板角山羊mtDNA Cyt b基因变异特点及系统进化研究

测定了板角山羊品种13个个体的细胞色素b基因全序列(1140 bp),比较分析了群体中细胞色素b基因的碱基组成和序列间碱基的变异情况,结果显示:在该品种(群体)中细胞色素b基因序列中6个变异位点上观察到11次T-C间和2次A-G间的碱基转换,除了有2次T-C间碱基转换发生在密码子第2位点为非同义突变以外,其余的11次碱基转换发生在密码子第3位点,均为同义突变;有1次T-G间碱基颠换发生在密码子第2位点,为非同义突变;观察到5种单倍型,单倍型多样度为0.8077.并以绵羊为外群,与山羊属其他种的同源区序列构建系统发生树.结果显示, 在系统地位上板角山羊与胃石山羊有较近的亲缘关系.     

雷州地区马尾藻系统进化分析

通过PCR扩增测序及种间比对,对获得的6种马尾藻18SrRNA、COI和ITS基因部分同源序列进行分析,结果显示:(1)18S rRNA基因同源序列长度为1 673 bp,其中保守位点1 668个,可变位点3个,简约信息位点1个,单变异多态位点2个;T、C、A、G的平均含量分别为26.4％、21.4％、24.4％、27.8％.(2)COI同源序列长度为643 bp,其中保守位点567个,可变位点76个,简约信息位点28个,单变异多态位点47个;T、C、A、G的平均含量分别为40.0％、17.6％、19.1％、23.3％.(3)ITS同源序列长度为1 539 bp,其中保守位点1 272个,可变位点193个,简约信息位点55个,单变异多态位点138个;T、C、A、G的平均含量分别为23.5％、26.5％、17.1％、32.9％.(4)基于Kimnra双参数模型计算遗传距离,选用褐藻纲部分物种为外源,构建18S rRNA,COI和ITS基因序列系统发育树.用COI和ITS基因构建的发育树与利用形态学进行的分类较为一致,真马尾藻亚属(Sargassum)的张氏马尾藻(Sargassum zhangii)、全缘马尾藻(Sargassum integerrimum)、匍枝马尾藻(Sargassum polycystum)、亨氏马尾藻(Sargassum henslowianum)、灰叶马尾藻(Sargassum cinereum)亲缘关系较近,先聚为一支,再与反曲叶亚属的半叶马尾藻(Sargassum hemiphyllum)聚为一支,然后与其他亚属的聚为一大支;而用18S rRNA基因构建的发育树中,亨氏马尾藻先与半叶马尾藻聚为一支,后与真马尾藻亚属的聚为一支,最后与其他亚属的聚为一大支.从序列的保守度分析,18SrRNA基因具有高度保守性,可用于属以上单元鉴别分类;COI和ITS基因多态性较高,可用于种间分类.     

大肠杆菌启动子特征参数的统计分析

首先统计了683条大肠杆菌sigrna70启动子序列的每个位点单碱基频率,并计算了每个位点单碱基体现保守性的M1（1）值和相应涨落限,从而获得多个大于涨落限的保守位点。其次,对大肠杆菌的转录起始位点到翻译起始位点的距离进行了统计,发现这个距离的范围是0-1000bp。大肠杆菌启动子还分布于一些特定的基因间和编码区,分别是的DIV基因间,55％的TAN基因间和6％的编码区。这些启动子的特征是启动子辨识的重要参数。     

原生动物柯立斯四膜虫(Tetrahymena corlissi)超高砷耐受性的分子机制研究

以原生动物柯立斯四膜虫(Tetrahymena corlissi)为实验对象,通过流式细胞仪测定细胞密度分析四膜虫相对生长率,得到砷暴露下T.corlissi 24 h的半数效应浓度(EC50)为110.7μg/m L,是梨形四膜虫(T.pyriformis)的3倍;在克隆到T.corlissi砷甲基转移酶(Ars M)基因的基础上,RT-PCR结果显示,该基因在砷暴露下表达;利用高效液相色谱-电感耦合等离子体质谱仪(HPLC-ICP-MS)联用方法对细胞内砷形态的检测显示,野生型T.corlissi具有比T.pyriformis强数倍的甲基化砷的能力,Ars M基因敲除的T.corlissi失去了甲基化砷的能力,从而推测T.corlissi的Ars M具有高效甲基化砷的能力,是其具有超高的砷耐受性的分子基础.     

