基于微卫星多重PCR技术的兰州鲇亲子鉴定

针对目前兰州鲇(Silurus lanzhouensis)种质资源救护保存和良种选育等研究工作中面临的亲子鉴定及系谱管理等问题, 研究应用微卫星荧光标记多重PCR与自动测序分型技术, 建立了2组四重PCR和2组三重PCR体系, 并成功应用于3个家系亲子鉴定中。利用Cervus v.3.0软件对110尾兰州鲇进行遗传多样性分析, 结果显示: 研究筛选的14个微卫星标记的平均观测杂合度(H_o)为0.750, 平均期望杂合度(H_e)为0.667, 平均多态信息含量(PIC)为0.624, 具有丰富的遗传多样性。对已知系谱信息的3个兰州鲇家系的90尾子代和20尾候选亲本进行亲子鉴定分析, 结果表明, 双亲基因型未知累积排除概率(CE-1P)、单亲基因型已知累积排除概率(CE-2P)和双亲基因型已知累积排除概率(CE-PP)分别为0.99753092、0.99983971和0.99999964。4组多重PCR累积模拟鉴定率为100%, 累积实际鉴定率为83%。采用50尾个体进行双盲验证, 利用MEGA7.0对3个家系50尾个体进行聚类分析, 结果表明同一家系94%的个体聚类分析结果与系谱关系一致。研究构建的兰州鲇4组微卫星多重PCR亲子鉴定技术为兰州鲇不同种质混养保存、种质选配扩繁、选育系谱管理和分子标记辅助选育等提供了重要技术支持。     

建鲤(Cyprinus carpio var. Jian)微卫星DNA亲权鉴定

利用16个微卫星座位对建鲤10个全同胞家系647个子代进行亲权鉴定。Cervus3.0分析表明,16个微卫星位点的平均多态信息含量为0.7025,平均等位基因数为6.63,期望杂合度平均为0.7405。当双亲未知时,累积排除概率为0.999225,已知单亲时的累积排除概率为0.999996,置信度为95%。进一步模拟分析表明,要达到亲权鉴定的要求在双亲未知时通常需要8～12个微卫星位点,已知单亲时需要5～8个微卫星位点。在双亲均未知的情况下进行亲权鉴定,94.6%的后裔找到了其父母本,真实鉴定率低于模拟分析预测值,分析可能是与候选亲本间存在亲缘关系、无效等位基因的存在以及分型错误等因素有关。9个建鲤全同胞家系的鉴定,为今后的遗传图谱构建、QTL定位及分子标记辅助育种研究奠定了基础。     

基于微卫星标记的鼋亲子鉴定技术

为研究鼋(Pelochelys cantorii)保种群体的遗传信息,利用鼋转录组文库, 筛选出了10对微卫星引物, 以此建立了基于 2 组各含5个微卫星位点多重 PCR 体系的鼋亲子鉴定技术, 并应用 2 组微卫星多重 PCR 体系, 通过 ABI3130 遗传分析仪及 PopGene3.2和CERVUS软件对56只子一代鼋进行个体基因型检测和群体遗传多样性分析, 结果显示, 鼋子一代群体的平均等位基因数为2.3, 微卫星位点的平均多态信息含量为0.3829, 平均观测杂合度和期望杂合度分别为0.6305和0.4767, 处中高度水平。对子代进行亲子分析, 10个微卫星位点的累积排除概率分别为73.14%(NE-1P)、90.98%(NE-2P)和98%(NE-3P), 能够满足亲子鉴定的需要。且4只亲本在生殖选择中表现出差异。鼋多重 PCR 亲子鉴定技术的建立为群体遗传多样性分析、家系鉴定管理和选择育种提供了有效的技术手段。     

茶树品种‘金牡丹’自然杂交后代遗传鉴定

为了对‘金牡丹’茶树自然杂交后代进行遗传鉴定,利用EST-SSR毛细管电泳荧光标记技术对65个金牡丹自然杂交后代进行研究。结果表明,28对SSR标记共扩增出192个等位片段,平均等位基因数(Na)、平均观测杂合度(Ho)及遗传多态信息量(PIC)分别为6.86、0.540、0.532。单亲基因型已知时的累积排除概率为0.999,说明选择的28对SSR标记位点具有高度的多态性和较高的排除概率,适用于遗传分析和个体的亲子鉴定。15个‘金牡丹’自然杂交后代的遗传鉴定结果表明,MD44、JMD45、JMD47、JMD32为早生绿茶类型;JMD51、JMD53为闽北肉桂乌龙茶类型;MD2、JMD56为闽南‘铁观音’乌龙茶类型;JMD24、JMD26、JMD29、JMD55、JMD59、JMD27、JMD61为‘黄棪’乌龙茶类型。     

