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。     

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。     

凡纳滨对虾SNP标记开发与家系亲缘关系验证分析

本研究基于转录组序列开发了37个SNP标记,并对22个凡纳滨对虾(Litopenaeus vannamei)家系进行了遗传多样性、家系聚类和亲子鉴定分析,探讨SNP标记在对虾家系选择育种中的应用途径。结果显示,37个SNP位点在凡纳滨对虾22个家系中的平均期望杂合度(He)和平均观测杂合度(Ho)分别为0.38和0.34;平均多态信息含量(PIC)为0.30,属于中度多态性(0.25相似文献   

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...     

中华绒螯蟹微卫星多重PCR体系的建立及其亲子鉴定应用

利用自主开发的微卫星标记对中华绒螯蟹(Eriocheir sinensis)进行微卫星多重PCR体系构建,最终成功建立4组多重体系,每组体系包含4个微卫星位点,并成功应用于3个家系的亲子鉴定中。结果显示:(1)本研究筛选的16个微卫星标记,平均观测杂合度为0.8206,平均期望杂合度为0.8164,平均多态信息含量为0.7927,具有丰富的多态性;(2)运用Cervus 3.0软件对已知系谱信息的3个中华绒螯蟹家系共95个子代个体进行亲子鉴定分析,结果显示,选用任意两组微卫星多重PCR体系时,累积实际正确鉴定率均超过94.74%;使用任意三组微卫星多重PCR体系时,累积实际正确鉴定率均大于98.95%;使用四组微卫星多重PCR体系时,累积实际正确鉴定率达到100%。并且当选用组合1、2和组合3,或者选用组合1、3和组合4时,对3个家系的累积正确鉴定率达到了100%,因此选用这些组合不但可以获得准确的系谱信息,还能减少工作量,降低成本。本研究构建的微卫星多重PCR体系能为中华绒螯蟹的种群选育和家系管理提供便捷、高效的途径。     

凡纳滨对虾遗传多样性的SSR分析

通过对5个SSR位点的聚合酶链式反应(PCR)扩增、聚丙烯酰胺凝胶电泳和银染色检测,探究凡纳滨对虾群体的遗传多样性.结果表明,供试的凡纳滨对虾群体在5个SSR位点上有2～4个等位基因,平均等位基因数3个,平均杂合度0.5755,平均多态信息量0.4864,平均有效等位基因数2.5053.全群基因纯合度0～0.8,平均0.3917.显示该凡纳滨对虾群体的遗传多样性水平处于中度多态.     

凡纳滨对虾养殖亲本群体遗传多样性分析

运用微卫星标记技术,选用8对微卫星引物对厦门市5个凡纳滨对虾亲虾群体(ZA1、HN、ZA2、ZP、DS)进行遗传多样性分析。结果显示,8对微卫星引物在5个凡纳滨对虾亲虾群体中共检测到75个等位基因,各群体的平均等位基因数(Na)为3. 875～7. 000、平均有效等位基因数(Ne)为2. 632～3. 719、平均PIC值范围在0. 498～0. 624。平均观测杂合度(Ho)为0. 410～0. 562、平均期望杂合度(He)为0. 589～0. 687。各群体的平均近交系数(Fis)为0. 147～0. 305,说明各群体内近交程度较高,存在杂合子缺失现象。除ZP与DS群体外,其他群体在多个微卫星位点上均显著偏离平衡(P 0. 05),说明大部分群体存在杂合子缺失现象。遗传变异分析结果显示,各群体间的遗传分化指数(Fst)为0. 028～0. 199;根据Fst计算得到各群体间的Slatkin’s遗传距离在0. 029～0. 249之间。本研究结果表明,采自厦门市的5个凡纳滨对虾亲虾群体均存在不同程度的近交现象。本文通过对厦门市5个凡纳滨对虾亲虾群体进行遗传多样性分析,旨在明确厦门市凡纳滨对虾亲虾群体的遗传结构、种质资源状况及近交程度,为该地区凡纳滨对虾种质资源的提纯、保优、复壮及遗传选育提供背景资料和建议。     

皱纹盘鲍微卫星多重PCR体系构建及其在家系鉴定中的应用

为提高微卫星分型效率,从以往报道的皱纹盘鲍微卫星中筛选出易扩增、特异性好的微卫星位点进行组合扩增,并通过优化退火温度、反应体系、引物浓度等条件,开发了4组多重PCR扩增体系.运用CERVUS3.0软件对12个皱纹盘鲍全同胞家系的372个子代进行家系鉴定,验证了这4组多重PCR在家系鉴定中的效率.结果发现,仅用1组微卫星多重PCR模拟和实际家系鉴定的成功率分别为86％和90％,两组则达到100％.结果表明,微卫星多重PCR技术能准确地把任意子代鉴定至其所属家系,可以进行大批量家系材料分析,具有较好的应用价值.     

