牙鲆渤海自然群体的遗传多样性分析

为分析牙鲆自然群体的遗传多样性并筛选出能有效鉴定群体遗传特征的特异性微卫星标记,实验收集了渤海流域74尾野生个体组成实验群体。选择牙鲆24个连锁群上不同区域的72个微卫星标记进行遗传分析,其中,近着丝粒区域包含17个标记,连锁群中部包含19个标记,远着丝粒区域包含36个标记。结果显示:在连锁群不同区域上等位基因数(A)介于6.400～7.389,有效等位基因数(N_e)介于4.469～5.129,Shannon多样性指数(I)介于1.565～1.683;观测杂合度(H_o)、无偏倚杂合度期望值(H_e)和多态信息含量(PIC)的范围分别为0.568～0.593、0.738～0.753和0.707～0.746;Hardy-Weinberg遗传偏离指数(d)在-0.200以下;群体内个体之间的遗传距离在0.620以上。各项遗传参数表明该实验群体具有较为丰富的遗传多样性,个体之间具有相对较远的遗传距离,适宜作为基础群体进行选育。位于连锁群不同区域的微卫星标记获得的遗传参数并无明显差异,验证了标记所在位置与标记多样性高低不存在必然联系,但这些多态性标记可以作为分析牙鲆群体遗传结构的候选标记。     

牙鲆连续四代减数分裂雌核发育家系的遗传特征分析

为了研究纯合度和遗传相似度在牙鲆(Paralichthys olivaceus)连续四代减数分裂雌核发育家系中的变化规律,本研究利用分布在不同连锁群上的24个高重组率微卫星标记对牙鲆连续减数分裂雌核发育二代(G2)、三代(G3)、四代(G4)家系及普通家系对照组进行了遗传分析。结果显示,24个微卫星位点在对照组、G2、G3、G4家系中,分别检测到96、42、32和32个等位基因,平均等位基因数分别为4.00、1.98、1.33和1.33;期望杂合度分别为0.6416、0.3472、0.1694和0.1492;纯合度分别为0.3503、0.6528、0.8306和0.8508;遗传相似系数分别为0.5822、0.9238、0.9890和0.9988。24个位点中已有17个纯合,但尚有7个保持杂合状态。同时,将上述结果和已发表的减数分裂雌核发育一代(G1)家系的数据进行分析,结果表明,诱导连续减数分裂雌核发育不仅能提高个体的纯合度,同时也可提高子代个体间的遗传相似度;纯合度和遗传相似度在G1、G2和G3家系中能够得到逐步提高,代际之间差异显著(P<0.05);但在G4家系中趋于稳定,与G3家系差异不显著。G4家系的遗传相似性(0.9988)已高于连续20代全同胞交配所获得的理论值(0.9860),连续诱导减数分裂雌核发育是快速建立鱼类近交系的良好方法。     

牙鲆遗传连锁图谱的构建

利用基因组测序得到的大量微卫星序列,以681383B为父本、6812E36为母本杂交获得的F1为作图群体,构建了牙鲆(Paralichthys olivaceus)微卫星标记(SSR)遗传连锁图谱。雌雄图谱共定位SSR标记529个,其中雄性连锁图谱包括418个标记,分布在24个连锁群上,总长度1 418.1 cM,标记平均间隔3.62 cM,图谱覆盖率为88.7%;雌性连锁图谱包括437个标记,分布在24个连锁群上,总长度1 298.1 cM,标记平均间隔为3.16 cM,图谱覆盖率为89.1%。牙鲆中密度遗传图谱的构建为QTL分析以及分子标记辅助育种进一步奠定基础,并可以有效推动牙鲆的遗传改良工作,推动牙鲆养殖业的可持续发展。     

有丝分裂雌核发育牙鲆不育的组织学观察及微卫星标记筛选

在有丝分裂雌核发育牙鲆(Paralichthys olivaceus)中出现数量较多的不育个体,利用组织切片观察比较不育牙鲆的组织学特点,利用分离群体分组分析法筛选不育相关的微卫星标记。结果表明:不育牙鲆的性腺指数为3.77%~4.17%,显著小于可育牙鲆(P0.05)。不育牙鲆的卵巢微血管较少,卵巢膜呈现白色,卵巢较硬,没有游离卵粒;切片结果显示,其卵巢处于Ⅲ期。通过对209个微卫星标记的筛选,共找到11个在可育和不育群体间有差异的标记;利用同一家系可育和不育的个体进行验证后发现,位于15号连锁群的4个标记与牙鲆的育性紧密相关。     

