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。     

Phylogenetic relationships of silver crucian carp Carassius auratus gibelio, C. auratus cuvieri, crucian carp C. carassius, and common carp Cyprinus carpio as inferred from mitochondrial DNA variation

PCR-RFLP analysis of the ND3/ND4L/ND4 and 12S/16S rRNA regions and nucleotide sequence variation of the cytochrome b gene were used to study the mtDNA divergence in species of the family Cyprinidae, to examine the phylogenetic relationships of the species, and to identify their taxonomic status. The results indicated that an ancestral form diverged into silver crucian carp and crucian carp after its separation from the common carp lineage. The divergence of continental Carassius auratus gibelio and Japanese C. auratus cuvieri occurred more recently. Two well distinguishable mtDNA phylogroups, suggesting divergent evolution, were observed in continental C. auratus gibelio populations. The divergence was possibly related to the formation of two silver crucian carp groups with different types of reproduction, triploid gynogenetic and diploid gonochoric. At the same time, the results supported the high probability of current genetic exchange between the forms. In view of these findings and high morphological similarity of the two forms, they were not considered to be separate species.     

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.     

Direct antibacterial activity of CD8+/CD4+ T-cells in ginbuna crucian carp,Carassius auratus langsdorfii

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8α⁺ T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8α⁺ T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1–2 h at 26 °C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8α⁺ T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8α⁺ T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4⁺ T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgM⁺ cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8α⁺, CD4⁺ T-cells and surface IgM⁺ cells in fish.     

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].     

Molecular characterization of MHC class I and beta-2 microglobulin in a clonal strain of ginbuna crucian carp, Carassius auratus langsdorfii

Clonal ginbuna crucian carp is, a naturally gynogenetic fish, and is a useful model animal for studying T-cell-mediated immunity. To gain molecular information on MHC class I molecules from this species, we have identified four types of MHC class I (caauUA-S3n, caauUF-S3n, caauZE-S3n, and caauZB-S3n) and five beta 2-microglobulin (β(2)m) (caauβ2m-1a, caauβ2m-1b, caauβ2m-2, caauβ2m-3a and caauβ2m-3b) by an expressed sequence tag (EST) analysis and using homology cloning with degenerated primers. Like UA class I genes in other cyprinid fish, the caauUA-S3n shows features of classical MHC class I, such as conservation of all key amino acids interacting with antigenic peptides, and ubiquitous tissue expression. A phylogenetic analysis shows that the β(2)m-1 and β(2)m-2 isoforms are clustered with those of other cyprinid fishes, while β(2)m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the β(2)m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and β(2)m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with β(2)m in teleosts.     

新疆伊犁河鲫鱼遗传多样性初步研究

为了解我国西北地区野生鲫鱼的遗传多样性,鲫鱼样品从新疆伊梨河中采用小型拖网捕获。通过解剖观察性腺鉴定性别;采用碘化丙啶（PI）染色,经流式细胞仪测定血细胞核DNA含量;采用非变性聚丙烯酰胺凝胶电泳,考马斯亮蓝染色分析血清转铁蛋白多态性。结果表明：新疆伊梨河鲫鱼群体为具雌、雄的两性个体的群体;伊犁河鲫鱼血细胞核DNA平均含量为(5.315±0.215) pg/个细胞核,血细胞核DNA含量约为二倍体彩鲫DNA含量的1.53倍,与银鲫血细胞核DNA含量相当;在所采集的鲫鱼样品中共发现9种转铁蛋白表现型, 由7个等位基因调控,等位基因频率依次为Tf a0.063、Tf b0.063、Tf f0.095、Tf g0.169、Tf c0.174、Tf e0.175和Tf d0.270。基因型频率分别为Tf cd0.043、Tf cdf0.043、Tf abdg0.043、Tf def 0.087、Tf ce0.13、Tf de0.13、Tf df0.13、Tf abeg0. 13和Tf cdg0.26。综合分析,伊犁河现存野生鲫鱼为银鲫,且具有两性融合生殖能力。伊犁河鲫鱼表现出的遗传特性可能与该特定水环境有关。     

