基于标记系谱的红鳍东方鲀生长性状遗传分析

利用分子标记辅助家系选育进行红鳍东方鲀生长性状的遗传评估,由红鳍东方鲀基础群体中选择性腺发育良好的雌雄亲本各11尾建立全同胞家系11个,每个全同胞家系随机采集10尾个体组建全同胞家系群体,从混合培育的家系中选择相对较大的个体400尾组建混合选育群体。在红鳍东方鲀遗传连锁图谱上挑选44个均匀分布于22个连锁群上的微卫星DNA标记,每个连锁群有2个标记。全同胞家系群体的遗传分析结果显示,10个高亲本排除概率微卫星标记的Excl(1)和Excl(2)为0.58~0.662和0.736~0.797,14个低亲本排除概率标记的Excl(1)和Excl(2)为0.054~0.43和0.177~0.608,剩余20个中等亲本排除概率标记的Excl(1)和Excl(2)为0.467~0.575和0.641~0.732。利用拥有高亲本排除概率的微卫星DNA标记进行混合选育群体的亲权鉴定,结果显示:不同父母本繁殖产生的子代数量存在明显差异,父本M2和母本F4产生了124个子代,占子代个体总数的32.89%。根据亲权鉴定结果建立的系谱估计主要生长性状的遗传参数,不同日龄的体质量和体长遗传力估计值为0.17~0.21和0.15~0.18。研究表明,利用具有高亲本排除概率的微卫星DNA标记能够有效建立红鳍东方鲀系谱,进行生长性状的遗传参数估计,分子标记辅助家系选育可以作为红鳍东方鲀目标性状遗传改良的新方法。     

荧光标记微卫星技术用于凡纳滨对虾不同家系亲权关系鉴定

应用荧光标记微卫星技术对凡纳滨对虾(Litopenaeus vannamei)家系进行亲权鉴定。挑选14个多态信息含量较高的微卫星位点,以人工选育建立的凡纳滨对虾5个全同胞家系为试验材料,采用Cervus 3.0进行亲权分析,并根据家系内个体间的遗传距离进行UPGMA聚类分析。结果表明,14个微卫星位点的平均等位基因数为6,平均多态信息含量为0.6896,平均期望杂合度为0.7309,平均观测杂合度为0.7661,第一亲本、第二亲本和双亲的累计排除率分别为0.99721733、0.99996559和0.99999997;进一步模拟分析表明,要达到亲权鉴定的要求,在双亲性别已知时至少需要5个微卫星位点,双亲性别未知时至少需要6个微卫星位点;模拟分析及试验验证所选用的14个微卫星位点最多可以鉴定1 954个已知性别的亲本或1 203个未知性别的亲本。所选用的14个微卫星标记可在生产及科研试验中用于获取凡纳滨对虾系谱信息。     

基于微卫星多重PCR的黄鳝亲子鉴定技术

为助力黄鳝(Monopterus albus)良种选育中的亲子鉴定和系谱管理问题,利用黄鳝全基因组预测并筛选获得的16个多态性较高的微卫星标记,建立了2组微卫星多重PCR体系,并成功用于11个家系的亲子鉴定。通过Cervus 3.0软件对132尾黄鳝进行遗传多样性分析,结果显示,16个微卫星标记的平均等位基因数(Na)为5.562,平均观测杂合度(Ho)和平均期望杂合度(He)分别为0.627和0.619,平均多态信息含量(PIC)为0.564。亲子鉴定结果表明,双亲基因型未知时单亲本的累积排除概率(CE-1P)为0.999 999 99,单亲基因型已知时另一亲本的累积排除概率(CE-2P)为0.999 999 91,双亲基因型未知时双亲的累积排除概率(CE-PP)为0.999 964 76。黄鳝11个家系的模拟鉴定率为99.96%,实际鉴定率为95%。模拟分析不同亲本数的结果显示,实验的16个微卫星标记在双亲性别已知的200对亲本和双亲性别未知的150对亲本的情况下,均可达到95%以上的鉴定率。利用NTSYS软件对11个家系的110尾子代个体进行聚类分析,结果显示除2尾子代外其余10...     

