百宜黑鸡与兴义矮脚鸡线粒体DNA 控制区遗传多样性分析

采用PCR和直接测序的方法测定百宜黑鸡和兴义矮脚鸡线粒体DNA控制区全序列,比较分析两种贵州地方鸡种的遗传变异。结果表明,百宜黑鸡、兴义矮脚鸡mtDNA控制区全序列长均为1231 bp;共发现26个变异位点(不包含种内变异位点),占分析位点总数的2.11%,其中12个转换,14个颠换;百宜黑鸡mtDNA控制区的A、T、G、C碱基含量分别为27.096%、33.628%、13.117%、26.159%,矮脚鸡的A、T、G、C碱基含量分别是A26.916%、T33.409%、G13.387%、C26.288%,t检验结果显示,两种鸡mtDNA控制区的A和T含量差异显著。百宜黑鸡和兴义矮脚鸡的遗传距离是0.0379,根据分子钟计算,二者约在190万年前分歧进化。     

香炉山鸡线粒体DNA D-Loop区遗传多样性及系统进化研究

试验旨在研究香炉山鸡的遗传多样性与系统进化。利用PCR扩增结合双向测序的方法对121只香炉山鸡线粒体DNA D-Loop(mtDNA D-Loop)区全长序列进行分析,并对mtDNA D-Loop区全长序列组成、变异与母系起源进行探讨。结果发现,香炉山鸡mtDNA D-Loop区全长序列为1 231～1 233 bp,A、T、C、G碱基含量分别为26.62%、33.55%、26.49%、13.34%,表现出T碱基含量最高,G碱基含量最低,A+T含量明显高于G+C含量,说明此区可能具有一定的碱基嗜好性;121条全长序列经分析发现共存在11种单倍型,26个变异位点,其中单一多态信息位点2个,简约信息位点24个,另外存在4处碱基插入与2处碱基缺失。遗传多样性分析发现,单倍型多样度为0.814,核苷酸多样度为0.00447,平均核酸差异数为5.494,表明香炉山鸡遗传多样性比较丰富,保种效果较好,具有一定的选育空间。经Tajima Test检测发现,Tajima’s D值为0.39378,检验结果不显著(P0.10),符合中性突变。聚类分析结果显示,香炉山鸡与红色原鸡聚为一支,说明香炉山鸡起源于红色原鸡;在分支内部又有4个分支,说明香炉山鸡存在多个母系起源。研究结果可为香炉山鸡种质资源保护与开发利用提供一定的参考数据。     

兰州大尾羊mtDNA D-loop和Cytb区序列分析与多态性研究

利用mtDNA序列分析技术对20只兰州大尾羊遗传多态性进行分析,兰州大尾羊mtDNA D-loop区序列长度为1 210 bp,碱基组成分析发现,A、T、G、C碱基含量分别为29.0%、32.3%、23.5%、15.2%,其中A＋T为61.3%,G＋C为38.7%,G＋C含量明显低于A＋T。通过对兰州大尾羊线粒体DNA D-loop区的碱基突变和同源性比较分析得出兰州大尾羊D-loop区核苷酸多样度（Nucleotide diversity）Pi为0.037 85,7只兰州大尾羊来自3个不同母本。兰州大尾羊mtDNA Cytb区序列长度为1 580 bp,其中A、T、G、C碱基含量分别为27.2%、33.7%、26.7%、12.4%;A＋T为60.9%,G＋C为39.1%,G＋C含量明显低于A＋T。应用计算机软件DNAsp4.10进行单倍型分析,17个兰州大尾羊个体mtDNA Cytb区序列共发现了12个单倍型（Haplotype）,其中第1号和第10号、第2号和第5号共用一种单倍型,单倍型比例在兰州大尾羊群体中较高,遗传多态性较低。     

