草鱼雌核发育后代不同群体的微卫星遗传分析及指纹识别

以紫外线灭活的团头鲂精子激活草鱼卵子,冷休克抑制第二极体排出的方法诱导出长江水系优良F2代草鱼减数雌核发育子代。在后代中不仅存在雌核发育后代,还存在草鲂杂交后代,雌核发育后代的体型与草鱼一致,而草鲂杂交后代的体型介于草鱼与团头鲂之间。Partec CyFlow倍性分析仪测定结果显示:普通草鱼与雌核发育草鱼的相对DNA含量分别为23.01和22.72,二者的DNA含量接近;而高体型子代的相对DNA含量为25.38,介于草鱼与团头鲂（DNA含量28.21）之间,属于草鲂杂交后代。选取17个微卫星标记对草鱼群体、雌核发育草鱼群体和草鲂杂交后代的遗传多样性进行了检测,共检测出59个等位基因,其中43.18个有效等位基因。草鱼对照群体、草鲂杂交后代和雌核发育草鱼群体的平均等位基因依次为3.57、2.86和2.79,平均有效等位基因依次为2.93、2.37和1.96,平均期望杂合度在依次为0.6502、0.5573和0.3775,多态信息含量（PIC）平均值依次为0.5738、0.4649和0.3791。与草鱼对照群体相比,雌核发育草鱼群体的遗传多样性显著下降,表明通过减数雌核发育方法可获得纯合性较高的草鱼个体。构建了草鱼后代不同群体的DNA指纹模式图,筛选到不同群体的9个特异微卫星标记,为草鱼优良群体的选育提供了基础资料。     

Genetic Diversity of and Differentiation among Five Populations of Blunt Snout Bream (Megalobrama amblycephala) Revealed by SRAP Markers: Implications for Conservation and Management

The blunt snout bream (Megalobrama amblycephala) is an important freshwater aquaculture fish throughout China. Because of widespread introductions of this species to many regions, the genetic diversity of wild and natural populations is now threatened. In the present study, SRAP (sequence-related amplified polymorphism) markers were used to assess genetic diversity of blunt snout bream. Three natural populations (Liangzi Lake, Poyang Lake and Yuni Lake, one cultured population (Nanxian) and one genetic strain (‘Pujiang No. 1’) of blunt snout bream were screened with 88 SRAP primer combinations, of which 13 primer pairs produced stable and reproducible amplification patterns. In total, 172 bands were produced, of which 132 bands were polymorphic. Nei''s gene diversity (h) and Shannon''s information index (I) values provided evidence of differences in genetic diversity among the five populations (Poyang Lake>Liangzi Lake>Nanxian>‘Pujiang No. 1’>Yuni Lake). Based on cluster analysis conducted on genetic distance values, the five blunt snout bream populations were divided into three groups, Poyang Lake and Liangzi Lake (natural populations), Nanxian and ‘Pujiang No. 1’ (cultured population and genetically selected strain), and Yuni Lake (natural population). Significant genetic differentiation was found among the five populations using analysis of molecular variance (AMOVA), with more genetic divergence existing among populations (55.49%), than within populations (44.51%). This molecular marker technique is a simple and efficient method to quantify genetic diversity within and among fish populations, and is employed here to help manage and conserve germplasm variability of blunt snout bream and to support the ongoing selective breeding programme for this fish.     

团头鲂MSTN基因cDNA结构、表达及过表达对胚胎发育的影响

研究通过cDNA末端快速扩增法(RACE)克隆得到团头鲂生长抑制素(MSTN)基因的cDNA全长并分析了MSTN基因在团头鲂胚胎、成鱼组织中表达以及MSTN基因在胚胎中过表达情况。结果表明团头鲂MSTN基因的cDNA全长为2187 bp, ORF(开放阅读框)大小为1128 bp, 编码376个氨基酸。组织逆转录PCR (RT-PCR)结果显示, MSTN基因在肌肉、脑和精巢组织中大量表达, 肝脏、脾脏和卵巢组织中的少量表达, 肠、腮、心、眼和肾组织中的微量表达。胚胎逆转录PCR (RT-PCR)结果显示, 在0—44 hpf胚胎发育阶段, MSTN基因表达量较低; 而在48—52 hpf胚胎发育阶段, MSTN基因表达量逐渐升高。整胚原位杂交(WISH)结果显示, 胚胎发育的16 hpf时期MSTN基因主要在脊索中表达, 胚胎发育的28 hpf和55 hpf时期MSTN基因在脑中表达。MSTN基因过表达结果显示, 胚胎在体节发生期出现前-后轴拉长, 背-腹轴变短; 脊索发生扭曲, 强烈抑制体节发育而导致不分化等现象。研究为后续团头鲂MSTN基因的功能研究及团头鲂分子育种提供相关参考依据。     

Molecular cloning and function analysis of insulin-like growth factorbinding protein 1a in blunt snout bream(Megalobrama amblycephala)

Insulin-like growth factor-binding protein 1(IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream(Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions.     

