Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Microsatellite DNA analysis proves nucleus of interspecies reconstructed blastocyst coming from that of donor giant panda

A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst and a giant panda specific microsatellite locus g(010) was successfully amplified. DNA sequencing of the PCR product showed that two sequences of reconstructed blastocysts are the same as that of positive control giant panda. Our results prove that the nucleus of interspecies reconstructed blastocyst comes from somatic nucleus of donor giant panda.

Origin of rabbit (Oryctolagus cuniculus) in China: evidence from mitochondrial DNA control region sequence analysis

A fragment of mitochondrial DNA (mtDNA) control region (similar to700 bp) was sequenced in 104 individuals from 20 breeds (three Chinese domestic breeds, five recently derived breeds and 12 introduced breeds) of domestic rabbits, Oryctolagus cuniculus . Nineteen sites were polymorphic, with 18 transitions and one insertion/deletion, and eight haplotypes (A1, A2, A3, A4, A5, A6, A7 and A8) were identified. Haplotype A1 was the most common and occurred in 89 individuals. In the 25 Chinese rabbits, only haplotype A1 was observed, while four haplotypes (A1, A3, A5 and A6) were found in 26 recently derived individuals. Haplotype A2 was shared by seven individuals among three introduced strains. The other six haplotypes accounted for 0. 96-1. 92% of the animals. Combined with the published sequences of European rabbits, a reduced median-joining network was constructed. The Chinese rabbit mtDNAs were scattered into two clusters of European rabbits. These results suggest that the (so-called) Chinese rabbits were introduced from Europe. Genetic diversity in Chinese rabbits was very low.

DNA sequence analysis of the triose phosphate isomerase gene from isolates of Giardia lamblia

Objective To confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries. Methods Genomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank. Results Of nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world. Conclusion Genotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.

乙醇浸泡蜜蜂标本DNA的提取纯化

利用RAPD、PCR、RFLP、分子杂交等分子生物学 技术研究生物的遗传变异、遗传多态、分子进化等，必 须以DNA作为模板，目前所见有关DNA提取纯化的 报道均用活体或冰冻蜜蜂，这给样品采集、保存带来不 便。本实验以采自云南不同地区以及马来西亚的蜜蜂 乙醇(75％)浸泡标本为材料，参照王文(1994年)的蛋 白酶K提取法并加以改进，对DNA进行提取纯化。

THE ORIGIN AND GENETIC DIFFERENTIATION OF NATIVE BREEDS OF PIGS IN SOUTHWEST CHINA - AN APPROACH FROM MITOCHONDRIAL-DNA POLYMORPHISM

Mitochondrial DNA (mtDNA) of six breeds of native domestic pigs from Yunnan province, southwest China, and two wild boars obtained from Sichuan, China, and Vietnam was analyzed using 20 restriction endonucleases that recognize six nucleotides. Restriction maps were made by double-digestion methods and polymorphic sites were located on the map. According to their mtDNA restriction types, all the breeds were classified into six groups. Genetic distances among groups were calculated to define their phylogenetic relationships. The relationship between the Sichuan wild boar and domestic pigs is close, while the Vietnamese wild boar is relatively far from them, so the domestic pigs in southwest China are likely to have originated from a wild pig which distributed in west China. We compare our results with previous reports in literature and discuss the relationship among Chinese pigs, Japanese pigs, and European pigs. The mtDNA cleavage pattern of the Mingguang pig digested by EcoRV was identical to that of Duroc; mutations at the EcoRI site, detected in the mtDNA of two Dahe pigs, are the same as in the Vietnamese wild boar, suggesting that mutational hot spots exist in the mtDNA of pigs.

牛山湖浮游生物群落DNA指纹结构与物种组成的关系

利用RAPD及DGGE指纹技术揭示牛山湖5个采样点浮游生物群落的DNA多态性,并定性地探讨其与物种组成的关系。结果如下:(1)从40条随机引物中筛选出9条引物,共获得93条谱带,多态率为58%;各采样点所得谱带平均为67条,其中Ⅰ站最少,为61条,Ⅴ站最多,为74条;(2)PCR-DGGE指纹图谱共含102条谱带,其中原核生物56条,真核生物46条,谱带总数以Ⅲ站、Ⅳ站和Ⅴ站较多,Ⅰ站和Ⅱ站较少;(3)5个采样点共观察到62种/类浮游生物,其中Ⅰ站和Ⅱ站种类较少,Ⅲ站、Ⅳ站和Ⅴ站种类较多,分布概率在100

丹江口水库赤眼鳟(Squaliobarbus curriculus)遗传多样性的RAPD和ISSR分析

利用RAPD和ISSR两种分子标记技术,分析了丹江口水库野生赤眼鳟30个个体的遗传多样性。用11个RAPD引物对其基因组DNA进行扩增,共获得101个重复性好且谱带清晰的扩增位点,片段大小在100—3000bp之间,其中多态性位点63个,多态位点比例为62.38%;个体间遗传距离在0.1049—0.3417之间,平均为0.1742。用10个ISSR引物共检测到88个位点,其中多态性位点61个,多态位点比例为69.32%;个体间遗传距离在0.1088—0.3847之间,平均为0.1907。结果显示,丹江口水

