SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Structure and evolution of teleost mitochondrial control regions

We amplified and sequenced the mitochondrial control region from 23 species representing six families of teleost fish. The length of this segment is highly variable among even closely related species due to the presence of tandemly repeated sequences and large insertions. The position of the repetitive sequences suggests that they arise during replication both near the origin of replication and at the site of termination of the D-loop strand. Many of the conserved sequence blocks (CSBs) observed in mammals are also found among fish. In particular, the mammalian CSB-D is present in all of the fish species studied. Study of potential secondary structures of RNAs from the conserved regions provides little insight into the functional constraints on these regions. The variable structure of these control regions suggests that particular care should be taken to identify the most appropriate segment for studies of intraspecific variation. Correspondence to: T.D. Kocher     

Mitochondrial DNA sequences of various species of the genus Equus with special reference to the phylogenetic relationship between Przewalskii's wild horse and domestic horse

The noncoding region between tRNA^Pro and the large conserved sequence block is the most variable region in the mammalian mitochondrial DNA D-loop region. This variable region (ca. 270 bp) of four species of Equus, including Mongolian and Japanese native domestic horses as well as Przewalskii's (or Mongolian) wild horse, were sequenced. These data were compared with our recently published Thoroughbred horse mitochondrial DNA sequences. The evolutionary rate of this region among the four species of Equus was estimated to be 2–4 × 10^–8 per site per year. Phylogenetic trees of Equus species demonstrate that Przewalskii's wild horse is within the genetic variation among the domestic horse. This suggests that the chromosome number change (probably increase) of the Przewalskii's wild horse occurred rather recently.Correspondence to: N. Ishida     

Genetic mapping of bovine mitochondrial DNA from a single animal

Using a physical map of bovine mitochondrial DNA derived from the liver of a single Holstein cow, we have determined the location of the genes specifying the large and small riibosomal RNAs by hybridization analysis and electron microscopic observations of R-loop forms. Also, the position of the origin of DNA replication (D-loop) has been located by electron microscopy. Additionally, the direction of D-loop expansion and the polarity of the large and small ribosomal RNA genes were determined.     

D-loop Dynamics and Near-Atomic-Resolution Cryo-EM Structure of Phalloidin-Bound F-Actin

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中国北鳅谱系地理学分析

北鳅(Lefua costata)为冷水性鱼类, 分布于淮河以北, 分析遗传结构能够反映其适应环境变迁的响应。基于线粒体D-loop区211条序列分析了我国北鳅的谱系地理学和遗传多样性, 样本采自9条水系共18个样点。单倍型分析显示共计55个单倍型, 呈高单倍型多样性(h=0.9304)和高核苷酸多样性(π=0.0087)。单倍型多样性和核苷酸多样性最高的为辽东半岛种群(LD) (h=0.8920, π=0.0074), 其他地理种群距辽东半岛越远, 单倍型多样性越低。遗传距离(K2P)、群体间的遗传分化指数(F_ST)及AMOVA分子方差分析均显示LD种群与其他地理种群存在显著分化。基于单倍型构建的分子系统发育树和单倍型网络关系图均显示9条水系的单倍型不能各自聚类, 形成3大分支: 分支A以LD种群为主; 分支B含所有地理种群; 分支C以淮黄河种群(HH)和海滦河种群(HL)为主。中性检验和错配分析显示不同地理种群有扩张现象。基于化石校准点和北鳅鱼类D-loop序列1.00%Ma的平均进化速率评估各地理种群的分歧时间, 为2.3784— 0.0477Ma前, LD种群与其他地理种群分化时间最早(2.3784 Ma前), 推测LD种群受第四纪冰期影响较小, 辽东半岛为我国北鳅起源地之一, 其他地理种群以辽东半岛为中心借助松辽古大湖和黄渤海平原河口等发生多次迁移。     

云南4个地区高山姬鼠线粒体控制区种群遗传分化研究

对云南4个种群38个个体的线粒体控制区（D-loop）905 bp的核苷酸序列遗传变异进行分析,探讨了高山姬鼠种群遗传结构和分化。在905 bp D-loop基因的碱基序列中,共发现了57个变异位点（全变异的6.30%）,共定义了23个单倍型,其中有一个单倍型（Hap1）为横断山3个种群（中甸、丽江和剑川）所共享,其余22个单倍型均为各个种群所特有。分子变异分析（AMOVA）表明,种群间的遗传变异占33.7%,种群内的遗传变异占66.3%。FST统计结果表明,除昆明种群和横断山种群之间差异显著（P〈0.05）,其它地理种群间的差异均不显著（P〉0.05）,说明昆明种群与横断山种群之间出现了明显的遗传分化。     

