有等位基因的频率和杂合子的比例,可以有效防止近交衰退,维持较高的种群遗传多样性和选择潜力。     

溢油的环境法医学鉴定:化学指纹分析与源识别

我国海洋溢油事故频发,给海洋环境带来重大损害。然而,目前海洋溢油环境污染诉讼因存在取证困难等一系列问题,使得利益相关者的合法权益得不到合理赔偿。环境法医学在溢油源识别中的应用对于因海洋溢油污染问题引起的环境责任追溯将起到至关重要的作用。本文通过对环境法医学在溢油源识别中的应用意义、溢油指纹鉴定原理和常用指纹特定目标物及新型源识别方法的综述,得出了溢油指纹分析较好的指示物以及溢油源识别的方法,并对未来溢油的环境法医学鉴定提出建议。     

Influence of the anaesthetic quinaldine on some tilapias

Three species of tilapia, Sarotherodon melanotheron and Sarotherodon guineensis from brackish water and Oreochromis (Sarotherodon) niloticus from fresh water, were exposed to different concentrations of the anaesthetic quinaldine to determine the safe level for handling and transportation of these species. It was observed that the time taken to lose balance increased with a decrease in the concentration of the anaesthetic. This was probably due to the very low concentration of the hydrogen ions in the progressively diluted test media. Dosages of quinaldine for handling fish for experimental work and for transportation are given with the proviso that the anaesthetization is carried out under conditions of salinity and temperature which are suitable for acclimation.     

The effects of introduced tilapias on native biodiversity

• 1. The common name ‘tilapia’ refers to a group of tropical freshwater fish in the family Cichlidae (Oreochromis, Tilapia, and Sarotherodon spp.) that are indigenous to Africa and the southwestern Middle East. Since the 1930s, tilapias have been intentionally dispersed worldwide for the biological control of aquatic weeds and insects, as baitfish for certain capture fisheries, for aquaria, and as a food fish. They have most recently been promoted as an important source of protein that could provide food security for developing countries without the environmental problems associated with terrestrial agriculture. In addition, market demand for tilapia in developed countries such as the United States is growing rapidly.
• 2. Tilapias are well‐suited to aquaculture because they are highly prolific and tolerant to a range of environmental conditions. They have come to be known as the ‘aquatic chicken’ because of their potential as an affordable, high‐yield source of protein that can be easily raised in a range of environments — from subsistence or ‘backyard’ units to intensive fish hatcheries. In some countries, particularly in Asia, nearly all of the introduced tilapias produced are consumed domestically; tilapias have contributed to basic food security for such societies.
• 3. This review indicates that tilapia species are highly invasive and exist under feral conditions in every nation in which they have been cultured or introduced. Thus, the authors have concluded that, despite potential or observed benefits to human society, tilapia aquaculture and open‐water introductions cannot continue unchecked without further exacerbating damage to native fish species and biodiversity. Recommendations include restricting tilapia culture to carefully managed, contained ponds, although exclusion is preferred when it is feasible. Research into culture of indigenous species is also recommended.

Copyright © 2005 John Wiley & Sons, Ltd.     

Genetic improvement of farmed tilapias: Biochemical characterization of strain differences in Nile tilapia

Four African wild strains (Egypt, Ghana, Senegal and Kenya) and four established Asian farmed strains of Nile tilapia, Oreochromis niloticus (popularly known in the Philippines as Taiwan, Thailand, Singapore and Israel) were analysed electrophoretically at 30 protein loci to estimate genetic differences among the strains. All strains shared alleles at 14 monomorphic and 16 variable loci. Among the African strains, characteristic allele frequency differences were observed at AAT-1 ^* 46 for Ghana and Senegal, ADH ^* 83 for Kenya, ADH ^* 120 for Senegal, G3PDH-2 ^* 300 for Egypt, IDDH ^* 67 for Senegal, sMDH-1 ^* 120 for Kenya and SOD ^* 150 for Senegal. Genetic distance values among the strains revealed a clustering of the farmed strains with Egypt and Ghana O. niloticus, a slight separation of the Senegal strain and a larger separation of the Kenya strain. This profile may reflect the origins of the few founder populations of this species previously introduced to Asia. It also confirms the wider genetic divergence of the Kenya strain (O. niloticus vulcani) from the others studied here, which are all O. n. niloticus. Observed heterozygosities of the strains ranged from 0.026 to 0.071, with the African wild strains the lower values (mean Ho = 0.036) and the farmed strains the higher ones (mean Ho = 0.056). The implications of these results to the ongoing tilapia genetic improvement programme in the Philippines are discussed.     

The pathology of Haplorchis pumilio (Looss, 1896) infections in cultured tilapias

Abstract. Haplorchis pumilio (Looss, 1896) is a common digenean parasite of many species of fish of economic importance and has been found to infect cultured Sarotherodon spp. Fry of S. spilurus (Günther), S. mossambicus (Peters) and S. galilaea (L.) were experimentally infected with cercariae of H. pumilio from natural infections of Melanoides tuberculata (Müller) from an East African fish farm. Large numbers of cercariae were lethal to the fry within a few hours. Histopathological examination showed that the most significant pathological effect was focal haemorrhages in skeletal muscles in heavily infected fish. Migration of cercariae was through connective tissue and the final location of the metacercariae was connective tissue associated with skeletal structures. Clusters of cysts occurred at the bases of fins and in the loose connective tissue of the head, but the reaction to encysted metacercariae was insignificant. The overall effect on growth performance and thus economic importance of H. pumilio infection was assessed. Growth studies showed no significant differences between specific growth rate, food conversion ratios and condition of infected and uninfected fish.     