基于微卫星多重PCR技术的黄喉拟水龟亲子鉴定

利用黄喉拟水龟(Mauremys mutica)微卫星标记,筛选出16对微卫星引物,通过优化各引物比例、荧光接头浓度、退火温度和循环次数,建立了基于2组各含8个微卫星位点多重PCR体系的黄喉拟水龟亲子鉴定技术。应用2组微卫星多重PCR体系,通过ABI3130遗传分析仪以及cervus3.0软件对428只黄喉拟水龟进行了个体基因型检测和遗传多样性分析,结果显示,群体的平均等位基因数为14.190,平均多态信息含量为0.748,平均观测杂合度和期望杂合度分别为0.687、0.771。对89只候选母本及296只子代进行亲子鉴定分析,结果显示:在父本信息未知时,母本鉴定率为87%;母本获得的子代个数范围为1-12,个体间表现出巨大的差异,这为选择育种提供了物质基础。黄喉拟水龟多重PCR亲子鉴定技术的建立为群体遗传多样性分析、家系鉴定管理和选择育种提供了有效的技术手段。       

微卫星标记在不同壳色虾夷扇贝家系亲权鉴定的适用性

实验选取8 个多态性微卫星位点, 用于虾夷扇贝4 个不同壳色全同胞和半同胞家系160 个子代的亲权鉴定。在亲本未知和一亲本已知的情况下, 8 个微卫星位点累积排除概率分别为0.823 和0.961。鉴于亲权分析时子代的亲本在已知和未知情况下位点的累积排除概率不同, 实验采用了两种方法用于家系的亲权鉴定。方法1: 当子代的父母本情况未知时, 根据子代基因型数据, 通过CERVUS 2.0 软件计算子代所对应的候选亲本的LOD 值, 直接将候选亲本中具有最大的两个LOD 值的亲本确定为子代的父母本; 方法2: 在子代的亲本未知情况下, 视具有最大LOD 值的候选亲本为子代的第一候选亲本, 然后将该亲本视为已知, 通过CERVUS 2.0 软件重新计算每个侯选亲本的LOD 值, 再从中选择具有最大LOD 值的候选亲本作为该子代的第二候选亲本。结果表明, 采用方法2 得到的家系亲权鉴定成功率达到95%以上, 确定了微卫星标记在虾夷扇贝家系鉴定中的可行性。所检测的微卫星位点在子代中出现无效等位基因现象, 而无效等位基因存在会引起子代与亲本的错配。实验在家系鉴定时采用了无效等位基因存在(情况1)和缺失(情况2)两种子代基因型文件进行分析, 不同情况下同一方法家系鉴定成功率相差无几。这表明了基于多个微卫星位点计算候选亲本LOD 值大小寻找子代真实父母本可以降低由无效等位基因引起的错配的几率。研究表明了微卫星标记适合于不同壳色虾夷扇贝家系亲权鉴定工作。       

微卫星标记对牙鲆有丝分裂雌核发育家系的亲子鉴定

利用18个微卫星标记,对6个家系的26尾有丝分裂雌核发育牙鲆进行亲子鉴定,PCR扩增产物经8％非变性聚丙烯酰胺凝胶电泳检测,结果表明：1个座位在母本中表现为相同的基因型,视为单态座位,其他17个座位为多态;多态座位在亲子鉴定中的累计排除概率和累计个体识别概率分别为0.9985、0.9999;根据被测个体在17个微卫星座位的基因型,最后确认26尾子代的母本,其中7尾子代在某些座位表现出与其母本不完全匹配的基因型。利用微卫星标记可确定雌核发育后代的亲子关系,从而构建牙鲆雌核发育家系系谱,对牙鲆雌核发育的深入研究具有重要意义。     

