草鱼连续两代的雌核发育群体及湘江流域群体基因组DNA的微卫星比较分析

对湘江流域草鱼群体,一代及连续两代极体型人工诱导雌核发育草鱼群体基因组DNA的微卫星引物统计分析结果表明:湘江流域草鱼群体存在一定程度的遗传多态性;大多数被检测的微卫星位点上存在两个以上的等位基因,但遗传多样性程度较低。一代人工诱导雌核发育草鱼群体中个体的基因位点已基本纯合,但就整个群体而言,个体之间的基因型还不完全一致,表现出一定的多态性。连续两代人工诱导雌核发育草鱼群体中不仅所检测个体的基因位点已完全纯合,并且各个体的基因型也完全相同。这些观察结果说明所检测的两代人工诱导雌核发育草鱼群体是纯合的,经连续两代人工诱导雌核发育可能建立起草鱼纯系。该实验结果还发现不仅草鱼的微卫星位点上存在等位基因的多态性,而且微卫星位点本身也存在多态性;在人工诱导草鱼雌核发育的过程中不仅存在微卫星等位基因快速丢失的现象,而且也存在微卫星位点丢失的现象。因此,加强对自然水体中草鱼种质资源多样性的保护和利用各种现代生物学技术纯化、筛选和组合优良性状基因,是草鱼遗传育种中同样重要和不可或缺的两个方面。     

翘嘴红鲌(Erythroculteri lishaeformis)精子诱导的雌核发育团头鲂(Megalobrama amblycephala)细胞及其分子遗传学分析

该研究运用紫外灭活的翘嘴红鲌(Erythroculter ilishaeformis)精子刺激团头鲂(Megalobrama amblycephala)卵子进行雌核发育,并于0~4℃冷水刺激条件下获得雌核发育团头鲂群体。在此基础上,于细胞遗传学(DNA含量测定、染色体数目检测及性腺发育观察)及分子遗传学水平(微卫星分析)研究了雌核发育团头鲂的细胞生物学以及遗传学性状。结果表明,雌核发育团头鲂DNA含量与普通团头鲂一致(2n=48),其外形特征与对照组极为相似,且均为雌性,为团头鲂性别决定方式(XY型)提供了细胞遗传学依据。同时,微卫星结果表明,在普通团头鲂、雌核发育团头鲂及翘嘴红鲌3个群体中共扩增出63个等位基因,雌核发育团头鲂群体平均观测杂合度和平均期望杂合度均显著低于亲本,即经过一代雌核发育,雌核发育团头鲂基因纯合度显著高于普通团头鲂和翘嘴红鲌,已达到快速建立纯系的目的,且雌核发育团头鲂群体与普通团头鲂群体遗传距离接近,即雌核发育为母系遗传。该研究为团头鲂遗传选育及品系改良提供了遗传材料。     

三角鲂×翘嘴鲌、团头鲂×翘嘴鲌两种杂交后代微卫星遗传结构分析

为了指导三角鲂(Megalobrama terminalis)、团头鲂(Megalobrama amblycephala) 与翘嘴鲌(Erythroculter ilishaeformis)的杂交育种工作, 利用筛选出的16对微卫星引物, 比较分析了团头鲂、三角鲂、翘嘴鲌、团头鲂♀×翘嘴鲌♂、三角鲂♀×翘嘴鲌♂后代群体的遗传结构; 结果显示, 平均等位基因数(N_a)分别为3.56、3.63、3.44、4.00和4.31, 平均观测杂合度(H_o)分别为0.3510、0.3757、0.3175、0.3818和0.4079, 平均期望杂合度(H_e)分别为0.6182、0.6290、0.5921、0.6490和0.6825, 平均多态信息含量(PIC)分别为0.5354、0.5367、0.5258、0.5785和0.6067。杂交群体的平均多态信息含量均大于他们的亲本团头鲂、三角鲂和翘嘴鲌, 表明杂交亲群体的遗传多样性较高。聚类分析显示团头鲂与三角鲂首先聚类, 团头鲂♀×翘嘴鲌♂与三角鲂♀×翘嘴鲌♂首先聚类, 然后这2大类聚为一支, 最后与翘嘴鲌聚类。其中团头鲂与翘嘴鲌遗传距离最远, 为0.5204, 团头鲂和三角鲂遗传距离最近, 为0.0853, 结合遗传相似度分析表明2种杂交子代均具有母本效应。基因型分析表明, 2种杂交后代的等位基因均来自于父母本。引物TTF3、TTF4、TTF10以及Mam25在5个群体中均可产生特异性条带, 可区分5个群体。研究结果对三角鲂×翘嘴鲌和团头鲂×翘嘴鲌的良种选育、种质资源保存以及种群鉴定具有重要意义。     

