应用微卫星标记对雌核发育银鲫的遗传多样性初探

利用Crooijmans et al.(1997)分离的包含CA重复单元的普通鲤鱼（Cyprinus carpino.L)的8个微卫星DNA标记,对银鲫（Carassius auratus gibelio Bloch)的5个不同雌核发育系的24尾个体进行PCR扩增。分析电泳结果发现,除MFW28未能在银鲫中稳定地扩增出相应的同源序列,其余的7对引物扩增的重复性和稳定性都很好,随引物不同,各等位基因数为1-14个,大小在100-506bp。在MFW1、MFW4、MFW19、MFW20、MFW23和MFW246个微卫星的扩增图谱中,不同的雌核发育系扩增出各自独特的图谱,而同一系内的不同个体间具有高度的遗传同质性,但仍然在个别个体中检测到少量的多态片段。不同系间的扩增图谱呈现出高度的遗传异质性,共鉴定出23个可以用于有效区分5个不同雌核发育系的分子标记。这5个微卫星标记反映了银鲫5个雌核发育系间的相互亲缘关系,其中P和A系同属一个雌核发育系,F系起源于E系,A、D和E系可能分别独立地起源于不同的杂交事件,鉴定的微卫星分子标记为进行银鲫群体遗传学和进化遗传学研究,以及银鲫的分子标记育种和进行基因组作图提供了理想的工具。     

转化生长因子β1对心脏移植排斥反应中穿孔素和颗粒酶B表达的影响

目的探讨转化生长因子β1（TGF-β1）对大鼠心脏移植排斥反应中穿孔素和颗粒酶B表达的影响。方法采用大鼠颈部心脏移植模型,实验动物随机分为3组：同系移植组、异系移植组和TGF-β1组。于术后第5d取移植心脏,用逆转录聚合酶链式反应（RT-PCR）法观察穿孔素和颗粒酶B的表达情况。结果异系移植组穿孔素和颗粒酶B表达明显升高（与同系移植组相比,P〈0．01）。TGF-β1组穿孔素和颗粒酶B表达明显降低（与异系移植组相比,P〈0．01）。结论TGF-β1对心脏移植排斥反应的免疫抑制作用可能与其抑制穿孔素和颗粒酶B的表达有关。     

人工诱导栉孔扇贝雌核发育后代的微卫星标记分析

用紫外线灭活栉孔扇贝精子和6-DMAP处理受精卵抑制第二极体,获得A、B、C 3组雌核发育栉孔扇贝。采用筛选获得的5对多态性微卫星引物对3个家系雌核发育组30个个体、双亲及对照组40个个体进行了遗传变异分析。结果显示：3个家系雌核发育组后代中均有部分个体出现父本基因,表明精子遗传物质失活不彻底,雌核发育组中存在正常受精个体;在具多态性的5个基因座位上均发生了基因-着丝点之间的重组,重组率在26.7%—55.0%之间,表明通过抑制第二极体排出获得的栉孔扇贝雌核发育后代在个体和群体水平上具有一定的基因杂合;3个家系后代的平均基因纯合率为59.1%,而其对照组家系为2.5%,雌核发育使基因的纯合率提高了56.6%。研究表明雌核发育是促进基因纯合的一个有效途径,微卫星标记技术是贝类雌核发育鉴定和遗传分析的有效方法。     

红白锦鲤人工雌核发育纯系的微卫星标记分析

利用连续两代人工雌核发育操作,构建了极具观赏价值的红白锦鲤人工雌核发育系。应用6对微卫星引物进行遗传标记研究,检测分析了该雌核发育系内个体间的遗传同质性及其基因座位的纯合度。实验结果显示,11尾经人工挑选具有相同表形、极具观赏价值的F1代性成熟个体的微卫星扩增图谱呈现出高度的一致性。同时,随机挑选的人工雌核发育F2代个体在所检测的基因座位均呈现纯合,扩增图谱呈现了高度的一致,从分子水平上证实了本实验所获得的红白人工雌核发育F2代是一个纯系。     

复合四倍体异育银鲫个体间遗传异质性的研究

用聚丙烯酰胺梯度凝胶电泳,分析比较了４个不同的银鲫雌核发育系,红鲤和复合四倍体异育银鲫鳍的５种不同的同工酶及蛋白表型的差异,表明复合四倍体异育银鲫表型上的差异主要是来自银鲫种内的遗传差异（不同的雌核发育系）。来源于同一个银鲫雌核发育系的复合四倍体异育银鲫,个体间的ＥＳＴ同工酶出现差异,并与父体红鲤的ＥＳＴ同工酶的多态性相关,因此,复合四倍体异育银鲫个体间的异质性也包含了来自父本的遗传影响。在所检测     

