银鲫两个雌核发育克隆间两性生殖子代的遗传多样性分析

本研究在揭示银鲫两性生殖方式的基础上,对以一尾人工雌核发育克隆F的卵子与一尾天然雌核发育克隆D的精子授精所获得的18尾FD后代及其亲本进行RAPD分析.扩增结果表明,在18尾FD子代中可检测到丰富的DNA多态片段,这些多态片段来自于银鲫两性生殖的重组.FD子代的扩增图谱不仅与母本的扩增图谱不同,而且个体间的扩增图谱也存在着较大的差异.这些差异的DNA片段根据其来源可基本分为四类.与异精刺激雌核发育的子代的情况不同,两性生殖FD子代间平均遗传距离高达0.23±O.123,远远高于异精刺激雌核发育的子代间的平均遗传距离(约＜0.01).在FD的三类表型中,SD个体间的平均遗传距离最大,为0.235±0.097;其次是SF间的平均遗传距离,为0.148±0.073;而NL间的平均遗传距离最小,为0.094±0.083.银鲫的遗传多样性和两性生殖方式为银鲫种群提供了丰富的遗传变异,创造了更多的遗传多样性.在分析了2320条(平均每个个体116条)可分辨的扩增带的基础上,采用NJTREE构建了呈现FD个体间及其与亲本相似性的分支图.NL3与母本的相似度最高,SD3与父本的相似度最高.在FD三种类型中,SD与NL和SF的平均遗传距离较大(＞0.31),而NL和SF的平均遗传距离仅为0.124.母本与父本的遗传距离约为0.35,与以前的研究结果类似,而FD 18尾个体与父母本的平均遗传距离均约为O.32,基本一致.但将18尾个体按体型分别统计平均遗传距离,不同的体型与父母本的相似性存在着较大差别.母本与SF的相似率最高,约为71%;父本与SD的相似率最高,约为70%.这可能由于扩增出一些与表型相关的DNA片段,从而导致了FD三种类型与亲本的相似性与表型相关.银鲫具有两种生殖方式的揭示将为解决长期困惑进化遗传学家的难题寻找到一个新的突破口,也进一步确立了银鲫在单性脊椎动物和多倍体脊椎动物进化遗传学研究中的特殊意义.     

复合四倍体异育银鲫产生的细胞学机制

银鲫是行天然雌核发育生殖的鱼类。以往对其繁殖生物学的研究一般都认为:异源父本的性状不能或仅能少量地在子代群体中得到表达。但桂建芳等(1991)在人工繁育的异育银鲫群体中发现了具有一整套父本染色体组的复合四倍体鱼,其出现率为0.2％左右。此外,Kobayasi     

对一例异常鲫鱼的同功酶和肌蛋白的电泳研究

淇鲫是生活于河南淇河的一种鲫鱼,由于其具有优良的经济性状而受到水产界的关注。与银鲫(Carassiusauratus gibelio)相似,淇鲫也以雌核发育(gynogenesis)的方式繁殖后代。在以淇鲫为母本,兴国红鲤(Cyprinuscarpio)为父本的“杂交”中,产生的子代绝大多数都呈现母本性状,但我们在其中偶尔发现一尾异常鱼:其体形似鲫鱼,但口角有须2对,显然是鲤鱼的特征。为了进一步确定其遗传组成,我们用4.5％聚丙烯酰胺凝胶平板电泳研究其乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)、酯酶(EST)和肌蛋白(muscle protein),并与淇鲫和兴国红鲤的电泳图谱作了比较。     

人工四倍体鲤鲫后代遗传同一性的FCM及RAPD证明

人工四倍体鲤鲫是本实验室采用人工染色体组叠加构建的一个以雌核发育为生殖模式的鲫克隆系,现已繁殖至第五代.它生长快、体型好、抗病力强、易捕捞,比普通二倍体鲫和三倍体鲫经济性状更优良,将具有广阔的生产应用前景.从表型看,子代是由四倍体鲤鲫原代母本拷贝而成,与母本极其相似,表明异源精子启动发育的后代无分离现象.我们曾对原代四倍体雌核发育生殖方式的遗传进行过基因组、形态学和同工酶分析研究[1-4],为了获得更进一步的证据,本文应用FCM和RAPD分子遗传标记方法进行验证.     