三角帆蚌微卫星多重PCR体系的建立及其应用

为了提高三角帆蚌微卫星分析效率,从已开发的微卫星标记中构建了三组四重PCR,并将稳定的多重PCR体系用于56个三角帆蚌紫色选育系的遗传多样性研究。结果表明该群体的平均等位基因数18.75,平均有效等位基因数为9.010,平均多态信息含量为0.857,平均观测杂合度和期望杂合度分别为0.809和0.876,香农多样性指数为2.422。运用Cervus v3.0软件对3个三角帆蚌全同胞家系共114个个体进行亲子鉴定,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%。该三角帆蚌微卫星多重PCR应用于三角帆蚌群体遗传多样性分析、亲子鉴定和家系管理等,可提高工作效率,降低实验成本。     

微卫星标记对牙鲆有丝分裂雌核发育家系的亲子鉴定

利用18个微卫星标记,对6个家系的26尾有丝分裂雌核发育牙鲆进行亲子鉴定,PCR扩增产物经8%非变性聚丙烯酰胺凝胶电泳检测,结果表明:1个座位在母本中表现为相同的基因型,视为单态座位,其他17个座位为多态;多态座位在亲子鉴定中的累计排除概率和累计个体识别概率分别为0.9985、0.9999;根据被测个体在17个微卫星座位的基因型,最后确认26尾子代的母本,其中7尾子代在某些座位表现出与其母本不完全匹配的基因型。利用微卫星标记可确定雌核发育后代的亲子关系,从而构建牙鲆雌核发育家系系谱,对牙鲆雌核发育的深入研究具有重要意义。     

微卫星技术在大耳白黑眼兔近交系培育中的监测分析

利用微卫星技术监测大耳白黑眼兔(white hair black eyes rabbits,WHBE兔)近交培育中第五代(F5)、第六代(F6)和第七代(F7)的遗传多样性.选取21个微卫譬座位,筛选出扩增产物稳定并且具有多态性的11对微卫星引物用于本研究.结果表明,F5代WHBE兔在每个座位上的等位基因数(Na)为3～9个不等,11个座位的平均有效等位基因数(Ne)为1.81个,平均观察杂合度(Ho)和平均多态信息含量(PIC)分别为0.381和0.524,累积个体识别率(CDP)达到100%,累积非父排除概率(CPE)在双亲信息都是未知情况下的为0.926,而在得知任一亲本信息的情况F,CPE值为0.993.F6代WHBE兔在每个座位上的Na为3～8个不等,11个座位的平均Ne为1.68个,平均Ho和PIC值分别为0.356和0.548,CDP达到100%,CPE在双亲信息都是未知情况下的为0.931,而在得知任一亲本信息的情况下,CPE值为0.994.F7代WHBE兔在每个座位上的Na为2～6个不等,11个座位的Ne为1.51个,平均Ho和PIC值分别为0.287和0.498,CDP达到100%,CPE在双亲信息都是未知情况下的为0.891,而在得知任一亲本信息的情况下,CPE值为0.986.在近交系培育过程中,从F5代到F7代,WHBE兔的平均Ne和平均Ho 都呈下降趋势,提示随着近交代数的增加,WHBE兔的基因纯合度越来越高.     

Parentage testing of racing camels (Camelus dromedarius) using microsatellite DNA typing

The camel racing industry would have added value in being able to assign parentage with high certainty. This study was aimed at assessing and applying microsatellite multiplexes to construct a parentage testing system for camels. An efficient system of 17 loci from 700 camel samples was used to construct a database of unrelated adults. Based on this, we estimated measures of polymorphism among the markers. In three multiplex reactions, we detected a total of 224 alleles, with 5–23 alleles/locus (mean = 13.18 ± 6.95 SD) and an average heterozygosity (H_E) of 0.54 (range 0.032–0.905). The total parentage exclusion probability was 0.99999 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.9999 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. We used 15 juveniles for parentage testing, as well as 17 sires (bull camels) and 21 dams (cows). In the case of parentage assignment, the microsatellite panel assigned all 15 offspring parentage with high confidence. Overall, these findings offer a set of microsatellite markers that are easy, simple and highly informative for parentage testing in camels.     