锦鲤4个人工雌核发育家系的微卫星标记研究

利用Crooijmans et al.(1997)分离的包含CA重复单元的普通鲤鱼（Cyprinus carpio L.)的8个微卫星DNA标记,对从锦鲤（Cyprinus carpio L.)的红白,大正和昭和3个不同品系中所获得的4个不同人工雌核发育家系的20尾个体进行PCR扩增。电泳结果表明,8对引物在20尾个体中均能重复稳定地扩增出相应的同源序列。随引物不同,各等位基因数为1－11个,大小在68－264bp。在MFW4,MFW7,MFW19,MFW20,MFW23和MFW24 6个微卫星的扩增结果中,20尾个体的扩增图谱呈现了高度的遗传多态性,不同雌核发育家系内个体的遗传异质性也较大。其中大正（TaS)和红白1（RW1）的个体不仅花色分化显著,而且个体间的平均遗传距离分别高达0．28。通过对微卫星等位基因和基因型分析发现,由于锦鲤品系中的每一个体是通过不断地杂交选育而获得,基因组来源复杂,基因高度杂合。因此,只进行1代的人工雌核发育,其家系内仅部分个体的部分座位出现纯合。所获得的人工雌核发育锦鲤为后续的色素遗传调控机制研究提供了必要的实验材料;同时,所鉴定的微卫星分子标记为进行锦鲤的分子标记育种的基因组作图提供了理想的工具。     

周围神经异体移植免疫机制的研究进展

周围神经异体移植由于移植物免疫原性的存在,移植后受体产生免疫排斥反应,造成移植失败。本文综述了移植物的免疫成分所在、SC及细胞因子在排斥反应中的作用、T淋巴细胞的激活途径及激活信号的转导过程,并讨论了减轻免疫排斥反应的治疗进展。     

鲤鱼鳞被和体色在人工诱导雌核发育中的遗传变异

于1986—1988年,先后人工诱导2尾红镜鲤和4尾荷包红鲤抗寒品系雌核发育,获得雌核发育2倍体当年鱼种2133尾,其中红镜鲤685尾,荷包红鲤1448尾。通过对这些后代鳞被和体色两个质量性状的分析,发现荷包红鲤抗寒品系雌核发育2倍体(1987)中有约三分之一个体表现散鳞型鳞被和桔红色(出现由深到浅一系列变化)及少量桔黄色、肉红色个体。红镜鲤散鳞型鳞被出现体表全部覆盖不规则大型鳞片、2/3、1/2覆盖不规则的大型鳞片和散鳞型个体。体色出现桔红色、桔黄色,上述二色都出现由深到浅一系列变化和肉红色。这两个质量性状在雌核发育代中表现出数量性状的特征。     

移植免疫简介

移植免疫简介刘年方（江苏省江都市小纪中学２２５２４１）在组织移植或器官移植中,受者接受供者的移植物后,受者的免疫系统与供者的移植物相互作用而发生的免疫应答“,称为移植免疫。研究移植免疫的主要目的是了解移植排斥反应发生的机制,以预防和控制排斥反应的发生...     

两个人工雌核发育红白锦鲤群体的RAPD标记分析

采用PAPD方法,对两个人工雌核发育红白锦鲤群体进行了多态性及分子标记分析。结果表明,同一雌核发育群体具有基本一致的扩增产物,而不同雌核发育群体间的扩增产物则有较大不同;并从30个随机引物的扩增谱带中找到了7个引物（Opo-7,Opo-9,Opo-12,Opo-14,Opj-4,Opj-8和Opj-10）的扩增谱带可以作为两个不同雌核发育群体间的遗传标记。由UPGMA聚类法构建的分支系统树清晰地反映了两个雌核发育群体及其个体间的相互关系。     

