应用RAPD标记对东方鲀属进行种类鉴别及其聚类分析

利用RAPD技术对红鳍东方鲀、假晴东方鲀、暗纹东方鲀和从日本引进的红鳍东方鲀4个种进行了遗传标记鉴别研究。在事先优化的条件下，20个随机引物共扩增出134条谱带清晰、重复性高的DNA片段，其中多态性片段77条。结果证实每一种均有其特异性扩增图谱，可作为种鉴定的依据；用UPGMA和NJ等方法进行聚类，构建的系统树与借助形态学和生化特征进行的传统分类结果一致。研究表明，RAPD作为一种新的分子标记，在海洋动物遗传鉴定方面具有巨大的潜力。     

基于标记系谱的红鳍东方鲀生长性状遗传分析

利用分子标记辅助家系选育进行红鳍东方鲀生长性状的遗传评估,由红鳍东方鲀基础群体中选择性腺发育良好的雌雄亲本各11尾建立全同胞家系11个,每个全同胞家系随机采集10尾个体组建全同胞家系群体,从混合培育的家系中选择相对较大的个体400尾组建混合选育群体。在红鳍东方鲀遗传连锁图谱上挑选44个均匀分布于22个连锁群上的微卫星DNA标记,每个连锁群有2个标记。全同胞家系群体的遗传分析结果显示,10个高亲本排除概率微卫星标记的Excl(1)和Excl(2)为0.58~0.662和0.736~0.797,14个低亲本排除概率标记的Excl(1)和Excl(2)为0.054~0.43和0.177~0.608,剩余20个中等亲本排除概率标记的Excl(1)和Excl(2)为0.467~0.575和0.641~0.732。利用拥有高亲本排除概率的微卫星DNA标记进行混合选育群体的亲权鉴定,结果显示:不同父母本繁殖产生的子代数量存在明显差异,父本M2和母本F4产生了124个子代,占子代个体总数的32.89%。根据亲权鉴定结果建立的系谱估计主要生长性状的遗传参数,不同日龄的体质量和体长遗传力估计值为0.17~0.21和0.15~0.18。研究表明,利用具有高亲本排除概率的微卫星DNA标记能够有效建立红鳍东方鲀系谱,进行生长性状的遗传参数估计,分子标记辅助家系选育可以作为红鳍东方鲀目标性状遗传改良的新方法。     

用RAPD技术探讨5种鲷科鱼类的亲缘关系

用RAPD技术对鲷科鱼类的黑鲷、真鲷、二长棘鲷、平鲷和黄鳍鲷进行亲缘关系的研究。在事先优化的反应条件下，在使用的60个随机引物中，有29个引物扩增出清晰稳定的片段，共计200条，大小在200～2500 bp之间。5种鲷均有其特异性扩增片段，可作为种类鉴别的依据。根据MEGA2.1软件的UPGMA和NJ程序构建的分子系统树显示：同属鲷属的黄鳍鲷和黑 鲷的亲缘关系最近，其次是二长棘鲷和真鲷，平鲷与二长棘鲷和真鲷的亲缘关系较近，与黄鳍鲷和黑鲷较远,黑鲷和二长棘鲷的亲缘关系最远。两种聚类的结果相一致。研究结果与借助形态学和生化特征进行的传统系统分析具有一致性。在40个个体之间遗传距离矩阵中，最大值达到0.8916。多数的遗传距离指数在0.50～0.85之间。通过分析，认为RAPD技术对分析属级间的亲缘关系具有一定的适用性。     