栲树微卫星分子标记的开发

采用基因组DNA富集法FIASCO对我国典型常绿阔叶林建群种栲树(Castanopsis fargesii Franch.)进行了微卫星分子标记的开发。从栲树基因组中分离和筛选了15个微卫星位点,并对江西九连山栲树自然分布居群的遗传多样性进行了分析。结果表明,栲树具有较高水平的遗传多样性,每个位点在28株栲树个体上的平均等位基因数(A)为6.7(4～8个),平均观察杂合度(HO)为0.690(0.250～1.000),平均预期杂合度(HE)为0.698(0.293～0.867)。每个位点的第一排除概率值Pr(Ex1)为0.043～0.527,位点综合值为0.9972。单个位点的第二排除概率Pr(EX2)为0.159～0.694,位点综合值为0.9999。这些微卫星标记可为研究栲树的遗传多样性及居群遗传结构提供有效的遗传工具。     

Paternity assessment: application on estimation of breeding value in body-weight at first egg trait of egg-laying duck (<Emphasis Type="Italic">Anas platyrhynchos</Emphasis>)

Paternity index was analyzed using five microsatellite loci among Chinese egg-laying ducks (Anas platyrhynchos). Based on the paternity relationship that was identified by paternity index analysis, the estimated breeding value (EBV) was calculated using BLUP (best linear unbiased predictor) method. Body weight at first egg (BWF) is the only considered trait in this study. In total, 12 sires, 31 dams and 77 daughters were involved in the EBV calculation. The results demonstrated that five microsatellite loci’s polymorphism information content (PIC) ranged from 0.795 in locus AY493338 to 0.957 in locus AY493264 with average 0.899; the parent–offspring relationships were built by these microsatellites’ genotype, 12 families of half sibling and 2 families of full sibling were involved, and the relationship error is smaller than 10⁻⁷. The EBV results suggest that the average EBV was significantly higher in females (average EBV is 10.234 and 0.1045 for mother and daughter, respectively) than males (average EBV is just −26.44). The EBV results on BWF were in good agreement with the principle of GH (growth hormone) expression in poultry. These results show that paternity analyses of Chinese egg-laying ducks were basically resolved using the five microsatellite loci selected. The paternity relationships can apply in Chinese egg-laying duck breeding to quicken the improvement of genetic progress.     

Microsatellite DNA markers for the pea aphid Acyrthosiphon pisum

Microsatellite loci were isolated from enriched partial genomic libraries of Acyrthosiphon loti and Acyrthosiphon pisum. Twenty of those loci were characterized in A. pisum. Fifteen of those loci were polymorphic. Genetic diversity varied across loci, allele repeat number ranging from two to 15, and observed heterozygosity from 0.1 and 0.96. An additional eight microsatellite loci originally isolated from other aphids but cross‐priming with A. pisum showed polymorphism as well. Allele size ranged from three to 9 and observed heterozygosity from 0.43 to 0.84. Overall, we present 23 microsatellite loci that can be used to reveal polymorphism in pea aphids.     

Parentage testing in alpacas (Vicugna pacos) using semi-automated fluorescent multiplex PCRs with 10 microsatellite markers

The aim of this study was to assess and apply a microsatellite multiplex system for parentage determination in alpacas. An approach for parentage testing based on 10 microsatellites was evaluated in a population of 329 unrelated alpacas from different geographical zones in Perú. All microsatellite markers, which amplified in two multiplex reactions, were highly polymorphic with a mean of 14.5 alleles per locus (six to 28 alleles per locus) and an average expected heterozygosity ( H _E) of 0.8185 (range of 0.698–0.946). The total parentage exclusion probability was 0.999456 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.999991 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. In a case test of parentage assignment, the microsatellite panel assigned 38 (from 45 cases) offspring parentage to 10 sires with LOD scores ranging from 2.19 × 10⁺¹³ to 1.34 × 10⁺¹⁵ and Δ values ranging from 2.80 × 10⁺¹² to 1.34 × 10⁺¹⁵ with an estimated pedigree error rate of 15.5%. The performance of this multiplex panel of markers suggests that it will be useful in parentage testing of alpacas.     

