鲤鱼微卫星分子标记的筛选

微卫星 (ｍｉｃｒｏｓａｔｅｌｌｉｔｅ)是近十几年来发展起来的一种新的分子标记 ,它是指以少数几个核苷酸 ( 1～ 6个 )为单位多次重复的简单序列 ,以双核苷酸重复最为常见 ,而其中又以 (ＣＡ/ＧＴ) ｎ 居多。由于微卫星在真核生物基因组中是随机分布的 ,而作为分子遗传标记又有着非常高的多态性和共显性 ,因此在构建遗传连锁图谱时备受青睐 (Ｂｒｏｏｋｅｔａｌ ,1994 )。目前 ,在人类和多种动物中已经构建了以微卫星为主的遗传连锁图谱。但在鲤鱼等水产动物的遗传连锁图谱中 ,微卫星分子标记还较少 (孙效文和梁利群 ,2 0 0 0 )。为了摸索…     

磁珠富集法筛选实验豚鼠微卫星分子标记

目的直接从实验豚鼠基因组DNA中筛选获得微卫星分子标记。方法应用磁珠和生物素标记的微卫星探针与豚鼠基因组酶切片段杂交,捕获200～1000 bp含有微卫星序列的DNA片段,连接到pMD-18V载体中,转化到感受态细胞E.coli DH5α中构建富集微卫星序列的小片段插入文库。然后用PCR法进行筛选。结果从约2000个转化子中获得240个阳性克隆。对其中98个进行了测序,并成功设计豚鼠微卫星引物17对。结论经过优化的磁珠富集法能够稳定、高效地获得豚鼠微卫星标记。本研究获得的微卫星位点将成为豚鼠遗传学研究的有力工具。     

基于转录组测序的大熊猫多态性微卫星标记筛选

结合已公布的大熊猫Ailuropoda melanoleuca基因组和本实验室所测6只大熊猫的转录组数据,筛选多态性微卫星位点并分析其组成及特征。结果显示:共获得326个多态性微卫星位点,其中二碱基多态性微卫星最多,共228个,占69.93%;三、四、五、六碱基所占比例分别为9.51%、14.11%、5.21%、1.22%。根据分析结果中缺失率与标准差2项指标以及位点序列长度,选取20个多态性二碱基微卫星位点,用于25只大熊猫个体血液DNA进行PCR验证并做后续分析。结果表明:不同位点的等位基因数为2～8,平均等位基因数为3.70,观测杂合度、期望杂合度分别为0～1.000、0.280～0.784,平均值分别为0.472和0.532。在Bonferroni校正后,证实4个位点显著偏离哈迪-温伯格平衡,所有位点未观察到显著连锁不平衡(P>0.01)。20个位点多态信息含量(PIC)在0.246～0.734,其中具有高度多态性的位点9个(PIC>0.50),11个位点呈中度多态性(0.25相似文献   

应用微卫星标记研究Dunkin Hartley豚鼠封闭群的遗传背景

目的检测我国现有Dunkin Hartley豚鼠封闭的遗传背景,分析评估其遗传多样性水平和遗传分离情况,为建立标准化的豚鼠封闭群监测方法提供基础资料。方法应用筛选获得的8个微卫星位点,从一个数量为1000的豚鼠封闭群中随机选择72个个体,通过PCR扩增和聚丙烯酰胺凝胶电泳的方法,进行等位基因检测。并根据检测结果分析评估了该豚鼠封闭群的遗传现状。结果共检测到28个等位基因,每个座位的等位基因数为2～5个,有效等位基因数为1.5191～3.4422,平均2.3093。平均期望杂合度为0.5294。各位点多态信息含量在0.3154～0.6545之间,平均值为0.4687。有5个位点显著偏离Hardy-Weinberg平衡。结论豚鼠封闭群的遗传多态性处于中等水平,遗传平衡检测结果提示种群的繁殖过程未能实现完全随机交配,近交现象一定程度上存在。本研究的结果将为豚鼠封闭群遗传监测方法和标准的建立提供基础。     

