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 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.     

Polymorphic microsatellites from silver crucian carp (Carassius auratus gibelio Bloch) and cross‐amplification in common carp (Cyprinus carpio L.)

Fifteen polymorphic microsatellites were isolated from the genome of silver crucian carp (Carassius auratus gibelio Bloch). Allele numbers ranged from two to four with an average of 2.7/locus, and the proportion of tri‐ and diallelic heterozygotes was 99.3%. The individuals tested seem to have originated from two different clonal lines: 14 of 16 showed the same genotype at all loci tested, whereas the remaining two were also identical, but different from the former ones. Eleven out of 15 primer pairs cross‐amplified products from the genome of common carp, whereas only five from that of zebrafish.     

Phylogenetic relationships of silver crusian carp Carassius auratus gibelio, C. auratus cuvieri, crucian carp Carassius 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.     

Inheritance of microsatellite DNA in bisexual gynogenesis complex of Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch)

Five gynogenetic progeny groups of silver crucian carp Carassius auratus gibelio were produced and sex ratios (males:total progeny) of each of the progeny groups were analysed. About 110 males and 366 females were genotyped at 15 microsatellite loci for comparison with their parents to (1) verify the gynogenesis status of Fangzheng C. auratus gibelio, (2) detect the incorporation of paternal genetic material into the offspring and (3) study the possible association of genetic exchange at microsatellite loci with the existence of sex. The sex ratios in progenies of five groups were highly variable, but all had significant female bias. The sex ratio ranged from 0 to 0·37. Significant differences in the sex ratio within and between groups were also found. Microsatellite genotyping at 15 loci showed that 100 and 97% of the progeny shared the same genotype with the mother in four groups and in one group, respectively, confirming that gynogenesis is the general mechanism of reproduction in C. auratus gibelio. However, 0·63% of all offspring did show incorporation of paternal genetic material. No single loci tested were associated with the occurrence of male progeny, indicating unknown genetic mechanisms for sex determination in C. auratus gibelio.     

三种鲫鱼品系同工酶比较研究

采用聚丙烯酰胺凝胶垂直板电泳技术,对彭泽鲫、银鲫D系以及野鲫三种鲫鱼品系的心、肝和肾脏组织的乳酸脱氢酶(LDH)、苹果酸脱氢酶(MDH)、苹果酸酶(ME)、酯酶(EST)和超氧化物歧化酶(SOD)同工酶表型进行了比较研究.结果表明彭泽鲫乳酸脱氢酶同工酶比银鲫D系在肝组织多出二条酶带(LDH7'和LDH8');超氧化物歧化酶在心和肾组织中分别多出一条酶带(SOD12'),表明彭泽鲫和银鲫已在生化水平产生明显的分化,推测它们可能起源于不同的地区,由不同的祖先,独立演化而形成.此外,彭泽鲫和银鲫D系的同工酶电泳图谱都包含野鲫的基本酶带,而彭泽鲫和野鲫的酯酶同工酶电泳图谱尤为相似,推测彭泽鲫和银鲫可能起源于野鲫,而彭泽鲫和野鲫的关系较近,银鲫和野鲫的关系较远.     

Genetic diversity analysis of crucian carp (Carassius auratus) based on cyt b and D-loop-containing region around Hongze Lake

Environmental Biology of Fishes - Crucian carp (Carassius auratus) is an important freshwater aquaculture species in China. The study of the genetic diversity of native crucian carp will provide...     

A Set of Polymorphic Trinucleotide and Tetranucleotide Microsatellite Markers for Silver Crucian Carp (Carassius auratus gibelio) and Cross-Amplification in Crucian Carp

Silver crucian carp (Carassius auratus gibelio Bloch), as a gynogenetic fish, is a promising model for the study of the evolutionary genetics of vertebrates. We have developed 59 polymorphic trinucleotide and tetranucleotide markers for the silver crucian carp through the biotin capture method and radioactive-labeling hybridization. The number of alleles ranged from 2 to 12 in the population, and the average proportion of heterozygotes (including tri- and diallelic) at polymorphic loci was 76.8%. In addition, these loci were successfully applied to a close relative, the crucian carp (Carassius auratus), by cross-amplification, as shown by the range of alleles (2–19), observed heterozygosity (0.1765–0.9706), expected heterozygosity (0.2392–0.9421), and polymorphism information content (0.2186–0.9236).     