黄鳝减数分裂雌核发育及子代遗传纯合度分析

为了培育性状优良、遗传稳定的黄鳝(Monopterus albus)群体,研究探索了人工诱导黄鳝减数分裂雌核发育方法。针对养殖性状优良的深黄大斑鳝进行种质纯合,创制出3个深黄大斑鳝雌核发育群体。流式细胞术和染色体计数分析表明雌核发育黄鳝的细胞DNA含量、染色体数目均与野生型相同。性腺组织学切片观察表明雌核发育黄鳝卵巢发育正常。微卫星多样性分析表明雌核发育黄鳝群体的有效等位基因(N_e)、平均香农指数(I)、平均多态信息指数(PIC)、平均观测杂合度(H_o)及平均期望杂合度(H_e)等参数均极显著低于野生群体,雌核发育群体的遗传纯合度显著提高。雌核发育群体之间的遗传距离增大,遗传相似系数变小。深黄大斑鳝雌核发育群体为培育性状优良的黄鳝养殖品种提供了育种材料。     

雌核发育团头鲂的形态和遗传特征分析

利用团头鲂(Megalobrama amblycephala)正常二倍体群体作为对照组, 对团头鲂减数分裂雌核发育二倍体群体的形态特征、染色体组型、性腺发育及遗传特征进行了分析. 结果表明: 雌核发育群体与正常群体在外部可数、可量性状上没有显著性差异(P0.05); 但雌核发育群体出现了尾鳍条数为12的畸形个体, 与正常个体之间有较大的差异; 两个群体的染色体条数都是48, 核型均为18 m+26 sm+4 st; 观察了20尾雌核发育个体的性腺, 均为雌性个体且卵巢发育良好; 采用10个微卫星标记对2个群体的遗传多样性分析, 结果表明正常群体和雌核发育群体平均等位基因数分别为3.8个和1.7个, 雌核发育群体的多态性显著低于正常群体, 表明减数分裂雌核发育二倍体具有高度的遗传相似性, 可作为一个很好的育种材料.       

草鱼种群SSR分析中样本量及标记数量对遗传多度的影响

利用45对微卫星分子标记（SSR）,以草鱼(Ctenopharyngodon idellus)自然群体为实验材料,探讨野生群体遗传多样性研究中所需的最适样本量与标记量。实验设置6个样本量梯度,9个标记量梯度。对等位基因数（Na）、有效等位基因数（Ne）、观察杂合度（Ho）、期望杂合度（He）等遗传多样性指标的变化趋势进行统计分析。结果表明,样本量、微卫星标记的数量和多态性水平对群体遗传多样性均有较大的影响,其中等位基因数与样本量大小呈显著正相关,而杂合度随标记量的增多而剧烈波动。当取样量大于40,标记量大于25时,各遗传参数值趋于稳定。因此,在应用微卫星标记对水产动物自然群体的遗传学研究中,要根据所研究种类的特点,尽可能采样40尾以上,采用25个以上标记,避免由人为选择的偏差对群体遗传多样性水平的正确评估所造成的影响。同时根据上述研究结果,对陕西草鱼自然群体进行了遗传多样性的评估,结果显示该群体平均等位基因数（MNA）、平均有效等位基因数、平均观测杂合度、平均期望杂合度分别为7.26、4.21、0.73、0.68,认为该群体具有较高的遗传多样性。     

两个不同的人工雌核发育草鱼群体基因组DNA的RAPD分析

采用随机扩增多态性DNA(RAPD)技术对连续两代人工诱导雌核发育草鱼群体和一代人工诱导雌核发育草鱼群体的群体内的遗传相似度、遗传距离以及群体多样性进行了分析 ,并用一个随机取样的普通草鱼群体作比较。用 2 6个多态性引物在 3个群体中共检测到了 2 91个扩增位点 ;在 3个群体中检测到的位点数分别为 2 6 5、2 72和2 82。其中多态性位点数分别为 15、19和 81。遗传学统计分析结果表明 :3个群体的多态位点比例分别为 5 6 6 %、6 99%、2 8 72 % ;香农表型多样性指数分别为 0 170 2、0 316 9和 0 84 5 0 ;按照Nei指数统计的三个群体的遗传相似度分别为 0 985 1、0 982 0、0 9114。这些结果表明 :两个雌核发育草鱼群体的遗传多样性远低于普通草鱼群体 ,而其群体的基因组DNA同质性远高于普通草鱼。而在两个雌核发育草鱼群体中 ,两代雌核发育群体的遗传多样性要低于一代雌核发育群体的遗传多样性 ;而群体的遗传纯合度则前者高于后者。     