mtDNA 和微卫星标记在放流牙鲆和非放流牙鲆鉴定中的应用

用mtDNA控制区特异性引物对牙鲆(Paralichthys olivaceus)亲鱼、放流牙鲆和回捕牙鲆的DNA进行扩增,获得了3个群体的mtDNA控制区第一高变区部分序列。分析结果表明:母本55尾具有26种单倍型,作为其子代的预备放流牙鲆129尾具有5种单倍型,与亲本的单倍型全部一致,验证了mtDNA方法鉴定子代的准确性。回捕牙鲆435尾具有70种单倍型,其中330尾具有17种单倍型,与母本的单倍型相一致,为疑似放流牙鲆;另外105尾具有53种单倍型,与母本的单倍型不一致,为野生牙鲆或非北戴河站母本后代放流牙鲆。利用4个高多态性微卫星标记对330尾疑似放流牙鲆做进一步鉴定。结果表明,330尾检测个体中有310尾等位基因与候选亲本等位基因全部对应,可确认其为放流牙鲆;另外20尾等位基因与候选亲本不对应,可确认其为非放流牙鲆。结合mtDNA和微卫星标记鉴定的结果,可以确认2013年的435尾回捕牙鲆中有310尾放流牙鲆,占71.26%;另外125尾为非放流牙鲆,占28.74%。本研究结果表明,利用mt DNA标记可快速排除回捕鱼中的非放流牙鲆,利用微卫星标记作进一步鉴定,可以排除疑似放流牙鲆中的非放流牙鲆,是区分放流牙鲆与非放流牙鲆,准确评价放流效果的好方法。     

基于M-C作图鉴定牙鲆不同二倍体的遗传特征

为了分析连锁群上不同位置的微卫星标记对鉴定牙鲆二倍体遗传特征的作用,实验以牙鲆选育基础群体为亲本,选择性腺发育良好的个体制备普通二倍体(ND),减数分裂雌核发育二倍体(MGD-1)、连续两代减数分裂雌核发育二倍体(MGD-2),并利用MGD-1发育达性成熟的雌鱼与同时诱导的性反转伪雄鱼交配制备近交二倍体(MGD1H)。从牙鲆遗传连锁图谱选择均匀分布于24个连锁群的72个微卫星标记,用4个MGD-1家系估计微卫星标记与着丝粒之间的相对距离。在假设无交叉干涉的情况下,17个标记位于着丝粒区域,19个标记位于连锁群中部,36个标记位于远着丝粒区域。分别选择上述区域的微卫星标记鉴定牙鲆4种二倍体的遗传特征。结果显示,4种二倍体的等位基因数(A)和多态信息含量(PIC)在不同区域变化范围较小,ND的A和PIC均为最高,MGD1H则表现为最低。随着标记与着丝粒之间距离的增加,4种二倍体的观测杂合度逐渐升高,纯合度逐渐降低。纯合个体比例在着丝粒区域最高,为8.8%～29.1%;在远着丝粒区域最低,为2.4%～23.2%。其中,MGD-1和MGD-2的变化幅度显著高于其余二倍体。结果表明,选择连锁群上不同位置的微卫星标记对鉴定牙鲆不同二倍体的遗传特征具有显著影响。     

利用微卫星标记高效鉴定松浦镜鲤的亲缘关系

用37尾雌和14尾雄松浦镜鲤Cyprinus carpio Songpu构建37个血统明确的全同胞家系,同池、同条件养殖。用筛选出的9个多态性微卫星标记和建立的多重PCR检测方法进行子代群体(1 318尾)的亲权鉴定。9个位点共检测到53个等位基因,各位点均为高度多态(PIC0.5),平均期望杂合度为0.742,观测杂合度为0.755。亲本基因型信息的模拟分析显示,利用9个标记将子代分配到亲本对的成功率可达100%。实际鉴定结果:1 287尾个体准确鉴定到配组的亲本对,鉴定成功率为97.6%。以上结果表明:本研究提供的标记和检测方法可用于松浦镜鲤的亲本遗传距离分析和家系亲缘追溯,也适用于群体遗传多样性监测。     

黄鳍棘鲷家系亲缘关系鉴定

为了建立黄鳍棘鲷微卫星亲子鉴定技术,利用荧光引物和自动测序技术检测了自主开发的12对微卫星分子标记在505尾黄鳍棘鲷个体中的遗传多态性,并构建了亲子鉴定技术。结果显示,该研究中筛选的12个微卫星标记共检测到119个等位基因,平均等位基因数(N_a)为9.91,平均观测杂合度(H_o)为0.651,平均期望杂合度(H_e)为0.661,平均多态性信息含量(PIC)为0.621,具有丰富的多态性。此外,运用Cervus 3.0软件对已知系谱信息的112尾黄鳍棘鲷亲本和393尾子代个体进行模拟分析,结果显示,当双亲未知且置信度为95%时,12个标记的累积排除概率达99.58%;当微卫星标记数量为8时,累积排除概率达到99.1%。因此确定AL49、AL37、AL01、AL20、AL14、AL18、AL15和AL51共8个多态性较高的微卫星标记为黄鳍棘鲷微卫星亲子鉴定的核心体系。在双亲性别未知的情况下,其双亲的累积排除率为99.1%。根据黄鳍棘鲷子代的实际基因分型数据,实际鉴定率为89.31%。该研究构建的微卫星标记组合能为黄鳍棘鲷不同家系混养后的亲子鉴定、种群选育和分子辅助家系管理提供科学的技术手段。     