白藜芦醇对高脂胁迫团头鲂抗氧化能力、非特异免疫机能和抗病力的影响

为探讨白藜芦醇（RSV）对高脂胁迫团头鲂特定生长率、抗氧化能力、非特异免疫机能和抗病力的影响,试验设计5组饲料:正常脂组（脂肪水平5%）、高脂组（脂肪水平11%）以及在高脂组中分别添加0.04%、0.36%、1.08% RSV。养殖试验持续10周,在采样结束后,进行嗜水气单胞菌攻毒试验,记录攻毒后96h的成活率。结果表明:团头鲂的特定生长率和日均采食量在添加1.08% RSV组出现最小值,并显著低于其他各组,且团头鲂的饲料效率表现出相似趋势。长期高脂饲喂可导致团头鲂血浆GSH含量显著下降,血浆MDA和NO含量显著升高,形成氧化应激。而长期氧化应激状态,可使团头鲂血浆溶菌酶活性和补体C3含量显著降低,肝脏中HSP70和HSP90应激调控基因表达上调,TNF-α炎症反应基因表达也上调。添加0.04% RSV组显著降低了血浆中SOD活性;添加0.36%和1.08% RSV组显著降低血浆MDA和NO含量,显著抑制了血浆SOD和CAT活性,且添加1.08% RSV组显著增加了鱼体血浆GSH含量。添加0.04%、0.36%和1.08% RSV组均显著提高了团头鲂血浆补体C3含量和溶菌酶活性,显著下调了高脂胁迫团头鲂肝脏中HSP70、HSP90与TNF-α mRNA的表达量。嗜水气单胞菌攻毒后团头鲂的成活率显著受到RSV的影响,并且在1.08% RSV添加组成活率最大。综上结果表明,团头鲂摄食高脂日粮之后,机体处于氧化应激状态,导致鱼体非特异免疫力和抗病力低下。而添加适宜剂量的RSV能够改善机体这种氧化应激的状态,提高鱼体的非特异免疫力和抗病力,其中以1.08%的添加量最优。     

饲料糖和脂水平对团头鲂生长性能及血浆代谢物的影响

实验以初重(6.20±0.01) g的团头鲂(Megalobrama amblycephala)幼鱼为研究对象,分别投喂高糖低脂(HCLL, 45%糖和2%脂)、中糖中脂(MCML, 30%糖和8%脂)和低糖高脂(LCHL, 15%糖和14%脂)的3种等氮饲料56d,以探究饲料不同糖和脂水平对团头鲂生长性能、鱼体生化组成、营养沉积及血浆代谢物的影响。实验结果显示,饲料糖和脂水平对团头鲂摄食率无显著性影响。随饲料糖水平降低和脂水平升高,团头鲂幼鱼特定生长率和饲料效率提高。高糖低脂饲料未造成肝脏糖原或甘油三酯含量的显著沉积,且各饲料组间肝体比、脏体比及肥满度均无显著性差异。饲料糖和脂水平对团头鲂鱼体生化组成无显著性影响,但随饲料糖水平降低和脂水平增加,团头鲂蛋白沉积率升高,脂肪沉积率降低。另外,血浆葡萄糖、甘油三酯、游离脂肪酸和胆固醇含量各组间均无显著性差异。低糖高脂组血浆低密度脂蛋白胆固醇显著升高,表明肝脏将脂质转运至外周组织以维持肝脏脂稳态。研究结果表明,团头鲂对饲料脂利用优于糖类,且具有较好地应答饲料糖和脂水平的代谢机制,研究结果可为团头鲂的饲料配方设计提供支撑。     

High-fat diet induces aberrant hepatic lipid secretion in blunt snout bream by activating endoplasmic reticulum stress-associated IRE1/XBP1 pathway