角类肥蛛Larinioides cornuta组蛋白H3基因保守序列克隆与分析

本文利用常规的PCR技术,克隆、测序园蛛科Araneidae角类肥蛛Larinioides cornuta组蛋白基因H3.将其H3基因与皿蛛科Linyphiidae 11个种的组蛋白H3基因的序列,在1级结构、2级结构、蛋白质水平进行了比较,分析发现:组蛋白基因H3的序列A、T、C、G碱基的百分含量比较平均,分别为24.9%、23.2%、25.7%、26.3%;简约信息位点数74,多数分布在第3位点;平均替换率为0.15.平均转换/颠换比(R)为1.8,大于0.5;G-C, C-T间的替换是该区域进化的另一个显著特征;DNA 1级结构高度保守的区段都不分布在2级结构"茎"区,不存在补偿性突变,2级结构的"环"(loop)区差异也很大;在氨基酸水平上所有成员非常保守.     

牛科动物HSL基因序列分析及其分子进化研究

在对牛科中4种动物即牦牛、瘤牛、普通牛和水牛HSL基因外显子Ⅰ部分核苷酸序列进行测定的基础上,与Gen-Bank中其他物种相应基因核苷酸序列、氨基酸序列进行了比对分析,并构建了牦牛与其他物种间分子系统进化树。结果表明:牦牛与普通牛、瘤牛、水牛、猪、人、小鼠、大鼠7个物种HSL基因外显子Ⅰ部分核苷酸序列间保守性较高,同源性大小依次为99.8%、99.6%、97.4%、90.6%、88.4%、83.5%、82.3%。相应氨基酸序列间保守性更高,同源性分别为100%、100%、98.2%、94.0%、92.2%、89.8%、89.8%。牦牛与各物种该基因部分核苷酸序列间碱基变异类型主要表现为碱基转换和颠换,无碱基插入和缺失发生,碱基转换的频率高于颠换的频率;在核苷酸水平上的多数碱基替换都是同义替换;序列间单碱基变异位点大多出现在同一位点,多发生在密码子第3位,其次是第1位,最少发生在第2位,符合分子进化的中性学说。HSL基因外显子Ⅰ部分核苷酸序列进行多序列对位排列构建的各物种间分子系统进化树结果表明,普通牛和瘤牛首先聚为一类,再分别与牦牛、水牛、猪、人聚类,最后与大鼠、小鼠聚为一类。该聚类结果与动物学上的分类结果一致,表明HSL基因外显子Ⅰ部分核苷酸序列适合于构建物种间分子系统进化树。研究表明,牦牛、普通牛和瘤牛3个物种间的遗传距离大小相近,牦牛和水牛间的遗传距离与普通牛、瘤牛和水牛间的遗传距离大小相当。牦牛、普通牛和瘤牛3个物种间的遗传距离远小于它们各自与水牛这一物种的遗传距离,它们三者之间的亲缘关系也相对于它们各自与水牛间的亲缘关系都较近,故将牦牛、普通牛和瘤牛划分在同一个属——牛属(Bos)更为合理。     

我国斑鳜六个群体mtDNA Cyt b序列的遗传变异

采用PCR技术扩增了鸭绿江、海河、长江、钱塘江、闽江、西江6个群体31尾斑鳜(Sinipercascherzeri)mtDNACytb基因的部分序列。在长度为781bp的同源序列中,共检测到78个变异位点,占分析位点数的9.6%,碱基替换大都发生在密码子的第三位点上;在31个体中共检出16种单倍型。鸭绿江、海河群体与长江、钱塘江、闽江、西江群体间的遗传距离较大。分子方差分析表明,斑鳜6个群体间总的遗传分化水平FST为0.9307(P<0.01),差异极显著。构建的NJ分子系统发育树表明,海河群体和鸭绿江群体构成了一支北方群体;长江群体、闽江群体和西江群体构成了南方群体;而钱塘江群体单独聚为一支。这表明我国斑鳜不同地理群体间已产生明显的遗传分化,但未与其自然地理分布格局呈完全对应关系。     