长江中上游两个鲢群体遗传变异的微卫星分析

对长江中上游2个鲢群体使用39个微卫星标记进行了遗传多样性分析, 计算并统计了平均观测等位基因数、平均有效等位基因数、多态信息含量、遗传杂合度、Hardy-Weinberg平衡偏离指数、遗传相似系数、遗传距离等遗传参数。结果表明: 万州鲢和监利鲢群体所检测微卫星位点的平均观测等位基因数分别为6.128和4.974; 平均有效等位基因数分别为4.107和3.395; 多态位点百分率分别为100和94.87; 39个微卫星标记共有等位基因259个, 173个等位基因为两群体所共有; 多态微卫星位点的PIC在0.077~0.865之间变动,平均为0.617; 两群体所检测位点平均观测杂合度为0.834和0.775, 平均期望杂合度为0.713和0.623; 两个群体间的遗传相似系数为0.618, 群体间的遗传距离为0.482。结果显示长江中上游两个鲢群体间存在显著遗传分化, 应隶属于不同的种群。     

异源精子遗传物质对雌核发育草鱼基因组的影响分析

为分析外源精子对人工诱导雌核发育草鱼基因组的影响,用随机扩增多态性和微卫星技术对经两代连续人工诱导,遗传背景一致的雌核发育草鱼以及母本草鱼和父本鲤鱼的基因组DNA进行了比较分析。10个随机引物在雌核发育草鱼中共检测到104个RAPD位点,在鲤鱼中检测到103个位点。7对微卫星引物在雌核发育草鱼中共扩增出4个微卫星位点,在鲤鱼中检测到22个位点。两种方法在雌核发育草鱼和鲤鱼中所检测到的位点均没有一个相同。根据RAPD和微卫星分析数据进行的遗传相似度分析表明二代人工雌核发育草鱼群体与其一代人工诱导雌核发育草鱼母本的遗传相似度从0.9903到1.000,与父本鲤鱼的遗传相似度为0.000。这些实验结果证明在适当的紫外线处理强度下,鲤鱼精子的遗传物质能够被完全破坏,不会对雌核发育草鱼的基因组造成遗传污染。     

微卫星DNA与生化标记分析对长爪沙鼠群体遗传分析的比较

目的比较生化标记和微卫星DNA标记方法对长爪沙鼠群体遗传分析的可靠性。方法应用27个生化位点和13个微卫星DNA位点,采用已建立的生化标记和微卫星DNA标记分析方法对国内2个长爪沙鼠群体进行遗传分析,计算并比较两种方法测得的各群体遗传参数。结果生化基因位点中有13个位点在整体中呈现遗传多态性,多态率为48.1%;微卫星位点中有11个位点在整体中表现出多态性,多态率均为84.6%。两种方法测得的平均有效等位基因数趋于一致,微卫星DNA的多态位点百分率和平均杂合度均明显高于生化标记方法。但生化标记和微卫星DNA检测对两个长爪沙鼠群体的遗传多样性差异反映一致,所反映的群体平衡状况也基本一致。结论生化标记分析和微卫星DNA方法均可较好地反映长爪沙鼠群体遗传结构。     

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.     

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.     

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.     

锦鲤4个人工雌核发育家系的微卫星标记研究

利用Crooijmans et al.(1997)分离的包含CA重复单元的普通鲤鱼（Cyprinus carpio L.)的8个微卫星DNA标记,对从锦鲤（Cyprinus carpio L.)的红白,大正和昭和3个不同品系中所获得的4个不同人工雌核发育家系的20尾个体进行PCR扩增。电泳结果表明,8对引物在20尾个体中均能重复稳定地扩增出相应的同源序列。随引物不同,各等位基因数为1－11个,大小在68－264bp。在MFW4,MFW7,MFW19,MFW20,MFW23和MFW24 6个微卫星的扩增结果中,20尾个体的扩增图谱呈现了高度的遗传多态性,不同雌核发育家系内个体的遗传异质性也较大。其中大正（TaS)和红白1（RW1）的个体不仅花色分化显著,而且个体间的平均遗传距离分别高达0．28。通过对微卫星等位基因和基因型分析发现,由于锦鲤品系中的每一个体是通过不断地杂交选育而获得,基因组来源复杂,基因高度杂合。因此,只进行1代的人工雌核发育,其家系内仅部分个体的部分座位出现纯合。所获得的人工雌核发育锦鲤为后续的色素遗传调控机制研究提供了必要的实验材料;同时,所鉴定的微卫星分子标记为进行锦鲤的分子标记育种的基因组作图提供了理想的工具。     