人工雌核发育鲢的遗传多样性及异源遗传物质整入的RAPD分析

运用RAPD技术对连续二代人工雌核发育鲢的遗传多样性及异源遗传物质的整入进行了分析 ,结果表明 :一代雌核发育鲢 ,个体间遗传相似度为 0 94 5— 0 995 6 ,多样性指数为 0 175 ;二代雌核发育鲢 ,个体间遗传相似度为0 96 15— 1 0 0 ,平均为 0 985 2 ,多样性指数为 0 0 6 2。研究揭示经过连续二代人工雌核发育后 ,其遗传多样性明显减少 ,种质进一步纯化。通过对雌核发育鲢二代、亲本鲢和雄鲤的RAPD扩增比较 ,发现雌核发育鲢含有少数与父本相同的特异DNA扩增带 ,而亲本鲢没有 ,在基因水平上表明雌核发育鲢整入了雄鲤的遗传物质     

Induction of meiotic gynogenesis in Atlantic cod,Gadus morhua (L.)

Gynogenesis is one of several chromosome‐manipulating techniques used in fish. In gynogenesis the male does not contribute to the genetic material of the offspring, and the sperm cells act only as stimulators in order for the egg to start development. This technique has several applications, both in aquaculture and in biological research: gynogenetic fish may be used as a step in the production of all‐female populations, the production of isogenetic‐ and inbred lines, revealing of the sex determination mechanism, construction of genetic maps, and testing of environmental vs genetic control of different traits. The aim of this study was to develop a simple protocol for production of gynogenetic cod (Gadus morhua L.) for further use in aquaculture research. Various milt dilutions and UV‐irradiation doses were tested, in order to inactivate the sperm without destroying its ability to induce egg development. This was followed by pressure treatment of the eggs shortly after ‘fertilization’ to suppress the completion of meiosis II, and thereby restoring diploidy. A dose of 9000 erg mm^?2, followed by a 5‐min pressure treatment (58.6 MPa) 180 min‐degrees after fertilization gave 100% gynogenetic larvae. Histologically, sexual differentiated fish were all females, possibly confirming female homogamety in Atlantic cod. No particular signs of reduced growth, survival or enhanced deformity rates were observed after the fish had reached the juvenile phase. Mortality was, however, high during the egg and larval stages. This protocol has made capable the production of gynogenetic cod juveniles in significant amounts using relatively simple means; the next step will be to elaborate on the technique in order to produce mitotic gynogenetic (double haploid) individuals, which are 100% homozygous.     

Genetic Evidence for Gonochoristic Reproduction in Gynogenetic Silver Crucian Carp (Carassius auratus gibelio Bloch) as Revealed by RAPD Assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp. Received: 5 January 2000 / Accepted: 3 August 2000     

Analysis of genetic heterogeneity among five gynogenetic clones of silver crucian carp, Carassius auratus gibelio Bloch, based on detection of RAPD molecular markers

The gynogenetic silver crucian carp, Carassius auratus gibelio, is a unique model system for studying evolutionary genetics and selective breeding, owing to its specific genetic background and reproductive modes. Five gynogenetic clones were analyzed by the random amplified polymorphic DNA (RAPD) technique, using 30 10-nucleotide-long primers. Twenty-six primers produced well-amplified DNA fragments with reproducible banding patterns, and 24 primers were polymorphic. Nearly identical banding patterns were observed among individuals within each clone, suggesting that each clone might possess a specific pattern owing to its gynogenesis. In contrast, the RAPD patterns of the five clones differed from each other. A phylogenetic tree was constructed using UPGMA cluster analysis based on a total of 3,744 distinguishable fragments (156 per individual). Average genetic distances within and among the five clones clearly indicated their intraclonal homogeneity, interclonal heterogeneity, and phylogenetic relationships. Clones A and P were the most closely related, whereas the most divergence was seen between clone D and clone E or F. A total of 88 polymorphic fragments were scored from 24 primers after excluding bands that were monomorphic for the five clones. Most primers corresponding to the polymorphic fragments amplified reproducible markers specific for one clone or that were shared by two, three, or four clones. Several primers (e.g., Opj-1, Opj-7, and Opp-10) produced abundant banding patterns that could be used to discriminate between the five clones. Markers specific for one or two clones were also identified. The RAPD markers identified in this study will likely benefit evolutionary genetics and selective breeding studies.     

