长苞铁杉nrDNA内转录间隔区及5.8S的二级结构分析研究

核糖体RNA及相邻区域的二级结构研究,作为一个重要的工具,已在一些分类等级上被应用于系统发育的分析。以长苞铁杉为实验材料,通过克隆、测序,利用最小自由能原理预测nrDNA内转录间隔区及5.8S转录本的二级结构,分析它们的结构特点,探讨假基因化拷贝与功能拷贝结构上的差异。分析结果表明:(1)ITS1区的二级结构主要由几个延展的发夹结构组成,配对的亚重复单位在松科植物特有的保守序列处有部分重叠,未配对的亚重复单位通常能自身折叠,保守序列的部分碱基出现在发夹结构的环中;(2)假基因化拷贝二级结构的自由能比正常拷贝高;(3)与正常拷贝的二级结构相比,假基因化拷贝在进化速率很低的5.8S功能区发生较大的变异,且在5.8 S末端没有和26 S连接配对。     

Ribosomal External and Internal Transcribed Spacers: Combined Use in the Phylogenetic Analysis of Medicago (Leguminosae)

We have estimated the potential phylogenetic utility of the ribosomal external transcribed spacer (ETS) from the nuclear ribosomal region. The ETS was sequenced from 13 annual Medicago (Fabaceae) species upstream a highly conserved motive which was found among many different organisms. In the genus Medicago, the ETS was found to evolve 1.5 times faster than the internal transcribed spacer and to be 1.5 times more informative. Reduced ribosomal maturation process constraints on ETS are proposed to explain the different evolutionary rates between the two spacers. Maximal phylogenetic resolution and support was obtained when the two spacers were analyzed together. No incongruence between the two spacers was found and ETS appears to be a valuable source of information for solidifying ITS plant phylogeny. The phylogeny obtained in Medicago suggests that none of the three subsections included in the study is monophyletic. Received: 15 April 1997 / Accepted: 29 July 1997     

利用核核糖体DNA内转录间隔区(ITS)探讨广义羽藓科系统发育

利用核核糖体DNA ITS序列,探讨了苔藓植物广义羽藓科的系统发育,摸索出适于扩增ITS片段的最适反应条件。实验共得到广义羽藓科6个种的ITS序列,它们分别是：Abieti-nela abietina(AJ417494),Anomodon minar(AJ344145),Chaopodium aciculum(AJ315968),Tuidium pristocalyx(AJ416443),Thuidium assimile(AJ416442),Herpetineuron toccoae(AJ315967),其中后5个种是国际上首次得到的。本文利用ITS序列构建羽藓科7属、11种植物的系统发育树,据Bootstrap严格一致树表明：广义的羽藓科为并系发育,可分为两个主要的分支,牛舌藓属Anomodon,羊角藓属Herpetineuron和多枝藓属Happohymenium等为一支,而山羽藓属Abietinella,羽藓属Thuuidium,沼羽藓属Helodium和麻羽藓属Claopodium等为另一主要分支,从分子水平上支持了据形态特征把原牛舌藓亚科的牛舌藓属,羊角藓属,多枝藓属提升为牛舌藓科的结论。     

Molecular Evolution and Phylogenetic Utility of the Internal Transcribed Spacer 2 (ITS2) in Calyptratae (Diptera: Brachycera)

The resolution potential of internal transcribed spacer 2 (ITS2) at deeper levels remains controversial. In this study, 105 ITS2 sequences of 55 species in Calyptratae were analyzed to examine the phylogenetic utility of the spacer above the subfamily level and to further understand its evolutionary characteristics. We predicted the secondary structure of each sequence using the minimum-energy algorithm and constructed two data matrixes for phylogenetic analysis. The ITS2 regions of Calyptratae display strong A-T bias and slight variation in length. The tandem and dispersed repeats embedded in the spacers possibly resulted from replication slippage or transposition. Most foldings conformed to the four-domain model. Sequence comparison in combination with the secondary structures revealed six conserved motifs. Covariation analysis from the conserved motifs indicated that the secondary structure restrains the sequence evolution of the spacer. The deep-level phylogeny derived from the ITS2 data largely agreed with the phylogenetic hypotheses from morphologic and other molecular evidence. Our analyses suggest that the accordant resolutions generated from different analyses can be used to infer deep-level phylogenetic relations.     