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.     

Evidence for the Formation of the Male Gynogenetic Fish

The females and unexpected males of gynogenetic red crucian carps (GRCC) with the 1:1 sex ratio were found in the progeny of the distant crossing of red crucian carp (RCC; ♀, 2n?=?100) × blunt snout bream (BSB; ♂, 2n?=?48). The females and males of GRCC were fertile, and they mated each other to generate the red crucian carps (GRCC₁) and another variational gray crucian carps (GGCC). The GRCC and their offspring were proved to be diploids (2n?=?100) with one to three microchromosomes by examining the chromosomal metaphases. The evidences for the male’s genetic effect in GRCC were provided by means of fluorescence in situ hybridization, Sox-HMG DNA markers, and microsatellite DNA markers. The genotypic variances of GRCC resulted in their phenotypic variances which were quite different from their maternal parent. It was concluded that the formation of the male gynogenetic fish in GRCC resulted from the genetic leakage of the paternal fish in the form of the microchromosomes including the paternal male-determining gene. After being activated by the sperm of BSB, which was inactivated and finally degraded but left the microchromosomes, the egg of RCC, in which the 50 chromosomes were spontaneously doubled to 100 chromosomes, developed into the diploid male gynogenetic fish. The formation of the bisexual GRCC and their progeny indicated that the distant hybridization could generate the bisexual diploid gynogenetic fish with genetic variation derived from the paternal fish, which is of great significance in both fish genetic breeding and evolutionary biology.     

Induced Gynogenesis in Grass Carp (Ctenopharyngodon idellus) Using Irradiated Sperm of Allotetraploid Hybrids

Grass carp (Ctenopharyngodon idellus) eggs were activated by UV-irradiated diploid sperm of allotetraploid hybrids derived from red crucian carp (♀)?×?common carp (♂) and then duplicated by cold shock in 4-6°C water for 10-12 min. Different cold shock initiation times resulted in two types of diploid gynogenetic grass carp: meiotic gynogenetic (meiG) and mitotic gynogenetic (mitG). Over a 5-year period, a total of 17,170 meiG and 1,080 mitG fry were produced and 6,862 meiG and 372 mitG grass carp survived. The gynogenetic fish were confirmed by morphological characteristics, chromosome examination, and microsatellite DNA analysis. The morphological traits of the gynogenetic grass carp were similar to those of wild diploid grass carp. Normal gynogenetic fish were identified as diploid with 48 chromosomes by chromosomal metaphases examination, while nonviable abnormal embryos were detected as haploid with 24 chromosomes. Microsatellite DNA analysis indicated that after one generation of gynogenesis, the genetic purity of meiG and mitG grass carp was significantly increased over that of wild grass carp. In addition, both meiG and mitG grass carp groups were 100% female, and 88% of these showed normal ovary development. Thus, the sex determination mechanism in female grass carp was homogamety. The ability to establish pure all-female groups of meiG and mitG grass carp should be a valuable contribution to both fish genetics and grass carp breeding.     

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].     

Development of microsatellite markers for blunt snout bream Megalobrama amblycephala using 5'-anchored PCR

A rapid method for isolating microsatellite loci in blunt snout bream, based on the 5'-anchored polymerase chain reaction technique, revealed 522 microsatellite loci (consisting of 442 dinucleotide, 4 trinucleotide and 76 tetranucleotide repeats). Of the 25 loci characterized, 10 turned out to be highly polymorphic. The number of alleles per locus ranged from 3 to 17 while the expected heterozygosity ranged from 0.4899 to 0.9355 in population of selected strain F(7 ) and from 0.5786 to 0.9556 in wild population from Lake Liangzi. These markers are useful as tools for the detection of genetic variation levels in selected strains and wild populations of blunt snout bream for germplasm conservation.