This study was conducted to understand the effect of high-fat diet challenge on lipid transport and endoplasmic reticulum stress in blunt snout bream. Ninety fish (average weight: 41.84 ± 0.07 g) were randomly fed a control diet (6% fat) or a high-fat diet (11% fat) for 9 weeks. The growth performance and feed utilization efficiency were evaluated at the end of the trial. The liver samples of both groups were harvested for molecular analysis and histological evaluation. Compared to the Control group, the high-fat diet group showed no effects on either growth performance or energy intake in blunt snout bream. However, high-fat diet resulted in a massive accumulation of lipid and pathological structural alternations, and disrupted expression of lipid transport-related genes and endoplasmic reticulum stress in the liver of the fish. In vitro, after exposure of the isolated primary hepatocytes from blunt snout bream to oleic acid, the cells showed increased intracellular TG accumulation, decreased VLDL secretion, which was attributed to altered expression levels of lipid transport-related genes through the activated IRE1/XBP1 signaling. The oleic acid-induced detrimental effects were alleviated by co-incubating the cells with an IER1 inhibitor, 4μ8c. In conclusion, high-fat diet could lead to aberrant lipid secretion by activating the ER stress-associated IRE1/XBP1 pathway. Inhibiting the activity of IRE1 represents a promising target to rescue the side-effects of high-fat diet on the liver function of blunt snout bream.     

人工诱导雌核发育日本白鲫

分别用遗传失活的散鳞镜鲤(Cyprinus carpio L.)、团头鲂(Megalobrama amblycephala)精子诱导日本白鲫(Carassius cuvieri)进行雌核发育.未经冷休克处理,用UV照射过的镜鲤的精子(其最佳UV照射剂量为4 200 mJ/cm2)与日本白鲫卵子受精获得(32.4±3.3)%雌核发育单倍体和(0.7±0.3)%杂交二倍体,而照射过的团头鲂精子(其最佳UV照射剂量为3 600mJ/cm2)与日本白鲫卵子受精得到了(33.8±1.4)%雌核发育单倍体和(0.5-±0.3)%杂交二倍体.日本白鲫卵子分别经灭活的镜鲤、团头鲂的精子激活(精子经各自最佳UV剂量处理),施以冷休克处理(受精后2min,0-4℃,40min),其孵化率分别为(27.8±2.1)%和(29.4±3.3)%,发育到摄食期,其正常的雌核发育二倍体鱼苗分别为(15.7±3.4)%和(23.6±4.1)%,在镜鲤实验组中,其正常的雌核发育二倍体鱼苗占孵化鱼苗总数的56%,这比团头鲂实验组中正常雌核发育二倍体鱼苗的比率(达到80%)低,结果表明诱导日本白鲫雌核发育,与母本遗传关系远的团头鲂精子较镜鲤的更有效.从染色体数目、外形特征和性腺结构等方面证实雌核发育后代的生物学特性.雌核发育日本白鲫全为雌性,这表明其雌性是同配XX型.雌核发育日本白鲫在生产上将有着潜在的价值,如比普通日本白鲫长得更快,抗病能力强等.所有的雌核发育日本白鲫的染色体数目都是100,而所有的团头鲂与日本白鲫的杂交后代都是三倍体(3n=124),新三倍体鱼在鱼类养殖和理论研究中将存在潜在的价值.     

Microsatellite analysis of different ploidy offspring of artificial gynogenesis in Cyprinus carpio

Gynogenesis was induced by using UV-irradiated spermatozoa of blunt snout bream Megalobrama amblycephala to activate eggs of common carp Cyprinus carpio. The maternal genome was then duplicated by cold shock in 0 to 4° C cold water to retain the second polar body. Two kinds of fry, normal fry and abnormal tortuous fry, were hatched. Their DNA content was measured by flow cytometry. The normal fry were identified as diploid, representing the successful gynogenesis in C. carpio whereas the abnormal tortuous fry were haploid. Ten microsatellite loci were used to study the genetic diversity among C. carpio, diploid gynogenetic C. carpio and unduplicated haploid tortuous fry. The results indicated that the genetic homozygosity of gynogenetic C. carpio was significantly higher than that of C. carpio. The genetic homozygosity of the haploid C. carpio was intermediate between that of gynogenetic C. carpio and C. carpio. It might be easier for the allogenetic DNA fragments to be integrated into the haploid genome than into diploid gynogenetic genome.     