牙鲆减数分裂与有丝分裂雌核发育的遗传差异

利用同一尾牙鲆亲鱼的同一批卵子诱导减数分裂雌核发育二倍体和有丝分裂雌核发育二倍体,同时用牙鲆精子做人工授精制备普通二倍体,作为对照组。利用10对微卫星引物对普通二倍体和两种雌核发育二倍体进行遗传分析。结果表明,母本基因型在8个位点为杂合,2个位点为纯合。普通二倍体有6种基因型,等位基因均来自父母本的随机结合,类型丰富;母本与子代、子代个体之间的遗传相似系数分别为0.452 8和0.560 3,接近随机交配群体的遗传相似度。减数分裂雌核发育二倍体有3种基因型,除在1个位点出现异于母本的纯合基因型外,其他所有位点的基因型与母本完全一致;母本与子代、子代个体之间遗传相似系数分别为0.976 6和0.959 5,接近近交系的遗传相似度。有丝分裂雌核发育二倍体有2种基因型,且全部为纯合型;母本与子代、子代个体之间遗传相似系数分别为0.806 2和0.742 5,有丝分裂雌核发育二倍体全部为纯合个体。减数分裂雌核发育二倍体具有高度的遗传相似性,适于固定母本性状;有丝分裂雌核发育二倍体纯合度高,适于作为制备克隆的亲本;两者具有明显不同的遗传特性,均可作为特征不同的育种材料。     

牙鲆连续两代减数分裂雌核发育家系的遗传特征

对牙鲆(Paralichthys olivaceus)减数分裂雌核发育二倍体(Meio-G1)再次诱导减数分裂雌核发育,获得连续两代雌核发育二倍体家系(Meio-G2),以Meio-G1亲本与1尾普通牙鲆雄鱼人工授精获得的家系作为对照组(control)。利用15个微卫星标记对3个家系进行遗传特征分析。结果显示,15个微卫星位点在Meio-G1、Meio-G2和对照组3个家系中,分别扩增到30、28、50个等位基因,平均等位基因数为2.0、1.9、3.3,平均观测杂合度(Ho)分别为0.875 3、0.774 2、0.908 3,平均纯合度分别为0.124 7、0.221 5、0.091 7。3个家系个体间的平均遗传相似系数分别为0.891 7、0.923 8、0.520 2,亲代与子代之间的平均相似系数分别为0.916 6、0.930 4、0.560 3。高重组率的Poli9-8tuf、Poli18tuf、Poli107tuf 3个位点在Meio-G1和Meio-G2中观测杂合度均为1.0,低重组率的Poli33tuf、Poli24MHFS两个位点在Meio-G1和Meio-G2中均全部纯合,观测杂合度为0。结果表明,Meio-G2的纯合度、个体间平均相似系数以及亲子之间的平均相似度均略高于Meio-G1,显著高于对照组家系,证明连续两代诱导减数分裂雌核发育,能提高鱼类纯合度和遗传相似度,具有固定母本遗传性状的作用。     

牙鲆家系亲权鉴定的微卫星DNA标记分析

为准确进行不同家系的亲权鉴定,筛选具有高亲本排除概率的微卫星DNA标记,从牙鲆选育基础群体中挑选性腺发育良好的雌雄亲本各10尾建立全同胞家系10个,从独立饲养的每个家系中随机选取30尾个体组成系谱结构已知的家系群体,从混合培育的子代群体中随机选取200尾个体组成系谱结构未知的混合群体。48个微卫星DNA标记选自牙鲆第二代遗传连锁图谱,且均匀分布于24个连锁群上,每个连锁群2个标记。家系群体的遗传分析结果发现,10个拥有丰富遗传多态性的微卫星DNA标记表现出高的亲本排除概率,其Excl1和Excl2的范围分别为0.655~0.719和0.792~0.837。随鉴定所用微卫星DNA标记数目的增加,累计排除概率逐步升高。当使用8个微卫星DNA标记鉴定时,累计排除概率达到100%。利用这些标记开展混合群体的亲权鉴定,显示共有13个雌雄个体参与繁殖过程,不同亲本配组产生的后代数量存在差异,亲本与后代群体之间的各项遗传学统计指标不存在明显差异。研究表明,筛选出具有高亲本排除概率的微卫星DNA标记能够有效进行牙鲆家系的亲权鉴定,可以作为今后开展家系选育与DNA分子标记联合育种的候选标记。     