合浦珠母贝3个养殖群体的RAPD分析

利用RAPD标记技术检测了广西省北海市、海南省三亚市、广东省深圳市等 3个地理种群养殖合浦珠母贝(Pinctadamartensii)基因组DNA的多态性 ,并对其遗传结构进行了初步分析。从 4 0个随机引物中筛选出 13个10bp引物 ,它们在 3种群中共扩增出了 14 0条DNA片段 ,3种群共有片段 16条 ,DNA片段数分别为 5 8、91和 71条 ,多态性位点比例分别为 4 1 4 % ,6 5 0 %和 5 8 4 %。遗传距离分析表明 ,广西种群与广东种群的亲缘关系较近 ,而与海南种群亲缘关系较远。 13个引物中 ,引物S11和S3 58扩增出的 3种群指纹图谱差异显著 ,可作为 3种群鉴定的分子标记。     

红鳍东方鲀亲本群体遗传多样性的微卫星分析

从日本进口的红鳍东方鲀Takifugu rubripes受精卵发育至性成熟的120尾亲鱼群体中,选择22个分布于每个连锁群的微卫星标记进行了遗传分析,以分析红鳍东方鲀亲本群体的遗传多样性,并筛选出有效鉴定群体遗传特征的特异性微卫星标记。结果显示:等位基因数(Na)和有效等位基因数(Ae)的范围分别介于4.000~13.000和1.834~7.706之间。观测杂合度(Ho)、期望杂合度(He)和多态信息含量(PIC)的范围分别为0.173~0.977、0.456~0.872和0.427~0.857。Hardy-Weinberg遗传偏离指数(d)介于-0.746~0.344。群体内个体间的遗传距离在0.51~0.86之间,依据遗传距离的远近将全部个体分成4个群体,每个群体23~38个体。各项遗传参数表明:该实验群体具有较为丰富的遗传多样性,不同个体之间拥有相对较远的亲缘关系,可以根据个体间的遗传距离制定育种计划,建立家系进行选育研究。实验所选的22个微卫星标记具有高度多态性,可作为分析红鳍东方鲀群体遗传多样性的候选标记。     

两种东方鲀的GH基因单核苷酸多态性分析

根据红鳍东方鲀生长激素基因全序列,设计13对引物,结合单链构象多态性(SSCP)技术和克隆测序技术对野生红鳍东方鲀、养殖红鳍东方鲀和养殖暗纹东方鲀群体进行了遗传多样性分析.结果表明:东方鲀整个生长激素基因的6个外显子中没有多态性,在其内含子2、内含子5以及3′侧翼区发现有多处序列变异位点,包括碱基的缺失、插入和一些单核苷酸突变.这些遗传多态性的存在为从分子水平上对东方鲀的鉴定提供了一定的依据.群体遗传结构分析结果表明,红鳍东方鲀养殖群体的一些等位基因丧失,造成其遗传多样性显著下降,应及早加强对其种质的保护.     

一个奥利亚罗非鱼雌性特异性SCAR标记的建立

以奥利亚罗非鱼Oreochromis aureus为实验材料,通过对800多条随机引物的筛选,获得了1个奥利亚罗非鱼雌性特异性的、长度为1 488 bp的RAPD标记片段RAPD71699-1488,经琼脂糖凝胶电泳后回收、克隆、测序,并根据测序结果设计PCR特异引物,再经PCR条件优化,成功地将该RAPD标记片段转化为实验结果稳定、操作简便的SCAR标记,即SCAROaF1488。采用双重PCR技术,以mtDNA 16S rRNA基因片段为PCR扩增阳性对照,对该标记的有效性在两个群体共200个个体(雌、雄各100个)中进行验证。结果显示,标记检测结果与表型性别的符合度为100%。SCA-ROaF1 488标记的获得为奥利亚罗非鱼遗传性别鉴定及标记辅助选择提供了有效工具,为深入研究鱼类性别相关基因及性别决定机制提供了重要线索和新的思路。     

建鲤与兴国红鲤RAPD分子标记的研究

利用随机扩增多态DNA(RAPD) 技术对鲤鱼的两个品系, 即建鲤和兴国红鲤进行了遗传分析。在所用20 个10 碱基随机引物扩增产物中, 找到了建鲤与兴国红鲤品系间的RAPD分子遗传标记, 为鲤鱼主要品系的保存和保护以及品种的选育工作提供了一种新的技术手段和基础资料。     