银鲫生殖方式多样性的转铁蛋白和同工酶标记的遗传分析

用银鲫克隆D、克隆A和鲤鱼的精子分别与银鲫克隆F的卵子受精产生了三种繁殖组合FD、FA和FL;再用转铁蛋白和同工酶标记对这三种组合的遗传模式进行比较研究.结果发现,FL组合的子代具有与其母本完全相同的体形和电泳图谱,表现出雌核生殖银鲫的克隆品性.而在FD组合中则出现了体形的分化和酶谱的多态性;在一些个体的蛋白座位上同时检测到了父本和母本各自特有的谱带,证明FD生殖过程中有性重组的发生.同时,在所研究的蛋白的不同座位上存在着极端的连锁不平衡现象,可以推断在FD的重组过程中多个基因座位组成的连锁群(甚至是染色体组)可能作为一个整体参与基因的传递.此外,不同的蛋白座位上都未观察到重组的纯合表型,暗示在不同的基因连锁群之间还可能存在一种平衡致死的机制.FA组合的F1代具有类似母本克隆F的体形和蛋白表型,FA组合个体近交产生的F2代却同时出现了克隆F和克隆A的体形和表型.即父本和母本的染色体组都能够通过有性生殖传递给FA子代,然而可能由于父母本基因的不相容性而使F1代父本的基因表达受到抑制.相对于雌核生殖的克隆遗传,本研究所揭示出来的有性生殖特性及伴随的有性重组能够使银鲫在一定程度上卸去积累的遗传负荷并从其它种群获取新的基因型,以维持其遗传多样性.多种可选择的生殖方式可能对于银鲫在自然条件下的生态适应有着重要意义,对于银鲫的遗传选育和养殖管理也有一定的参考价值.     

雌核发育银鲫的受精生物学研究——天然雌核发育银鲫繁殖方式的讨论

本文研究了同源雌核发育银鲫精子在4种类型的雌核发育银鲫卵中的发育特征。初步揭示了天然雌核发育银鲫根据精子的来源不同而分别具有二种不同的繁殖方式,对其在维持雌核发育银鲫种群生存,促使克隆分化等方面的独特的生物学意义进行了讨论。     

银鲫种内的遗传标记及其在选种中的应用

用聚丙烯酰胺梯度凝胶电泳分析了银鲫和异育银鲫的血清蛋白和肝脏酯酶,发现这两种鱼的血清蛋白和肝脏酯酶各具有4种不同的表型。由同一母本银鲫繁殖的子代——银鲫或异育银鲫,具有与母本相同的表型,只是在异育银鲫的肝脏酯酶谱型中另有一些无规律的变化,显然这是异源精子的作用所致。此外,这4种不同表型的鱼具有不同的生长能力,其差距最大可达50％．这些结果表明,天然雌核发育的银鲫种内含有若干个不同的雌核发育系,这对于开展银鲫的选育研究具有重要的实践意义。     

雌核发育银鲫微卫星突变速率和突变模式

银鲫是天然雌核发育的三倍体两性型种群, 因其遗传背景和生殖方式的特殊性, 已经成为研究单性和多倍体脊椎动物进化遗传学的理想模式鱼类。利用33个微卫星序列对雌核发育银鲫的突变速率和突变模式进行研究, 结果表明: (1) 22个子代个体中, 检测到1尾个体在15个微卫星位点具有18个突变等位基因; (2) 每个微卫星位点每代总体平均突变率是1.16×10-2, 95%置信区间是6.87×10-3~1.83×10-2, 与其他鱼类相比, 雌核发育银鲫的突变率明显偏高, 这与天然雌核发育鱼类处在单性生殖和两性生殖的过度阶段有密切关系; (3) 具有突变等位基因的13个位点的重复单元数目都在10次以上, 11个复合型微卫星位点的突变率(1.31×10-2)与21个完美型位点(1.00×10-2)的突变率没有明显差异(P = 0.67), 微卫星突变率受到重复单元数目的影响, 然而与重复结构类型和侧翼序列GC碱基含量无相关性; (4) 序列分析表明, 雌核发育银鲫的微卫星突变模式并不严格遵守逐步突变模型。     

