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。     

中国对虾微卫星家系鉴定的模拟分析与应用

本研究基于中国对虾群体所获的微卫星标记等位基因频率进行了计算机模拟分析,并选择5个微卫星标记,就单独养殖家系群体微卫星标记家系鉴定的准确性及混养家系群体微卫星标记家系鉴定的应用价值做了研究.模拟分析表明4个微卫星标记可以鉴定95%的后裔.而单独养殖的家系鉴定准确率达到92.9%,在30个可能的父母对,215尾中国对虾组成的混养家系群体中,90.7%的后裔可以鉴定其父母.本研究结果表明微卫星分子标记可以应用于中国对虾的家系鉴定.模拟分析与实际应用的差异及父母与子代间的错配部分原因是由于无效等位基因的出现,基因分型错误也是一个重要原因.基于父母LOD值的分析可以降低错配的几率.     

基于微卫星标记的斑点叉尾鮰家系鉴定技术及应用

研究旨在建立准确、高效且经济的斑点叉尾鮰(Ictalures punctatus)家系亲缘鉴定体系, 以期为斑点叉尾鮰的遗传评估及家系育种提供科学依据。选用10对具有较高多态性SSR标记, 建立两个5重PCR反应体系。应用建立的斑点叉尾鮰家系亲缘鉴定体系对来源于13个全同胞家系和8个半同胞家系的333尾个体进行亲权鉴定。结果表明: 10个位点平均等位基因数为9.8、平均观测杂合度0.8591、平均期望杂合度0.8092、平均多态信息含量0.7845; 3种情况下的累积排除概率分别为: 0.99996806、0.99833267和0.99999998; 验证群体的鉴定结果与系谱高度一致, 真实鉴定率达到99.1%, 子代与父母本三者之间配对平均LOD值介于13.30—24.70, 且置信度均达到95%。研究选择的微卫星位点等位基因数目较多, 多态性较高, 可以快速、准确地对斑点叉尾鮰混养群体进行家系鉴定。     

虾夷扇贝遗传结构及微卫星标记与经济性状相关分析

选用30个多态性微卫星分子标记对大连大长山(96个)、大连獐子岛(96个)及日本青森县陆奥湾(96个)3个地区虾夷扇贝养殖群体(Patinopecten yessoensis)进行遗传多样性分析。结果显示,30个基因座共检测到198个等位基因(Na),平均等位基因数为6.63个,每个座位检测到的等位基因数为3-12,有效等位基因数(Ne)每个位点平均为4.70个,观测杂合度(Ho)平均值0.58,期望杂合度(He)的平均值为0.75,30个位点的平均多态信息含量为0.703,说明3个地区虾夷扇贝群体遗传多样性水平较高。运用统计软件SPSS11.5对30个微卫星标记与大长山群体生长性状相关性进行了分析。结果表明,位点DQ221714和FJ262378与壳长、壳高、壳宽和鲜重显著相关(P0.05)位点;位点FJ262372与壳宽、软体部重和闭壳肌重显著相关;位点FJ262369与软体部重及闭壳肌重显著相关。对差异显著的位点进行不同基因型间的多重比较,找到了与虾夷扇贝生长性状相关的基因型,进一步将所得显著性标记对大长山虾夷扇贝个体最大的17个和个体最小的13个基因组DNA进行检测,验证其准确性。结果表明,位点DQ221714在两组虾夷扇贝中存在特异性条带,可作为QTL定位候选标记。     

微卫星标记应用于凡那滨对虾家系鉴别的研究

以人工选育建立的凡那滨对虾（Litopenaeus vannamei）全同胞家系为实验材料,探讨了微卫星标记对混养家系进行亲权鉴定的可能性。Cervus模拟分析表明从10个微卫星基因座组合中选取的多态性信息含量最高的6个进行组合与原组合的累积排除概率均在0.99,多态性信息含量最高的6个基因座的组合判定正确率为0.97,置信度为95%。在家系混养的模拟实验中使用这6个高多态性的微卫星基因座从20个候选雌虾中找到真正母亲的概率为88%,从30个候选雄虾中找到真正父亲的概率为78%,低于理论预测值,分析可能与微卫星基因座中的无效等位基因,等位基因的突变以及PCR过程中Taq酶发生链滑移等因素有关。      