Phylogenetic Analysis ofBambusa (Poaceae: Bambusoideae) Based on Internal Transcribed Spacer Sequences of Nuclear Ribosomal DNA

Phylogenetic analyses of Bambusa species were performed using internal transcribed spacer sequences of nuclear ribosomal DNA. The 21 species sampled included members of Bambusa (sensu stricto), Dendrocalamopsis, Dendrocalamus, Guadua, Leleba, and Lingnania. Arundinaria gigantea was used as an outgroup. Using the maximum parsimony method with PAUP*, gaps were treated as missing states or new states. Parsimonious analysis revealed that Dendrocalamus latiflorus was closely related to the members of Dendrocalamopsis. Dendrocalamus membranaceus was a sister species to Dendrocalamus strictus. Three Dendrocalamus species were closely related to and nested in a polyphyletic Bambusa. Bambusa subaequalis was a sister species to B. multiplex, B. emeiensis to B. chungii, B. contracta to B. hainanensis, and B. flexuosa was a sister species to B. sinospinosa, B. tuldoides, B. surrecta, B. intermedia, and B. valida group, which raised doubts about the monophyly of the subgenera Bambusa (sensu stricto), Dendrocalamopsis, Leleba, and Lingnania under the genus Bambusa.     

基于细胞核rDNA ITS片段的水青冈属的分子系统发育

对山毛榉科水青冈属6种、1亚种、1栽培变种的ITS区片段进行了测序和分析,并对其中2个具有ITS序列多态性的分类群进行了ITS区克隆。水青冈属ITS系统发育树聚成两支,位于基部的是分布于北美的大叶水青冈,另一分支则包括了欧洲和东亚的类群。在欧洲和东亚分支中,又包括两支,其中日本北部的波叶水青冈位于基部,台湾水青冈和欧亚大陆的水青冈形成另外一支。ITS区分析与现行的水青冈属基于形态学性状的属下分类系统有一定差异,而与本属现存物种的地理分布格局较为一致。各类群间TIS区序列差异较小,显示属内现存物种的分化时间不是太长。     

Sequence Variation of Ribosomal Internal Transcribed Spacers (ITS) in Commercially Important Phytoseiidae Mites

Preliminary work is needed to assess the usefulness of different markers at different taxonomic scales when a new group is analyzed, such as the commercially important Phytoseiidae mites. We investigate here the level of sequence variation of the nuclear ribosomal spacers ITS 1 and 2 and the 5.8S gene in six species of Phytoseiidae: Neoseiulus californicus, N. fallacis, Euseius concordis, Metaseiulus occidentalis, Typhlodromus pyri and Phytoseiulus persimilis. As expected, the 5.8S gene (148 base pairs) is markedly conserved and displays little variation in between genera comparisons. ITS1 and ITS2 show contrasting patterns: while the ITS2 is short (80–89 bp) and shows little variation, the ITS1 is longer (303–404 bp) and is very variable in sequence. This fact compromises reliable nucleotide homologies when comparing the genera. The comparison of ITS1 sequence similarity at the species level might be useful for species identification, however, the value of ITS in taxonomic studies does not extend to the level of the family. The intraspecific variations of ITS were investigated in three species: N. californicus, N. fallacis and E. concordis. The first species has identical ITS1 sequences and the last two display low polymorphism (2 nucleotide substitutions). The ITS2 and 5.8S sequences were identical in all three subspecies comparisons.     

