华南虎、东北虎、孟加拉虎的D-loop和ND5序列及其在系统进化分析中的应用

本文采用PCR和质粒克隆测序方法,获得了华南虎线粒体D-loop区的480 bp序列和东北虎、孟加拉虎线粒体D-loop区的503 bp序列;同时还获得了这三个虎亚种和金钱豹线粒体ND5基因5'端309 bp的部分序列.根据D-loop序列分析,华南虎与孟加拉虎、东北虎的平均距离(p-distance)分别为0.110 88和0.110 87,而东北虎与孟加拉虎间的平均距离为0.009 94;根据ND5序列分析,华南虎与孟加拉虎、东北虎的平均距离分别为0.114 34和0.117 58,而东北虎与孟加拉虎间的平均距离为0.003 24.三个虎亚种的mtDNA D-loop和ND5序列比较表明,华南虎是这三个虎亚种中最为古老的亚种.     

我国的珍贵兽类

我国珍贵的哺乳动物,种类繁多,除著名的大熊猫、金丝猴和白暨豚等以外,现介绍以下几种。东北虎虎和狮是最凶猛最大形的食肉类动物,常被称为“百兽之王”。虎只产在亚洲,但分布广泛,由西伯利亚到南洋群岛都有。虎有八个亚种,其中东北虎、华南虎、孟加拉虎三个亚种,我国都有分布。东北虎的产区包括我国的东北地区,朝鲜的最北部山区和苏联西伯利亚的远东地区。由     

华南虎

我国的虎有四个亚种:东北虎、华南虎、孟加拉虎和东南亚虎,是世界上虎亚种最多的国家华南虎(Panthera tigris amoyensis)是我国四个亚种虎中的一个特有种。体型较小,毛呈桔黄色、斑纹黑而     

福建梅花山圈养华南虎遗传状况分析

华南虎是世界上密切关注的旗舰物种,在过去的10 年间没有发现野生华南虎存在的证据,因此它是极度濒危的虎亚种。福建梅花山圈养华南虎群体是整个圈养华南虎群体的重要组成部分,拥有 12 只华南虎。基于组合长度为 3934 bp 的线粒体序列分析发现梅花山圈养华南虎拥有 3 种线粒体单倍型; 而基于 20 个微卫星位点基因型分析显示梅花山圈养华南虎一共有71 个等位基因,平均等位基因数是3. 55,等位基因丰度的平均值是3. 32,平均期望杂合度和多态信息含量( PIC) 分别为0. 513 和0. 445。这些提示梅花山华南虎圈养群体维持着较高的遗传多样性。     

成绩·危机·希望 中俄东北虎保护

在世界的13个老虎分布国中,中国拥有世界上最多的老虎亚种,即东北虎、华南虎、印度支那虎和孟加拉虎四个亚种。据估计,东北虎约18-22只,主要分布在黑龙江、吉林省的东部,沿中俄边界一线;华南虎在新中国成立初曾经分布于中国的福建、广东、江西、浙江、湖北、湖南等省,现在已经野外功能性灭绝,所能见的华南虎多为人工饲养的,     

孟加拉笛鲷线粒体基因组序列结构及其进化

采用Long-PCR扩增线粒体全基因组方法得到了孟加拉笛鲷线粒体基因组全序列.序列分析结果表明,孟加拉笛鲷线粒体基因组序列全长16 511 bp,共有13个编码蛋白质基因、22个tRNA基因、2个rRNA基因和1个D-loop区.在编码蛋白质基因中,除COⅠ是以GTG作为起始密码子外,其它均是以ATG起始,NDⅠ、COⅡ、ND3以TAG作为终止密码子,而ND4、Cyt b则以不完全的T为终止密码子,其余8个蛋白质基因的终止密码子均为TAA.孟加拉笛鲷线粒体基因组各基因长度、位置与典型的脊椎动物相似,其编码蛋白质基因和rRNA基因与其它硬骨鱼类具有很高的同源性.基于14种笛鲷线粒体区段COⅠ、COⅡ和Cyt b基因的全序列合并成的一个组合数据集构建系统进化树,显示孟加拉笛鲷与四带笛鲷关系最为密切.     

