Sequence Analysis of Mitochondrial DNA D-loop Region in Xinjiang Goose

[Objective] The sequences of mitochondrial DNA D-loop region of Xinjiang Goose with three different colors of plumage were analyzed in order to study the genetic diversity of Xinjiang Goose, as well as the phylogeny and evolution. [Method] Ten geese were selected randomly from the core populations of grey-, mosaic- and white-plumaged Xinjiang Goose respectively with a total number of thirty as experi- mental materials, of which the blood samples were collected from the largest vein under the wing （brachial vein） for DNA extraction. Sequences of mitochondrial DNA D-loop regions were determined using DNA sequencing technology to analyze the polymorphism. In addition, the genetic distances among different populations were estimated through the comparison with the reference sequences. [Resull] The con- tents of A, G, C and T nucleotides in the D-loop region of Xinjiang Goose were 28.85%, 17.05%, 25.38% and 28.72%, respectively. The average haplotype diversity and nucleotide diversity of Xinjiang Goose were 0.583 and 0.056. Xinjiang Goose and Greylag Goose were clustered into the same group. [Conclusion] The results showed that Xinjiang Geese with three different colors of plumage all descend from Greylag Goose （Anser anser）.

Molecular Taxonomy of Conogethes punctiferalis and Conogethes pinicolalis(Lepidoptera: Crambidae) Based on Mitochondrial DNA Sequences

Conogethes punctiferalis（Guenée）（Lepidoptera： Crambidae） was originally considered as one species with fruit-feeding type（FFT） and pinaceae-feeding type（PFT）, but it has subsequently been divided into two different species of Conogethes punctiferalis and Conogethes pinicolalis. The relationship between the two species was investigated by phylogenetic reconstruction using maximum-likelihood（ML） parameter estimations. The phylogenetic tree and network were constructed based upon sequence data from concatenation of three genes of mitochondrial cytochrome c oxidase subunits I, II and cytochrome b which were derived from 118 samples of C. punctiferalis and 24 samples of C. pinicolalis. The phylogenetic tree and network showed that conspecific sequences were clustering together despite intraspecific variability. Here we report the results of a combined analysis of mitochondrial DNA sequences from three genes and morphological data representing powerful evidence that C. pinicolalisand C. punctiferalis are significantly different.

Complete Nucleotide Sequence of the Mitochondrial DNA of <i>Halyomorpha halys</i>(Stal) (Hemiptera: Pentatomidae) Specimens Collected Across Georgia

The brown marmorated stink bug, Halyomorpha halys (Stal) (Hemiptera: Pentatomidae) is an invasive species native to East Asia that has spread across Asia, Europe, and North America. H. halys causes damages to various grains, fruits, and vegetables, which is exemplified by the significant damage to the hazelnut harvest in Georgia (during 2016). This report describes the first attempted genetic study of the spread of H. halys in Georgia. The first main goal of this research was to identify the haplotype of an invasive population in Georgia. For this purpose, the mitochondrial cytochrome c oxidase I subunit (COI) gene fragment from 65 samples of H. halys collected from different regions across Georgia was sequenced on an Applied Biosystems 3100 or 3700 genetic analyzer. In all cases, only the H1 haplotype, which is native to China, was identified. The second goal of this research was to determine the complete mitochondrial DNA sequence of H. halys (Stal) specimens collected across Georgia. The complete mitochondrial DNA of H1 haplotype sequenced on an Illumina MiSeq platform. The mitochondrial DNA of the Georgian H1 haplotype has a length of 15,478 base pairs. Using the sequence of the H22 haplotype of H. halys (native to Korea) as a reference, 62 single nucleotide polymorphisms (SNPs), three inversions, and four single T insertions were identified. Furthermore, 60 SNPs and four insertions in two tRNA and one rRNA genes were identified among 18 mitochondrial genes from the Georgian H1 haplotype. Nine of these SNPs resulted in amino acid substitutions. Furthermore, the detection of SNPs revealed many other polymorphic sites beyond the COI gene, which can be used to detect new haplotypes.