白鲫×红鲫杂交后代的遗传变异

以雌性日本白鲫和雄性红鲫为亲本进行杂交,获得了杂交子代合方鲫.对该杂交子代及其亲本的形态特征、繁殖特性和遗传特性进行了较系统的研究.合方鲫的体色为青灰色,外形特征介于父母本之间,表现出杂交特征.合方鲫性腺发育正常,雌雄两性可育,并且具有较高的受精率(90.2%)和孵化率(80.5%),为新品系的繁衍和群体扩大奠定了基础.合方鲫的染色体数为2n=100,DNA含量与亲本一致,是一种二倍体鱼.合方鲫的45S r DNA的ITS区包括ITS-1,5.8S和ITS-2三部分,总长度为884 bp,与亲本序列相似度较高并表现出偏向于父本遗传的特征.同时,杂交子代序列兼有双亲特异碱基位点,表现出重组现象.合方鲫5S r DNA的NTS区具有3种长度类型,序列全长为654 bp,与母本相似度为94.2%,与父本似度为95.1%,杂交子代序列具有明显的重组和突变特征,这些基因重组和突变是品种改良和新品系形成的遗传基础.本研究证明了合方鲫在遗传组成和外形方面都与其亲本有本质区别,是一种有杂种特性的后代,该杂交品种的形成在鱼类遗传育种方面具有重要意义.     

异源精子诱导稀有鮈鲫的人工雌核发育

用经过紫外线灭活的异源精子启动稀有鮈鲫卵子发育,再经过热休克分别抑制极体排放和第一次卵裂,得到了极体雌核发育和有丝分裂雌核发育的存活个体。通过正交试验确定诱导极体雌核发育的最佳参数为受精后2min、40℃休克处理2min;诱导有丝分裂雌核发育的最佳参数为受精后17min、40℃休克处理2min。雌核发育个体的形态学特征没有显示出受到父本影响的迹象。RAPD分析表明雌核发育个体扩增片段全部来自于母本,没有发现异源父本DNA成分进入稀有鮈鲫基因组的迹象。实验中还发现,鲤×稀有鮈鲫的正常杂交组合能以极低的几率产生出雌核发育的稀有鮈鲫。     

银鲫生殖方式多样性的转铁蛋白的同工酶标记的遗传分析

用银鲫克隆D,克隆A和鲤鱼的精子分别与银鲫克隆F的卵子受精产生了三种繁殖组合FD,FA和FL;再用转铁蛋白和同工酶标记对这三种组合的遗传模式进行比较研究。结果发现,FL组合的子代具有其母本完全相同的体形和电泳图谱,表现出雌核生殖银鲫的克隆品性。而在FD组合中则出现了体形的分化和酶谱的多态性;在一些个体的蛋白座位上同时检测到了父本和母本各自特有的谱带,证明FD生殖过程中有性重组的发生。同时,在所研究的蛋白的不同座位上存在着极端的连锁不平衡现象,可以推断在FD的重组过程中多个基因座位组成的连锁群（甚至是染色体组）可能作为一个整体参与基因的传递。此外,不同的蛋白座位上都未观察到重组的纯合表型,暗示在不同的基因连锁群之间还可能存在一种平衡致死的机制。FA组合的F1代具有类似母本克隆F的体形和蛋白表型,FA组合俱本近交产生的F2代却同时出现了克隆F和克隆A的体形和表型。即父本和母本的染色体组都能够通过有性生殖传递给FA子代,然而可能由于父母本基因的不相容性而使F1代父本的基因表达受到抑制。相对于雌核生殖的克隆遗传 ,本研究的揭示出来的有性生殖特性及伴随的有性质组能够使银鲫在一定程度上卸去积累的遗传负荷并从其它种群获取新的基因型,以维持其遗传多样性。多种可选择的生殖方式可能对于银鲫在自然条件下的生态适应有着重要意义,对于银鲫的遗传选育和养殖管理也有一定的参考价值。     

Genetic analysis of offspring from intra‐ and interspecific crosses of Carassius auratus gibelio by chromosome and RAPD analysis

The ploidy of silver crucian carp Carassius auratus gibelio individuals, originating from nine natural habitats of Hungary, was estimated by erythrocyte nucleus area analysis. On the basis of DNA polymorphism, the genetic homogeneity or heterogeneity and the chromosome number of different offspring derived from the crossing of triploid and diploid populations and of two types of silver crucian carp females with other cyprinid males ( Cyprinus carpio, Carassius carassius, Carassius auratus and Barbus conchonius ) were determined. The results of chromosome and RAPD analysis demonstrated that diploid females could reproduce sexually with silver crucian carp and other cyprinid males and that the offspring of intra‐ and interspecific crosses contained the paternal DNA. Triploid females usually reproduced by gynogenesis and their offspring were clones, however, in very rare cases paternal genes were actually transmitted ( i.e . paternal leakage) to the offspring and the progeny were triploid interspecific hybrids. RAPD analysis showed that while the paternal DNA appeared in the offspring, the maternal phenotype was strongly expressed.     