基于线粒体COⅠ基因的齿小蠹属昆虫DNA条形码研究

齿小蠹属（鞘翅目： 小蠹科）昆虫是植物检疫中经常截获的类群, 为探讨线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因的特定区段作为DNA条形码快速准确鉴定齿小蠹种类的可行性, 以齿小蠹属昆虫为研究对象, 测定分析了线粒体COⅠ基因462 bp碱基序列。序列分析结果显示： 变异位点为259个, 保守位点203个, 简约信息位点181个, 自裔位点78个。所有位点中, A, G, C和T碱基平均含量分别为30.7%, 16.5%, 17.0%和35.8%。A+T含量较高, 为66.5%, 明显高于G+C含量, 表现明显的A+T碱基偏嗜, 且A与T含量相当, 符合昆虫线粒体基因碱基组成的基本特征。转换与颠换结果显示： 该段序列未达到饱和, 可以得到准确的进化分析。利用Kimura 2-parameter模型分析遗传距离得到, 同物种间的遗传距离介于0.002~0.007之间, 不同种间的遗传距离介于0.056~0.431间, 平均遗传距离为0.199, 说明该段序列能够区分不同物种。基于COⅠ基因序列构建的邻接法系统发育树（NJ树）显示, 同一物种聚为同一小支, 且分支自展值均为100%; 近缘种能聚集在一起, 且置信度很高（≥97%）。结果表明应用基于COⅠ基因片段的DNA条形码进行齿小蠹属昆虫分类鉴定具有可行性。     

六种灵猫科动物的分子系统关系研究

对六种灵猫科物种线粒体12 S rRNA基因及其中四种的Cytb基因部分序列进行了测定,并从Gen-Bank获得斑林狸(Prionodon pardicolor)、熊狸(Arctictis binturong)的Cytb基因同源序列。两基因整合序列比对后长755 bp,12 S rRNA基因序列中有70个变异位点,31个简约信息位点,在Cytb基因序列中,共有120个位点呈现变异,60个简约信息位点,Cytb基因的碱基变异百分比高于12 S rRNA基因的碱基变异百分比。使用邻接法(NJ)、最大似然法(ML)重建的分子系统树显示:斑林狸从灵猫亚科中分离出来,支持灵猫亚科的多系起源,而且斑林狸可能是中国起源最早且最特化的灵猫科动物。另外,同属于灵猫亚科的大灵猫(Viverra zibe-tha)、小灵猫(Viverricula indica)聚为一支,同属于棕榈狸亚科的果子狸(Viverricula indica)、熊狸聚为姐妹群,这些与传统形态学分类观点一致。     

拟南芥和水稻金属蛋白酶ftsH基因家族的基因组比较分析

FtsH(Filamentation temperature-sensitive H)是一种广泛存在于原核生物和真核生物中的ATP依赖型金属蛋白酶。同源性分析表明,在拟南芥和水稻基因组中分别有12个和9个ftsH基因。ftsH基因在染色体上的分布有明显的偏爱性,如拟南芥的1、2、5号染色体和水稻的1、5号染色体。亚细胞定位分析表明,所有FtsH蛋白均定位于叶绿体或线粒体中。系统进化分析表明,21个FtsH蛋白成员可分为8个类群,其中AtFtsH12在水稻中没有发现种间同源物。每个类群成员的蛋白序列高度保守,种内同源物显示出大于80%的相似性,而种间同源物的相似性也大于70%。类群内的同源基因并非平行进化产生的,拟南芥基因组中进化出AtftsH1/5、AtftsH2/8、AtftsH3/10和AtftsH7/9共4个同源基因对,而水稻基因组中只有OsftsH3/8和OsftsH4/5两个同源基因对。每一类群中的成员在基因外显子-内含子边界分布上表现出高度保守性,在蛋白功能结构域的可变残基上具有偏爱性,而内含子在碱基组成和序列长度上表现出广泛的变异。拟南芥和水稻ftsH基因家族的比较分析为其他物种ftsH基因的特...     

MetMap Enables Genome-Scale Methyltyping for Determining Methylation States in Populations

The ability to assay genome-scale methylation patterns using high-throughput sequencing makes it possible to carry out association studies to determine the relationship between epigenetic variation and phenotype. While bisulfite sequencing can determine a methylome at high resolution, cost inhibits its use in comparative and population studies. MethylSeq, based on sequencing of fragment ends produced by a methylation-sensitive restriction enzyme, is a method for methyltyping (survey of methylation states) and is a site-specific and cost-effective alternative to whole-genome bisulfite sequencing. Despite its advantages, the use of MethylSeq has been restricted by biases in MethylSeq data that complicate the determination of methyltypes. Here we introduce a statistical method, MetMap, that produces corrected site-specific methylation states from MethylSeq experiments and annotates unmethylated islands across the genome. MetMap integrates genome sequence information with experimental data, in a statistically sound and cohesive Bayesian Network. It infers the extent of methylation at individual CGs and across regions, and serves as a framework for comparative methylation analysis within and among species. We validated MetMap''s inferences with direct bisulfite sequencing, showing that the methylation status of sites and islands is accurately inferred. We used MetMap to analyze MethylSeq data from four human neutrophil samples, identifying novel, highly unmethylated islands that are invisible to sequence-based annotation strategies. The combination of MethylSeq and MetMap is a powerful and cost-effective tool for determining genome-scale methyltypes suitable for comparative and association studies.     

Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N⁶-methyladenine (6 mA) and N⁴-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5′-CTAT-3′), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5′-GAN₇TAY-3′/3′-CTN₇ ATR-5′). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.     

Conservation and divergence of DNA methylation in eukaryotes: New insights from single base-resolution DNA methylomes

DNA methylation is one of the most important heritable epigenetic modifications of the genome and is involved in the regulation of many cellular processes. Aberrant DNA methylation has been frequently reported to influence gene expression and subsequently cause various human diseases, including cancer. Recent rapid advances in next-generation sequencing technologies have enabled investigators to profile genome methylation patterns at singlebase resolution. Remarkably, more than 20 eukaryotic methylomes have been generated thus far, with a majority published since November 2009. Analysis of this vast amount of data has dramatically enriched our knowledge of biological function, conservation and divergence of DNA methylation in eukaryotes. Even so, many specific functions of DNA methylation and their underlying regulatory systems still remain unknown to us. Here, we briefly introduce current approaches for DNA methylation profiling and then systematically review the features of whole genome DNA methylation patterns in eight animals, six plants and five fungi. Our systematic comparison provides new insights into the conservation and divergence of DNA methylation in eukaryotes and their regulation of gene expression. This work aims to summarize the current state of available methylome data and features informatively.Key words: DNA methylation, methylome, single-base resolution, CpG, gene body, broadness, deepness, promoter     

Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation

Aspergillus flavus first gained scientific attention for its production of aflatoxin. The underlying regulation of aflatoxin biosynthesis has been serving as a theoretical model for biosynthesis of other microbial secondary metabolites. Nevertheless, for several decades, the DNA methylation status, one of the important epigenomic modifications involved in gene regulation, in A. flavus remains to be controversial. Here, we applied bisulfite sequencing in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Both the bisulfite sequencing data and the methylome comparisons with other fungi confirm that the DNA methylation level of this fungus is negligible. Further investigation into the DNA methyltransferase of Aspergillus uncovers its close relationship with RID-like enzymes as well as its divergence with the methyltransferase of species with validated DNA methylation. The lack of repeat contents of the A. flavus' genome and the high RIP-index of the small amount of remanent repeat potentially support our speculation that DNA methylation may be absent in A. flavus or that it may possess de novo DNA methylation which occurs very transiently during the obscure sexual stage of this fungal species. This work contributes to our understanding on the DNA methylation status of A. flavus, as well as reinforces our views on the DNA methylation in fungal species. In addition, our strategy of applying bisulfite sequencing to DNA methylation detection in species with low DNA methylation may serve as a reference for later scientific investigations in other hypomethylated species.     

Whole genome sequencing and methylome analysis of the wild guinea pig

Background

DNA methylation is a heritable mechanism that acts in response to environmental changes, lifestyle and diseases by influencing gene expression in eukaryotes. Epigenetic studies of wild organisms are mandatory to understand their role in e.g. adaptational processes in the great variety of ecological niches. However, strategies to address those questions on a methylome scale are widely missing. In this study we present such a strategy and describe a whole genome sequence and methylome analysis of the wild guinea pig.

Results

We generated a full Wild guinea pig (Cavia aperea) genome sequence with enhanced coverage of methylated regions, benefiting from the available sequence of the domesticated relative Cavia porcellus. This new genome sequence was then used as reference to map the sequence reads of bisulfite treated Wild guinea pig sequencing libraries to investigate DNA-methylation patterns at nucleotide-specific level, by using our here described method, named ‘DNA-enrichment-bisulfite-sequencing’ (MEBS). The results achieved using MEBS matched those of standard methods in other mammalian model species. The technique is cost efficient, and incorporates both methylation enrichment results and a nucleotide-specific resolution even without a whole genome sequence available. Thus MEBS can be easily applied to extend methylation enrichment studies to a nucleotide-specific level.