7种珍珠贝RAPD鉴别标记的初步研究

采用RAPD分子标记技术对珠母贝属的大珠母贝、珠母贝、黑珠母贝、白珠母贝、合浦珠母贝、长耳珠母贝和珍珠贝属的企鹅珍珠贝的基因组DNA的特异性遗传标记进行分析。从21个OPM和S系列中筛选出4个引物,共扩增出57个位点,每条引物平均产生14·3个位点。扩增片段大小在250~2000bp间,平均每种贝每条引物产生4·9条带。其中引物S10对7种珍珠贝的RAPD产物呈现出物种的特异性,可同时将7种珍珠贝分开,其余引物可以将2种或2种以上的珍珠贝区别开来。引物S10可以作为种间鉴定的标记。     

红鳍东方鲀体型性状选育指标的综合判定

对红鳍东方鲀的18个体型性状进行了测定,并计算各性状的表型参数和相关系数,探讨应用主成分分析和聚类分析确定红鳍东方鲀体型性状的选育指标.结果表明,系统聚类分析与主成分分析的结果基本上是一致的,红鳍东方鲀体型性状选育的适宜指标中,可选择包括体重、体周长1、体长、眼后头长和眼径5个性状作为代表性的性状指标.研究结果为红鳍东方鲀体型性状的选育提供了参考资料.     

Screening and characterization of sex-specific DNA fragments in the freshwater fish matrinchã, Brycon amazonicus (Teleostei: Characiformes: Characidae)

The matrinch? Brycon amazonicus, a commercially important freshwater fish resource, has no heteromorphic sex chromosomes so far described. In the present study, we performed a screening of sex-associated DNA markers in this species, through the use of a random amplified polymorphic DNA (RAPD) assay and a genomic DNA restriction digestion analysis. DNA digestions evidenced no differences between sexes. Sixty-six random primers were used in pooled and individual DNA samples of males and females, and the analysis of the RAPD fingerprints revealed one female sex-associated band. Cloning and sequencing of this band led to the identification of two distinct DNA segments. While one of the isolated fragments showed a significant identity with a described protein gene (phosphatidylinositol glycan anchor biosynthesis, class W), the other fragment, composed of 535?bp, corresponds to a novel DNA marker. Further experiments were performed with this second DNA fragment in order to verify its sex-specificity. Data on dot blot hybridization, using total DNA of both sexes, confirmed its female-specificity in B. amazonicus. A primer set was designed based on its sequence data and used in PCR with DNA samples of this species, leading to diagnose the animals' sexes with a 100?% overall accuracy through a sequence characterized amplified region approach. No amplification results were found for two other species of the genus-B. orbignyanus and B. lundii. The obtained data can lead to the hypothesis that B. amazonicus may present heteromorphic sex chromosomes that should be in an early phase of differentiation.     

Morphological and genetic characteristics of Flavobacterium columnare isolates: correlations with virulence in fish

Variability in pathogenicity of Flavobacterium columnare makes disease treatment difficult because there is currently no way to easily recognize those strains that warrant aggressive treatments. In order to identify suitable virulence markers, 17 isolates of F. columnare were cultured from six different fish species. The DNA from all isolates was analysed using random amplified polymorphic DNA (RAPD). Bootstrap analysis of the RAPD data produced a tree with three major groups supported by bootstrap scores of 80-100%. Virulence of the isolates was determined by bath exposure of channel catfish, Ictaluruspunctatus (Rafinesque), and golden shiners, Notemigonus crysoleucas (Mitchill), to broth cultures of F. columnare. In channel catfish, 13 of 17 isolates produced 100% mortality within 48 h post-exposure. All isolates of cyprinid fish origin clustered in a single RAPD group. At least two of the four isolates that were not virulent in channel catfish were of cyprinid fish origin. There was a wide variation in cell morphology between isolates with lengths of cells or cell chains ranging from 3 to 11 microm, even under identical culture conditions. Most of the shorter or single cell isolates fell into a single RAPD group. No clear association was identified between virulence and any other characteristic, including RAPD group.     