锦鲤4个人工雌核发育家系的微卫星标记研究

利用Crooijmans et al.(1997)分离的包含CA重复单元的普通鲤鱼（Cyprinus carpio L.)的8个微卫星DNA标记,对从锦鲤（Cyprinus carpio L.)的红白,大正和昭和3个不同品系中所获得的4个不同人工雌核发育家系的20尾个体进行PCR扩增。电泳结果表明,8对引物在20尾个体中均能重复稳定地扩增出相应的同源序列。随引物不同,各等位基因数为1－11个,大小在68－264bp。在MFW4,MFW7,MFW19,MFW20,MFW23和MFW24 6个微卫星的扩增结果中,20尾个体的扩增图谱呈现了高度的遗传多态性,不同雌核发育家系内个体的遗传异质性也较大。其中大正（TaS)和红白1（RW1）的个体不仅花色分化显著,而且个体间的平均遗传距离分别高达0．28。通过对微卫星等位基因和基因型分析发现,由于锦鲤品系中的每一个体是通过不断地杂交选育而获得,基因组来源复杂,基因高度杂合。因此,只进行1代的人工雌核发育,其家系内仅部分个体的部分座位出现纯合。所获得的人工雌核发育锦鲤为后续的色素遗传调控机制研究提供了必要的实验材料;同时,所鉴定的微卫星分子标记为进行锦鲤的分子标记育种的基因组作图提供了理想的工具。     

大熊猫多态性微卫星标记筛选

选用前人分离得到的42对大熊猫微卫星引物,分别用圈养大熊猫的血液DNA和野生大熊猫的粪便DNA对其进行PCR扩增,并比较琼脂糖凝胶电泳和变性聚丙烯酰胺凝胶电泳的检测结果。结果表明,不同标记的多态性差异较大,筛选出13对能较好地应用于大熊猫遗传多样性研究的微卫星引物。     

东北虎微卫星DNA遗传标记的筛选及在亲子鉴定中的应用

利用18个家猫微卫星基因座，在东北虎(Panthera tigris sibilia)DNA中扩增结果有4个基因座没有产物，8个基因座为单态，6个基因座为多态性。同时利用苏门答腊虎的微卫星序列设计了8对引物，在东北虎DNA中有4对具有多态性。微卫星基因座的多态性百分率为38．5％。在供试的27只东北虎中，发现等位基因间的变异均为偶数碱基长度变化，对有准确谱系记录的个体研究表明，这10个微卫星DNA遗传标记符合孟德尔遗传规律，所以这些微卫星DNA可以有效的应用于东北虎的亲子鉴定。利用这10对多态性引物，我们成功地鉴定了7个父子关系不清的后代。收集的样品包括23只毛发样品和4只血液样品，实验结果表明，毛发和血液样品均可以得到清晰的微卫星条带[动物学报49(1)：118—123．2003]。     

微卫星荧光标记基因组扫描

微卫星荧光标记基因组扫描为 2 0世纪 90年代发展起来的一项分子生物学技术 ,在基因定位、个体识别等方面有广泛的应用 .文中对其原理、策略、分析方法及应用作了论述 ,并探讨了它的发展前景 .     

藏鸡微卫星文库的构建与微卫星标记筛选

通过磁珠富集法构建了藏鸡基因组微卫星富集文库,分离微卫星序列并对其进行分析.将藏鸡基因组DNA经Sau3AI酶切后纯化回收,连接特定接头.用生物素标记的(CA)12探针与藏鸡基因组酶切回收片段杂交,捕获200～900 bp片段,随后将获得的片段连接到pMD 18-T载体上,转化至JM109中,成功构建藏鸡微卫星富集文库.从1200个转化子中获得了353个阳性克隆,随机挑选53个测序,根据测序结果成功设计了18对藏鸡微卫星引物,最终筛选出6个具有多态性的微卫星标记,其PIC值均大于0.5,同时可以用于研究藏鸡遗传多样性.实验结果表明磁珠富集法能够有效提高分离微卫星标记的效率.     