凡纳滨对虾微卫星位点在两个选育家系中遗传的初步研究

利用两个选育凡纳滨对虾全同胞家系研究了10个微卫星位点的遗传特征。通过ABI310或3100测序仪检测, 在所观察到的20个基因型比例（genotypic ratios）(10个微卫星位点 X 2个家系)中,有17个基因型比例符合孟德尔遗传。微卫星位点TUMXLv8.220在两个家系中均存在无效等位基因,从而3个不符合孟德尔遗传基因型中2个可由无效等位基因来解释。TUMXLv 3.1在06家系偏离了1：1：1：1的孟德尔预期比。3个微卫星位点（TUMXLv5.66,TUMXLv7.74,TUMXLv8.224）在两个家系中均表现单态。3个微卫星位点（TUMXLv5.45,TUMXLv7.56,TUMXLv8.256）在两个家系均既表现多态又遵循孟德尔共显性遗传, 是亲子鉴定和种群遗传分析的较好选择。结果显示在应用微卫星标记进行遗传分析之前利用全同胞家系进行遗传模式研究是非常必要的。     

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。     

鳙基于10个微卫星标记的亲子鉴定分析

为开展鳙(Hypophthalmichthys nobilis)家系选育工作,本研究进行了基于微卫星标记的亲子鉴定研究。试验中筛选了10个扩增效率较高的微卫星标记,通过引物荧光修饰,引物结合毛细管电泳分型技术,对鳙48尾亲本及384尾子代进行了基因分型,并计算了等位基因频率和模拟分析和亲子鉴定等分析。结果发现,各位点的等位基因数介于4~13之间,其中9个位点均具有较高的多态性和杂合度(PIC>0.5,He>0.5),研究发现位点的多态性信息含量(PIC)与亲本对排除率(E-PP)存在显著正相关(p<0.01)。模拟分析结果显示,该10个标记预计可用于已知性别的50组亲本(100尾)或未知性别的50尾亲本的鉴定分析(鉴别成功率>95%)。亲子鉴定发现,对试验中2个交配组(每组12对亲本)的鉴别成功率分别为98.96%和100%;且父母本对子代的贡献率存在极显著差异(p<0.01)。通过累积位点的鉴定分析发现,当标记数为7个和9个时分别能满足试验中12组和24组亲本对应子代的鉴定分析(鉴别率>95%),模拟分析和亲子鉴定分析成功率趋势基本符合。本研究所开发的亲子鉴定技术可为鳙家系选育提供技术支持。     

应用微卫星标记分析野生中国明对虾的亲权关系

利用5个高度多态性的微卫星座位(Fc04、Fc06、Fc18、Fc24、Fc27),分析了不同年份中建立的10个野生中国明对虾家系中的雌性亲虾及其后代基因型分离的情况,表明每个家系中只有一个雄性野生中国明对虾对子代的遗传有贡献.从其中3个家系的雌性亲虾纳精囊中提取了雄虾精子DNA,微卫星标记显示各家系的子代个体均有一个等位基因与雄性亲虾的基因型相符,且符合孟德尔遗传规律,这为野生中国明对虾雌虾在繁殖季节一对一的繁殖行为提供了遗传学的证据.用UPMGA的方法随机对3个家系的40尾子代个体进行聚类分析,每个家系都能被单独聚成一类.上述工作表明微卫星标记在对虾育种中作为一种有效的亲子关系分析工具,在种群的遗传结构、亲缘关系、繁殖行为等方面具有广阔的应用前景.     

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。       

Characterization of 13 polymorphic microsatellite loci for an evergreen tree,Myrica rubra

We developed 13 microsatellite markers from a genomic library enriched for dinucleotide (CT) repeats in a dioecious evergreen tree, Myrica rubra. We screened loci from 32 adult trees. The number of alleles ranged from two to 14, and the expected heterozygosity ranged from 0.324 to 0.884. The total paternity exclusionary power where the mother was known equalled 0.99965. These loci are of clear value in studying mating system and parentage analysis of this species.     

Isolation and characterization of 13 microsatellite loci for Neochamaelea pulverulenta (Cneoraceae)

We report 13 polymorphic nuclear microsatellite loci for Neochamaelea pulverulenta (Cneoraceae) using an enriched-library approach. Although this plant species is tetraploid, expected patterns for tetrasomic segregation were completely absent, and all loci analysed showed a diploid pattern of inheritance. We detected a total of 102 alleles in 57 individuals genotyped (mean number of alleles per locus was 7.85). The values of observed and expected heterozygosities ranged from 0.193 to 0.737 and 0.425 to 0.812 respectively. Disomic segregation, levels of polymorphism and the exclusionary power of the developed markers render them readily applicable for parentage assignment of dispersed seeds, and for analyses of spatial genetic structure and population connectivity.     

Development and characterization of 11 microsatellite markers in the rock sparrow,Petronia petronia

Eleven polymorphic microsatellite loci were isolated in the passeriform Petronia petronia using the enrichment protocol FIASCO (fast isolation by AFLP of sequences containing repeats). We detected three to 13 alleles per locus in 25 specimens collected from an Italian population. The level of expected heterozygosity ranged from 0.439 to 0.856. One locus is sex linked to the Z chromosome. The total exclusionary probabilities using these loci for the first and the second parent were 0.978 and 0.999, respectively. These are the first microsatellite loci characterized from the rock sparrow that can be used for estimating population structure and for large‐scale parentage analysis.     

