雌核发育二倍体鲫鲤杂交克隆品系建立

研究了雌核发育二倍体鲫鲤第2代(G₂)产生的二倍体卵子在无染色体加倍情况下形成第3代(G3)的雌核发育细胞学行为,G₃,G₂×散鳞镜鲤和G₂×四倍体鲫鲤的染色体数目.研究结果表明:(ⅰ)G₂产生的二倍体卵子无需染色体加倍处理,仅在灭活散鳞镜鲤精子激活下,形成了大量G₃.(ⅱ)G₃和雌核发育二倍体鲫鲤第1代(G₁)、G₂一样,也表现出杂交特征,并且都是二倍体(2n=100);G₂与二倍体散鳞镜鲤和四倍体鲫鲤分别交配形成了三倍体(3n=150)和四倍体(4n=200)鱼;(ⅲ)二倍体G₂产生的二倍体卵子在雌核发育过程中,有明显第二极体排出,排除了二倍体卵子源于第二极体保留的可能.另外,还对二倍体鲫鲤产生二倍体卵子的机制进行了讨论.雌核发育二倍体鲫鲤杂交克隆品系建立证明二倍体卵子通过雌核发育形式可形成一个能产生二倍体卵子的新型二倍体鲫鲤品系,二倍体鲫鲤产生二倍体卵子的特殊繁殖方式在生物进化和生产应用方面都具有重要意义.     

远缘杂交形成的二倍体鱼和多倍体鱼生殖细胞染色体研究

本文采用性腺染色体制片及组织学切片方法,系统地研究了不同发育时期的鲫鲤杂交第二代(F2) (2n=100)、异源四倍体鲫鲤(4n=200)、三倍体鲫鱼(3n=150))、雌核发育二倍体鲫鲤第二代(G2)(2n=100)及鲤鱼(Cypninus carpio L)(2n=100)(对照组)生殖细胞的染色体特征.研究结果表明,对照组中鲤鱼精原细胞染色体数与体细胞染色体数一致,为二倍体精原细胞(2n=100),而远缘杂交形成的二倍体鱼和多倍体鱼的生殖细胞中则观察到明显的染色体数加倍现象,其中,鲫鲤杂交第二代(F2)精巢生殖细胞染色体数加倍现象特别丰富,占检测的染色体分裂相的21.6%,为其产生不减半的二倍体配子提供了直接的细胞学证据,同时也说明远缘杂交是导致生殖细胞染色体数加倍的一个重要因素.该研究在探讨多倍体鱼的发生及鱼类遗传育种方面具有重要意义.     

不同倍性鱼的血细胞和精子DNA含量比较

我们以前的研究表明, 以红鲫 (2n=100) 为母本及湘江野鲤 (2n=100) 为父本的杂交后代的F1-F2 为二倍体 (2n= 100)。在二倍体 F2 个体中, 存在能分别产生二倍体卵子和二倍体精子的雌、雄个体, 二倍体卵子和二倍体精子结合, 形成了两性可育的四倍体鱼 (F3)。目前四倍体鲫鲤已连续繁殖了 12 代 (F3-F14), 形成了一个遗传性状稳定的四倍体鱼群体 (4n= 200) (Liu et al.,2001; 孙远东等, 2003)。雌性四倍体鲫鲤产生的二倍体卵子经紫外线照射的散鳞镜鲤精子激活后,无须染色体加倍处理, 可发育为全雌性二倍体雌核发育后代 (G1) (2n=10…     

湖南师范大学鱼类发育学研究室最新研究成果展示

湖南师范大学鱼类发育生物学研究室在成功研制了四倍体鲫鲤群体(F3-F18)的基础上,利用四倍体鲫鲤群体产生的二倍体卵子本身具有2套染色体组的特点,通过雌核发育技术,在没有染色体加倍处理情况下,经灭活的散鳞镜鲤精子刺激,建立了一个能大量产生二倍体卵子的雌核发育二倍体克隆体系(G1-G5)。有关该研究     