Sequence Divergence of Mitochondrial 12S rRNA Gene and Phylogenetic Relationship Between Rattus rattus Sladeni and Rattus rattus Hainanicus

The appearance and hair color between these two subspecies Rattus rattus Sladeni and R.r. Hainanicus are similar to each other. Their most major distinctive characteristic is the length ratio of tail to body. However, this characteristic was unstable in some measuring records. In Guangdong, R.r. Hainanicus is restrictedly distributed in the west region, and R.r. Sladeni is widely distributed in the other regions of this province. In this study, we detected the sequences of mitochondrial 12S rRNA gene fragments of 9 samples from R.r. Hainanicus and R.r. Sladeni (Longmen and Hong Kong populations). These 385 nucleotide positions of 12S rRNA gene fragment include 26 variable sites and 14 parsimony-informative sites. 3 insertion/deletion sites are observed. The phylogenetic relationships among these samples were constructed by Neighbor-joining and Maximum parsimony methods. The analysis shows that R.r. Hainanicus is more closely relative to the Longmen population of R.r. Sladeni than to the Hong Kong population of R.r. Sladeni. The sequencing analysis of 12S rRNA gene fragments is not agreement on the classification of subspecies R. r. Hainanicus inferred from the morphology and geographical distribution. The morphological variation of R.r. Hainanicus should result from the natural selection, which causes local adaptation and geographic isolation.

Mutations in the D-loop region of mitochondrial DNA in gastric cancer

Objective: To investigate the mutati ons in the D-loop region of mitochondrial DNA (mtDNA) in gastric cancer. Methods: The mtDNA of D-loop region was amplified by PCR and sequence d in 20 samples from gastric cancer tissue and adjacent normal membrane. Results: There were 7/20(35%) mutations in the mtDNA of D-loop regio n in gastric cancer patients. There were four microsatellite instabilities among the 18 mutations. Nine new polymorphisms were identified in 20 patients. Conclusion: The mtDNA of D-loop region might be highly polymorphoric and the mutation rate is high in patients with gastric cancer.

Isolation and Purification of Carp Mitochondrial DNA and Structural Analysis of Its tRNA^(Cys) Gene and the Light Strand Origin

A new method is presented with which we isolated milochondrial DNA from fresh carp liver usingdifferential centrifugation and DNase treatment that gave high yield of purified product with an easyand economical procedure. Highly distinct bands were displayed in agarose gel electrophoresls ofthe product digested with restrictlon enzymes, which were successfully used in constructingrestriction map and molecular clone of mitochondrial genes. With DNAs thus obtained, we havecloned cysteine tRNA gene (tRNA^(Cys) gene) of carp mitochondria, determined the nucleotide sequenceof it and the light strand origin, and depicted the cloverleaf secondary structure of tDNA^(Cya) and thelight strand origin. Analysis of nucleotide sequences of tRNA^(Cy) genes of 5 vertebrates has revealedunusual features of carp mitochondrial tRNA^(Cy) gene as compared with their cytoplasmic counter-parts, Altogether 36 bases were found in the light strand origin of carp mitochondriaf: 11 pairs in thestem; and 14 bases in the loop. As compared with those of other 11 vertebrate species, the sequenceof the stem is very conservative while both sequence and length of the loop are quite variable. Thestructure of the stem-loop may play an important role in light strand replication.

根据mtDNAD-loop序列分析东海银鲳群体遗传多样性

根据线粒体D-loop序列对舟山群岛附近海域的银鲳(Pampus argenteus)群体(n=24)的遗传多样性进行了研究。通过PCR技术对线粒体D-loop序列进行扩增,获得大小约为500bp的扩增产物。PCR产物经纯化并进行序列测定后,得到了357bp的核苷酸片段。在24个个体中,共检测到14个变异位点,其中8个转换位点,5个颠换位点,1个转换与颠换同时存在的位点。运用MEGA软件计算出不同个体间的遗传距离,并根据其遗传距离构建了UPGMA和NJ系统树。DNASP软件计算出的单倍型多样性(h)、核苷酸多样性(π)及平均核苷酸差异数(k)分别为0.89、0.007与2.57。此外,岐点分布及中性检验显示,东海银鲳群体在历史上可能经历过种群扩张。研究结果表明,线粒体D-loop基因可用于银鲳群体内及群体间遗传多样性的分析。