西藏昌都两个地方绵羊品种(类群)遗传多样性分析

应用PCR和序列分析技术,分析了测定西藏昌都地区8只阿旺绵羊和8只藏绵羊mtDNA控制区全序列,并结合GeneBank中绵羊mtDNA的两种变异类型A和B序列进行了比对和系统进化分析.结果表明阿旺绵羊mtDNA控制区长度为1180～1183 bp,而藏绵羊为1 180 bp,序列中A+T含量明显高于G+C含量.将两品种(类群)9种单倍型序列与mtDNA的两种变异类型A和B序列进行比对,共发现106个变异(突变)位点,占分析位点总数的8.945%.两品种(类群)核苷酸多样性和单倍型多样性分别为1.755%、0.928%和0.857±0.082、0.893±0.086;序列的分歧度为3.8%,Kimura 2-parameter距离为0.0344.系统发育NJ树表明藏绵羊和A类线粒体聚为一类,而阿旺绵羊mtDNA单倍型序列同B型线粒体聚为一枝,说明藏系绵羊mtDNA存在一定程度的分化.     

略阳乌鸡线粒体DNA控制区(D-loop)的遗传多样性分析

为了研究略阳乌鸡线粒体DNA控制区（mtDNA D-loop）的遗传多样性和起源,本研究对30只略阳乌鸡样品的mtDNA D-loop全序列进行了PCR扩增和测序,结合GenBank中公布的其他鸡的D-loop区序列,分析略阳乌鸡线粒体多态性及其起源。结果表明,略阳乌鸡mtDNA D-loop区全序列中,A、C、G、T平均含量分别为26.6%、26.6%、13.4%和33.4%,26个核苷酸多态位点均为转换位点,核苷酸多样度（Pi）为0.00705,单倍型变异度（Hd）为1.000,中性检验Tajima's D值为-0.47272。通过群体构建的系统进化树发现,略阳乌鸡样品在系统进化树上聚为4大分支。研究结果表明,略阳乌鸡群体内个体序列变异程度较大,遗传多样性丰富,揭示略阳乌鸡在遗传组成上具有4个母系来源。     

兴义矮脚鸡GH基因的多态性研究

兴义矮脚鸡为贵州特有的地方品种鸡.本研究对兴义矮脚鸡GH基因上第一外显子和部分第一内含子序列直接测序进行多态性分析.结果表明,在扩增出来的777 bp序列中,A、T、G、C碱基的平均含量分别为28.1%、23.9%、24.9%和23.1%.A+T的含量(52%)略高于G+C的含量(48%);总共发现10个变异位点,占分析位点总数的1.287%,这些变异位点分别为C124T、T319G、T321C、G333A、A364G、A464C、A507C、C508A、T541C和T630C,其中C124T突变点位于5'端调控区,其余突变点都集中在第一内含子上,均为转换,表现较高的转换偏向.     

宁夏滩羊mtDNA Cyt b基因遗传多样性分析

为了分析宁夏滩羊线粒体DNA(mtDNA)细胞色素b脱复辅基酶(Cytb)基因的遗传多样性,试验采用PCR扩增和基因测序的方法对线粒体Cyt b基因在宁夏地区3个绵羊品种(滩羊、小尾寒羊和蒙古羊)群体的遗传多样性进行检测,分析其碱基组成及序列间碱基的变异。结果表明:滩羊线粒体Cyt b基因部分序列长度为363 bp,A、C、G、T的平均含量分别为39.11%、19.84%、19.26%、21.79%,碱基组成的百分比显示,G相对缺乏,A+T平均含量(60.90%)比G+C(39.10%)平均含量高。在滩羊与蒙古羊、小尾寒羊品种间存在1个变异位点,在47位点处发生了A/G的转换,可以作为候选基因用于辅助检测。     

兴义矮脚鸡GH基因的多态性研究

兴义矮脚鸡为贵州特有的地方品种鸡。本研究对兴义矮脚鸡GH基因上第一外显子和部分第一内含子序列直接测序进行多态性分析。结果表明,在扩增出来的777 bp序列中,A、T、G、C碱基的平均含量分别为28.1%、23.9%、24.9%和23.1%。A+T的含量(52%)略高于G+C的含量(48%);总共发现10个变异位点,占分析位点总数的1.287%,这些变异位点分别为C124T、T319G、T321C、G333A、A364G、A464C、A507C、C508A、T541C和T630C,其中C124T突变点位于5′端调控区,其余突变点都集中在第一内含子上,均为转换,表现较高的转换偏向。     