东北虎幼体葡萄糖、四种离子、尿素氮及肌酐含量的测定与分析

用HITACHI 7600-20型全自动生化分析仪对13只东北虎幼体进行了8项生化指标的测定,运用生物统计学分析方法,分别对东北虎幼体雌雄个体间及东北虎幼体与华南虎成体间进行了比较分析.结果表明,东北虎雌雄个体间血清中K 含量呈极差异显著(P<0.01),而葡萄糖、Na 、Ca2 、HCO3-、尿素及肌酐含量并无显著差异;在Cl-、肌酐含量上,雌性东北虎幼体与雌性华南虎有显著差异(P<0.05);雄性东北虎幼体与雄性华南虎在Cl-含量上无显著差异,在葡萄糖及肌酐含量上有极显著差异(P<0.01).     

赤麂线粒体全基因组的序列和结构

提取赤麂细胞株总DNA,参照我们实验室已测定的同属动物小麂线粒体全基因组序列设计引物,PCR扩增、测序、拼接,获得赤麂线粒体全基因组序列并进行生物信息学分析。赤麂线粒体全基因组序列全长16354bp。定位了22个tRNA基因、2个rRNA基因、13个蛋白编码基因和1个D-loop区。赤麂与小麂及其它哺乳动物线粒体的基因组结构相同,它们的序列同源性都较高。     

基于线粒体12S rRNA和ND5基因序列的中国飞蝗属3亚种系统发育关系研究

对分布于中国的飞蝗Locusta migratoria Linnaeus 3亚种的线粒体12SrDNA(487bp)和ND5(451bp)基因的部分序列进行测定,与已知全线粒体基因组全序列的非洲飞蝗亚种进行序列比较,并在此基础上对ND5基因,以斑腿蝗科瘤喉蝗Parapodisma mikado为外群,重建MP树。基于序列长度的限制,将两基因联合分析重建了1棵最简约无根树。在对准的4个亚种共有的481bp的12SrDNA和451bp的ND5片断序列比较中,AT含量明显高于GC含量。由序列差异来看,哑洲飞蝗和东亚飞蝗序列相似度高,非洲飞蝗和西藏飞蝗相似度高,ND5基因的MP树和联合树也支持这个结论。由于大陆漂移和青藏高原隆起的特殊地质事件,正好解释了非洲飞蝗和西藏飞蝗的相似性,支持西藏飞蝗为单独1亚种,与非洲飞蝗为同一起源地,与其它2亚种相区别。     

从分子水平探讨中国东北狍的分类地位

国内外对中国狍的分类地位意见不一。本测定了来自中国东北完达山、小兴安岭、大兴安岭、三江和吉林向海等5个地区的15只野生狍的线粒体DNA Cyt 6基因(415 bp)和来自完达山、三江和吉林向海等3个地区的6只野生狍的线粒体控制区部分序列(764 bp),分析了中国东北狍与西伯利亚狍、欧洲狍的系统进化关系,得出中国东北狍与欧洲狍之间mtDNA Cyt 6和mtDNA控制区序列差异平均值分别为4．12%,和4．89％,达到了鹿类动物的种间差异水平;而与西伯利亚狍的mtDNA Cyt 6和mtDNA控制区序列差异平均值仅为1.61％和2．44％,只达到亚种水平。结合形态学的差异,本认为中国东北的狍与欧洲狍为两个独立的物种,与西伯利亚狍为同种。并提出中国东北狍与欧洲狍的分歧时间约在200万年前,与西伯利亚狍的分歧时间约在80万年前。     

Phylogeography and genetic ancestry of tigers (Panthera tigris)