有丝分裂雌核发育牙鲆的微卫星鉴定

通过静水压抑制卵裂方法获得多个牙鲆(Paralichthys olivaceus)有丝分裂雌核发育家系,在仔鱼阶段经历大批死亡后,将各个家系残存的个体混合饲养,24个月后获得1批存活个体。利用11对微卫星分子标记,对173尾有丝分裂雌核发育牙鲆进行分析,结果表明,其中111尾在11位点全部纯合,62尾在部分位点杂合,平均杂合子比例为0.2338。11个位点的等位基因数为4～8,平均等位基因数为5.0336,有效等位基因数为2.0421～5.1268,平均有效等位基因数为3.2815。采用NJ法对111尾纯合牙鲆的亲缘关系进行聚类分析,可分为4群。微卫星标记鉴定结果表明,通过静水压抑制卵裂法可以获得完全纯合的牙鲆,但有部分杂合个体出现。分析其原因,可能是由于某些卵子发育不同步,静水压抑制其第二极体排出而非全部抑制卵裂,导致减数分裂雌核发育所致。     

三倍体牙鲆(Paralichthys olivaceus)群体遗传多样性的微卫星分析

采用微卫星遗传标记技术对三倍体牙鲆群体及二倍体对照群体进行比较分析。取2012年冷休克诱导和培育至11月龄的三倍体及同期二倍体对照牙鲆个体各32尾,通过高盐法提取肌肉组织总 DNA;利用筛选得到的21对微卫星引物进行 PCR 扩增,并经14%聚丙烯酰胺凝胶电泳分型后,进行人工判读和分析。结果显示,21个微卫星位点均为多态,二倍体和三倍体群体的平均等位基因数(A)、基因型总数、平均有效等位基因数(ae)、平均观测杂合度(HO)和平均期望杂合度(HE)分别为5.6和5.0、190和142、3.7和2.8、0.613和0.868、0.681和0.629。二倍体和三倍体群体的多态信息含量(PIC)分别为0.642和0.589,个体识别率(DP)为0.660和0.609,非父排除率(PPE)为0.482和0.399,其累积个体识别率和非父排除率均达到0.999,表明所选座位均属中高识别力的遗传标记,可以将它们应用于今后牙鲆雌核发育群体的遗传变异分析以及进一步的遗传育种的研究中。二倍体和三倍体群体间的遗传距离(D)为0.312,遗传相似系数(I)为0.732,基因分化系数(GST)为0.971。以上结果说明,三倍体诱导对牙鲆的遗传结构造成了一定影响,导致三倍体遗传多样性水平相对二倍体有所下降,二倍体和三倍体牙鲆的基因型也存在一定差异。     

三倍体牙鲆(Paralichthys olivaceus)群体遗传多样性的微卫星分析

采用微卫星遗传标记技术对三倍体牙鲆群体及二倍体对照群体进行比较分析。取2012年冷休克诱导和培育至11月龄的三倍体及同期二倍体对照牙鲆个体各32尾,通过高盐法提取肌肉组织总DNA;利用筛选得到的21对微卫星引物进行PCR扩增,并经14%聚丙烯酰胺凝胶电泳分型后,进行人工判读和分析。结果显示,21个微卫星位点均为多态,二倍体和三倍体群体的平均等位基因数(A)、基因型总数、平均有效等位基因数(ae)、平均观测杂合度(HO)和平均期望杂合度(HE)分别为5.6和5.0、190和142、3.7和2.8、0.613和0.868、0.681和0.629。二倍体和三倍体群体的多态信息含量(PIC)分别为0.642和0.589,个体识别率(DP)为0.660和0.609,非父排除率(PPE)为0.482和0.399,其累积个体识别率和非父排除率均达到0.999,表明所选座位均属中高识别力的遗传标记,可以将它们应用于今后牙鲆雌核发育群体的遗传变异分析以及进一步的遗传育种的研究中。二倍体和三倍体群体间的遗传距离(D)为0.312,遗传相似系数(I)为0.732,基因分化系数(GST)为0.971。以上结果说明,三倍体诱导对牙鲆的遗传结构造成了一定影响,导致三倍体遗传多样性水平相对二倍体有所下降,二倍体和三倍体牙鲆的基因型也存在一定差异。     