Hormonal sex reversal of tilapias: implications of hormone treatment application in closed water systems

The phenomenon of incidental sex reversal was observed in experiments originally designed to determine the importance of feeding duration and feed quality in the optimization of masculinization and feminization treatments of Oreochromis niloticus (L.) fry by oral application of hormones. Four separate experiments were conducted: (1) masculinization and (2) feminization using three feeding durations and (3) masculinization and (4) feminization using three alternative feed mixtures. Masculinization experiments incorporating 17 α-methyltestosterone (MT) into the feed at 40 mg kg^?1 were applied to putative monosex male progeny whilst feminization treatments were applied to putative all female fry using diethylstilboestrol (DES) at 1000 mg kg^?1. Treatments were applied outdoors in small cages suspended in 900-l circular concrete tanks with a flow through rate of 1-l min^?1. Replicate cages for all treatments and the controls in each experiment were reared together in the same tank. Only the first experiment produced significant differences in sex ratio, with the control treatment producing a slightly lower proportion of males than the three MT treatments at different durations. The most significant finding was that in all experiments the sex ratio of the control differed significantly from expectations and in all but experiment 1, did not differ from those of the treated fish in which sex reversal had clearly been effected. It appears likely that incidental sex reversal occurred in the control fish as a result of the accumulation of active metabolites from the treated fish and/or leachates from uneaten food given to fish in the same water body. The results are discussed in the context of improving the efficiency and safety of hormonal sex reversal treatments.     

The life history of Haplorchis pumilio (Looss, 1896) from cultured tilapias

Abstract. The metacercaria of Haplorchis pumilio (Looss, 1896) (Digenea: Hetero-phyidae) was found to occur commonly in intensively reared tilapias ( Tilapia, Sarotherodon ) in an East African fish farm. The life cycle on the farm was found to involve the gastropod Melanoides tuberculata (Müller) as the first intermediate host. The infection was experimentally transmitted to fish in fresh water from naturally infected molluscs. The salinity of the water on the farm varied with season from 0 to 3%₀, but cercariae were shown to remain infective up to 10%₀ although longevity was reduced at 7.5%₀. Piscivorous predators, Ardea cinerea L., Phalocrocorax sp. and Varanus sp., which were resident or regular visitors to the farm were heavily infected with the parasite. Mice, hamsters and pigeons were found to be convenient laboratory hosts. H. pumilio shows a lack of specificity at the second intermediate and final host level as well as a wide geographic range and has been reported to infect man. Its potential as a possible cause of zoonoses, and control methods are discussed.     

罗非鱼温和气单胞菌病的病原研究和药敏试验

从患病濒死尼罗罗非鱼肝脏分离到99-5-A和99-7-28菌株.经细菌学鉴定均为温和气单胞菌(Aeromonas sobria).用这2菌株对尼罗罗非鱼、 奥利亚罗非鱼及尼奥罗非鱼(尼罗罗非鱼♀×奥利亚罗非鱼♂)进行人工感染,均可使其致病.发病鱼症状与自然病鱼症状一致,呈出血性败血症.再分离菌株的各种特性与原分离菌株相同,确认这2菌株皆为温和气单胞菌.采用纸片扩散法,用35 种药物对上述2菌株进行药物抑菌试验,其中,丁胺卡那霉素、庆大霉素、多粘菌素、 妥布霉素、氟哌酸、氯霉素、氨曲南抑菌效果最佳;99-5-A菌株对呋喃妥因敏感,但 99-7-28菌株对其耐药;99-7-28对复方新诺明敏感,而99-5-A菌株对其则无反应.     

新引进萨罗罗非鱼与其它8种罗非鱼耐盐差异

以萨罗罗非鱼、尼罗罗非鱼、以色列红罗非鱼和奥利亚罗非鱼自交和双列杂交后代共9种罗非鱼为实验材料,通过耐盐致死实验、半致死盐度实验和慢性耐盐驯化实验,对萨罗罗非鱼与其他几种罗非鱼耐盐生态特点的差异进行了比较分析,结果表明,(1)萨罗罗非鱼MST为(9.24±1.353)h,ST50为(6.20±1.235)h,MLS-96理论值为30.2,观察值为31.2,远高于另外8种罗非鱼。(2)根据耐盐能力,9种罗非鱼可分为4类:第一类是萨罗罗非鱼,耐盐极强,各项耐盐指标都远高于其他罗非鱼;第二类是以色列红罗非鱼和尼罗罗非鱼♀×奥利亚罗非鱼♂,耐盐强,离差值为正值;第三类是奥利亚罗非鱼、尼罗罗非鱼♀×以色列红罗非鱼♂、以色列红罗非鱼♀×尼罗罗非鱼♂、尼罗罗非鱼♀×奥利亚罗非鱼♂和奥利亚罗非鱼♀×尼罗罗非鱼♂,耐盐较强,离差值为负值;第四类是尼罗罗非鱼,耐盐较弱。(3)当进行慢性耐盐驯化时,萨罗罗非鱼在5d内,死亡率由0达到30%,以后缓慢增长,14～15d有个死亡率的高增长期,然后即进入缓慢增长期;第11天,盐度32.8时,死亡率达到50%,第39天,盐度为102时全部死亡。死亡率与驯化时间之间的线性回归关系为LnY=3...