Induction of Gynogenesis in Japanese Crucian Carp (Carassius cuvieri)

Diploid gynogenesis was induced in Japanese crucian carp (Carassius cuvieri) eggs using UV-irradiated genetically inactive spermatozoa from mirror carp (Cyprinus carpio L.) or blunt snout bream (Megalobrama amblycephala), with or without cold shock. The optimal radiation dosage was 4200 mJ/cm² and 3600 mJ/cm² for mirror carp and blunt snout bream sperm, respectively. At this dosage and without cold shock, the yields were (32.4±3.3)% vs. (33.8±1.4)% gynogenetic haploids and (0.7±0.3)% vs. (0.5±0.3)% hybrid diploids, respectively. At the optimal UV dosage but with cold shock (2 min after fertilization, 0-4°C for 40 min), the hatching rates were (27.8±2.1)% and (29.4±3.3)%, respectively. From hatching to feeding, (15.7±3.4)% and (23.6±4.1)% normal gynogenetic diploids were recorded, respectively. Survival of normal gynogenetic diploids was 56% out of the hatched fry when using irradiated spermatozoa of mirror carp, which was lower than that (up to 80%) when using irradiated spermatozoa of blunt snout bream. This indicated that the sperm of blunt snout bream, with distant genetic relation to the maternal Japanese crucian carp, was more effective than that of mirror carp to induce diploid gynogenesis. The nature of the gynogenetic progeny was identified with external appearance, chromosome number and gonad structure. The presence of only females in gynogenetic progeny probably suggested XX genotype in the female Japanese crucian carp. The gynogenetic diploids have potential values such as faster growth and stronger disease resistance than the normal Japanese crucian carp. All gynogenetic progeny possessed 100 chromosomes whereas all J x B crosses were triploid with 124 chromosomes. The formation of the new triploid hybrids in J x B crosses may be useful in aquaculture.     

Isolation and characterization of microsatellite DNA loci in sea bass,Lates calcarifer Bloch

Polymerase chain reaction (PCR)‐based isolation of microsatellite arrays (PIMA) technique was used to isolate seven polymorphic microsatellite loci in sea bass, Lates calcarifer Bloch. A total of 62 samples of wild and cultivated sea bass collected from a few populations within Peninsular Malaysia were used in the study. For seven polymorphic loci, the number of alleles ranged from four to nine and locus heterozygosities ranged from 0.710 to 1.000. The loci will be useful for studying population structure, genetic variability of wild and hatchery stocks of L. calcarifer and broodstock management purposes.     

Isolation and characterization of dinucleotide microsatellite loci in the Asian elephant (Elephas maximus)

The endangered Asian elephant is found today primarily in protected areas. We characterized 18 dinucleotide microsatellite loci in this species. Allelic diversity ranged from three to eight per locus, and observed heterozygosity ranged from 0.200 to 0.842 in a wild population. All loci were in Hardy-Weinberg equilibrium, but linkage disequilibrium was detected between two loci in the wild, but not in the zoo elephants. These loci will be useful for the population-level studies of this species.     

Isolation and characterization of nine polymorphic microsatellite loci in the rock carp, Procypris rabaudi (Tchang)

Nine highly polymorphic microsatellite loci were isolated from AC- and GATA-repeat microsatellite enrichment DNA libraries in the rock carp, Procypris rabaudi (Tchang). The number of alleles for these loci ranged from eight to 18 in tested individuals. Polymorphism information content ranged from 0.712 to 0.908 with an average of 0.837. Average observed and expected heterozygosities were 0.719 and 0.870, respectively. These molecular markers will be useful for the assessment of genetic diversity and analysis of population structure in wild rock carp.     

团头鲂GPR43基因克隆、组织分布及黄连素对其mRNA表达量的影响

试验采用RACE技术克隆了团头鲂(Megalobrama amblycephala)G蛋白偶联受体43(GPR43)基因的cDNA序列, 并探究了不同组织中的GPR43 mRNA表达量及黄连素对其表达量的影响。结果显示, 克隆得到的团头鲂GPR43基因的cDNA序列全长为2026 bp, 含有1个长度为 981 bp的开放阅读框, 编码了326个氨基酸。RT-PCR检测发现GPR43在团头鲂的肠道、肌肉、鳃和肝胰腺中具有较高的表达。为期8周的养殖试验选取均重为(80.00±0.90) g的团头鲂320尾, 随机分于16个网箱中, 饲喂4种不同的试验日粮, 分别为正常日粮(脂肪含量为5%)、正常日粮+50 mg/kg黄连素、高脂日粮(脂肪含量为10%)、高脂日粮+50 mg/kg黄连素。结果显示: 在肠道组织中, 与正常日粮组相比, 高脂组的GPR43表达量降低, 添加黄连素能够显著升高其表达水平(P<0.05)。与正常日粮组相比, 高脂组的胆固醇(CHO)含量以及细胞分裂素蛋白激酶(p38)的表达量均呈现了显著上升(P<0.05)的趋势, 添加黄连素后其含量及表达量显著下降(P<0.05)。肝胰腺组织和肌肉组织中的多不饱和脂肪酸(PUFA)含量变化也有着相似的趋势, 而肉碱棕榈酰基转移酶Ⅰ(CPT Ⅰ)、过氧化物酶体增值因子α&β (PPARα&β)、AMP依赖性蛋白激酶(AMPK)的表达量以及2个组织中的饱和脂肪酸(SFA)和单不饱和脂肪酸(MUFA)含量呈现出了相反的趋势。此外, 在正常日粮中添加黄连素并不能对上述各指标产生明显的调控效应, 有时反而会导致轻微的负调控效应。综上结果表明, 黄连素能够显著上调GPR43在高脂抑制下的表达量, 同时能够缓解高脂诱导的团头鲂肝胰腺脂肪沉积, 改善其脂肪代谢性能。黄连素对于脂肪代谢的调控作用可能通过GPR43受体来实现。     

