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。       

用基因诱捕法制作Ayu17-449基因敲除小鼠模型

为了探明Ayu17-449基因在小鼠生长发育过程中的功能, 用特殊的诱捕载体（Gene trapping vector）导入小鼠ES细胞中,5′RACE、Southern blot方法鉴定成功地单一捕获Ayu17-449基因后,由这种ES制作了Ayu17-449 敲除小鼠并用Northern blot方法该基因在突变小鼠体内的表达。结果在Ayu17-449 敲除小鼠体内,诱捕载体位于Ayu17-449基因的翻译起始密码上游,Ayu17-449基因的转录被抑制。表明Ayu17-449敲除小鼠为分析Ayu17-449基因的功能提供了可靠的实验材料。      

藏羚羊mtDNA D-Loop区遗传多样性研究

该研究采用非损伤性DNA基因分型技术,对可可西里地区10个藏羚羊（Pantholops hodgsonii）个体的mtDNA非编码区部分片段（444~446bp）进行了序列分析,结果显示A、T%含量（61.8%）明显高于G、C%含量（38.2%）,共发现10种单倍型,包括48个多态位点,其中转换位点44个、颠换位点1个、插入位点1个、缺失位点2个。单倍型间平均遗传距离为0.031,单倍型多态性（h）为1.000,核苷酸多态性（π）为2.96%。说明藏羚羊线粒体控制区存在着丰富变异,最后从藏羚羊的生态习性及地理分布两方面对这一结果进行了分析探讨。      

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。     

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.     

利用SRY基因和微卫星标记鉴定反刍动物性别

以反刍动物为研究对象,应用多重PCR技术扩增绵羊基因组中X、Y染色体上的4个微卫星标记和SRY基因, 根据基因型进行性别鉴定,试图通过一次DNA扩增同时提供性别鉴定和基因分型的信息。结果表明所设计SRY基因的引物具有高度特异性,是性别鉴定的主要依据,而Y染色体上的MCM158、MAF45两标记由于特异性不好,因此不适用于性别鉴定,对于X染色体上所选的两标记MILVET09和AE25只能进一步验证所鉴定的雄性个体。得出结论在被检个体中,能同时扩增出SRY基因、MCM158、MAF45,X染色体上MILVET09和AE25,且X染色体上的MILVET09、AE25基因型为纯合子的个体为正常的雄性;被检个体中只有Y染色体上MCM158、MAF45和X染色体上MILVET09、AE25的扩增产物,而没有SRY基因的扩增产物,则被检个体为雌性,且MILVET09、AE25的基因型对雌性个体的性别判断无影响。MCM158、MAF45两标记基因型不影响个体的性别鉴定结果。
     

小麦条锈菌国际鉴别寄主Vilmorin23的抗条锈病基因YrV23微卫星标记

Vilmorin23是小麦条锈菌国际鉴别寄主和国际上重要抗源材料。采用SSR技术,利用由Vilmorin23为基因供体转育而成的小麦抗条锈近等基因系Taichung29*6/YrV23,选用YrV23所在2B染色体上的55对SSR引物,对Taichung29*6/ YrV23及其轮回亲本Taichung29和抗性基因供体Vilmorin23的基因组DNA进行PCR扩增和聚丙烯酰胺凝胶电泳分析。结果显示,引物Xwmc356在近等基因系与轮回亲本间扩增出特异性DNA片段,经F₂代群体150个抗、感单株检测证实,该片段位点与抗条锈病基因YrV23有连锁关系,遗传距离为9.4 cM。Xwmc356可作为抗条锈基因YrV23的SSR标记。      

乌骨鸡蛋用系群体遗传结构的微卫星标记分析

用20对微卫星引物对丝羽乌骨鸡BM、BF两个蛋用新品系的基因组DNA进行扩增,应用多重PCR结合全自动电泳技术分析群体遗传结构。试验结果表明：18个微卫星标记表现出丰富的多态性,每个标记平均检测到7.444个等位基因（3～15个）,平均观测杂合度为0.3962,平均预期杂合度为0.7301,平均多态信息含量为0.669。本研究的标记检测结果比以前的研究报道值要高,说明全自动电泳分析技术比聚丙烯酰胺凝胶电泳结合银染法检测电泳结果的传统方法要精确,能更好地用于群体遗传结构的分析。      

羊FSHR基因5′端转录启动调控区生物学特性

文章对小尾寒羊、滩羊和澳洲绵羊等繁殖性状不同的3种绵羊与排卵有关的FSHR基因5′端转录启动调控区进行了克隆和分析,通过对FSHR基因的15个转录调控元件序列进行比较,结果表明,羊不同品种FSHR基因的转录调控元件序列之间没有差异。这说明绵羊的品种与FSHR基因5′端转录启动调控区的相关性不强,排除了因转录调控元件突变而影响转录调节能力的可能性。      

Parentage determination of mandarin fish (Siniperca chuatsi) based on microsatellite DNA markers

In this study, ten microsatellite loci were chosen to estimate the parentage of 260 progeny in five mandarin fish (Siniperca chuatsi) full-sib families. Simulation based on allele frequency date demonstrated the combined exclusion power would be over 97% if the number of loci was up to nine. Based on the information from these nine loci, 98% of progeny were unambiguously allocated to their putative parental pairs in the parentage analysis. The assignment success rate by the real data set was lower than that predicted by the simulations, with 94% of progeny assigned correctly. The discrepancies might be caused by a scoring error or allelic dropout caused by poor quality genomic DNA. Moreover, 69 progeny were selected randomly for the double-blind test, the result indicated that 95% of the progeny can be correctly assigned to their families. This study provided a microsatellite-based approach for parentage assignment in S. chuatsi, and that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this species.     