中国五个绵羊群体mtDNA D-环遗传多样性研究

为探讨中国绵羊mtDNA D-环遗传多样性,通过PCR扩增和测序技术,对中国五个绵羊群体mtDNA D-环全序列进行研究,发现这五个绵羊群体mtDNA D-环碱基组成:A、T、G、C平均含量分别为34.66%、28.94%、13.90%、22.50%,(A+T)%含量明显高于(G+C)%含量,碱基突变以转换/颠换为主,且在绵羊mtDNA D-环区存在75bp的重复序列,含有4个重复序列是中国绵羊的基本特征;遗传距离分析结果显示,甘肃滩羊与其他中国绵羊群体之间的遗传距离较大,为0.0459～0.0573,其他四个绵羊群体之间遗传距离相对较小,为0.0246～0.0342,核苷酸多样度Pi为0.0323%,说明这五个绵羊群体mtDNA D-环遗传多样性均较贫乏,应该对其遗传资源进行保护.     

绿头鸭线粒体DNA控制区全序列测定及分析

用PCR产物直接测序法,获得5只绿头鸭线粒体DNA控制区(D-loop)全序列。序列分析显示:绿头鸭D-loop区全长为1051bp,碱基A、G、T、C含量分别为27.97%、15.51%、25.21%、31.30%,A+T含量大于C+G;共检测到11个多态位点,其中转换10个,颠换1个,没有发现插入和缺失,核苷酸多样度为0.0046。结合GenBank已公布河鸭属鸭类D-loop区序列,基于邻接法和最小进化法构建河鸭属鸭类系统进化树。结果表明,绿头鸭、斑嘴鸭及家鸭三者关系密切,它们共同构成进化枝A,推测在家鸭的形成过程中,绿头鸭和斑嘴鸭都作出了贡献;其它河鸭类聚为进化枝B。     

Survey of Cryptosporidium spp. and Giardia spp. infections in various animals at a zoo in Japan.

A total of 284 fecal samples of 89 species (43 mammalian species and 46 avian species) were examined for Cryptosporidium oocysts and Giardia cysts from 1999 to 2002. Each sample was collected at the zoo located at Osaka in Japan and examined by microscopy after performing the sucrose flotation method and by two immunofluorescent assay kits for detection of Cryptosporidium oocysts and Giardia cysts. Cryptosporidium spp. was found only in a raccoon dog (Nyctereutes procyonoides), and Giardia spp. was detected in a mandarin duck (Aix galericulata) and two ruddy shelducks (Tadorna ferruginea). In this study, the prevalences of these parasites were found to be low. However, these results suggested that the infected animals could serve as a source of contamination for surface water. This is the first report about the survey of Cryptosporidium spp. and Giardia spp. at a zoo in Japan.     

四川7个地方山羊品种(类群)mtDNA遗传多样性研究

测定了四川7个地方山羊品种（类群）43个个体的mtDNA控制区全序列，结果表明：山羊mtDNA控制区全序列长度为1212bp或1213bp，A＋T含量（59．9％）明显高于G＋c含量（40．1％）。共检测到74个变异位点，序列均为中性突变，核苷酸多样度为1．686％，这些差异共定义了27种单倍型，单倍型多样度为0．966，遗传多样性较为丰富，品种（类群）间存在不同程度的遗传分化。7个地方山羊品种（类群）间的遗传距离变异范围为0．0017～0．0306。用MEGA软件的NJ法构建单倍型序列的系统发育无根树，结果表明四川地方山羊品种（类群）有两个母系来源，但是否就对应于角笋骨羊（Capra aegagrus）和捻角山羊（Capra falconeri）两个野生祖先，还有待于进一步研究。     