Eight traditional subspecies of tiger (Panthera tigris),of which three recently became extinct, are commonly recognized on the basis of geographic isolation and morphological characteristics. To investigate the species' evolutionary history and to establish objective methods for subspecies recognition, voucher specimens of blood, skin, hair, and/or skin biopsies from 134 tigers with verified geographic origins or heritage across the whole distribution range were examined for three molecular markers: (1) 4.0 kb of mitochondrial DNA (mtDNA) sequence; (2) allele variation in the nuclear major histocompatibility complex class II DRB gene; and (3) composite nuclear microsatellite genotypes based on 30 loci. Relatively low genetic variation with mtDNA,DRB,and microsatellite loci was found, but significant population subdivision was nonetheless apparent among five living subspecies. In addition, a distinct partition of the Indochinese subspecies P. t. corbetti in to northern Indochinese and Malayan Peninsula populations was discovered. Population genetic structure would suggest recognition of six taxonomic units or subspecies: (1) Amur tiger P. t. altaica; (2) northern Indochinese tiger P. t. corbetti; (3) South China tiger P. t. amoyensis; (4) Malayan tiger P. t. jacksoni, named for the tiger conservationist Peter Jackson; (5) Sumatran tiger P. t. sumatrae; and (6) Bengal tiger P. t. tigris. The proposed South China tiger lineage is tentative due to limited sampling. The age of the most recent common ancestor for tiger mtDNA was estimated to be 72,000-108,000 y, relatively younger than some other Panthera species. A combination of population expansions, reduced gene flow, and genetic drift following the last genetic diminution, and the recent anthropogenic range contraction, have led to the distinct genetic partitions. These results provide an explicit basis for subspecies recognition and will lead to the improved management and conservation of these recently isolated but distinct geographic populations of tigers.     

虎物种特异性鉴定的 PCR 方法研究

为了建立可对虎DNA 进行特异性检测的PCR 方法, 应用在野外调查中获得的虎疑似样品和保护执法工作中难以检查辨认的虎产品进行物种鉴定, 从Genbank 数据库下载5 个虎亚种及其它6 种猫科动物和6 种鹿科动物的mtDNA 细胞色素b 基因序列, 并用Wdnasis (V2.5) 软件对上述不同动物的该基因碱基序列进行比较。在此基础上, 综合考虑了设计引物的基本原则, 选择了虎与其它动物碱基序列上差异位点较多的两个片段, 设计出PCR 引物(引物1、2) 。用该对引物分别对从东北虎、华南虎及8 种猫科动物和6 种非猫科动物的肌肉、脏器组织、皮或毛发中提取的DNA 进行PCR (聚合酶链反应) 扩增。结果表明, 所设计的引物对虎DNA 具有特异性,从而达到了对该物种进行特异性检测鉴定的目的。     

Highly conserved D-loop-like nuclear mitochondrial sequences (Numts) in tiger (Panthera tigris)

Using oligonucleotide primers designed to match hypervariable segments I (HVS-1) ofPanthera tigris mitochondrial DNA (mtDNA), we amplified two different PCR products (500 bp and 287 bp) in the tiger (Panthera tigris), but got only one PCR product (287 bp) in the leopard (Panthera pardus). Sequence analyses indicated that the sequence of 287 bp was a D-loop-like nuclear mitochondrial sequence (Numts), indicating a nuclear transfer that occurred approximately 4.8–17 million years ago in the tiger and 4.6–16 million years ago in the leopard. Although the mtDNA D-loop sequence has a rapid rate of evolution, the 287-bp Numts are highly conserved; they are nearly identical in tiger subspecies and only 1.742% different between tiger and leopard. Thus, such sequences represent molecular ‘fossils’ that can shed light on evolution of the mitochondrial genome and may be the most appropriate outgroup for phylogenetic analysis. This is also proved by comparing the phylogenetic trees reconstructed using the D-loop sequence of snow leopard and the 287-bp Numts as outgroup.     

Analysis of variable sites between two complete South China tiger (Panthera tigris amoyensis) mitochondrial genomes

In order to investigate the mitochondrial genome of Panthera tigris amoyensis, two South China tigers (P25 and P27) were analyzed following 15 cymt-specific primer sets. The entire mtDNA sequence was found to be 16,957 bp and 17,001 bp long for P25 and P27 respectively, and this difference in length between P25 and P27 occurred in the number of tandem repeats in the RS-3 segment of the control region. The structural characteristics of complete P. t. amoyensis mitochondrial genomes were also highly similar to those of P. uncia. Additionally, the rate of point mutation was only 0.3% and a total of 59 variable sites between P25 and P27 were found. Out of the 59 variable sites, 6 were located in 6 different tRNA genes, 6 in the 2 rRNA genes, 7 in non-coding regions (one located between tRNA-Asn and tRNA-Tyr and six in the D-loop), and 40 in 10 protein-coding genes. COI held the largest amount of variable sites (9 sites) and Cytb contained the highest variable rate (0.7%) in the complete sequences. Moreover, out of the 40 variable sites located in 10 protein-coding genes, 12 sites were nonsynonymous.     