9个地方绵羊品种mtDNA D-loop高变区序列分析

对中国9个绵羊品种的103个个体mtDNA D-loop高变区（763 bp）进行了研究,结果表明：获得的序列长度范围为522-683 bp,可归为21种分子类型,各分子类型在绵羊品种间分布趋于一致,不存在特异性,而在品种内却存在异质性;绵羊mtDNA D-loop区序列长度变异主要是由于75 bp基元重复序列重复次数的不同（2、3或4个）和少数碱基的插入或缺失造成的;103个个体mtDNA D-loop区序列中A＋T碱基组成比例（64.7%）明显高于G＋C碱基组成比例（35.3%）,说明了绵羊mtDNA D-loop区是A、T碱基富集区,且为高突变区,碱基突变类型包括转换、颠换、插入和缺失,且碱基转换频率远高于碱基颠换频率。

基于线粒体DNAD-loop全序列的麒麟鸡遗传多样性研究

【目的】从线粒体DNA(mtDNA)D-loop全序列角度评估麒麟鸡的遗传多样性水平,以期阐明麒麟鸡的品种形成。【方法】通过PCR产物直接测序法对麒麟鸡保种群的黄羽、白羽和黑羽3个资源群及8个地方鸡品种进行mtDNA D-loop全序列测定,分析遗传变异,并进行中性检测,构建单倍型中介网络图,探究麒麟鸡的群体历史。【结果】麒麟鸡mtDNAD-loop全序列为1231~1232bp,C+G含量(39.8%)低于T+A含量(60.2%),总体核苷酸多样性为0.00630±0.00054,明显高于其他8个地方鸡品种。3个资源群黄羽、白羽和黑羽麒麟鸡的单倍型多样性分别为0.777±0.076、0.816±0.060、0.710±0.057,核苷酸多样性分别为0.00699±0.00115、0.00662±0.00090、0.00546±0.00062,遗传多样性水平基本上与群体规模呈正相关。麒麟鸡与8个地方鸡的双参数遗传距离为0.008~0.009,净遗传距离为0.003~0.005,均大于其他地方鸡之间的遗传距离。90份麒麟鸡样本共检测到45个突变位点,定义了17个单倍型,其中独享型单倍型9个,单倍型多样性为0.773±0.039。黄羽、白羽和黑羽麒麟鸡的单倍型数量分别为12、7、5个。8个地方鸡品种定义了19个单倍型,其中河田鸡的单倍型数量最多(8个),宁都黄鸡的最少(3个),没有与麒麟鸡共享的单倍型。中性检测结果显示,除了黑羽麒麟鸡外,供试鸡种在品种水平上近期均未经历明显的种群扩张。麒麟鸡单倍型类群主要分布在进化枝A、B、C和E,优势单倍型类群是B(30.0%)和E(64.4%);8个地方鸡单倍型类群主要为A和B。【结论】独特的单倍型提示麒麟鸡具有独立的品种形成历史,相对较高的遗传多样性水平将有利于其保护和利用工作的开展。

鸳鸯mtDNA D-loop序列分析

采用PCR和直接测序方法测定鸳鸯线粒体DNA(mtDNA)控制区全序列,与GenBank上已知序列相比较分析mtDNA D-loop 3个区的序列变异。结果发现,鸳鸯mtDNA控制区序列长1035bp;与欧洲鸳鸯相比,鸳鸯(样品采集来自贵州省石阡县)mtDNA控制区共存在25处转换、12处颠换、2处插入和12处缺失;两种鸳鸯之间mtDNA控制区Ⅰ、Ⅱ和Ⅲ区的序列变异率分别为14.5%、0.64%和0.8%,Ⅰ区的变异速率最快;mtDNA控制区的A、C、T、G碱基含量分别是26.6%、16.0%、26.6%和30.8%,A+T含量稍高于C+G含量,G含量最高,符合序列组成的碱基偏倚性。与其他鸭科物种进行了mtDNA控制区序列一致性的比较,结果均高于80%。