4个引进山羊品种mtDNA控制区序列变异和系统发生关系研究

本研究测定了四川4个引进山羊品种24个个体的线粒体控制区全序列,并从GenBank获得山羊属2个野山羊种的2每控制区序列。利用MEGA2．0软件构建分子系统发育无根树。序列分析表明:山羊控制区线粒体控制全序列长度为1 212bp或1 213 bp,A+T含量占59．9％,其中64个核苷酸位点存在变异(约占5．28％),核苷酸多样度为 1．731％,这些差异共定义了16种单倍型,单倍型多样性为0．913±0．048。安哥拉山羊、波尔山羊都有自己独特的单倍型,与其他品种间都没有共享类型。使用NJ法构建了系统发育树,结果表明:2个野山羊种中,角(?)羊与家养山羊的关系相对较近,4个家养山羊品种有2个母系来源,在4个家养山羊品种内部,安哥拉山羊的分化要比其他3个品种早。     

Distribution of nuclear mitochondrial DNA in cattle nuclear genome

The nuclear mitochondrial pseudogenes (numts), originated from mtDNA insertions into the nuclear genome, have been detected to exist in many species. However, the distribution of numts in cattle nuclear genome yet has not been fully reported. By referring to the whole cattle mtDNA sequence and to the recently released cattle nuclear genome by Human Genome Sequencing Center (HGSC), 303 numts were identified by BLAST with 55 numts unmapped to cattle nuclear genome. Further analysis found that the size of the numts ranges from 37 to 1932 bp, and the homologous identity between numts and their corresponding mtDNA fragments varies from 73 to 98%. Furthermore, the identified cattle numts cover nearly all the mitochondrial genes including mtDNA control region, distributing over all the chromosomes with the exception of the chromosome 23 and Y chromosome (in the latter the sequence data are not available). In the discovered numts, 29 relatively complete mitochondrial genes, which were distributed in 72 numts, were detected. Undoubtedly, this research would provide some valuable information for successive research related to mitochondrial genes and the evolution of cattle.     

Investigation of the genetic diversity among native Turkish sheep breeds using mtDNA polymorphisms

A total of 135 unrelated sheep from nine Turkish native sheep breeds (Dagl?c, Kivircik, Imroz, Chios, Morkaraman, Ivesi, Hemsin, Karayaka and Akkaraman) were investigated to determinate the maternal genetic diversity using a sequence of a 531-bp segment of the mtDNA control region. Analysis of the mtDNA control region sequence revealed 63 haplotypes and 53 polymorphic sites. Haplotype diversity, nucleotide diversity and the average number of nucleotide differences were estimated to be 0.9496?±?0.011, 0.01407?±?0.00060 and 7.456, respectively. The sequence analysis also revealed high level of genetic diversity among the native Turkish breeds. These breeds were grouped into three major maternal haplogroups: A, B and C, with one animal belonging from the Akkaraman breed to the rare haplogroup E. Irregular shape of mismatch distribution of haplogroup C could be an indicator that haplogroup C may represent different haplogroups. Contrarily to previous studies carried out on Turkish native breeds, majority of animals grouped in haplogroup A in the present study. This result and the irregular shape of mismatch curve of haplogroup C indicate that genetic structure of Turkish native sheep breeds could be more complicated than it is thought.     

Phylogenetic analysis for the Seoul National University (Minnesota) miniature pig by mitochondrial DNA sequence polymorphism

Seoul National University (SNU) miniature pigs are originated from the Minnesota miniature pig. This study was conducted to investigate the maternal origin of SNU (Minnesota) miniature pigs and their phylogenetic relationships by analyzing the mitochondrial DNA (mtDNA) D‐loop (control region) sequence. Two mtDNA D‐loop sequences of the SNU miniature pigs were identified. On an unweighted pair‐group method with an arithmetic mean (UPGMA) phylogenetic tree analysis, the large white was the pig breed closest to the SNU miniature pig, and the pairwise distance analysis showed the same result. While mtDNA sequences of 4 pig breeds which were used to establish Minnesota miniature pig were not known, our result might be different from the history of the Minnesota miniature pig development. In conclusion, we thought that some haplotypes of the Minnesota miniature pig maternally were originated from the Large white pig, or that wild pigs had similar mtDNA sequences to the Large white pig, and all SNU miniature pigs were derived from this colony.     