DNA fingerprinting: application to conservation of the CITES-listed dragon fish, Scleropages formosus (Osteoglossidae)

Since 1975,CITES has listed the dragon fish, Scleropages formosus, as anendangered species. In 1995, a captive-bred population was set upby a commercial fish farm with assistance from the PrimaryProduction Department in Singapore. Other farms in Indonesia andMalaysia followed suit. These populations have contributed to animmediate conservation of the species. Due to very high demandfor this ornamental fish, these venues may be its last sanctuary.DNA fingerprints of the dragon fish were obtained by different methods from the green, red and gold varieties grown in a Singapore fish farm to determine which method was most suitable in providing information on genetic variability. Because a DNA fingerprint is a pattern made up of DNA fragments that are resolved by electrophoresis, each individual has its own unique fingerprint due to a genetic make-up different from another individual. Thus, genetic variability was best studied by developing DNA fingerprints.Firstly, restriction fragment length polymorphisms (RFLPs) were obtained. DNA fragments formed by cleavage with nine restriction endonucleases used singly were hybridized individually to four non-radioactively labelled probes to give RFLPs. The RFLPs for each variety were similar and genomic DNA from each variety had many binding sites to the probes. This made differentiating RFLPs specific to individual varieties difficult. Secondly, random amplified polymorphic DNA (RAPD) fingerprints were developed. DNA fragments that were resolved on a denaturing polyacrylamide gel were hybridized to seven arbitrary primers used singly. RAPD fingerprints for each variety were different for each primer tested. The similarity index indicated low genetic variability between varieties. Lastly, DNA was screened for microsatellite loci which refer to short tandem repeats of two or three bases. The occurrence of other microsatellite loci, their chromosome location and frequency is being investigated while primers have been designed to detect more loci by the polymerase chain reaction. As this method provides undisputed and reproducible evidence of relatedness and stock identification, and can be applied for long-term management of domesticated populations through pedigree construction and evaluation of heterozygosity, it is the preferred choice to determine genetic variability     

Biochemical and molecular typing of Streptococcus iniae isolated from fish and human cases

Streptococcus iniae is an important bacterial pathogen of fish, causing up to 50% mortality in stocks, which has recently been associated with human infections. To determine whether S. iniae isolates from humans and fish are similar, the present authors examined the biochemical profiles and genetic relatedness of these isolates by random amplified polymorphic DNA (RAPD) analysis and repetitive primer polymerase chain reaction(REP PCR). The biochemical profiles differentiated between the human and fish isolates of S. iniae using pyrrolidonyl arylamidase, arginine dehydrogenase, ribose, β-glucoronidase and glycogen as markers. These biochemical results suggest that the fish and human S. iniae isolates are genetically different. However, RAPD and REP PCR do not have the discriminatory power to differentiate between these streptococcus isolates using five different RAPD primers and BoxA primer.     

十种常见淡水鱼类的RAPD鉴定

用１４种１０ｂｐ长的随机引物对青鱼、草鱼、鲢鱼、鳙鱼、鲫鱼、鲤鱼、团头鲂、胭脂鱼、土鲶和黄颡十种鱼进行ＲＡＰＤＰＣＲ扩增。除引物ＯＰＫ０８外,其它的１３种引物都得到较清晰的ＤＮＡ带。１３种引物共产生６７２条带,平均每种鱼产生６７条带,每种引物产生５２条扩增带。鲤鱼扩增带最多,胭脂鱼扩增带最少。引物ＯＰＱ０１、ＯＰＱ０４、ＯＰＱ０６、ＯＰＱ１１、ＯＰＱ１２、ＯＰＫ０７、ＯＰＰ０１、ＯＰＰ１５、ＯＰＰ１７扩增的带谱可以作为种间鉴定的标记。本文结果显示ＲＡＰＤ是一种非常灵敏的种间鉴定技术,特别是对鱼卵和鱼苗的鉴定有重要意义。     