异源四倍体鲫鲤F9～F11染色体和性腺观察

采用肾细胞染色体制片技术,检测了异源四倍体鲫鲤F9-F11代的染色体数目及组型,结果表明：其染色体数目为4n=200,核型公式为44m 68sm 44st 44t,证明F9—F11继续保持四倍体性。观察异源四倍体鲫鲤F9—F11成熟性腺,在这3代四倍体鱼中仍然保持正常卵巢和精巢,分别形成正常二倍体卵子和二倍体精于。在自然环境下,观察了异源四倍体鲫鲤自行产卵受精井产生存活后代过程,证明该四倍体鱼群体在自然环境下能够自行繁殖传代。异源四倍体鲫鲤稳定的染色体数目和正常的性腺结构以及自然条件下的生殖传代行为,说明该异源四倍体鲫鲤已成为一个染色体数目为4n＝200、遗传性状稳定的新型四倍体鱼群体,具备形成新种所需的关键因素。     

用雄核发育方法制备改良异源四倍体鲫鲤群体

用UV照射金鱼的卵子使其卵核的遗传物质失活, 再与异源四倍体鲫鲤(AT)产生的二倍体精子受精, 在无雄核染色体加倍处理情况下, 成功地获得了两性可育的二倍体雄核发育鱼(A₀). Ⅱ龄性成熟的A₀自交形成了雄核发育鱼自交子一代(A₁). 本研究对10月龄A₁的染色体数目、性腺的显微和亚显微结构以及外型特征进行了观察, 实验结果表明: (ⅰ) A₁中包含有四倍体(A₁-4n)、三倍体(A₁-3n)以及二倍体后代(A₁-2n), 他们所占比例分别为85%, 10%和5%, 其染色体数目分别为4n=200, 3n=150和2n=100. 其中四倍体和三倍体的形成证明二倍体A₀能产生二倍体配子. 二倍体雄核发育鲫鲤杂交鱼产生二倍体配子的原因与早期生殖细胞的核内复制机制有关. (ⅱ) A₁-4n的性腺为两性型且发育正常. 其中雄性个体能挤出白色精液, 其中的二倍体精子头部明显比红鲫的单倍体精子头部大. 这些二倍体精子具有正常结构, 由头部和尾部组成, 头部与尾部交接处有多个线粒体, 精子尾巴的中央轴有典型的“9+2”微管结构. A₁-4n雌性个体的卵巢发育饱满, 其中含有大量Ⅱ, Ⅲ和Ⅳ时相的卵母细胞. 在Ⅳ时相卵母细胞的放射膜上能观察到受精孔. 同时期的A₁-2n, A₁-3n的性腺发育异常, 均表现为不育. A₁-2n的不育性与其为远源杂交二倍体有关, A₁-3n的不育性与其为远源杂交三倍体有关. (ⅲ) 与AT相比, A₁-4n不仅具有生长速度快、抗逆性强的优点, 而且在外型上具有体背高、尾柄短、头部小等优良性状. 本实验说明运用雄核发育技术不仅能获得两性可育的四倍体鱼, 而且能对异源四倍体鲫鲤进行有效的遗传改良, 这在细胞遗传研究和鱼类育种方面都具有重要意义.     

二倍体鲫鲤F2产生不同倍性卵子的证据

在检测到鲫鲤F2产生3种不同大小(直径分别为0.13 cm,0.17cm和0.2 cm)类型的卵子基础上,进行了F2(♀)×红鲫(♂)及F2(♀)×四倍体鲫鲤(♂)的交配实验.通过染色体计数和流式细胞仪分析,在F2(♀)×红鲫(♂)后代中获得了四倍体、三倍体、二倍体鱼;在F2(♀)×四倍体鲫鲤(♂)后代中获得了四倍体和三倍体鱼.这两个交配组合后代中出现的不同倍性的鱼类为证明鲫鲤F2能产生三倍体、二倍体和单倍体卵子提供了进一步证据.F2(♀)×红鲫(♂)中雄性四倍体鱼的存在说明在四倍体后代中存在基因型为XXXY的个体.对上述两个交配组合后代的四倍体鱼和三倍体鱼的性腺结构观察表明四倍体鱼是可育的,而三倍体鱼是不育的.作者认为鲫鲤F2能够产生二倍体和三倍体卵子与核内复制机制和生殖细胞的融合有关.     