鄱阳湖南矶山自然保护区鲤、鲫的随机扩增多态DNA分析

利用RAPD(随机扩增多态)标记分析湖内鲤、鲫群体遗传多样性,从40个随机引物中各筛选出8个引物适合鲤、鲫群体RAPD扩增。在鲤群体中,共检测出60条带,其中多态性带42条,多态位点比率为70.00%;而在鲫群体中,共检测出61条带,其中多态性带40条,多态位点比率为65.57%。用POPGENE软件分析实验数据,结果显示:湖内鲤群体的遗传多样性水平(He=0.230 1,H0=0.391 0)和鲫群体的遗传多样性水平(He=0.218 6,H0=0.375 8)都较高,都有较大的遗传变异。而在鲤、鲫群体

利用RAPD-PCR探讨天蓝喇叭虫生物群系分化

选择天蓝喇叭虫(Stentor coeruleus)作为研究对象,对武汉市南湖、月湖、关桥3个水体共5个样点天蓝喇叭虫(S.coeruleus)样本的总DNA进行随机扩增多态DNA聚类分析,以检测各个样本的遗传相似性和趋异程度,借以评估样本间的遗传变异度。结果如下:(1)从98条随机引物中筛选12条引物共扩增出89条大小为100～1500bp的清晰条带,平均每条引物扩增出7.4条片段。(2)用Rapdistance1.04分析显示,不同样点样本之间存在着一定的变异,其遗传距离在0.076～0.416之间。

乌龟遗传多样性的RAPD分析

运用RAPD技术对乌龟的遗传多样性进行了分析。用 2 0个随机引物对 2 4个个体的基因组DNA进行了PCR扩增 ,一共扩增出 32 88条DNA片段 ,平均每个个体扩增出 137条带。在检测到的 137个位点中 ,多态位点数为 119个 ,占 86 9% ,标记的分子量在 0 .2kb— 3kb之间。个体间最大的遗传距离为 0 4 6 7,个体间最小的遗传距离为0 16 8。 2 4个个体的平均遗传距离为 0 32 4 +0 0 6 31。表明乌龟的遗传多样性水平较高。采用类平均聚类法(NJTREE)构

低等鲤科鱼类RAPD分析及系统发育研究

通过对鲤科鱼类30个个体所代表的18个种的随机扩增，作者得到了大量有系统发育信息的DNA多态片段。通过Rapdplot程序将DNA的多态片段转换成遗传距离（d=1－S，S＝2NxNy／Nx＋Ny）。该遗传距离的矩阵经PHYLIP软件包中的Neighbor（option=NJ）程序处理后，生成了低等鲤科鱼类代表属种的分支系统图。从该系统图可以看出：RAPD分析方法在鲤科鱼类的系统发育研究中有一定的局限性，它比较适合于亚科内属间系统发育的研究。结果显示亚科并不是一个单元类群，其中的马口鱼类和细鲫类各自形成单元

中华鲟随机扩增多态性DNA及遗传多样性研究

采用随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术进行了连续３年（１９９５－１９９７）共７０尾来源于长江水系中华鲟样本遗传分析。共用了４０个 １０ｂｐ长的随机引物，在 ２６种可供分析的引物中，只有ＯＰＫ０１、ＯＰＫ０２、ＯＰＫ０３、ＯＰＫ０９、ＯＰＫ１４和ＯＰＱ０８ＲＡＰＤ－ＰＣＲ产物有多态现象，多态引物占２３％。２６个引物中共扩增出１０８条稳定的ＤＮＡ带。其中１２条带为多态带，多态座位比例为１１．１％。个体间遗传距离变动为０．９５１　０－１．０００ ０，平均为０．９７４ ３。 １９９５、１９９６和１９９７年的遗传

四种十足目甲壳动物遗传差异的RAPD分析

采用RAPD技术，对分类阶元不同的四种十足目甲壳动物：克氏原螯虾、汉水华溪蟹、日本绒螯蟹合浦亚种和中华绒螯蟹，进行了遗传多态性的研究。四个物种DNA库的扩增结果表明，随着亲缘关系由近变远，物种间的相似率依次减小，由56．1％下降到22．3％。用UPGMA方法作聚类分析构建出物种的系统树图，反映出四个物种对应的种、科间、科间以上遗传差异逐步增大，这与以形态标记为主的分类结果相一致。在同工酶分析难以揭示差异的两种绒螯蟹中，用RAPD分析观察到明显的种间差异。两种绒螯蟹个体的扩增图谱分析结果为，中华绒螯蟹个体间

银鲫复合种外源遗传物质整入的RAPD分析

在优化RAPD检测条件的基础上，采用40对随机引物，比较分析了银鲫复合种、异育银鲫和兴国红鲤相互间扩增DNA片段的异同性。总的来说，银鲫复合种和异育银鲫具有基本一致的扩增产物，而与兴国红鲤的扩增产物多数不同。相似率分析表明，银鲫复合种与兴国红鲤之间的相似率为31.6％，异育银鲫与兴国红鲤之间的相似率为28．6％。在分析中，除发现银鲫复合种、异育银鲫与兴国红鲤间共有的扩增片段外，还发现了银鲫复合种与兴国红鲤间共有的扩增片段以及银鲫复合种所特有的DNA扩增带。本研究不但为银鲫复合种异源遗传成分的整人提供了新的