尼罗罗非鱼(♀)×萨罗罗非鱼(♂)F_1家系亲权关系微卫星分析

利用尼罗罗非鱼(Oreochromis niloticus)第2代遗传连锁图谱标记,对3组不同尼罗罗非鱼(♀)×萨罗罗非鱼(Sarotherodon melanotheron)(♂)杂交F1家系内亲权关系进行分析。结果显示,86个微卫星位点中共筛选出20个在尼罗罗非鱼、萨罗罗非鱼中存在差异的扩增位点,含13个种间特异性和7个共享带差异位点。尼萨杂交F1中,平均等位基因2.90,平均多态信息含量0.439,位点多态性较高。3个尼萨杂交F1家系组间遗传距离0.362~0.504,组内个体间遗传距离0.245~0.316,组内遗传距离明显小于组间。利用3个种间特异位点组合,可对3个不同家系组父、母本个体进行鉴别。通过对各组亲本与子代位点基因型分析,家系A、B和C组分别使用4、8和12个特异位点组合进行亲权鉴定,累积排除概率分别为99.99%、99.99%和99.91%,家系A、B组分别含3个半同胞家系,家系C组含2对非同胞或4个半同胞家系。     

凡纳滨对虾在氯化物型盐碱水养殖环境下不同家系间生长、存活性能分析

本研究以凡纳滨对虾(Litopenaeus vannamei)61个家系为材料,开展了为期50 d的氯化物型盐碱水混合养殖测试,分析了各个家系的生长和存活性能。研究结果显示,凡纳滨对虾家系在氯化物型盐碱水养殖环境下的体重和存活率均存在显著差异,且家系间体重和存活率差异较大,变异系数分别高达36.26%和46.82%;凡纳滨对虾家系的绝对增重率均值和特定增重率均值分别为0.09g/d和1.82%/d,绝对增重最快的家系比绝对增重率均值高7.54个百分点,比增重最慢家系高12.95个百分点;凡纳滨对虾家系存活率范围为1.00%~63.33%,家系平均存活率为26.61%,家系最高存活率比家系最低存活率高了62.33个百分点,比家系存活率均值高了36.72个百分点。本研究结果表明:凡纳滨对虾家系在氯化物型盐碱水养殖条件下生长、存活存在较大差异,具有较大的遗传改良空间和选育潜力,本研究结果可为下一步的凡纳滨对虾盐碱水选育工作提供数据支持。     

Multiplex microsatellite PCR sets for parentage assignment of grass carp (Ctenopharyngodon idella)

Pedigree information is essential for the genetic improvement of traits of interests in breeding programs. In this study, twelve microsatellite loci were selected to optimize three multiplex PCR protocols for parentage assignment in grass carp (Ctenopharyngodon idella). One hundred and fifty adult fish and 252 progenies produced from three pilot groups (P1, P2, and P3) were used to examine the power of the three multiplex microsatellite PCR sets for parentage analysis. The average number of alleles (Na) per locus, observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism (PIC) were 21.83, 0.883, 0.882, and 0.869, respectively. The combined exclusion power using all loci was greater than 99.99 %. Simulation analysis revealed a high assignment success rate (100 %). Parentage analysis of real offspring demonstrated that 99.6 % of all offspring were unambiguously allocated to single pairs of parents. Our results indicate that these three multiplex PCR sets could be used in pedigree reconstruction for grass carp breeding.     

大黄鱼微卫星多重PCR体系的建立及其应用

利用本实验室开发的微卫星标记,通过优化退火温度、引物浓度、循环次数等条件,建立了3组大黄鱼微卫星多重PCR体系,每组包含3个微卫星位点,用3组微卫星多重PCR分析了一个大黄鱼选育群体JD-01的遗传多样性。结果显示,该群体的平均等位基因为12.111,平均有效等位基因为7.408,平均多态信息含量为0.846,平均观测杂合度和期望杂合度分别为0.825、0.868,香农多样性指数为2.165。运用Cervus 3.0软件,对已知系谱关系的9个大黄鱼家系及对应亲本进行了亲子鉴定分析,以验证3组微卫星多重PCR在亲子鉴定中的准确性。结果显示,使用该3组微卫星多重PCR体系进行亲子鉴定准确率为100%。大黄鱼微卫星多重PCR体系的建立为分析群体的遗传多样性、亲子鉴定和辅助家系管理提供了一种高效的技术手段。     

Development of a Multiplex Microsatellite PCR Assay Based on Microsatellite Markers for the Mud Carp,Cirrhinus molitorella

In this study, we developed a multiplex microsatellite polymerase chain reaction (PCR) assay that consisted of 13 pairs of primers selected from previously developed microsatellite markers in mud carp data obtained in our laboratory. We adjusted the number of cycles, the time of extension, and the primer ratios to make the multiplex PCR system as valuable as possible. Using this system, we performed a genetic analysis and parentage assignment for 2 male and 5 female mud carps and 146 of their progeny. The results showed a range of alleles of 2 < K < 5, with an average of 3.54. The polymorphism information content ranged from 0.271 to 0.697, with an average of 0.4954. The observed heterozygosity ranged from 0.183 to 0.810, with an average of 0.6008. The expected heterozygosity ranged from 0.324 to 0.741, with an average of 0.5572. The null allele frequency was 1.29–27.68%. The computer‐simulated identification was 99%, while the combined exclusion power using all loci was 100% for actual offspring. Thus, this multiplex PCR assay is a new technological tool for selective breeding and a genetic resource for studying the mud carp, Cirrhinus molitorella.     