POPU金钱豹mtDNA ND5基因序列及其遗传变异

测定山西7个不同地域金钱豹9个体mtDNA ND5基因部分序列,结合GenBank下载的37条序列,进行遗传多样性分析,用豹属的狮Panthera leo和虎Panthera tigris作外群,构建其不同单倍型的NJ分子聚类关系。46条序列共产生18个单倍型,单倍型多样度和核苷酸多样度指数表明金钱豹的遗传多样性比较丰富,AMOVA分析显示其种群间出现显著的遗传分化。单倍型聚类分析表明,亚洲和非洲种群尽管有一定的分化,但其置信度很低,不足以形成各自独立的分支。     

mtDNA indicates profound population structure in Indian tiger (Panthera tigris tigris)

We analyzed mtDNA polymorphisms (parts of control region, ND5, ND2, Cytb, 12S, together 902 bp) in 59 scat and 18 tissue samples from 13 Indian populations of the critically endangered Indian tiger (Panthera tigris tigris), along with zoo animals as reference. Northern tiger populations exhibit two unique haplotypes suggesting genetic isolation. Western populations from Sariska (extinct in 2004) and Ranthambore are genetically similar, such that Ranthambore could serve as a source for reintroduction in Sariska. Zoo populations maintain mitochondrial lineages that are rare or absent in the wild.     

Twelve polymorphic microsatellite loci for the South China tiger Panthera tigris amoyensis

Twelve microsatellite loci were found from genomic DNA‐enriched libraries of the South China tiger (Panthera tigris amoyensis). The number of observed alleles for each locus in 53 individuals ranged from five to 14; the expected and observed heterozygosity was 0.480 to 0.883 and 0.333 to 0.956, respectively; and the mean polymorphic information content (PIC) was 0.729. These markers would be considered a useful tool for the study of genetic variation in South China tiger in the future.     

东北虎源等孢球虫的发现

对东北虎粪便进行虫卵检查,分离获得了自然感染的球虫卵囊。对球虫卵囊的形态和经培养发育后的各阶段的卵囊形态进行了观察。东北虎源球虫卵囊符合等孢属球虫的形态学特征。根据Genbank 上发表的猫等孢球虫18S rDNA 序列(L74671)设计一对特异性引物,利用PCR 技术对东北虎源球虫的18S rDNA 进行了扩增。扩增得到一段360 bp 的DNA 片段,并对该片段进行了序列测定。测序结果和其它17 种原虫的相应序列进行序列同源性比较分析,分析显示东北虎源球虫和其它等孢属球虫单独聚为一类。结合对东北虎源球虫的卵囊形态、卵囊发育情况及序列分析结果,确定其为等孢属球虫。      

Subspecies genetic assignments of worldwide captive tigers increase conservation value of captive populations

Tigers (Panthera tigris) are disappearing rapidly from the wild, from over 100,000 in the 1900s to as few as 3000. Javan (P.t. sondaica), Bali (P.t. balica), and Caspian (P.t. virgata) subspecies are extinct, whereas the South China tiger (P.t. amoyensis) persists only in zoos. By contrast, captive tigers are flourishing, with 15,000-20,000 individuals worldwide, outnumbering their wild relatives five to seven times. We assessed subspecies genetic ancestry of 105 captive tigers from 14 countries and regions by using Bayesian analysis and diagnostic genetic markers defined by a prior analysis of 134 voucher tigers of significant genetic distinctiveness. We assigned 49 tigers to one of five subspecies (Bengal P.t. tigris, Sumatran P.t. sumatrae, Indochinese P.t. corbetti, Amur P.t. altaica, and Malayan P.t. jacksoni tigers) and determined 52 had admixed subspecies origins. The tested captive tigers retain appreciable genomic diversity unobserved in their wild counterparts, perhaps a consequence of large population size, century-long introduction of new founders, and managed-breeding strategies to retain genetic variability. Assessment of verified subspecies ancestry offers a powerful tool that, if applied to tigers of uncertain background, may considerably increase the number of purebred tigers suitable for conservation management.