Conclusions

The approach is suited to study methylomes of not yet sequenced mammals at single nucleotide resolution. The strategy is transferable to other mammalian species by applying the nuclear genome sequence of a close relative. It is therefore of interest for studies on a variety of wild species trying to answer evolutionary, adaptational, ecological or medical questions by epigenetic mechanisms.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1036) contains supplementary material, which is available to authorized users.     

Endonuclease enrichment TAPS for cost-effective genome-wide base-resolution DNA methylation detection

Whole genome base-resolution methylome sequencing allows for the most comprehensive analysis of DNA methylation, however, the considerable sequencing cost often limits its applications. While reduced representation sequencing can be an affordable alternative, over 80% of CpGs in the genome are not covered. Building on our recently developed TET-assisted pyridine borane sequencing (TAPS) method, we here described endonuclease enrichment TAPS (eeTAPS), which utilizes dihydrouracil (DHU)-cleaving endonuclease digestion of TAPS-converted DNA to enrich methylated CpG sites (mCpGs). eeTAPS can accurately detect 87% of mCpGs in the mouse genome with a sequencing depth equivalent to 4× whole genome sequencing. In comparison, reduced representation TAPS (rrTAPS) detected less than 4% of mCpGs with 2.5× sequencing depth. Our results demonstrate eeTAPS to be a new strategy for cost-effective genome-wide methylation analysis at single-CpG resolution that can fill the gap between whole-genome and reduced representation sequencing.     

Exploring the Roles of DNA Methylation in the Metal-Reducing Bacterium Shewanella oneidensis MR-1

We performed whole-genome analyses of DNA methylation in Shewanella oneidensis MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-molecule real-time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found that S. oneidensis MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes under these conditions, suggesting that DNA methylation is not a major regulator of gene expression in S. oneidensis MR-1. The enrichment of methylated GATC motifs in the origin of replication indicates that DNA methylation may regulate genome replication in a manner similar to that seen in Escherichia coli. Furthermore, comparative analyses suggest that many Gammaproteobacteria, including all members of the Shewanellaceae family, may also utilize DNA methylation to regulate genome replication.     

Advanced methylome analysis after bisulfite deep sequencing: an example in Arabidopsis

Deep sequencing after bisulfite conversion (BS-Seq) is the method of choice to generate whole genome maps of cytosine methylation at single base-pair resolution. Its application to genomic DNA of Arabidopsis flower bud tissue resulted in the first complete methylome, determining a methylation rate of 6.7% in this tissue. BS-Seq reads were mapped onto an in silico converted reference genome, applying the so-called 3-letter genome method. Here, we present BiSS (Bisufite Sequencing Scorer), a new method applying Smith-Waterman alignment to map bisulfite-converted reads to a reference genome. In addition, we introduce a comprehensive adaptive error estimate that accounts for sequencing errors, erroneous bisulfite conversion and also wrongly mapped reads. The re-analysis of the Arabidopsis methylome data with BiSS mapped substantially more reads to the genome. As a result, it determines the methylation status of an extra 10% of cytosines and estimates the methylation rate to be 7.7%. We validated the results by individual traditional bisulfite sequencing for selected genomic regions. In addition to predicting the methylation status of each cytosine, BiSS also provides an estimate of the methylation degree at each genomic site. Thus, BiSS explores BS-Seq data more extensively and provides more information for downstream analysis.     

Conservation and divergence of DNA methylation in eukaryotes

DNA methylation is one of the most important heritable epigenetic modifications of the genome and is involved in the regulation of many cellular processes. Aberrant DNA methylation has been frequently reported to influence gene expression and subsequently cause various human diseases, including cancer. Recent rapid advances in next-generation sequencing technologies have enabled investigators to profile genome methylation patterns at single-base resolution. Remarkably, more than 20 eukaryotic methylomes have been generated thus far, with a majority published since November 2009. Analysis of this vast amount of data has dramatically enriched our knowledge of biological function, conservation and divergence of DNA methylation in eukaryotes. Even so, many specific functions of DNA methylation and their underlying regulatory systems still remain unknown to us. Here, we briefly introduce current approaches for DNA methylation profiling and then systematically review the features of whole genome DNA methylation patterns in eight animals, six plants and five fungi. Our systematic comparison provides new insights into the conservation and divergence of DNA methylation in eukaryotes and their regulation of gene expression. This work aims to summarize the current state of available methylome data and features informatively.