Isolation and characterization of 12 microsatellite loci for Rhamnus alaternus (Rhamnaceae)

Twelve polymorphic microsatellite loci were developed for Rhamnus alaternus using an enriched-library approach. We detected 69 alleles in 49 individuals genotyped (mean number of alleles per locus was 4.79) in two different populations. The values of observed and expected heterozygosities ranged from 0.045 to 0.963 and 0.089 to 0.873 respectively. Levels of polymorphism and the exclusionary power of the developed markers render them readily applicable for studies of contemporary pollen and seed gene flow through parentage analyses.     

Microsatellite loci for the basal angiosperm Austrobaileya scandens (Austrobaileyaceae)

We developed nine microsatellite loci for the basal angiosperm Austrobaileya scandens, a vine endemic to northeastern Australia. High levels of polymorphism were detected in 30 individuals from a single population, with an average of 14.78 alleles per locus and an average expected heterozygosity of 0.81. Historical inbreeding levels were significantly different from zero (f = 0.136), consistent with genetic evidence for self‐parentage in open‐pollinated progeny. The high exclusionary power of these loci (> 99.98%) indicates they will be useful in future parentage analyses designed to explore mechanisms of mate choice in Austrobaileya.     

Isolation of seven polymorphic microsatellites in Ophioblennius atlanticus atlanticus (Perciformes,Blenniidae)

We isolated and characterized seven polymorphic microsatellite loci of Ophioblennius atlanticus atlanticus (Valenciennes, 1836) using an optimized protocol to construct and screen a microsatellite‐enriched genomic library. The analysis of variability was performed in 16 specimens from Faial Island (Azores, Portugal). The mean number of alleles was 8.71 ± 2.43 and the level of expected heterozygosity ranged from 0.764 to 0.903. The total exclusionary probabilities using these loci for the first and the second parent were 0.985 and 0.998, respectively, suggesting that these microsatellites are a useful tool for large‐scale parentage analysis.     

Parentage assignment of the mud crab (Scylla paramamosain) based on microsatellite markers

In this study, microsatellite markers were employed to identify the parentage relationship in Scylla paramamosain. The exclusion probability of loci was found to be related with the level of their heterozygosity. When no parent information or only one parent information was available, the exclusion probability ranged from 22.0% to 56.6% and from 41.2% to 73.1%, with the combined exclusion probability for ten loci being 97.0% and 99.8%, respectively. The cumulative assignment success rate was 100% when no parent information was available using seven most informative microsatellite markers. Moreover, the power of the seven microsatellite markers for parentage assignment was tested by a double-blind test, which indicated that 95% of the progeny can be correctly assigned to their parents. This study provided a microsatellite-based approach for parentage assignment in S. paramamosain that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this important crab species.     

Development and characterization of microsatellite markers in the Colorado potato beetle,Leptinotarsa decemlineata

The characterization of 11 Leptinotarsa decemlineata microsatellite loci isolated using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol is reported. Three to 15 alleles per locus were detected in 54 beetles collected from four populations of L. decemlineata. The mean number of alleles was 7.4 ± 3.2 and the level of expected heterozygosity ranged from 0.451 to 0.798. The total exclusionary probabilities using these loci for the first and the second parent were 0.982 and 0.999, respectively. These are the first microsatellite loci characterized from the Colorado potato beetles that can be used for estimating genetic diversity, population structure and parentage analysis.     

Can microsatellite markers replace PIT tags in rohu (Labeo rohita Hamilton, 1822) selective breeding programmes?

This study was undertaken to assess the feasibility of microsatellite markers for resolving parentage in Labeo rohita (rohu) selective breeding programmes in lieu of conventional PIT tag marking. Ten microsatellite markers were used to assign offspring parentage. A total of 160 individuals were tested from eight full‐sibling families. Eight highly polymorphic loci with allele numbers ranging from 2 to 8 and polymorphism information content (PIC) values in the 0.47–0.82 range were utilized. With the use of a real data set from a breeding programme, all but 12 individuals out of 160 could be assigned exclusively to their correct parental pairs. The matching rate was 92.5%, which was lower than in a simulated study (98.44%, Excl‐2). Discrepancies between simulations and real data sets may be due to several factors such as genetic relatedness among candidate parents (half‐sib and full‐sib), presence of null alleles, and sampling variance of the parents. The results of this study suggest that a marker panel probably with a wider genome coverage may be necessary for near‐100% accuracy in assigning parentage in rohu selective breeding programmes.