Evaluation of the genetic diversity of Laiwu pigs using twenty-seven microsatellite markers

The Laiwu pig, an indigenous pig breed known for extremely high intramuscular fat content, is a well-preserved ancient breed due to long-term natural and artificial selections. In this study, using 27 microsatellite markers jointly recommended by the International Society of Animal Genetics (ISAG) and the Food and Agriculture Organization (FAO), we investigated the genetic diversity of the Laiwu pig breed. The genetic diversity of Laiwu pigs is dramatically low, with the observed heterozygosity ranging from 0.067 to 0.767. Among the 27 microsatellite markers, 10 were high polymorphic loci, 10 were moderate polymorphic loci, six were low polymorphic loci, and no polymorphism was detected at one locus (IGFI). Further analyses with the 10 high polymorphic loci and five moderate polymorphic loci revealed that the Laiwu pig breed was inbred and heterozygous deficient to some extent, but not severely, and that the Laiwu pigs were relatively pure, with almost no hybridization with other breeds. Two subgroups of the current 13 Laiwu pig pedigrees were identified. These results suggest that the Laiwu pig breed has a low diversity and a conservation program must be developed to preserve the “Laiwu pig” gene pool.     

Development and characterisation of microsatellite markers for the medicinal plant Scutellaria baicalensis (Lamiaceae)

Scutellaria baicalensis is a popular medicinal plant that is on the verge of extinction due to uncontrolled harvesting, habitat destruction and deterioration of its ecosystem. We isolated and characterised 21 microsatellite loci in this species. Ninety-four individuals from six populations were used to test the polymorphism of the microsatellite loci. The number of alleles per locus ranged from 1 to 13, with a mean of 7.2. Observed and expected heterozygosities varied from 0.000 to 1.000 and 0.000 to 0.938, respectively. Among these new microsatellite markers, only two loci showed significant deviation from Hardy–Weinberg equilibrium. No locus pairs showed significant linkage disequilibrium. The 21 primer pairs were tested in other Scutellaria species. Most of these primer pairs worked successfully, except for Scut18. These new microsatellite markers could be applied to investigate the genetic diversity and population genetic structure of S. baicalensis and its closely related species.     

Application of two microsatellite sequences in wheat storage proteins as molecular markers

In eukaryotes, tandem arrays of simple-sequence repeat sequences can find applications as highly variable and multi-allelic PCR-based genetic markers. In hexaploid bread wheat, a large-genome inbreeding species with low levels of RFLP, di- and trinucleotide tandem repeats were found in 22 published gene sequences, two of which were converted to PCR-based markers. These were shown to be genome-specific and displayed high levels of variation. These characteristics make them especially suitable for intervarietal breeding applications.     

Identification of microsatellite markers in the rye genome

The rye genomic library, which consists of DNA fragments in the range of 0.5–1.1 kb, was screened for the presence of tri-and tetranucleotide and compound microsatellites. Of the 1,600,000 clones analysed, 102 clones were positive and 41 were suitable for SSR primer pair design. Twenty-six primer pairs amplified specific products, and six of them were capable of detecting polymorphism among 30 rye accessions of different genetic backgrounds. Using a set of Chinese Spring-Imperial wheat-rye addition lines, it was possible to locate 3 newly identified microsatellites on chromosomes 3R, 4R and 7R.     

Ten polymorphic microsatellite loci for the Atlantic halibut (Hippoglossus hippoglossus) and cross-species application in related species

In the present study, 10 polymorphic microsatellite DNA loci from Atlantic halibut (Hippoglossus hippoglossus) were isolated and characterized. The number of alleles for these loci ranged from 2 to 4 in tested 24 individuals. Observed and expected heterozygosities per locus varied from 0.21 to 0.70 and from 0.31 to 0.65, respectively. Most of these 10 microsatellite loci were successfully amplified and showed polymorphic in five related species. These loci will be useful for the assessment of genetic diversity and population structure of Atlantic halibut. H. Ding and C. Shao contributed equally to this work.     