远缘杂交导致不同倍性鱼的形成

远缘杂交可以使基因组从一个物种转移到另一个物种中,从而导致杂交后代的表现型和基因型都发生改变.本文描述了在红鲫(♀)与鲤鱼(♂)的远缘杂交后代中,形成了F3~F18两性可育异源四倍体鲫鲤群体(4n=200,简称为4nAT).4nAT的雌、雄个体分别产生的二倍体卵子和二倍体精子,经过雌核发育和雄核发育,在没有染色体加倍处理情况下,分别发育成雌核发育二倍体后代和雄核发育二倍体后代.其中雌核发育体系衍生出具有遗传变异的改良四倍体鲫鲤和改良二倍体鱼,二倍体杂交鱼产生不减数的配子的现象与减数分裂前核内复制或者核内有丝分裂或者生殖细胞融合有关.用雄性4nAT与雌性二倍体鱼进行倍间交配大规模制备了不育三倍体鱼.在红鲫(♀)与团头鲂(♂)的远缘杂交后代中,成功地获得两性可育的天然雌核发育红鲫(2n=100),不育的三倍体鲫鲂(3n=124)以及两性可育的四倍体鲫鲂(4n=148),此外,还制备了两种五倍体鲫鲂(5n=172;5n=198).该文在细胞和分子水平上对不同倍性鱼的生物学特点和形成机制进行了比较,揭示了远缘杂交或者将远缘杂交与雌核发育和雄核发育相结合的方法在具有遗传变异的不同倍性鱼的形成中发挥着积极作用,这在生物进化和鱼类遗传育种方面都具有重要意义.     

MAPK在不同生殖特性鲫鲤杂交鱼性腺组织中的表达分析

运用Western-blot技术和免疫组织化学来检测丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)家族成员细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)和c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)在不同生殖特性鲫鲤杂交鱼性腺组织中的表达。研究表明,JNK、ERK在鱼类性腺组织中均有较高量的表达,但在鱼类卵巢和精巢间、雌核发育二倍体鲫鲤和不同倍性鱼的性腺组织间,JNK或ERK的表达量并不存在明显的差异,而P-JNK在雌核发育二倍体鲫鲤性腺中的表达量远高于三倍体和四倍体鲫鲤的卵巢组织。此外,JNK和ERK在雌核发育二倍体鲫鲤早期性腺性原细胞中均有阳性表达。其中,JNK在10月龄雌核发育二倍体鲫鲤性腺中的阳性反应强度高于6、8月龄;而ERK在8月龄和10月龄的性腺中的阳性反应则弱于6月龄。结果表明JNK通路可能对雌核发育二倍体鲫鲤产生不减数配子具有重要调控作用。     

新型高背型鲫鱼的形成及其生物学特征研究

利用鱼类远缘杂交技术和雌核发育方法获得了新型四倍体鲫鲤(G1×AT). 在G1×AT中发现2%的高背型个体, 其自交后代性状发生分离并形成3种两性可育的二倍体鱼: 高背型红鲫、高背型双尾金鱼和青灰色鲤鱼. 其中高背型红鲫自交, 后代性状继续发生分离, 形成高背型红鲫、花鲫和青鲫. 本文主要对这3种高背型鲫鱼及其自交后代的外形特征、染色体数目、性腺显微和超微结构以及繁殖力等方面进行了研究. 结果表明: (ⅰ) 这3种高背型鲫鱼在外形上都具有体背高、尾柄短、头部小的优良特性. 高背型红鲫、花鲫和青鲫的体高/体长值分别为0.54, 0.51和0.54, 三者明显高于普通红鲫的体高/体长值0. 41(P<0. 01); (ⅱ) 3种高背型鲫鱼染色体数目与普通红鲫染色体数目一致, 都为2n=100; (ⅲ) 这3种高背型鲫鱼都具有正常的卵巢和精巢, 它们分别能产生成熟的卵子和精子, 为二倍体高背型鲫鱼品系的形成奠定了基础; (ⅳ) 与普通红鲫相比, 3种高背型鲫鱼具有产卵(产精)量大、繁殖期长、受精率和孵化率高等优点. 它们通过自交培育出了具有高背特征的二倍体鲫鱼群体; (ⅴ) 3种高背型鲫鱼既具有较高的观赏和食用价值, 也可以作为优良的二倍体亲本与四倍体鱼交配来制备高背型三倍体鲫鱼. 这3种高背型鲫鱼的形成在生物进化研究和鱼类遗传育种研究方面都具有重要意义.     