Evaluation of genetic relationship among six Labeo species using randomly amplified polymorphic DNA (RAPD)

Labeo species constitute an important group of fish with intense diversity and potential for commercial aquaculture in many Southeast Asian nations including the Indian subcontinent. The present investigation involves the comparative analysis of randomly amplified polymorphic DNA (RAPD) profiles of six Labeo species viz., L. bata (bata), L. calbasu (calbasu), L. dyocheilus (dyocheilus), L. fimbriatus (fimbriatus), L. gonius (gonius) and L. rohita (rohu) at the nuclear DNA variation level. Fifteen decamer random primers were chosen from 40, which amplified a total of 449 DNA fragments ranging in size from 400 to 3000 bp. Both monomorphic and polymorphic DNA bands were identified based on their presence or absence that could be used for specieswise differentiation. Similarity coefficients were calculated to quantify the genetic variation within and between species. On an average, the highest intra‐species genetic similarity value was found in calbasu (0.93) followed by rohu and fimbriatus (0.91), bata (0.87), gonius (0.86) and dyocheilus (0.77). The interspecies genetic similarity estimates among the species of Labeo were used to deduce their phylogenetic relationships. The cluster analysis showed two main clusters, one with calbasu, rohu, fimbriatus and gonius and another with bata and dyocheilus. The study provides evidence that RAPD could be used for genetic differentiation of closely related species.     

Genetic variation of Rutilus rutilus caspicus (Jakowlew 1870) populations in Iran based on random amplified polymorphic DNA markers: a preliminary study

Rutilus rutilus caspicus is regarded as a valuable fish species both for angling and commercial food in Iran. This fish is also considered as a significant food source for beluga sturgeon. The genetic diversity of this fish species collected from two geographical areas (Gorgan Bay and Anzali Wetland) along the Iranian coastline of the Caspian Sea was examined using the analysis of randomly amplified polymorphic DNA (RAPD). Using 10 decamer primers, the total number of RAPD bands produced in both Gorgan Bay and Anzali Wetland populations were 94 bands. The percentages of polymorphic bands were comparable in Anzali Wetland (41.48%) and Gorgan Bay (43.61%), suggesting similar levels of polymorphism of the two populations to be used for establishing selective breeding programmes. The value of Nei's genetic distance (d=0.04) among populations was small, despite the large geographic separation. The data serve as a baseline analysis of current genetic diversity found among R. rutilus caspicus populations in Iran.     

三种黄鳝RAPD分析的引物筛选

试验用随机扩增多态DNA(RAPD)技术对黄鳝属中产自缅甸的山黄鳝、印度尼西亚的穴黄鳝及中国的黄鳝的混合DNA进行引物筛选。40条引物有35条引物扩增出多态性片段。35条引物共获得334个扩增片段,长度为250～5417bp。     

中国对虾5个地理群体的RAPD分析

采用随机扩增多态DNA(RAPD)技术对取自辽东湾、渤海湾、海州湾、乳山湾及海洋岛5个群体的中国对虾共95个个体的遗传多样性进行分析。14个随机引物共检测出103个位点(200～2500bp),各群体的多态位点比例在31．07％～34．95％之间。遗传距离显示中国对虾群体间有一定遗传分化,其中辽东湾和乳山湾群体问的遗传距离最大(0．0742),而辽东湾和渤海湾群体间的遗传距离最小(为0．0243),群体间的遗传分化系数为0．2083。整体来看,中国对虾的遗传变异水平较低,遗传多态度为0．1621。用UPGMA对5个群体进行聚类分析,构建谱系关系图。