基于微卫星标记评估凡纳滨对虾收获体长、体质量以及抗WSSV性状的遗传参数

为更加准确地评估凡纳滨对虾体长、体质量以及抗WSSV性状的遗传参数,实验利用8个微卫星位点对凡纳滨对虾69个家系母本和子代进行基因分型,并利用分型信息进行系谱重构和分子亲缘关系相关度的计算,以重构系谱、分子亲缘关系度以及物理系谱分别构建加性遗传相关矩阵,进而对体长、体质量和抗WSSV性状的遗传参数进行评估.最终通过交叉...     

Development and evaluation of microsatellite markers for identification of individual Greenshell™ mussels (Perna canaliculus) in a selective breeding programme

A set of 49 microsatellite loci isolated from the endemic New Zealand Greenshell™ mussel, Perna canaliculus, were evaluated for inclusion in a parentage assignment marker suite by assessing their ease of PCR amplification, allele scoring and conformity to Mendelian inheritance in hatchery-produced families. Ten polymorphic loci (mean H_e = 0.78 and polymorphic information content (PIC) = 0.72) were identified as being suitable for parentage assignment. These 10 microsatellite loci gave a combined non-exclusion probability of < 0.001 (probability that an unrelated parent pair will not be excluded from parentage of an arbitrary offspring), based on allele frequencies from 16 broodstock mussels. Simulations predicted an assignment success rate of 99.9% with all 10 loci and > 95% with the best 5 or more loci (mean PIC = 0.84). In actual parentage assignments, 124 offspring from 8 full-sib families were assigned to the correct parent pair with 4 or more loci. We found evidence for null alleles and extensive size homoplasy in many loci, highlighting the importance of thoroughly characterizing and evaluating microsatellite markers prior to parentage assignment and other applications.     

A highly accurate, single PCR reaction for parentage assignment in Senegal sole based on eight informative microsatellite loci

From a battery of microsatellite markers (100 loci), recently identified by our group, we have selected eight for parentage assignment in Senegal sole ( Solea senegalensis ). This tool is based on microsatellite loci obtained from four genomic DNA libraries and one cDNA library. Within the eight loci (six from anonymous genomic DNA sequences and two located in expressed sequence tags of known genes), we have found, in an analysis of a reproductive broodstock, between nine and 16 alleles. The expected heterozygosity was between 0.616 and 0.860. In addition, we have optimized the polymerase chain reaction (PCR) conditions to amplify all loci simultaneously in a single multiplex PCR reaction, and we have tested three lots of male and female (five to six individuals) and three offspring (50–60 larvae each). The use of the eight microsatellite loci, the possibility of amplifying them in a single PCR reaction and the high value of the exclusion probability (0.9992) make this multiplex PCR method a unique tool for parentage assignment.
Finally, analysing one meiotic gynogenetic progeny, we have determined the relative distance of six of these loci to the centromere, and we have also found that all of them are unlinked. All these characteristics confer this tool with a high accuracy for parentage studies and genetic population analyses of Senegal sole.     

Use of microsatellite loci and AFLP markers to verify gynogenesis and clonal lines in Nile tilapia Oreochromis niloticus L.

To develop an effective system for parentage analysis in gynogenetic and clonal progeny of Nile tilapia, Oreochromis niloticus L., polymorphic microsatellite loci and amplified fragment length polymorphisms (AFLPs) were investigated in several gynogenetic families and clonal lines. Six microsatellite loci were screened in two meiotic gynogenetic families to look for loci with high gene–centromere recombination rates, which can be used to discriminate meiotic from mitotic gynogenetics. Microsatellite loci UNH189 and UNH211 showed 96.7% and 92.0% heterozygosity, respectively, in these families, while other loci showed lower recombination frequencies. Scoring both UNH189 and UNH211 would give a very low probability of an individual meiotic gynogenetic being homozygous for both loci. Multiplex polymerase chain reaction of microsatellite loci was used to verify parentage in four families of mitotic gynogenetics and five fully inbred clonal lines. The genotype of each clonal line should serve as a unique identifier. Twelve AFLP primers were also investigated and 26 diagnostic AFLP bands were identified to follow inheritance in mitotic gynogenetic individuals. Amplified fragment length polymorphisms were found to be effective for this purpose but microsatellites were more appropriate since they are co‐dominant, while AFLPs are dominant markers. A multiplex of the microsatellite loci used in this study would be useful for general parental assignment as well as for the analysis of the products of chromosome set manipulations.