简单重复DNA序列在哺乳动物mtDNA D-loop区的分布及进化特征

简单重复序列广泛分布于从原核到真核生物的基因组中,其形成的分子机理目前尚不明确。对NCBI数据库中已有256种哺乳动物线粒体DNA(mtDNA)D-loop区进行序列比对分析,根据其所含有的简单重复序列类型分为3组,分别是53种哺乳动物含有六核苷酸重复序列;104种哺乳动物含有非六核苷酸重复序列(>6bp);99种哺乳动物不含有任何重复序列。通过碱基序列分析比对,发现六核苷酸重复序列集中分布在CSB1-CSB2间隔区,而非六核苷酸重复可以分布于终止区(TAS)、中央保守区(Central domain)以及CSB(Central sequence block)区。通过比较含有重复序列与不含重复序列的功能保守区发现,简单重复序列的存在并不明确影响D-loop区内的中央保守区以及CSB1、CSB2、CSB3三个功能保守区的碱基序列保守性。在此基础上,利用N-J法构建了256种哺乳动物的进化树,分析了哺乳动物D-Loop区内重复序列在进化过程中的可能变化规律,发现简单重复序列随着物种的进化地位的升高而呈现消失趋势。

Comparative analysis of mitochondrial fragments transferred to the nucleus in vertebrate

Mitochondrial DNA sequences transferred to the nucleus give rise to the so-called nuclear mitochondrial DNA （numt）. In the GenBank database, 244 numts have been found in six orders of birds （Anseriformes, Columbiformes, Falconiformes, Charadriiformes, Galliformes and Passeriformes）. Sequences alignment （NCBI-BLASTN） was carded out with mitochondrial and corresponding nuclear genome sequences in nine vertebrate species. The sequences with high homology were considered as numts. The number of numts ranged from 15 in chicken to 159 in chimpanzee. The sequences of numts in macaque, chimpanzee, and human spanned 100% of the entire mammalian mitochondrial genome. The reconstructed frequency of the mitochondrial gene transferred to the nucleus demonstrated that the rRNA genes had high frequencies than other mitochondrial genes. Using the RepeatMasker program, the transposable elements were detected in the flanking regions of each numt. The results showed that less than 5% of the flanking sequences were made up of repetitive elements in chicken. The GC content of 5＇- and 3＇-flanking regions of numts in nine species was less than 44%. The analysis of the flanking sequences provided a valuable understanding for future study on mechanism of mitochondrial gene transfer to the nucleus and the site of numt integration.

Mutations of mitochondrion DNA in mouse tumors

Objective： To ascertain the variations of mitochondrion DNA （mtDNA） in mouse tumors and to inquire into the relationship between mutations of mtDNA and carcinogenesis Methods： The variations of D-loop, ND3 and tRNA^Met＋Glu＋Ile gene fragments of mtDNA from six tumor cell lines of mice were analyzed by PCR technology with restriction fragment length polymorphism analysis （polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP） and single strand conformation polymorphism analysis （SSCP-PCR） method. Results： ND3 and tRNA^Met＋Glu＋Ile gene fragments ofmtDNA from the tumors showed no variation in 27 endonuclease sites; D-loop ofmtDNA from Hca-F had an additional endonuclease sites of Hinf I in contrast to that of the inbred mouse. Deeply analyzed by PCR-SSCP, the D-loop ofmtDNA was found to possess mutations in 4 of 6 tumors. Conclusion： D-loop is the hot spot of tumor mtDNA mutation which can act as contributors to the carcinogenic

The Genetic Structure and Diversity of Repomucenus curvicornis Inhabiting Liaoning Coast Based on Mitochondrial COⅠ Gene and Control Region