Isolation and characterization of polymorphic trinucleotide microsatellites of the polyploid crucian carp (Carassius auratus)

In this study, we isolated and characterized nine polymorphic trinucleotide microsatellites (CAG and CCT) from the crucian carp (Carassius auratus). The number of alleles per locus ranged from two to six. Five loci showed a significantly excess homozygosity, and a genetic linkage between CAL0102 and CAL0495 was strongly suggested. Our results confirmed the triploidy of Korean individuals, and the microsatellites were found to be useful for analysing the allelic state of the polyploid crucian carp.     

A molecular approach to detect hybridisation between crucian carp (Carassius carassius) and non-indigenous carp species (Carassius spp. and Cyprinus carpio)

1. Releases of non‐native fish into the wild is an increasing problem posing considerable ecological and genetic threats through direct competition and hybridisation. 2. We employed six microsatellite markers to identify first generation hybrids and backcrosses between native crucian carp (Carassius carassius) and introduced goldfish (C. auratus) and common carp (Cyprinus carpio) in the U.K. We also investigated the genetic characteristics of the taxonomically controversial gibel carp (Carassius spp.) from sites across Europe. 3. Natural hybridisation between goldfish and crucian carp occurs frequently, although hybrids between all other species pairs were observed. Only 62% of British crucian carp populations (n = 21) consisted exclusively of pure crucian carp. In some populations hybrids were so frequent, that no pure crucian carp were caught, indicating a high competitive ability of hybrids. 4. Most hybrids belonged to the F1 generation but backcrossing was evident at a low frequency in goldfish × crucian carp hybrids and goldfish × common carp hybrids. Furthermore, some local populations had high frequencies of backcrosses, raising the opportunity for introgression. 5. Gibel carp from Germany and Italy belonged to two triploid clonal lineages that were genetically closely related to goldfish, whereas all individuals identified from British populations proved to be crucian carp × goldfish hybrids. 6. Our study suggests that the release of closely related exotic cyprinids not only poses a threat to the genetic integrity and associated local adaptations of native species, but may also contribute to shifts in community structure through competitive interactions.     

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     

Subcellular localization and functional characterization of a fish IRF9 from crucian carp Carassius auratus

Mammalian interferon (IFN) regulatory factor 9 (IRF-9) has long been recognized as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex, which is critical for type I IFN to induce the expression of IFN-stimulated genes (ISGs) against viral infection. Recent studies have shown that fish IFN exerts antiviral effects by induction of a number of ISGs and also of itself; however, little is known about the role of fish IRF9 in IFN signaling. Here we identify a fish IRF9 orthologue (CaIRF9) from IFN-producing cell line, crucian carp Carassius auratus blastulae embryonic (CAB) cells. Analysis of subcellular distribution of CaIRF9-green fluorescent protein indicates that CaIRF9 is constitutively present in the nucleus, which is driven by two nuclear localization signals (NLS), one locating within DNA-binding domain (DBD) of CaIRF9 and the other immediately behind DBD, although human IRF9 contains only one NLS analogous to the former of CaIRF9. Overexpression of CaIRF9 together with CaSTAT2 not only activates ISRE-containing promoter but also upregulates the expression of fish ISGs. Strikingly, CaIRF9 together with CaSTAT2 also exhibits an ability to activate crucian carp IFN promoter, and blockade of cellular CaIRF9 attenuates IFN itself-induced activation of crucian carp IFN promoter. Taken together, these data suggest that crucian carp IFN induces the expression of ISGs and also of itself possibly by the JAK-STAT signaling pathway that is conserved from fish to mammals.     

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.     

Detection of Myxobolus rotundus (Myxozoa: Myxosporea) in skin mucus of crucian carp Carassius auratus auratus using a monoclonal antibody

Diagnosis of myxosporean Myxobolus rotundus infection was conducted by examining skin mucus from the infected crucian carp Carassius auratus auratus with a monoclonal antibody, MAb 2D12, raised previously against the parasite. A positive reaction was observed in skin mucus collected from infected fish, and spores and pre-spore stages of the parasite were identified by the MAb 2D12. It was also demonstrated that M. rotundus infection can be successfully detected by a simple method, enzyme-linked immunosorbent assay (ELISA), and that skin mucus collected from infected fish skin had a significantly higher optical density (OD) value than that from uninfected fish.     