Heterozygosity and extra-pair paternity: biased tests result from the use of shared markers

Recent studies of extra-pair paternity have found support for the idea that heterozygous males have an advantage in siring offspring. Most studies use DNA microsatellite loci to determine paternity and then use the same loci to estimate individual heterozygosity. However, because the likelihood of detecting extra-pair offspring depends on the combinations of parental alleles, it is possible that biases arise from particular allele combinations. This might produce false support for the influence of heterozygosity on mating behaviour. We used a simulation model to assess how large this bias might be. We found two sources of bias. First, we found a bias in the null hypothesis of a simple statistical test commonly used to test several predictions of the heterozygosity hypothesis. The use of randomization tests could eliminate this bias. Second, we found that using the same loci for both paternity and heterozygosity can cause an increase in results supporting the heterozygosity hypothesis when no effect of heterozygosity actually exists. This bias is reduced through the use of more markers with higher levels of polymorphism and heterozygosity, but can be eliminated entirely by using a separate set of markers to determine paternity and assess heterozygosity. The two sources of bias reduce evidence favouring the heterozygosity hypothesis, but do not negate all of the studies that support it. We suggest that further studies of heterozygosity and extra-pair paternity are important and likely to be informative, but our recommendations should be incorporated by researchers to improve the reliability of their conclusions.     

利用微卫星技术分析不同类群鸡的遗传多样性

利用8个微卫星标记分析了6个生产类群鸡的遗传多样性。计算了各群体在各位点上的等位基因频率,并据此计算出各群体的平均遗传杂合度(h)、多态信息含量(PIC)和有效等位基因数(Ne)。结果表明:6个鸡群在8个微卫星座位上的基因频率存在明显的差异,其平均基因杂合度为0.7317,平均多态信息含量为0.6815。其中,群体平均杂合度最高的是安卡红鸡,为0.7716;平均杂合度最低的是新罗曼鸡,为0.7073。所选的8个微卫星座位均为高度多态,可作为有效的遗传标记用于鸡群体遗传多样性的分析。     

泥蚶G2代快速生长家系遗传结构的微卫星分析及其与生长性状的关联

利用微卫星标记对泥蚶(Tegillarca granosa Linnaeus) G2代家系的遗传结构和遗传多样性进行了分析, 并结合家系的表型生长数据, 关联分析筛选可用于育种的候选标记。遗传结构分析表明, 18对引物共检测出59个等位基因, 其中家系F19、F21、F22的平均等位基因数(N_a)分别是2.500、2.722和2.722; 平均观测杂合度(H_o)为0.446、0.510和0.628; 平均期望杂合度(H_e)为0.394、0.433和0.464; 多态信息含量(PIC)分别是0.346、0.379和0.403。标记与生长性状的相关性分析显示, 有3个位点与壳高、壳长、壳宽和总质量显著相关, 其中F19家系的Teg-30的BB基因型与壳高、总质量显著相关, F21家系的Teg-03的BB基因型和Teg-20的BC基因型与壳高、壳长、壳宽和总质量均显著相关, 筛选出的与生长性状相关的标记为开展泥蚶分子标记辅助育种提供了有价值的遗传信息和参考依据。     

Power of exclusion of 19 microsatellite markers for parentage testing in river buffalo (Bubalus bubalis)

In the present study, 19 microsatellite markers were assessed for their power of exclusion to test parentage in river buffalo. Microsatellite genotypes of 216 unrelated buffaloes belonging to five different breeds were utilized for the study. The probabilities of exclusion were calculated for three hypothetical situations viz. paternity testing (PE1), one parental genotype unavailable (PE2) and exclusion of both parents i.e. substituted offspring (PE3). The mean probability of exclusion across 19 investigated markers in buffalo was 0.578 (PE1), 0.405 (PE2) and 0.764 (PE3) respectively. The probability of exclusion for paternity (PE1) ranged between 0.297 and 0.814 across different markers. The exclusion probability for the cases one parent unavailable (PE2) and substituted offspring (PE3) varied from 0.143 to 0.688 and 0.465 to 0.946 respectively. Polymorphism information content and expected heterozygosity were found to have significantly high correlation with probability of exclusion of microsatellite markers. The cumulative PE1 of nine marker loci was estimated to be 0.9999 while in case of absence of one of the parental genotypes, a minimum of 11 markers were required to achieve a cumulative PE2 of 0.999. In conclusion, the present study proposes two multiplex sets with four and five markers respectively for routine parentage testing in buffalo and an additional set of four markers for doubtful cases of paternity.