[Object] This study was conducted to explore the genetic diversity and structure of the wild Repomucenus curvicornis inhabiting Liaoning Coast, China. [Method] The mitochondrial COⅠ gene and control region（CR） were PCR amplified from the wild R. curvicornis populations from the Liaodong Bay（n=22） and the northern Yellow Sea（n=18）, sequenced and analyzed for genetic diversity. [Result] The contents of A, T, C and G of 624 bp COⅠ gene were 24.09%, 31.04%, 25.28%, and 19.59%, and those of 460 bp CR fragment were 32.96%, 32.80%, 14.86% and 19.38%, respectively. The total number of variable sites, average number of nucleotide differences（ k）, haplotype diversity（H） and nucleotide diversity（π） based on COⅠ gene were 38, 4.67,（0.96±0.02） and（0.007 5±0.004 2）, and those based on CR fragment were 26, 3.35,（0.97 ±0.02） and（0.007 3±0.004 3）, respectively. Based on mitochondrial COⅠ gene and CR, the genetic diversity of Liaodong Bay population was lower than that of the northern Yellow Sea population. The AMOVA analysis based on CR fragments revealed almost significant genetic divergence between the Liaodong Bay and the northern Yellow Sea populations, while there was no significant genetic divergence based on COⅠ gene. The results showed that CR and COⅠ gene are effective molecular markers for detecting the genetic diversity of R. curvicornis population, while CR is more reliable than COⅠ gene in detecting the genetic structure. [Conclusion] CR is an appropriate marker for genetic analysis of marine fish population.

基于线粒体DNA控制区序列的极边扁咽齿鱼群体遗传研究

极边扁咽齿鱼是黄河上游特有的裂腹鱼类,由于人类活动的加剧,使其野生数量急剧下降,近年来通过增殖放流的方式为野生资源量进行补充。为评价增殖放流过程中人工繁育群体对野生群体的种质资源的影响,笔者利用线粒体DNA控制区(D-loop)序列来探究极边扁咽齿鱼野生和养殖的4个群体遗传多样性、遗传分化及遗传结构的差异。研究结果显示,4个群体均表现出了较高的单倍型多样性(0.950±0.011)和较低的核苷酸多样性(0.00987±0.00041),并且野生群体的单倍型数目、单倍型多样性指数、核苷酸多样性指数均高于人工繁育群体。遗传结构分析结果显示,野生群体与人工繁育群体之间已产生了一定的遗传分化。中性检验表明,2个野生群体可能经历过种群扩张事件。研究结果表明,极边扁咽齿鱼人工繁育群体可能受到亲本数量有限、人工选择等影响,遗传多样性略有降低,有必要定期开展增殖放流效果的评估,并采用科学的人工繁育方法,丰富极边扁咽齿鱼种群的遗传多样性。

The complete mitochondrial DNA sequence and the phylogenetic position of Achalinus meiguensis (Reptilia: Squamata)

The mitochondrial genome complete sequence of Achalinus meiguensis was reported for the first time in the present study. The complete mitochondrial genome of A. meiguensis is 17239 bp in length and contains 13 protein-coding genes, 22 tRNA, 2 rRNA, and 2 non-coding regions (Control regions). On the basis of comparison with the other complete mitochondrial sequences reported, we explored the characteristic of structure and evolution. For example, duplication control regions independently occurred in the evolutionary history of reptiles; the pseudo-tRNA of snakes occurred in the Caenophidia; snake is shorter than other vertebrates in the length of tRNA because of the truncations of TΨC arm (less than 5 bp) and "DHU" arm. The phylogenic analysis by MP and BI analysis showed that the phylogenetic position of A. meiguensis was placed in Caenophidia as a sister group to other advanced snakes with the exclusion of Acrochordus granulatus which was rooted in the Caenophidia. Therefore we suggested that the subfamily Xenodermatinae, which contains A. meiguensis, should be raised to a family rank or higher rank. At the same time, based on the phylogenic statistic test, the tree of Bayesian was used for estimating the divergence time. The results showed that the divergence time between Henophidia and Caenophidia was 109.50 Mya; 106.18 Mya for divergence between Acrochordus granulatus and the other snakes of the Caenophidia; the divergence time of A. meiguensis was 103 Mya, and Viperidae diverged from the unilateral of Elapidae and Colubridae was 96.06 Mya.