雌核发育团头鲂的形态和遗传特征分析

利用团头鲂(Megalobrama amblycephala)正常二倍体群体作为对照组, 对团头鲂减数分裂雌核发育二倍体群体的形态特征、染色体组型、性腺发育及遗传特征进行了分析. 结果表明: 雌核发育群体与正常群体在外部可数、可量性状上没有显著性差异(P0.05); 但雌核发育群体出现了尾鳍条数为12的畸形个体, 与正常个体之间有较大的差异; 两个群体的染色体条数都是48, 核型均为18 m+26 sm+4 st; 观察了20尾雌核发育个体的性腺, 均为雌性个体且卵巢发育良好; 采用10个微卫星标记对2个群体的遗传多样性分析, 结果表明正常群体和雌核发育群体平均等位基因数分别为3.8个和1.7个, 雌核发育群体的多态性显著低于正常群体, 表明减数分裂雌核发育二倍体具有高度的遗传相似性, 可作为一个很好的育种材料.       

Breeding Value and Variance Component Estimation from Data Containing Inbred Individuals: Application to Gynogenetic Families in Common Carp (Cyprinus Carpio L.)

Under gynogenetic reproduction, offspring receive genes only from their dams and completely homozygous offspring are produced within one generation. When gynogenetic reproduction is applied to fully inbred individuals, homozygous clone lines are produced. A mixed model method was developed for breeding value and variance component estimation in gynogenetic families, which requires the inverse of the numerator relationship matrix. A general method for creating the inverse for a population with unusual relationships between animals is presented, which reduces to simple rules as is illustrated for gynogenetic populations. The presence of clones in gynogenetic populations causes singularity of the numerator relationship matrix. However, clones can be regarded as repeated observations of the same genotype, which can be accommodated by modifying the incidence matrix, and by considering only unique genotypes in the estimation procedure. Optimum gynogenetic sib family sizes for estimating heritabilities and estimates of their accuracy were derived and compared to those for conventional full-sib designs. This was done by means of a deterministic derivation and by stochastic simulation using Gibbs sampling. Optimum family sizes were smallest for gynogenetic families. Only for low heritabilities, there was a small advantage in accuracy under the gynogenetic design.     

Genetic analysis of androgenetic rainbow trout.

We analyzed a number of genetic characteristics in androgenetic rainbow trout (Oncorhynchus mykiss) and their progeny. The androgenetic progeny of individual androgenetic males appeared genetically identical to each other based on eight enzyme loci. Their viability was no higher than that of androgenetic progeny of outbred males. Homozygous androgenetic female rainbow trout produced very poor quality eggs. When common eggs and sperm from outbred individuals were used to produce androgenetic and gynogenetic progeny, the yield of gynogenetic progeny was higher but some were heterozygous at protein loci, while no androgenetic progeny were heterozygous. Some androgenetic diploid rainbow trout were successfully produced from cryopreserved sperm. The progeny of some androgenetic males crossed to normal females were virtually all males, while the progeny of other males were virtually all females. This suggests that both XX and YY androgenetic individuals may develop as males. Androgenesis is likely to be useful for generating homozygous clones for research and for recovering strains from cryopreserved sperm.     

草鱼雌核发育后代不同群体的微卫星遗传分析及指纹识别

以紫外线灭活的团头鲂精子激活草鱼卵子,冷休克抑制第二极体排出的方法诱导出长江水系优良F2代草鱼减数雌核发育子代。在后代中不仅存在雌核发育后代,还存在草鲂杂交后代,雌核发育后代的体型与草鱼一致,而草鲂杂交后代的体型介于草鱼与团头鲂之间。Partec CyFlow倍性分析仪测定结果显示:普通草鱼与雌核发育草鱼的相对DNA含量分别为23.01和22.72,二者的DNA含量接近;而高体型子代的相对DNA含量为25.38,介于草鱼与团头鲂（DNA含量28.21）之间,属于草鲂杂交后代。选取17个微卫星标记对草鱼群体、雌核发育草鱼群体和草鲂杂交后代的遗传多样性进行了检测,共检测出59个等位基因,其中43.18个有效等位基因。草鱼对照群体、草鲂杂交后代和雌核发育草鱼群体的平均等位基因依次为3.57、2.86和2.79,平均有效等位基因依次为2.93、2.37和1.96,平均期望杂合度在依次为0.6502、0.5573和0.3775,多态信息含量（PIC）平均值依次为0.5738、0.4649和0.3791。与草鱼对照群体相比,雌核发育草鱼群体的遗传多样性显著下降,表明通过减数雌核发育方法可获得纯合性较高的草鱼个体。构建了草鱼后代不同群体的DNA指纹模式图,筛选到不同群体的9个特异微卫星标记,为草鱼优良群体的选育提供了基础资料。