二倍体与二基体

DiploidandDihasoidShenShuxiang(NantongAgricultureSchoolofJiangsurovince226007)在作物遗传学的教科书上,二倍体的概念都是这样的：在相物体细胞的染色体数目中,包含着两套染色体,一套来自父本的花粉细胞,另一套来自母本的卵细胞。凡是体细胞中含有步套染色体的植物（2n）统称为二倍体。按照这个定义,普通小麦、陆地棉等与水稻、玉米一样,都是二倍体。而实际上,普通小麦是“六倍体”,陆地棉是“四倍体”、由此可见,上述二倍体的概念是不够确切的．为了弄清利么是二倍体,我们先来了解一厂一倍体的概念、在遗传学的染色体变…     

Diploid Gynogenesis in the Mexican Axolotl

Gynogenetic diploid axolotls were produced by activating eggs with ultraviolet-inactivated sperm, and then subjecting the activated eggs to heat shock. Optimal conditions for ultraviolet inactivation of the sperm, and for suppression of the second meiotic division by heat shock, were established. Gynogenetic diploids produced by these procedures included progeny homozygous for recessive alleles carried by a heterozygous mother. Gynogenesis could, therefore, be used to uncover new mutations more rapidly than by conventional inbreeding techniques. However, some difficulty was encountered in recognizing mutant phenotypes because of the high incidence of abnormalities and deaths. Defective embryos probably resulted from a combination of heat-shock-induced damage to the eggs and the expression of deleterious recessive alleles carried by the mother.     

Testing Differentiation in Diploid Populations

We examine the power of different exact tests of differentiation for diploid populations. Since there is not necessarily random mating within populations, the appropriate hypothesis to construct exact tests is that of independent sampling of genotypes. There are two categories of tests, F(ST)-estimator tests and goodness of fit tests. In this latter category, we distinguish ``allelic statistics', which account for the nature of alleles within genotypes, from ``genotypic statistics' that do not. We show that the power of F(ST)-estimator tests and of allelic goodness of fit tests are similar when sampling is balanced, and higher than the power of genotypic goodness of fit tests. When sampling is unbalanced, the most powerful tests are shown to belong to the allelic goodness of fit group.     

Deciphering the Diploid Ancestral Genome of the Mesohexaploid Brassica rapa

The genus Brassica includes several important agricultural and horticultural crops. Their current genome structures were shaped by whole-genome triplication followed by extensive diploidization. The availability of several crucifer genome sequences, especially that of Chinese cabbage (Brassica rapa), enables study of the evolution of the mesohexaploid Brassica genomes from their diploid progenitors. We reconstructed three ancestral subgenomes of B. rapa (n = 10) by comparing its whole-genome sequence to ancestral and extant Brassicaceae genomes. All three B. rapa paleogenomes apparently consisted of seven chromosomes, similar to the ancestral translocation Proto-Calepineae Karyotype (tPCK; n = 7), which is the evolutionarily younger variant of the Proto-Calepineae Karyotype (n = 7). Based on comparative analysis of genome sequences or linkage maps of Brassica oleracea, Brassica nigra, radish (Raphanus sativus), and other closely related species, we propose a two-step merging of three tPCK-like genomes to form the hexaploid ancestor of the tribe Brassiceae with 42 chromosomes. Subsequent diversification of the Brassiceae was marked by extensive genome reshuffling and chromosome number reduction mediated by translocation events and followed by loss and/or inactivation of centromeres. Furthermore, via interspecies genome comparison, we refined intervals for seven of the genomic blocks of the Ancestral Crucifer Karyotype (n = 8), thus revising the key reference genome for evolutionary genomics of crucifers.     

