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.     

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.     

多倍体银鲫克隆多样性和双重生殖方式的遗传基础和育种应用

单性物种一般是多倍体,且通过单性生殖方式如雌核生殖、杂种生殖或孤雌生殖繁殖.与其他单性和多倍体物种相比,银鲫具有更高的倍性,为六倍体.它经历了几轮连续的基因组多倍化,还经历了一次额外的、在较近年代发生的基因组复制事件.更为重要的是,银鲫已被证实同时存在雌核生殖和有性生殖双重生殖方式.本文综述了银鲫的多倍化起源、克隆多样性和双重生殖方式的遗传基础,概述了其新品种培育和有关其生殖与早期发育相关基因鉴定的研究进展.已有实验证据表明,银鲫正处于二倍化的进化轨道中.作为一个新的进化发育(Evo-Devo)生物学模型,重点论述了银鲫在重复基因的功能歧化和单性动物的有性起源和进化等方面的研究前景.     

雌核发育银鲫子代中微卫星特异序列分析

雌核发育个体的基因型基本上完全与母本相同,这是源于卵子发生过程中没有经过减数分裂.父本的遗传物质是在随机水平、亚基因组水平或基因组水平参与到子代的遗传重组过程,从而对长期突变积累的雌核发育生物基因组进行补偿,一直是遗传学家关注的问题.本文对雌核发育银鲫特异个体及父母本5个微卫星位点的扩增条带进行了克隆测序,相似性比对结果显示,特异个体所表现的父咎匾霥NA条带,序列结果与父本完全相同或相似(SCM4、SCM9、YJ5),并在某些位点上保留了母本的特异条带(YJ5),而个体本身特异的DNA条带与父母本的相似性均较高(SCM13).同时,所检测到的个体经越冬后查验为雌性个体,进一步进行同源繁育,研究变异条带在繁殖中的命运.连续2代的繁育检测结果表明,融合了父本特异性条带的银鲫个体在繁殖过程中仍行雌核发育的生殖方式,变异来的条带能够传递给子代,进一步证实同源雌核发育银鲫通过小概率两性融合事件丰富银鲫种群的遗传多样性[动物学报 53(3):537-544,2007].     

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.     

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。     

外源精子基因组整入雌核生殖银鲫卵子创制异源八倍体的有效方法

银鲫(Carassius auratus gibelio Bloth)具有将其他鱼类精子的染色体组、染色体片段并入到卵核中协同发育的能力, 但通过异精雌核生殖自发形成异源八倍体子代的概率极低。研究采用0.25%、0.5%、1%、2%和4%胰蛋白酶溶液处理白鲫精子10min后以及1%胰蛋白溶液处理白鲫精子5min、10min、15min、20min和25min后, 分别与异育银鲫A+系成熟卵子受精; 接着通过比较对照组和不同处理组合中精子结构和活力的变化, 兼顾受精率、孵化率、成活率等繁育指标与较高的子代八倍体率, 建立了一种将外源精子基因组整入雌核生殖银鲫卵子创制异源八倍体的有效方法。即采用1%胰蛋白溶液处理白鲫精子15min后与异育银鲫A+系成熟卵子受精, 子代的平均存活率为(2.4±0.7)%, 平均八倍体率可达(16.3±0.5)%。批量处理并结合流式细胞术筛选6月龄子代, 获得57尾融入有白鲫精子染色体组的异源八倍体成鱼。研究为创制异育银鲫优异异源八倍体种质提供了一个简单易行的方法, 创制的异源八倍体可作为后续培育生长快、抗病能力强的银鲫新品种的核心育种材料。     

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。     

银鲫HIRA多克隆抗体的制备及组织特异性表达分析

Hir/Hira基因家族的成员广泛存在于多种生物体中,但有关其在生物体发育过程中的具体功能还不甚清楚.对果蝇的研究表明,dHira基因产物可能在受精时精核的解凝过程和雄性原核的正常形成过程中起重要作用.本研究组前期已经分别克隆出雌核发育银鲫和两性生殖彩鲫的Hira基因(cagHira 和caHira),本实验在银鲫cagHira基因的特异区域,设计一对引物,以银鲫成熟卵母细胞总RNA逆转录出的cDNA为模板,扩增出cagHira的特异片段.再将该片段克隆到原核表达载体pET-32a上,转化BL21(DE3)菌株,经诱导后表达出融合蛋白.分析表明,该融合蛋白主要以包涵体形式表达.以纯化的融合蛋白作为抗原去免疫小鼠,制备多克隆抗血清,经蛋白质印迹分析检测,该抗血清(稀释到1:2000)与包涵体蛋白识别反应良好,确定获得了具有高效价的特异性银鲫CAGHIRA多克隆抗体,为进一步研究HIRA在鱼类发育和雌核生殖过程中的作用奠定了基础.对银鲫HIRA蛋白的组织特异性表达分析发现,该蛋白仅在成熟卵巢组织中特异表达,故表明HIRA可能对鱼类卵子发生和/或早期胚胎发育具有重要作用.     