Phylogenetic relationships of silver crucian carp in Carassius auratus complex based on mtDNA analysis

The phylogenetic relationships of Carassius genus subspecies were investigated based on the data of the variability of nucleotide sequences of the mtDNA cytochrome b (cyt b) and control region (CR). Dendrograms constructed based on the BA, ML, NJ, and MP methods revealed five clusters of the congruent topologies that substantially corresponded to geographical localities and taxonomic conception of the C. auratus complex. An analysis of two mtDNA fragment topologies demonstrated that the island forms of Japanese crucian carps C. cuvieri and C. auratus langsdorfii diverged later compared to the divergence of continental C. auratus forms (4.0?C4.5 mln years ago, by molecular calibration). Among the continental silver crucian carps, C. a. gibelio forms two clusters corresponding to two phylogroups with a mean uncorrected genetic distance p = 0.044. The genealogical combination of haplotypes with the first C. a. gibelio phylogroup was observed in C. auratus clade. According to the data of mtDNA analysis, these subspecies represent sister lineages with a level of intergroup divergence of p = 0.022?C0.036. No genetic differences were observed between diploid (except for the two C. a. gibelio phylogroups) and polyploid C a. auratus, as well as monophyly in polyploid forms. New approaches based on a comparative study of the nuclear markers might help to unravel the origin of gynogenetic forms and phylogenetic relationships within the C. auratus complex.     

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

Based on the discovery of gonochoristic reproductive mode in silver crucian carp (Carassius auratus gibelio), 18 individuals from the mated offspring between clone F and clone D and their parents were analyzed using 11 RAPD primers. The mated offspring differentiated into three phenotypes. One phenotype is similar to that of clone F (SF). The second is similar to that of clone D (SD). The third shows a novel longer and thinner body type (NL). Electrophoretogram results show abundant polymorhic DNA fragments among individuals in the FD mated offspring. Obviously, the polymorphic DNA fragments originate from the genome recombination owing to the gonochoristic reproductive mode. The FD mated offspring amplify different fingerprints from maternal, and their fingerprints are different from each other. These different DNA fragments can be divided into four groups according to their origin. The average genetic distance among individuals from the FD offspring (0.23 +/- 0.123) is much higher than that of allogynogenetic offspring, which is only about 0.01. Of the three phenotypes in the FD offspring, the average distances among SD is the highest (0.235 +/- 0.097). The next is SF phenotype, in which the average distances is 0.148 +/- 0.073. The average distances among NL is the lowest (0.094 +/- 0.083). The gonochoristic reproductive mode introduces high genotypic variability and underlines genetic diversity. A dendrogram was constructed by NJTREE cluster analysis based on a total of 2320 distinguishable fragments (116 per individual). NL3 and maternal are most closely related, and SD3 and paternal are most closely related. The average genetic distances between SD and SF or NL (> 0.31) is higher than that of between SF and NL (0.124). Consistent with previous study, the genetic distance between maternal and paternal is 0.35. The average genetic distances between FD offspring and parents are both about 0. 32. But the similarities among the three phenotypes in mated group FD offspring and parents are different. The similarity between SF and maternal (71%) is higher than similarities among other phenotypes and maternal. The similarity between SD and paternal (70%) is higher than similarities among other phenotypes and paternal. The results suggest the similarities among three phenotypes in the FD offspring and parents are related with their phenotypes because some DNA fragments specific for phenotypes were amplified in this study. The revelation about reproductive diversity will be able to open the door in which complicated mechanism has been locked in unisexual organisms for long time, and establish the important roles of silver crucian carp in the studies on evolutionary genetics in unisexual or polyploid vertebrates.     

Ecological and genetic mechanisms of the dynamics of population abundance in silver crucian carp (Carassius auratus gibelio (Bloch, 1782)) from the Tsimlyansk Reservoir

Silver crucian carp is the dominant commercial fish of the Tsimlyansk Reservoir (the Don River). A retrospective analysis of the ecological and genetic factors determining the dynamics of the silver crucian carp population at different stages of reservoir functioning was performed. At the most recent stage, the lake-swamp environmental conditions suitable for crucian carp and a lack of any dependence on other cyprinid fishes during the reproduction of this species determine its domination in the reservoir??s fish community. The size, sex, and genetic structures of the crucian carp population, characterized by numerous age groups and low linear and weight growth rates, are presented.     

Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii

The gynogenetic fish, Carassius auratus langsdorfii (the ginbuna, a crucian carp), provides an interesting model for the study of the mechanisms controlling male pronucleus formation. When the sperm nucleus of a different subspecies (C. a. cuvieri) is incorporated into the gynogenetic egg, the nuclear envelope of the spermatozoon is not broken down, and the pronucleus fails to develop, although dispersion of the sperm chromatin occurs to some extent within the space limited by the nuclear envelope. When spermatozoa without plasma membranes and nuclear envelopes were microinjected into mature activated eggs, the sperm nuclei underwent chromatin dispersion, nuclear envelope formation, DNA synthesis, and transformation into male pronuclei. These results indicate that the failure of the male pronucleus to form in ginbuna is primarily due to the failure of sperm nuclear envelope breakdown. We conclude that sperm nuclear envelope breakdown is an indispensable step for the development of the male pronucleus.     

Mitochondrial DNA variation in diploid and triploid forms of silver crucian carp Carassius auratus gibelio

Variation of two mtDNA fragments amplified in polymerase chain reaction was compared by the RFLP method in a population of the sympatric bisexual (diploid) and gynogenetic (triploid) forms of silver crucian carp. The mtDNA haplotypes of all individuals differed in at least 2.5% of nucleotide substitutions and fall into two phylogroups. All gynogenetic individuals have haplotypes of a single phylogroup, whereas haplotypes of diploid fish belonged to both mtDNA phylogroups. This testifies to a possibility of transformation of gynogenetic individuals into a bisexual form, whereas the reverse process is either extremely rare or impossible.     

Predator recognition and defence strategies in crucian carp, Carassius carassius

Crucian carp from populations that lack piscivores are extremely vulnerable to predation. However, in the presence of piscivores these fish develop an inducible morphological defence, a deep body. This switch from a vulnerable, shallow-bodied morph to a morphologically defended morph makes this species very suitable for investigations of anti-predator strategies, and trade-offs between morphological and behavioural defences. To address these questions, we performed eight different experiments. We found that crucian carp exhibited fright responses to chemical cues from unfamiliar predators (northern pike, perch) when these were fed prey that contained alarm substance (for northern pike: crucian carp, roach; for perch: crucian carp). Cues from small pike that were fed prey that lacked alarm substance (swordtails) caused no significant fright response whereas cues from larger pike with the same diet did. Perch on a chironomid diet elicited weaker but significant fright responses. Starved predators caused as strong fright reactions as recently fed ones did, whereas no response was exhibited towards nonpredatory fish (roach, crucian carp). Crucian carp were able to detect the presence of pike after cues had been diluted to an equivalent of 21 000 l, and larger predators elicited stronger fright responses. Prior experience of predators decreased fright responses. In particular, individuals from populations that coexisted with northern pike responded less to chemical cues from northern pike than individuals without prior experience did. Thus, crucian carp may use both alarm-substance related and predator-related cues to identify predators. Further, they were able to discriminate between large and small predators. Finally, individuals from populations that coexist with predators exhibit less pronounced fright responses. These fish have an induced morphological defence, a deep body, which most likely decreases the need for strong antipredator behaviour.     