萍乡肉红鲫的性腺发育研究

萍乡肉红鲫(Pingxiang red-transparent crucian carp,Carassius auratus L.)是在江西省萍乡地区分布的天然三倍体鲫突变体经人工选育后获得的遗传性状基本稳定的后代,具有两性生殖和雌核生殖两种生殖方式.研究以F5代萍乡肉红鲫为材料,自孵化后每满1个月开始取性腺,观察了其卵巢1周年性成熟和精巢的发育过程,结果表明萍乡肉红鲫的性腺为1年成熟类型.卵巢发育进程町以分为6个时期,卵母细胞发育相应可分为6个时相.统计了卵巢成熟系数周年变化,体重为95 g左右的雌性萍乡肉红鲫,其成熟卵巢的成熟系数约为(11.73±2.8)%,成熟的卵母细胞内充满卵黄,相对怀卵量为(3018±310)粒/g.萍乡肉红鲫精巢属于小叶型,在精小叶中可观察到不同发育阶段的生殖细胞.由精原细胞分裂而来的仞级精母细胞经分裂增殖,产生次级精母细胞并最终发育成为精子.萍乡肉红鲫的精巢发育程序与普通鲫鱼和鲤鱼相似,卵巢和精巢的发育过程基本同步,孵化后50日龄内性腺分化不明显,到70日龄左右开始出现雌雄分化,3月龄发育为第1期,4-5月龄发育为第2期,6-7月龄发育至第3期,7-10月龄可见第4期卵巢,1年即可成熟产卵,精巢可排出精液.结果表明,该鲫鱼突变体的性腺发育与普通二倍体鲤(鲫)鱼的性腺发育方式类似.     

Difference in the hypoxia tolerance of the round crucian carp and largemouth bass: implications for physiological refugia in the macrophyte zone

The hypoxia tolerance of larval and juvenile round crucian carp, Carassius auratus grandoculis, and largemouth bass, Micropterus salmoides, was determined using respirometry to examine the potential of hypoxic areas in the macrophyte zone as physiological refugia for round crucian carp. The tolerance, which was measured as the critical oxygen concentration (Pc), was 1.32 mg O₂/l in the round crucian carp and 1.93 mg O₂/l in the largemouth bass. As the round crucian carp tolerated hypoxia better than the largemouth bass, hypoxic areas in the macrophyte zone might function as physiological refugia for round crucian carp.     

Studies on resistance characteristic and cDNA sequence conservation of transferrin from crucian carp, Carassius auratus

Transferrin (Tf) is a kind of non-heme β-globulin with two iron ions (Fe³⁺)-binding sites. To prove Tf’s physiological functions, Fe³⁺-proteins, serum iron contents, and total iron-binding capabilities were tested for Tfs of crucian carps (Carassius auratus) and sliver carps (Hypophthalmichthys molitrix). The above results demonstrated that sliver carps shared 1/3 Tf alleles with crucian carps; Tf of crucian carps had stronger Fe³⁺-binding ability and transportation ability in plasma than that of sliver carps. In addition, the results of oxygen consumption experiments indicated that crucian carps had the higher oxygen utility rate than sliver carps. For acute hypoxia exposure assay, normoxic gas mixture, hypoxic gas mixture A, and hypoxic gas mixture B were used to induce oxygen-regulated gene expression of crucian carps in acute hypoxia. The results of quantitative real-time PCR revealed that mRNA levels of Tf gene, Tfr gene and ATPase gene were down-regulated in acute hypoxia but mRNA level of LDHa gene was up-regulated in acute hypoxia. The results of crucian carp Tf-cDNA sequence analysis showed that cDNA regions of two Fe³⁺-binding sites were T₇₄₇–T₁₀₂₆ and T₁₇₃₇–A₁₈₈₄ based on the principle of bioinformatics. The sequence conservation of two Fe³⁺-binding sites was higher than that of the other five regions, which were confirmed according to the subregion model of Tf-cDNA sequence.