两对互补的显性基因控制着柑桔属和枳属的无融合生殖

无融合生殖具有重要的学术价值和应用价值。这种生殖方式普遍存在于 柑桔属及其近缘属植物中,对母本分别为单胚（即有性生殖）的宽皮柑桔品种克力迈丁和韦尔金,父本分别为多胚（即无融合生殖）的甜橙品种锦橙,新会橙,桃叶橙和哈姆林的杂种F1代群体总计8个杂交组合的229个开花结果后代,进行了胚性分离的调查,发现F1代中既有有性生殖,也有无融合生殖,有性生殖与无融合生殖的分离比例,在韦尔金后代中接近1：2,而在克力迈丁后代中接近1：1。根据该结果,结合前人的研究资料,提出在柑桔属和枳属或者还包括其他柑桔近缘属中,无融合生殖受到位于细胞核的两对互补的显性基因A1和A2的调控,且其中一对基因,设为A1表现显性纯合致死效应,两对基因的分离重组符合孟德尔的分离定律和自由组合定律,按该模式可以较为合理地解释已有的大部分常规杂交资料。     

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     

Two unisexual artificial polyploid clones constructed by genome addition of common carp (<Emphasis Type="Italic">Cyprinus carp</Emphasis>) and crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.     

Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

The application of hybrid vigor and crossbreeding is conventional and proved effective. Nevertheless, the phenotype of the progeny of hybrids, which carry hybrid vigor and produce their offspring through bisexual reproduction, will segregate inevitably and their hybrid vigor will de-crease in subsequent generations. The more serious consequence might result in destroying com-pletely those endemic populations when hybrids are released into open water bodies, because hy-brids will cross with the…     

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     

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2)of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes.Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp, and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gynogenetic progeny of allotetrapioid hybrids of red crucian carp × common carp had important significances in both biological evolution and production application.     

Genetic Diversity among Different Clones of the Gynogenetic Silver Crucian Carp, Carassius auratus gibelio, Revealed by Transferrin and Isozyme Markers

Genetic diversity among four clones (A, D, E, F) of gynogenetic silver crucian carp was studied using transferrin and isozymes in the blood as markers. Of the five proteins investigated, three (transferrin, esterase and superoxide dismutase) indicated polymorphism and eight polymorphic loci were detected. These loci were probably encoded by codominant alleles and their inheritance patterns were analyzed. Intraclonal homogeneity and interclonal heterogeneity were observed in these clones, which allowed us to infer the clonal nature and evolutionary relationship between them. Clonal diversity in this population of silver crucian carp in China was also compared with data reported from gynogenetic crucian carp in Germany.     

异源精子在银鲫雌核发育子代中的生物学效应

黑龙江省方正县双凤水库的两性型银鲫群体是三倍体雌核发育种群。异源精子不仅能刺激银鲫卵雌核发育,而且还能影响雌核发育子代的某些性状,如对于子代的生长、性比、体色和肝脏LDH同工酶等都产生了影响。为区别于原有术语“雌核发育gynogenesis”,我们把这种表现了异源精子生物学效应的雌核发育称之为“异精雌核发育allogynogenesis”,发育的子代称之为“异育银鲫”。异育银鲫已以其明显的生长优势在生产上显示了优良的经济性状。       

四类不同倍性鲫鱼在胚胎发育期间同工酶基因表达的比较分析

Isozyme zymograms of esterase (EST), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and superoxide dismutase (SOD) were analysed by polyacrylamide gradient gel electrophoresis at different developmental stages of embryogenesis in 4 types of various ploidy crucian carp embryos, including haploids, diploids, natural triploids, and multiple tetraploids, and 2 types of haploid and diploid common carp embryos. Haploid embryos of crucian carp (Carassius auratus) and common carp (Cyprinus carpio) were produced by treating eggs with UV-irradiated milt from blunt snout bream (Megalobrama amblycephala). Natural triploid embryos were obtained from the eggs of gynogenetic silver crucian carp (Carassius auratus gibelio) inseminated with milt from red common carp. Multiple tetraploid embryos were also produced by gynogenesis from eggs of the newly discovered multiple tetraploid females inseminated with milt from red common carp. Gradient gel electrophoresis indicated that the band types and staining intensity of 4 isozymes expressed in haploid embryos of crucian carp and red common carp were similar to that in the correlative diploid embryos. In natural triploid silver crucian carp embryos, the zymograms of MDH and SOD isozymes were identical with that of diploid crucian carp embryos, but the EST and LDH isozymes manifested more new enzyme bands in comparison with diploid embryos. The corresponding expressed products of some bands in the triploid embryos, such as EST5 and EST6, could be observed also in red common carp embryos, which provided evidence for hybrid origin about the gynogenetic fish. The multiple tetraploids incorporated one foreign genome of red common carp, therefore, the effects of genes from the foreign genome could be observed in the multiple tetraploid embryos. Gene expression of the isozymes in the tetraploid embryos was somewhat similar to that in hybrids. Owing to interaction of triploid silver crucian carp genomes and common carp haploid genome, some isozyme bands, such as EST5 and EST6, changed in quantity, and some bands increased, such as s-SOD1, s-SOD2, s-SOD3 and s-SOD4 in the tetraploid embryos. Moreover, the heterogeneity was revealed among embryos developed from gynogenetic eggs of 3 different multiple tetraploid individuals.     

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.