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。     

柴达木地区野生黑果枸杞种群遗传多样性的AFLP分析

采用扩增片段长度多态性(AFLP)分子标记技术对青海省柴达木地区5个野生黑果枸杞(Lycium ruthenicum)种群的120份样品的遗传多样性进行分析。结果表明: 柴达木地区野生黑果枸杞具有很高的遗传多样性, 9对选扩引物共得到1691条清晰条带, 其中多态性条带1678条, 多态性变异率为99.23%, 种群间的有效等位基因数为1.4712, Nei’s基因多样性为0.3245, Shannon信息指数为0.4367。分子方差分析(AMOVA)结果表明: 柴达木地区5个黑果枸杞种群的遗传变异主要存在于种群内部(92%), 种群间的遗传分化较小(8%, 遗传分化系数0.08)。黑果枸杞种群间的遗传相似系数介于0.9709-0.9922之间, 平均值为0.9835。种群间的聚类及Mantel检验(γ = 0.3368, p = 0.8064)均表明柴达木地区黑果枸杞种群地理距离与遗传距离之间的相关性不明显; 黑果枸杞个体间的聚类表明同一种群的个体不能完全聚在一起。对同一种源的遗传多样性分析发现, 诺木洪奥斯勒草场的种源内部的遗传变异更为丰富, 这或许可以推断诺木洪可能为柴达木地区野生黑果枸杞种质资源的中心产区。     

Clonal diversity in a Rhododendron ferrugineum L. (Ericaceae) population inferred from AFLP markers

In the European Alps, Rhododendron ferrugineum can constitute dense populations with almost 100% of cover. The developmental pattern by layering and the resulting complexity of population structure make it difficult to identify distinct clones even by excavation. Therefore genotypic structure of a R. ferrugineum population, in the French Alps, was inferred from AFLP markers. In a first step, we analysed 400 samples using AFLP profiles generated by one selective primer pair. Seventeen bands out of 25 were polymorphic (68%). We identified a total of 32 multilocus genotypes. In a second step, the 32 genotypes were verified by applying two additional primer pairs to the two most distant samples from each genotype. The mean similarity (proportion of band sharing) between pairs of clones was 0.85 (range from 0.52 to 0.94). The spatial distribution of clones showed that vegetative spreading mainly occurred down a slope. Based on an annual shoot mean growth of 2.6 cm/year and the size of the widest clone, we estimated the age of the oldest individual to be at least 300 years. A single genotype can occupy a large surface and sometimes form a dense patch, suggesting that this species adopts a phalanx growth form with limited intermingling of some genets.     

黑龙江水系不同倍性鲫鱼的遗传多样性

摘要: 利用12对微卫星标记对黑龙江水系6个野生鲫鱼不同倍性群体进行遗传结构分析。结果表明: 6个采集群体的平均等位基因数为5.8~6.8, 平均有效等位基因数为2.8~4.6, 平均期望杂合度为0.5592~0.6962, 平均多态信息含量为0.5962~0.6481, 说明这几个群体遗传多样性水平较高。根据d值, 各群体均有不同程度的偏离Hardy-Weinberg平衡的现象, 均表现为杂合度过度。Kruskal-Wallis 检验表明两种倍性、6个采集群体鲫鱼遗传多样性差异不显著, 没有发现三倍体鲫由于倍性增加而出现额外的等位基因片段。群体间基因分化系数(GST)为0.0398, 表明群体间存在轻度遗传分化。聚类分析表明, 同水体两种鲫鱼亲缘关系最近, 不同采集群体间松花江与乌苏里江分化最小, 月亮湾与新荒泡其次, 双凤水库群体分化最大     

New cell types of the pancreatic islets in the crucian carp,Carassius carassius

Summary The pancreatic islets ofCarassius carassius have been studied by electron microscopy. 1. Besides A-, B- and D-cells, two new cell types, the fourth and the fifth, have been identified. The fourth cell type is numerous; it occurs interposed among the other types of islet cells or in small clusters. The secretory granules (90–280 mg in diameter) are round or oval and usually with much lower electron density than α- and δ-granules. The secretory granules of the fifth type of cell (approximately 140–240 mμ in diameter) contain finely granular material and an electron dense core that is round or often tetra- or hexagonal. 2. The islet cells with clear cytoplasmic matrix generally contain large numbers of fine, agranular and cored vesicles 400–680 ? in diameter. They appear, in bead-like chains, or randomly scattered throughout the cytoplasm, or often clustered in aggregates close to the secretory granules and show evidence of incorporation into the secretory granules. The two types of vesicles may be formed by constriction or pinching-off of the tubular smooth endoplasmic reticulum.