Fast development of genetically uniform strains by gynogenesis in Japanese flounder (Paralichthys olivaceus)

The Japanese flounder is a major species in both aquaculture and research. Inbred strains of Japanese flounder were developed efficiently in our laboratory by meiotic and mitotic gynogenetic reproduction techniques. To determine the induction efficiency of gynogenesis and the rate at which full homozygosity is produced, six meiotic gynogenetic females (G1–G6) that had experienced meiotic gynogenesis once and three common females (C1–C3) were selected for production of mitotic gynogenesis in our experiments. Of the nine adult females, all six gynogenetic fish successfully produced viable offspring. However, only one of the three common fish did. Using microsatellite markers, we estimated the homozygosity of gynogenetic fish induced by mitotic gynogenesis. We found that the homozygosity of seven lines (C1, G1–G6) increased quickly, individually from 43%, 72%, 69%, 71%, 78%, 69% and 58% to 100%, 100%, 100%, 90%, 100%, 100% and 90% offspring complete homozygosity. Under mitotic gynogenesis, individuals with higher homozygosity had a higher induction rate. The G4 line showed the highest induction rate, achieving 44.59% convert hatching rate and 29.28% convert normality rate. We conclude that meiotic gynogenesis may be a feasible method to produce DH genetic material in Japanese flounder.     

黄姑鱼异质雌核发育子代的遗传分析

为了解黄姑鱼（Nibea albiflora）异质雌核发育子代的基因纯合情况,利用微卫星标记（SSR）和扩增片段长度多态性标记（AFLP）对黄姑鱼异质雌核发育家系进行遗传鉴定和分析。结果显示：（1）雌核发育家系在4个SSR位点和5对AFLP引物组合扩增出的位点均未发现父本特异性等位条带,表明雌核发育体比率为100%。（2）用于遗传分析的7个SSR位点在雌核发育家系和正常交配家系中均未见完全纯合的情况,雌核发育家系7个SSR位点的平均纯合度为0.382,是正常交配家系（0.161）的2.37倍。雌核发育家系各个体的纯合位点数为0~6个,纯合位点所占比例为0~85.7%。（3）5对AFLP引物共扩增出182条清晰的扩增条带,其中有21条父本特异性条带和16条母本特异性条带。16条母本特异性条带中有7个条带在雌核发育家系中显著偏分离（P<0.05）。雌核发育家系和正常交配家系多态性条带比例分别为14.7%和20.3%。（4）雌核发育家系与母本的遗传相似度高于与正常交配家系的遗传相似度,正常交配家系同父本和母本的遗传距离大致相同。研究结果表明,黄姑鱼异质雌核发育二倍体家系的遗传纯合度显著高于正常交配家系,人工诱导雌核发育是促进基因纯合的一个有效途径,它不仅可以加速有利基因的纯合固定,还可以加速有害基因的淘汰,从而有效提高育种效率。     

Artificial gynogenesis and assessment of homozygosity in meiotic gynogens of spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>)

In the present study, methodology of gynogenetic induction in spotted halibut were developed and optimized; the sex ratio of putative meiotic gynogenetic diploids was determined using AFLP-based molecular sexing technique; the homozygosity of gynogenetic population was assessed as opposed to cultivated population. The results showed that high percentage of meiotic gynogenetic diploids were generated when the eggs fertilized with irradiated heterologous sea perch frozen sperm (30–50 mJ cm⁻²) were cold shocked in sea water of −1°C for 40–75 min at 5 min after fertilization. About 15,200 diploid gynogenetic larvae were achieved and they exhibited normal morphology similar to diploid control. The gynogenetic diploids were 100% female, which first confirmed the female homogamete (XX/XY) sex determination in spotted halibut. The genetic analysis showed that the average H _O was, respectively, 0.404 and 0.724 in gynogenetic population and cultivated population, indicating an increase of homozygosity in gynogenetic population.     