利用微卫星标记进行马氏珠母贝家系遗传结构分析与系谱认证

为改良马氏珠母贝养殖群体性状,2010年4月,从马氏珠母贝选育系F3选择性腺成熟个体为亲本,建立了36个家系,按照常规技术进行幼体培育和海区养成.2010年11月,从36个家系中随机选取4个家系,每个家系取样30个个体,利用13对微卫星引物进行家系遗传结构和系谱鉴别分析.结果显示,(1) 13个微卫星引物在4个家系中共检测到39个等位基因,每个位点的等位基因数为2～5,4个家系的平均观测杂合度(Ho)为0.531 ～0.597,平均期望杂合度(He)为0.474 ～0.507;(2)根据子代基因型成功地推断出4个家系的亲本基因型,据此鉴别各个家系;用UPGMA法对120个样本进行聚类分析,98.3％的同一家系的子代个体能够聚到一起,分类结果与系谱来源基本一致.结果说明,这4个家系具有较高的遗传多样性和家系间具有明显的遗传分化;微卫星标记能有效地为马氏珠母贝群体的系谱分析提供技术支持.     

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。     

红鳍东方鲀亲本群体遗传多样性的微卫星分析

从日本进口的红鳍东方鲀Takifugu rubripes受精卵发育至性成熟的120尾亲鱼群体中,选择22个分布于每个连锁群的微卫星标记进行了遗传分析,以分析红鳍东方鲀亲本群体的遗传多样性,并筛选出有效鉴定群体遗传特征的特异性微卫星标记。结果显示:等位基因数(Na)和有效等位基因数(Ae)的范围分别介于4.000~13.000和1.834~7.706之间。观测杂合度(Ho)、期望杂合度(He)和多态信息含量(PIC)的范围分别为0.173~0.977、0.456~0.872和0.427~0.857。Hardy-Weinberg遗传偏离指数(d)介于-0.746~0.344。群体内个体间的遗传距离在0.51~0.86之间,依据遗传距离的远近将全部个体分成4个群体,每个群体23~38个体。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,不同个体之间拥有相对较远的亲缘关系,可以根据个体间的遗传距离制定育种计划,建立家系进行选育研究。实验所选的22个微卫星标记具有高度多态性,可作为分析红鳍东方鲀群体遗传多样性的候选标记。     

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.     

微卫星DNA标记用于中国对虾亲子关系的鉴定

在水产养殖中应用微卫星标记来确定亲缘关系已经得到认同和广泛使用。本研究通过控制交尾,利用微卫星标记确定中国对虾后代的亲缘关系,对微卫星标记应用于中国对虾的家系识别进行初步探讨。作者以4个谱系清晰的家系为基础,检测两对微卫星标记EN0033和RS062在中国对虾家系鉴别中的应用。结果表明,利用两对微卫星引物产生的DNA指纹图谱,能够有效地区分中国对虾4个家系。根据微卫星数据计算各家系之间的遗传距离,构建UPGMA图,得到的结果与各家系来源情况一致,进一步证明了微卫星技术在进行家系间的遗传分析中的可行性。EN0033和RS062两对微卫星标记的累积个体识别率高达0.989,属于高识别力的遗传标记系统,可以利用它们进行中国对虾的亲缘关系鉴定。     

Parentage assignment in hybrid abalones (Haliotis rufescens × Haliotis discus hannai) based on microsatellite DNA markers

Parentage analysis in aquaculture determines genealogical relationships between broodstock and progeny when the parents are unknown. Thus, parentage analysis is a useful tool to establish pedigree reports in molecular‐assisted selection programs. Here, we evaluated 10 heterologous microsatellite markers for parentage assignment in abalone hybrids produced from 43 abalone broodstocks of red abalone (Haliotis rufescens) and Japanese abalone (H. discus hannai). The allele frequencies, exclusion probabilities and broodstock contributions were calculated using CERVUS, PAPA and GERUD software. The polymorphic information content (PIC) values showed that most of the microsatellite loci were highly informative (>0.7) and more than 90% of parentage assignment was possible with a minimum of 5–6 microsatellite markers. Parentage assignment for hybrid and pure‐red progeny showed a better performance than pure‐Japanese progeny. This result could be due to the high level of allele loss in the parental genotypes. In addition, results indicated that only two sires contributed over 80% and 90% of red and hybrid progenies, respectively. This study gives a new molecular tool to support marker‐assisted selection in abalone hybrids produced in Chile.     