An AFLP-based approach for the identification of sex-linked markers in blunt snout bream, Megalobrama amblycephala (Cyprinidae)

Sex-specific DNA markers are useful for studying sex-determination mechanisms and establishment of monosex populations. Three widely spaced geographical populations (Liangzi, Poyang and Yuni Lakes in China) of blunt snout bream (Megalobrama amblycephala) were screened with AFLPs to search for sex-linked markers. Female and male pools (10 individuals in each pool) from each population were screened using 64 different primer combinations. A total of 4789 genomic fragments were produced, with a mean frequency of 75 bands per primer pair. Three different primer combinations produced putative sex-associated amplifications and were selected for individual screening in the three populations. However, none showed sex specificity when we converted these three markers into sequence characterized amplified region markers and evaluated all the individuals from the three populations.     

Genetic Analysis of Selected Strains of Eastern Oyster (Crassostrea virginica Gmelin) Using AFLP and Microsatellite Markers

Amplified fragment length polymorphisms (AFLPs) and microsatellite markers were used to examine genetic variation and divergence in 4 selected strains (DBH, NEH, FMF, and CTS) and 1 wild population (DBW) of the eastern oyster Crassostrea virginica Gmelin. Eighty-six AFLP markers (from 3 primer pairs) and 5 microsatellite loci were used for the analysis of 30 oysters from each of the 5 populations. Microsatellite loci were considerably more variable than AFLPs. The observed heterozygosity ranged from 0.560 to 0.640 across populations for microsatellites, and from 0.186 to 0.207 for AFLPs. Both F_st and _PT of microsatellite data and _PT statistics of AFLP data revealed significant divergence between all pairs of populations. There was no significant reduction in heterozygosity in all 4 selected strains; however, the number of alleles per locus was considerably lower in the selected strains than in the wild population. Two strains subjected to long-term selection for disease resistance shared frequency shifts at a few loci, which deserve further analysis to determine if they are linked to disease-resistance genes.     

Development of microsatellite loci in Pacific herring (Clupea pallasi)

Fifteen polymorphic di‐, tri‐ and tetranucleotide microsatellite markers were developed in Pacific herring (Clupea pallasi) to aid in the delineation of population structure of herring in British Columbia. Twelve of the microsatellite loci were analysed in over 4000 wild herring. Expected heterozygosities ranged 0.73–0.95, and only two loci significantly deviated from Hardy–Weinberg equilibrium.     

Isolation and Characterization of Microsatellite Loci for Striped Bass (Morone saxatilis)

Genetic variation has been difficult to detect in striped bass (Morone saxatilis). Therefore, we identified and characterized 13 microsatellite loci to provide additional genetic markers for striped bass. Microsatellites were identified by screening a striped bass genomic library or by using primers developed for European sea bass (Dicentrarchus labrax) microsatellite loci. We found that 6 of the 13 microsatellite loci were polymorphic in DNA samples obtained from wild populations of striped bass. The number of alleles per locus varied from 3 to 12, and the observed heterozygosities ranged from 0.55 to 0.78. These results indicate that microsatellite loci provide more alleles and higher heterozygosities than other genetic markers developed for striped bass. Received November 9, 1999; accepted February 11, 2000.     

Isolation and characterization of polymorphic microsatellite markers from the mosquito Anopheles moucheti,malaria vector in Africa

Anopheles moucheti is a major human malaria vector in Equatorial Africa. The screening of an Anopheles moucheti genomic microsatellite library allowed us to select 36 sequences with AC/GT dinucleotide tandem repeats. Primer pairs were designed to amplify the loci and 25 out of 36 gave a repeatable and scorable amplification. In total, 17 loci were selected for their high degree of polymorphism (the number of alleles per locus ranged from four to 16, and observed heterozygosity from 0.43 to 0.87) and suspicion of absence of null alleles, using 30 wild females from South‐Cameroon. No linkage disequilibrium was found between the loci.