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.     

Isolation and characterization of microsatellite loci from the endangered New Zealand takahe (Gruiformes; Rallidae; Porphyrio hochstetteri)

Nineteen polymorphic microsatellite loci were characterized from the endangered takahe (Porphyrio hochstetteri). Like many of New Zealand's other native avian species, levels of polymorphism were low, with variation detected at only 19 of 110 (17.3%) loci, and most polymorphic loci (78.9%) were diallelic (mean number of alleles = 2.3). Despite these low levels of variation, the microsatellites developed here will be useful for parentage assignment for confirming pedigrees, and investigating relationships between genetic variation, pedigree‐based inbreeding and reproductive success in this highly endangered species.     

Nonamplifying alleles at microsatellite loci: a caution for parentage and population studies

While genotyping wild red deer (Cervus elaphus) at microsatellite loci for paternity assignment, we found three loci (MAP65, BOVIRBP and CelJP23) with segregating nonamplifying alleles. Nonamplifying alleles were detected through mismatches between known mother-offspring pairs and by significant deviations from Hardy-Weinberg equilibria. In a wide range of molecular ecology applications, and especially in parentage assignment, the possible existence of undetectable alleles must be taken into account; this may be particularly important for microsatellite data.     

基于粪便DNA的贺兰山岩羊亲权鉴定和婚配制研究

2017年7-8月和11-12月在贺兰山岩画风景区采集到369份岩羊新鲜粪便样本,用多态性较高的10个微卫星位点进行基因分型,共识别出282只岩羊个体。通过复合扩増和牙釉蛋白基因PCR法共鉴定出152只雄性,130只雌性。微卫星分析得到平均等位基因（Na）为10.1,平均观测杂合度（Ho）为0.7737,平均期望杂合度（He）为0.6512,平均多态信息（PIC）为0.6129。运用Cervus3.0软件进行亲权鉴定,并用Kingroup V2计算个体间的亲缘系数（r）,作为亲权鉴定的补充,共鉴定出父-母-子7对,父-子21对,母-子19对。根据亲缘关系对岩羊的婚配制进行推测分析,表明岩羊婚配制应属一雄多雌。     

Microsatellite evidence for monogamy and sex-biased recombination in the Western Australian seahorse Hippocampus angustus

Four polymorphic microsatellite loci were used to assess biological parentage of 453 offspring from 15 pregnant males from a natural population of the Western Australian seahorse Hippocampus angustus . Microsatellite genotypes in the progeny arrays were consistent with a monogamous mating system in which both females and males had a single mate during a male brooding period. Multilocus genotypes implicated four females in the adult population sample as contributors of eggs to the broods of collected males, but there was no evidence for multiple mating by females. Based on genotypic data from the progeny arrays, two loci were linked tightly and the recombination rate appeared to be ≈ 10-fold higher in females than in males. The utility of linked loci for parentage analyses is discussed.     

Effectiveness of 10 polymorphic microsatellite markers for parentage and pedigree analysis in plateau pika (Ochotona curzoniae)

Background

The plateau pika (Ochotona curzoniae) is an underground-dwelling mammal, native to the Tibetan plateau of China. A set of 10 polymorphic microsatellite loci has been developed earlier. Its reliability for parentage assignment has been tested in a plateau pika population. Two family groups with a known pedigree were used to validate the power of this set of markers.

Results

The error in parentage assignment using a combination of these 10 loci was very low as indicated by their power of discrimination (0.803 - 0.932), power of exclusion (0.351 - 0.887), and an effectiveness of the combined probability of exclusion in parentage assignment of 99.999%.

Conclusion

All the offspring of a family could be assigned to their biological mother; and their father or relatives could also be identified. This set of markers therefore provides a powerful and efficient tool for parentage assignment and other population analyses in the plateau pika.     

An evaluation of allowing for mismatches as a way to manage genotyping errors in parentage assignment by exclusion

In parentage assignment by exclusion, using multiple and very polymorphic loci, genotyping errors are a major cause of non‐assignment. Using stochastic simulations, we tested the possibility to allow for mismatches at one or more allele as a way to recover assignment power. This was very efficient provided the set of loci used had a high assignment power (> 99%) and the error rate was not too high (below 3–4%). In these cases, most of the theoretical assignment power could be recovered. We also showed the efficiency of the method in a practical experiment with rainbow trout.     

Analysis of microsatellites and parentage testing in saltwater crocodiles

Fifteen microsatellite loci were evaluated in farmed saltwater crocodiles for use in parentage testing. One marker (C391) could not be amplied. For the remaining 14, the number of alleles per locus ranged from two to 16, and the observed heterozygosities ranged from 0.219 to 0.875. The cumulative exclusion probability for all 14 loci was .9988. the 11 loci that showed the greatest level of polymorphism were used for parentage testing, with an exclusion probability of .9980. With these 11 markers on 107 juveniles from 16 known breeding pairs, a 5.6% pedigree error rate was detected. This level of pedigree error, if consistent, could have an impact on the accuracy of gentic parameter and breeding value estimation. The usefulness of these markers was also evaluated for assigning parentage in situations where maternity, paternity, or both may not be known. In these situations, a 2% error in parentage assignment was predicted. It is therefore recommended that more micro-satellite markers be used in these situations. The use of these microsatellite markers will broaden the scope of a breeding program, allowing progeny to be tested from adults maintained in large breeding lagoons for selection as future breeding animals.