豚鼠眼形觉剥夺后恢复期的生物学参数变化规律探讨

目的探讨豚鼠眼球形觉剥夺后恢复期的生物学参数变化规律。方法普通级2～3周龄豚鼠30只,随机分为两组：①实验组：20只,右眼采用半透明眼罩遮盖进行形觉剥夺4周,随后去遮盖3周,左眼作为自身对照;②正常对照组：10只,双眼不进行任何干预,开放饲养7周。形觉剥夺前、形觉剥夺4周后及去遮盖后第2、6、10、14和21天,测量豚鼠双眼生物学参数：睫状肌麻痹后行带状光检影测量屈光度;A超测量前房深度、晶体厚度和眼轴长度,计算出玻璃体腔长度。结果经过4周形觉剥夺,实验组豚鼠右眼向近视漂移,屈光度为（-2.88±2.30）D,诱导了（-5.50±1.9）D相对近视。去遮盖后,豚鼠右眼重新正视化,屈光度恢复的快速期发生在6 d内,14 d时双眼屈光度差值差异无显著性（t=-2.049,P=0.080）,为（-0.18±0.26）D;右眼玻璃体腔长度缩短,14 d时双眼玻璃体腔长度差值差异无显著性（t=1.652,P=0.14）,为（0.0234±0.0400）mm;右眼眼轴长度缩短,14 d时双眼眼轴长度差值差差异无显著性（t=1.443,P=0.192）,为（0.0183±0.0359）mm。与正常对照组右眼相比,去遮盖6 d,屈光度差异为（-0.48±0.36）D,差异无显著性（t=-1.325,P=0.206）,而2 d时玻璃体腔和眼轴长度差异分别为（0.0961±0.0630）mm、（0.0621±0.0386）mm,差异无显著性（t=1.652,P=0.14;t=1.607,P=0.125）。结论 2～3周龄豚鼠去除形觉剥夺后可以重新进行正视化,伴随玻璃体腔和眼轴长度缩短;去遮盖6 d内为眼生物学参数恢复的主要时期。     

Six new polymorphic microsatellite loci isolated and characterized from the African savannah elephant genome

The African savannah elephant (Loxodonta africana) is a ‘keystone’ species that plays a vital role in regulating the dynamics of both plant and animal communities and yet it is endangered and its numbers have been reduced to approximately 500 000 across their entire continental range. Molecular genetic markers are important tools for providing genetic information useful in formulating effective management and conservation strategies for the surviving elephant populations. We describe the isolation and characterization of six new polymorphic microsatellite markers in the African savannah elephant and demonstrate that these loci can be PCR (polymerase chain reaction)‐multiplexed, a desirable attribute that saves costs in large‐scale microsatellite screening.     

Development and characterisation of nine polymorphic microsatellite markers for Tephrosia calophylla Bedd. (Fabaceae)

Tephrosia calophylla Bedd. (Fabaceae) is an endangered tropical plant endemic to southwestern Ghats, India. The objective of this study was to contribute to the characterisation of the diversity of this rare species, which is necessary for its future conservation. Accordingly, microsatellite markers were designed, and their ability to detect polymorphisms was determined. Nine microsatellite markers were developed using genomic libraries, and all of the markers were successfully amplified in 42 individuals. Three to nine alleles per locus were observed, and the heterozygosity of the loci ranged from 0.381 to 0.905. The nine newly developed polymorphic markers recognise a sufficient number of varying loci to perform further studies on the conservation and breeding of this medicinal cultivar.     

Isolation of polymorphic microsatellite markers from <Emphasis Type="Italic">Przewalskia tangutica</Emphasis> (Solanaceae)

Microsatellite-containing regions were isolated and characterized in Przewalskia tangutica Maxim (Solanaceae), an endemic and endangered species to the Qinghai-Tibetan Plateau of China. An enrichment protocol yielded 200 positive clones. We designed primers to amplify 29 unique microsatellites, 12 of which amplified cleanly and were polymorphic. A survey of 17 individuals showed that these loci are highly variable with the number of alleles ranging from 3 to 12, and expected heterozygosity ranged from 0.2929 to 0.4947. Those markers will be useful for studies of population structure and intraspecific variation in P. tangutica.