Standardization of Human Diploid Cell Cultivation

Human embryonic diploid lung fibroblasts grown in Eagle's medium were exposed continually to a variety of environmental conditions over a large number of passages to observe how these conditions affected the growth and longevity of these cells in vitro. The cells grew well at temperatures between 34 and 37 C and some cells could be adapted to grow at 40 C. Very limited growth occurred at 30 to 31 C; however, confluent monolayers of cells could be maintained for months at 30 C and still give rise to actively growing cultures. Increasing the amino acid concentration in Eagle's medium or the calf serum concentration above 10% had no effect on the growth rate or longevity. One per cent calf serum could not support prolonged active growth. Trypsin concentrations between 1 and 0.1% and crystalline trypsin at 50 μg/ml showed no influence on cell growth. Ethylenediaminetetraacetic acid treatment and scraping, however, destroyed many of the cells, and the survivors grew poorly. The clonal morphology varied with age. Young cells frequently gave rise to densely packed clones, whereas older cells gave rise to clones with widely scattered cells. The cloning efficiency was high when the cells were young but decreased rapidly with successive passage. It was relatively constant from the 7th to 20th passage at about 15%.     

Regeneration of Diploid and Polyploid Plants from the Stem Pith Explants of Diploid Marrow Stem Kale (Brassica oleracea L.)

Viable plants of kale (Brassica oleracea L.) have been regeneratedfrom stem pith explants grown on complex agar media. About 80per cent of kale plants cv. Krasa gave explants which differentiatedroots and shoots. Analysis of stomatal length, pollen grainmorphology and estimation of chromosome number in PMC and somaticcells showed that a set of 71 regenerated plants derived fromfive diploid mother plants contained 6 di-, 54 tetra-, and 11octoploid regenerates. Utilization of this method in plant breedingis discussed.     

DNA Replication Error-Induced Extinction of Diploid Yeast

Genetic defects in DNA polymerase accuracy, proofreading, or mismatch repair (MMR) induce mutator phenotypes that accelerate adaptation of microbes and tumor cells. Certain combinations of mutator alleles synergistically increase mutation rates to levels that drive extinction of haploid cells. The maximum tolerated mutation rate of diploid cells is unknown. Here, we define the threshold for replication error-induced extinction (EEX) of diploid Saccharomyces cerevisiae. Double-mutant pol3 alleles that carry mutations for defective DNA polymerase-δ proofreading (pol3-01) and accuracy (pol3-L612M or pol3-L612G) induce strong mutator phenotypes in heterozygous diploids (POL3/pol3-01,L612M or POL3/pol3-01,L612G). Both pol3-01,L612M and pol3-01,L612G alleles are lethal in the homozygous state; cells with pol3-01,L612M divide up to 10 times before arresting at random stages in the cell cycle. Antimutator eex mutations in the pol3 alleles suppress this lethality (pol3-01,L612M,eex or pol3-01,L612G,eex). MMR defects synergize with pol3-01,L612M,eex and pol3-01,L612G,eex alleles, increasing mutation rates and impairing growth. Conversely, inactivation of the Dun1 S-phase checkpoint kinase suppresses strong pol3-01,L612M,eex and pol3-01,L612G,eex mutator phenotypes as well as the lethal pol3-01,L612M phenotype. Our results reveal that the lethal error threshold in diploids is 10 times higher than in haploids and likely determined by homozygous inactivation of essential genes. Pronounced loss of fitness occurs at mutation rates well below the lethal threshold, suggesting that mutator-driven cancers may be susceptible to drugs that exacerbate replication errors.