野桑蚕12S rRNA基因的克隆及序列分析

通过PCR技术,对中国山东青州野桑蚕mtDNA的12SrRNA基因进行了克隆和序列分析。结果表明,青州野桑蚕12SrRNA基因为788nt,在DNA水平上,与家蚕12SrRNA基因的同源性达到99%,与中国陕西安康、日本筑波来源的野桑蚕12SrRNA基因的同源性分别为97%、98%;分子进化分析显示,家蚕可能是起源于中国野桑蚕。     

家蚕地方品种线粒体基因组A+T丰富区的序列及分子进化分析

为了研究家蚕线粒体基因组A+T丰富区的结构和家蚕品种的进化,用PCR方法扩增了12个家蚕(Bombyxmori)地方品种线粒体基因组A+T丰富区及其侧翼序列,分离纯化后克隆到pMD18-T载体进行测序。序列分析表明,克隆片段长度约1.1 kb,基因排列顺序与C108线粒体相同,依次为12S rRNA基因3′端、A+T丰富区、tRNAMet、tRNAIle、tRNAGln和ND2基因5′端,在tRNAGln和ND2基因之间有47 bp的非编码区。以日本野桑蚕(Bombyxmandarina)为外群,用Phylip软件包构建了基于12个家蚕品种线粒体基因组A+T丰富区序列的NJ进化树。结果显示,甘肃种单独聚为一群,其进化早于其它11个品种聚成的类群,说明甘肃种是供试家蚕品种中进化最早的品种。这一结果在分子水平上为黄河流域是家蚕品种的发祥地之一提供了证据,也进一步支持了家蚕品种的中国起源说。对A+T丰富区及其侧翼基因的结构分析表明,家蚕线粒体12S rRNA和ND2基因都十分保守,14个品种统计只分别发生1个和2个碱基转换;A+T丰富区中(A+T)比例高达94.9%以上,第27 nt开始有1个T-串结构,长度为16~19 bp不等;同时还根据3个tRNA基因的核苷酸序列推定了其二级结构。     

An assessment of the clonality of the components of canine mixed mammary tumours by mitochondrial DNA analysis

The aim of this study was to investigate if mutations in the mitochondrial DNA (mtDNA) D-loop fragment control region of canine mammary mixed tumours could be used as clonal markers that identified the cell population of origin. Ten benign mixed mammary tumours and nine carcinomas arising from benign mixed tumours were microdissected and DNA from epithelial and mesenchymal tumour cells and from normal mammary tissue was examined for sequence variations in a fragment of the hypervariable control region.Identical sequence variants in both the epithelial and mesenchymal components (as well as in the corresponding normal tissue) were found in 80% of the benign mixed tumours and in 89% of the carcinomas arising from benign mixed tumours suggesting a shared clonal origin. The distinctive sequence alterations identified in the epithelial and mesenchymal components of 15.8% of all 19 tumours examined, suggests the possibility that a minority of mammary tumours are polyclonal in origin or that early clonal divergence occurs. Increased mutation within the mtDNA D-loop fragment of mixed tumour components was not observed.     

Genetic diversity of Chinese domestic goat based on the mitochondrial DNA sequence variation

The aim of this study was to characterize the genetic diversity of domestic goat in China. For this purpose, we determined the sequence of the mitochondrial DNA (mtDNA) control region in 72 individuals of the Yangtze River delta white goat, and reanalysed 723 published samples from 31 breeds/populations across China. All goat haplotypes were classified into four haplogroups (A–D) previously described. The phylogenetic pattern that emerged from the mtDNA control region sequence was confirmed by the analysis of the entire cytochrome b sequence of eight goats representative of the four haplogroups. It appeared that in Chinese domestic goat, haplogroups A and B were dominant and distributed in nearly all breeds/populations, while haplogroups C and D were only found in seven breeds/populations. Four breeds/populations contained all four haplogroups. When grouping the breeds/populations into five geographic groups based on their geographic distributions and ecological conditions, the southern pasturing area had the highest diversity whereas the northern farming area had the lowest diversity. 84.29% and 11.37% of the genetic variation were distributed within breeds and among breeds within the ecologically geographical areas, respectively; only 4% of genetic variation was observed among the five geographic areas. We speculate that the traditional seasonal pastoralism, the annual long-distance migrations that occurred in the past, and the commercial trade would account for the observed pattern by having favoured gene flows.