Isolation and characterization of microsatellite markers from Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch), and cross‐amplification in the closely related species crucian carp,Carassius auratus auratus (Linnaeus)

We report the development of 20 microsatellite markers for Fangzheng silver crucian carp, Carassius auratus gibelio (Bloch). Nineteen out of 20 showed polymorphism with alleles ranging from two to 14. These loci were screened to amplify the closely related species crucian carp, Carassius auratus auratus (Linnaeus) and all of them can amplify DNA with the size similar to the former. The origin of silver crucian carp is in issue and the population genetic structure is still unclear. Microsatellite markers isolated from the silver crucian carp and their utility in the crucian carp will be useful for these researches.     

异源四倍体鲫鲤及其原始亲本遗传变异的微卫星标记分析

采用从鲤中分离出来的32对微卫星DNA标记,对异源四倍体鲫鲤、红鲫和野鲤的基因组DNA进行了研究。在筛选出的15对微卫星引物中,随引物不同,各等位基因数为1～8个,大小在100～420bp之间。从3个不同群体内部的遗传相似系数来看,异源四倍体鲫鲤个体之间的遗传相似系数最大,说明异源四倍体鲫鲤群体内部的遗传变异程度最低,已经形成了一个遗传性状稳定的群体。从3个不同群体之间的遗传相似系数来看,异源四倍体鲫鲤和红鲫遗传相似系数为0．5625,和野鲤的遗传相似系数为0．5125,说明异源四倍体鲫鲤接受原始母本的遗传物质比原始父本野鲤要多一些。微卫星标记与以前报道的RAPD标记的检测结果是相似的,然而由微卫星标记获得的种群内和种群间的遗传距离均大于RAPD,说明微卫星标记比RAPD标记显示出更高的个体多态性。     

鲤鲫人工多倍体谱系中同工酶和蛋白的基因表达

通过对红鲤、红鲫、镜鲤、鲤鲫杂种二倍体一代,二代,鲤鲫杂种三倍体,鲤鲫复合三倍体,鲤鲫杂种四倍体一代,二代的同工酶及蛋白电泳谱型和扫描数据分析表明,在鲤鲫人工多倍体谱系中,亲代的等位基因在杂交子代中共有四种表达模式;（1）两亲本基因在子代中共同表达,即共显表达;（2）父本的基因表达受到部分或完全的抑制,即母本的基因优先得到表达;（3）母本的基因表达受到抑制,父本的基因得到表达;（4）双亲本的基因表达均受到一定程度的抑制或都不表达。其中第一种表达模式是主要的模式。根据以上基因在杂交子代中的表达特点,可用同工酶和蛋白电泳图谱将鲤鲫人工多倍体谱系的各种生物型逐一加以区分。     

Genetic Stability of Progeny from an Artificial Allotetraploid Carp Using Sperm from Five Fish Species

Microsatellite markers and D-loop sequences of mtDNA from a female allotetraploid parent carp and her progenies of generations 1 and 2 induced by sperm of five distant fish species were analyzed. Eleven microsatellite markers were used to identify 48 alleles from the allotetraploid female. The same number of alleles (48) appeared in the first and second generations of the gynogenetic offspring, regardless of the source of the sperm used as an activator. The mtDNA D-loop analysis was performed on the female tetraploid parent, 25 gynogenetic offspring, and 5 sperm-donor species. Fourteen variable sites from the 1,018 bp sequences were observed in the offspring as compared to the female tetraploid parent. Results from D-loop sequence and microsatellite marker analysis showed exclusive maternal transmission, and no genetic information was derived from the father. Our study suggests that progenies of artificial tetraploid carp are genetically stable, which is important for genetic breeding of this tetraploid fish.     

Development of microsatellite DNA markers of silver carp (Hypophthalmichthys molitrix) and their cross‐species application in bighead carp (Aristichthys nobilis)

Forty‐four microsatellite DNA markers were developed for silver carp, and used to investigate polymorphisms of 41 wild silver carps and seven wild bighead carps collected from Jingzhou fragment of Yangtze River. In silver carp, 40 markers were polymorphic. A total of 297 alleles were detected at 40 polymorphic loci. The number of alleles per locus varied from two to 16 with an average of 7.4 and the expected heterozygosities of each locus ranged from 0.07 to 0.91 with an average of 0.69. All markers amplified both silver carp and bighead carp DNAs.     

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