Microsatellite–centromere mapping in bighead carp ( Aristichthys nobilis) using gynogenetic diploid families

Gene–centromere mapping using half‐tetrad analysis is a powerful tool for understanding chromosomal behaviour and determining the position of centromeres in relation to genes or markers in fish. In this study, eight gynogenetic diploid families induced by inhibiting of the second meiotic division were genotyped at 66 microsatellite loci for microsatellite–centromere (M‐C) mapping in bighead carp ( Aristichthys nobilis). The absence of paternal alleles verified the success of gynogenetic development in all gynogenetic families. All loci were consistent with Mendelian segregation in control families. The estimated recombination frequency (y) ranged from 0.057 to 0.875 with an average of 0.477 ± 0.222. Seventeen loci (25.76%) showed high M‐C recombination frequencies of over 0.667. M‐C distances ranged from 2.85 to 43.75 cM under the assumption of complete interference. Thus, these loci are distributed from the centromeres to the telomeres of their respective chromosomes, while mainly in the intermediate region. Information on centromere mapping could serve as a starting point to consolidate the genetic linkage groups in bighead carp.     

Phenotypic variation in meiotic and mitotic gynogenetic diploids of the Nile tilapia, Oreochromis niloticus (L.)

A range of phenotypic characters were measured and their mean and coefficient of variation (CV) calculated in meiotic and mitotic gynogenetic individuals and normal diploids produced from the same full-sib family of Oreochromis niloticus (L.). The traits studied in all three groups were weight, length, various fin ray and scale counts, gonadosomatic index (GSI) and reproductive response. The study revealed two main trends in the majority of the traits studied in the gynogenetic fish: a decrease in the mean of each trait in the ranked order normal diploid > meiotic gynogenetic > mitotic gynogenetic, and an expansion of its phenotypic variation (CV) in the order normal diploid < meiotic gynogenetic < mitotic gynogenetic. This could be related to the levels of genetic homozygosity and a possible reduction in developmental homeostasis in the gynogenetics. The utility and potential of gynogenetic individuals in research and the use of the technique in the improvement of fish strains for aquaculture are discussed.     

ENU诱变草鱼及其雌核发育后代的微卫星遗传分析

为了获得雌核发育ENU诱变草鱼(Cetpharyngodon idellus)群体的相关遗传参数,实验采用Partec Cy Flow倍性分析仪测定ENU诱变草鱼群体(Q群体)和雌核发育ENU诱变草鱼群体(E群体)相对DNA含量分别为24.02和23.80,二者的DNA含量接近,均为二倍体。选取28个微卫星标记对Q群体和E群体多样性进行了检测。结果表明,E群体和Q群体的平均等位基因分别为3.7143、5.1786,平均有效等位基因分别为2.1857、4.0028,平均期望纯合度分别为0.5122、0.2814,平均期望杂合度分别为0.4878、0.7186,多态信息含量(PIC)平均值分别为0.4282、0.6606。从个体在微卫星位点的纯合率分析,在E群体中,每个个体的纯合度均小于1.00,说明没有完全纯合的个体。从每个微卫星位点在群体的纯合率分析,除了微卫星位点5476,HLJC118和HLJC81外,其他位点的纯合度以不同的速率得到明显的提高。综上所述,经过减数雌核发育方法,ENU诱变草鱼群体的各微卫星位点的纯合度以不同的速率得到提升,遗传多样性明显降低,此方法可以获得纯合度较高的雌核发育ENU诱变草鱼个体,为ENU诱变草鱼良种选育提供了重要的遗传数据资料。     

Use of Microsatellite DNA Profiling to Identify Japanese Flounder,Paralichthys olivaceus of Hatchery Origin

Japanese flounder, Paralichthys olivaceus, is an economically important marine fish species in Asia. A suite of 18 microsatellite markers chosen from published genetic linkage maps was used to carry out parentage assignments of 188 hatchery‐reared juveniles from a small number of breeders. The probabilities of exclusion for the 18 microsatellite markers were 0.604–0.913, and the effectiveness of combined probability of exclusion reached 100% when using the eight microsatellite markers with higher Excl 1 probabilities. The cultured and wild stocks (WSs) were differentiated in a release‐recapture population based on these markers. Of the 321 recaptured offspring, 28.34% were assigned to their parental pairs in our broodstock, whereas the remaining offspring could not be traced back to a possible sire or dam. Significant reduction in genetic diversity of the cultured stock (CS) had not been found compared with that of the WS. The results suggest that CSs released into the wild will not adversely affect the genetic structure of natural populations. Our results demonstrate that these markers provide an efficient tool for parentage assignments and genetic analysis of Japanese flounder.