凡纳滨对虾(Litopenaeus vannamei)微卫星多重PCR体系的建立及其在家系亲权鉴定中的应用

为了在遗传分析研究中提高效率并节约成本,本研究从已报道的凡纳滨对虾(Litopenaeus vannamei)微卫星位点中选取多态性较高的位点,基于各位点的扩增片段大小及退火温度等因素进行微卫星位点的组合.通过不断优化位点组合、反应体系及反应程序,成功建立了1个五重、2个四重和1个三重PCR反应体系,并将其应用于11个凡纳滨对虾家系的亲权鉴定.结果显示,四组多重PCR体系中的16个微卫星位点在11个凡纳滨对虾家系中的平均等位基因数(Na)为6,平均多态信息含量(PIC)为0.5813,平均观测杂合度(Ho)为0.513,平均期望杂合度(He)为0.636.利用Cervus3.0软件,对已知系谱关系的11个凡纳滨对虾家系进行亲权分析,其第一亲本累积排除率(CE-1P)、第二亲本累积排除率(CE-2P)和双亲累积排除率(CE-PP)分别为0.99525487、0.99990862和0.99999986.进一步分析表明,当同时使用四组多重PCR体系进行亲权分析时,其模拟配对率和亲权鉴定准确率均为100％,全同胞和半同胞家系鉴别效果良好,表现出准确的鉴别能力.本研究所建立的四组凡纳滨对虾微卫星多重PCR体系均可为后续的凡纳滨对虾遗传多样性分析和亲权鉴定提供高效、准确的检测手段.     

Parentage testing for hatchery-produced abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> based on microsatellite markers: preliminary evaluation of early growth of selected strains in mixed family farming

This study dealt with the detection of parentage in a mixed family tank of abalone Haliotis discus hannai using microsatellite DNA markers. As a preliminary investigation, the early growth rate between selected and non-selected abalone families, which were reared together in the mixed family tank from settlement stage to juvenile stage (7 months of age), was also compared. The selected families were derived from seven parents (three females and four males) selected for larger size in shell length at about 1-year-old, and the non-selected families originated from five wild captives (three females and two males). Parentage analysis based on eight microsatellite markers unambiguously allocated the 170 juveniles sampled from the single tank to the 17 parental pairs. The family size was highly heterogeneous among families, as two males in the selected families and one male in the non-selected families dominated the contribution to the offspring pool (>80%). The mean shell length of the selected families was approximately 23% larger than that of the non-selected families (Student’s t-test, P<0.001). This study demonstrated that the use of microsatellite markers is effective for parentage determination in the mixed family farming, commonly used in abalone hatcheries, and selective operations for larger size could improve the growth of the next generation.     

A microsatellite marker tool for parentage analysis in Senegal sole (Solea senegalensis): Genotyping errors, null alleles and conformance to theoretical assumptions

A set of 50 potential microsatellite loci previously described in several flatfish species was evaluated for designing a suitable tool for parentage assessment in Senegal sole (Solea senegalensis). Low cross-amplification within Pleuronectiformes, even between congeneric species, was achieved. This suggests a low conservation of microsatellite flanking regions in this order regarding other fish orders of similar evolutionary age. Twelve loci showed appropriate amplification and polymorphism to acquire enough statistical power for parentage analysis. All forty-one individuals from one broodstock and 320 offspring were analyzed using this set of markers. The high potential of microsatellites for parentage exclusion (Excl1 = 0.9999; Excl2 = 1) permitted us to trace back all offspring to single pairs of parents. The families obtained were used to analyze the performances of the loci applied and to identify possible sources of error for parentage analysis in Senegal sole. The 2.5% of single mismatches in the families studied were attributed mostly to genotyping errors and to a lesser extent to null alleles. The average mutation rate estimated over 7680 gametes was in the range reported within fish (3.9 × 10^− 4) and did not compromise parentage analysis. All loci conformed to theoretical assumptions (Mendelian segregation, independent segregation), excluding the F13-II8/4/7 and Sol 9A loci, which showed strong genotypic association in the families analyzed. A set of 4 microsatellites (Sol CA13, F13-II8/4/7, Smax-02 and Sse GATA38) was finally selected for parentage evaluation in S. senegalensis combining both enough exclusion potential (> 0.970) and the lowest economic cost.     

Application of microsatellite DNA parentage markers in the swimming crab Portunus trituberculatus

Microsatellites have been a powerful genetic tool in determining pedigree relationships among individuals. A microsatellite marker suite can be used to reliably assign parentage in the Portunus trituberculatus selective breeding programs. Thirty published microsatellites were screened for their suitability to be included in a DNA parentage marker suite. Nineteen microsatellite markers that exhibited significant stutter, poor allele resolution, null allele, and significant deviation from Mendelian expectations were eliminated from the marker suite, and the left eleven markers were ranked by PIC. Six most polymorphic markers with mean He?=?0.928 and PIC?=?0.901 from the original eleven were further assessed for the efficiency of pedigree identification. Total predicted power of the markers to exclude false parents was higher than 99%. Both simulations and real data analysis confirmed the high statistical power of six microsatellites suite in parentage exclusion, which permit us to trace back all offspring to single pairs of parents.