新疆罗布诺尔地区铜器时代古代居民mtDNA多态性分析

目的 :通过新疆罗布诺尔地区铜器时代古代居民 m t DNA多态性分析 ,探讨新疆境内古代欧洲人种成分的来源。方法 :从孔雀河古墓沟墓地古代居民人骨中提取出古 DNA。用 4对套叠引物对线粒体基因组的调控区(36 3bp)进行扩增、测序。结果 :10个个体中具有 10个 DNA序列 ,系统发育分析表明罗布诺尔古代居民的核酸多样性及平均配对差异均与欧洲群体接近。结论 :早在 380 0年前的铜器时代 ,罗布诺尔地区已有单一的欧洲人种构成的人群存在。

中国境内不同地理型东方蜜蜂线粒体DNA tRNA^(leu)～COⅡ基因多态性研究

【目的】从线粒体水平上探讨中国境内不同地理型东方蜜蜂的系统发育关系,为东方蜜蜂亚种分化的研究及我国境内东方蜜蜂资源的保护与合理开发利用提供基础资料。【方法】采用公开的E2、H2引物对11个不同地理型东方蜜蜂线粒体DNA tRNAleu～COⅡ基因进行了PCR扩增、测序,并利用相关软件和网站进行了序列比较分析。【结果】该序列长度为471bp;序列中共有9个位点发生变异;序列相似性均在99%以上;限制性酶切分析表明:云南保山东方蜜蜂缺少一个SwaⅠ酶切位点;部分编码蛋白序列比对表明,海南海口和吉林安图东方蜜蜂各有一个氨基酸发生变异。与GeneBank上公开登录的国内外有关东方蜜蜂相关序列的比较表明,单纯利用非编码区序列对比,不能把日本蜜蜂同中国大陆的东方蜜蜂区别开来;COⅡ基因部分序列对比显示,东方蜜蜂的线粒体类型包括:日本-韩国型、中国大陆型、中国台湾型、马来西亚沙巴州-印度黑色蜜蜂型、中国海南型、印度黄色蜜蜂型、泰国南部型和印度黑色蜜蜂型。【结论】中国境内不同地理型东方蜜蜂存在着较明显的遗传分化,其中海南东方蜜蜂由于长期的海岛隔离形成了一个独特的类群,支持了通过形态学认定的海南东方蜜蜂为东方蜜蜂的一个新亚种的观点。本研究部分测序结果已在美国国家生物信息中心(NCBI)网站GeneBank上登录,登录号为:DQ385854,DQ388602～DQ388609。

我国耐力运动员线粒体DNA高变区Ⅰ序列多态性分析

为了探讨人类运动能力相关的基因标记与分子机制 ,对mtDNA高变区I作了序列多态性分析。研究选取汉族耐力运动员 95人 ,相应汉人对照 92人 ,对其mtDNA高变区I特异性片段进行扩增、测序 ,分析其序列多态性改变。结果显示 :中国汉族耐力运动员的多态位点有 83个 ,其中 ,碱基替换位点 6 8,缺失位点 5个 ,插入位点 10个 ,位点 16 2 2 8碱基缺失及 16 113- 16 114和 16 335 -16 336碱基插入为运动员独有 ;中国汉族运动员mtDNA高变区I同质性多态主要表现为碱基转换、碱基颠换、缺失及插入四种类型 ,其中 ,碱基转换发生率最高 ,碱基颠换发生率次之 ,碱基缺失及插入频率最低。运动员碱基颠换频率明显高于常人 (P <0 0 5 ) ,而碱基缺失及插入频率则显著低于常人 (P <0 0 5 )。运动员T -C转换频率显著高于常人 (P <0 0 5 ) ,运动员A -G转换频率则显著低于常人 (P <0 0 1)。研究结果还提示 ,mtDNA扩增产物直接测序分析作为一种简便快捷的mtD NA序列多态性和单核苷酸多态性研究方法 ,为运动能力遗传标记的筛选与研究提供了有效方法 。

家蚕蛹线粒体DNA EcoRⅠ 1.9kb片段的克隆及序列分析

克隆了二化性家蚕蛹体的线粒体DNAEcoRⅠ酶解 1 .9kb片段 ,并进行了序列测定。结果表明 :克隆片段中包含完整的线粒体赖氨酸转移RNA(tRNA Lys)基因、天冬氨酸转移RNA(tRNA Asp)基因、ATP合酶亚基Ⅷ基因和细胞色素C氧化酶亚基Ⅱ、ATP合酶亚基Ⅵ基因的部分序列。并与果蝇线粒体DNA的同源性和参照果蝇线粒体DNA密码子翻译的氨基酸顺序进行了比较 。