环境因子对新疆鹅喉羚群体遗传多样性的影响

环境因素在物种进化和遗传变异过程中起着非常重要的作用。为探讨新疆鹅喉羚遗传多样性与环境因子的关系,本研究采用聚合酶链式反应（PCR）和直接测序的方法,测定了新疆鹅喉羚11个群体84份样本的线粒体DNA Cyt b基因（1140 bp）和D-loop区（1100 bp）序列,分析各群体遗传多样性及环境因子对遗传多样性的影响。结果显示新疆鹅喉羚具有较高的单倍型多样性,较低的核苷酸多样性,表明其遗传多样性处于较低水平。环境因子与群体遗传多样性的相关性分析结果表明,海拔、年均降水量、年均气温、人口数量是影响新疆鹅喉羚遗传多样性的主要环境因子,其中海拔是最关键的环境因子。本研究结论为新疆鹅喉羚群体有效合理的保护与管理提供理论依据。     

Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx

Noninvasive methods using genetic markers have been suggested as ways to overcome difficulties associated with documenting the presence of elusive species. We present and assess a novel, reliable and effective molecular genetic technique for the unequivocal genetic identification of faeces from the endangered Iberian lynx (Lynx pardinus). From mitochondrial DNA (mtDNA) cytochrome b and D-loop region sequences, we designed four species-specific primers (for products 130-161 bp long) that were considered to be likely to amplify degraded DNA. We compared two DNA extraction methods, various DNA amplification conditions and the robustness and specificity of the primer pairs with 87 lynx samples from 5 potentially different lynx populations and with 328 samples of other carnivore species. The utility of the identification technique was tested with faeces of different ages, with faeces from controlled field experiments, and with faeces collected from locales with possible lynx populations from throughout the state of Andalusia, Spain (8052 km2). Faecal mtDNA extraction was more efficient using PBS wash of the faeces instead of a faeces homogenate. Our assay increased from 92.6 to 99% efficiency with a second amplification and a reduction in template concentration to overcome polymerase chain reaction (PCR) inhibition. Our assay never produced false positives, and correctly identified all lynx faeces. Of 252 faeces samples of unknown species collected throughout Andalusia, 26.6% (from three different areas) were classified as Iberian lynx, 1.4% showed evidence of PCR inhibition and 1.2% were of uncertain origin. This method has proven to be a reliable technique that can be incorporated into large-scale surveys of Iberian lynx populations and exemplifies an approach that can easily be extended to other species.     

Genetic diversity of Cheju horses (Equus caballus) determined by using mitochondrial DNA D-loop polymorphism

We used sequence polymorphism of the mitochondrial DNA D-loop (968 bp excluding the tandem repeat region) to determine genetic diversity of horses inhabiting Cheju (a southern island of Korea). Seventeen haplotypes with frequencies from 1.5 to 21.5% were found among 65 Cheju horse samples. Genetic diversity (h) of the 17 haplotypes was calculated to be 0.91, indicating that the extant Cheju horse population consists of diverse genetic groups in their maternal lineage. Phylogenetic analysis showed that 17 types of Cheju (D-loop sequences determined), 5 Mongolian, 6 Arabian, 3 Belgian, 2 Tsushima, 2 Yunnan, 1 Przewalskii, and 3 Thoroughbred horses (published sequences for the latter seven breeds) showed that Cheju horses were distributed into many different clusters in the tree. Four Mongolian horses clustered with separate Cheju horse groups, showing that some Cheju horses are clearly of Mongolian origin. The analysis of partial sequences (284 bp) of the D-loop of 109 horses showed that Thoroughbred, Mongolian, Lipizzan, and Arabian breeds are as diverse as Cheju horses. Our data together with others' suggest that most horse breeds tested with reasonably sufficient numbers of samples are diverse in their maternal lineages and also are not uniquely different from each other.