珍珠菜rDNA ITS序列扩增及分析

以贵州境内珍珠菜属植物为材料,对其rDNA转录间隔区(ITS)序列进行PCR扩增和序列测定。实验共得到7个种的ITS序列,它们分别是:过路黄(Lysi machia christinae,GenBank登录号FJ362382),矮桃(L.cle-throides,GenBank登录号FJ362383),叶头过路黄(L.phyllocephala,GenBank登录号FJ362386),临时救(L.con-gestiflora,GenBank登录号FJ362387),显苞过路黄(L.rubiginosa,GenBank登录号FJ362388),茂汶过路黄(L.stellarioides,GenBank登录号FJ362384),腺药珍珠菜(L.stenosepala,GenBank登录号FJ362385),其中后2个种是国际上首次得到的。采用Blast方法将测序结果进行同源搜索,采用邻接法构建与其相关植物的ITS序列系统发育树。结果表明,珍珠菜属7种植物ITS序列总长度为613~620 bp;ITS1区序列长度为234~239 bp,5.8SrDNA区序列长度163 bp,ITS2区序列长度216~219 bp,7种植物的ITS序列差异主要集中在ITS1与ITS2区。聚类分析将茂汶过路黄聚为一支,其它6种植物聚为一支,表明茂汶过路黄与其它6种植物的碱基差异较大,从分子水平上支持据形态特征把花辐射生长的茂汶过路黄另立一类。     

海参核糖体基因间隔区2(ITS2)的克隆及序列分析

为探讨刺参科海参和海参科海参的系统进化关系,本研究通过PCR技术获取19种刺参科和海参科海参的ITS2序列,从NCBI上获取瓜参(C. salma)的ITS2序列。结果表明ITS2序列具有长度多态性,从318 bp (绿刺参)到591 bp (白尼参属)。海参属的ITS2序列长度多态性高,ITS2的GC含量从56.7%(糙海参)到70.6%(瓜参)。海参ITS2序列保守性不高,仅有48个保守位点,其余均为变异位点。基于ITS2的系统进化树结果显示进化树主要分成两支,一支包括海参科的4个属:海参属、白尼参属、辐肛参属和格皮氏海参属。辐肛参属和格皮氏海参属为姐妹关系,二者聚在一起后与白尼参属聚为一支,随后再与海参属聚在一起。白尼参属和辐肛参属为单系,海参属为复系。另一支为C. salma和刺参科。梅花参属与刺参属聚为一支后,再与仿刺参属聚在一起,3个属都是单系。在20种海参中,S. naso与B. argus的遗传距离最大(6.415)。刺参属中,S. monotuberculatus和S. horrens遗传距离最近(0.012),海参属中,糙海参与H. fuscopunctata的遗传距离最大(3.24)。本研究为从分子水平上研究海参科和刺参科之间的系统进化关系奠定了基础。     

节瓜及近缘葫芦科作物种质资源rDNA-ITS序列分析与系统进化研究

通过PCR扩增获得了56份节瓜种质资源及7份相关瓜类种质资源的rDNA-ITS序列,测序后结合GenBank中34份葫芦科作物的ITS序列,利用生物信息学软件分析序列长度、变异位点、G+C含量、遗传分歧、同源性百分比差异、系统进化关系。节瓜种质资源ITS基本序列全长612~617 bp,G+C含量59.97%~61.07%,供试材料间共96个变异位点,其中一些变异位点有明显的种性特征,可作为特异DNA指纹鉴别位点。基于ITS序列差异,56份节瓜材料聚为5大类,其中12-2-3材料为单独一个分支,黑毛节瓜H2251为第二分支,H5FA为第三分支,剩余节瓜材料聚为第四、五分支。节瓜材料12-2-3与其他材料遗传分歧最大,农艺性状与抗性也差异较大,可用作节瓜杂交育种的亲本以扩大选择范围。节瓜的系统位置排列在Indomelothria blumei(GU799496)与Dactyliandra welwitschii(HQ201973)之间,与Indomelothria blumei亲缘关系最近;Mrbayes软件分析表明,葫芦科作物Zehneria thwaitesii(AM981145)、Ctenolepis cerasiformis(AM981142)、Cucumis melo(AM36377)、Dactyliandra welwitschii(HQ201973)、Trochomeria macrocarpa(AM981141)最原始,系统进化顺序为甜瓜→南瓜、栝楼、苦瓜、丝瓜、西瓜、蒲瓜→冬瓜、节瓜→黄瓜。本研究通过分析节瓜及近缘葫芦科作物种质资源的ITS序列,为其系统进化、DNA指纹鉴别、育种亲本选择及比较组学分析等提供了分子生物学依据。     

大豆疫霉根腐病菌的rDNA ITS序列分析

采用真菌核糖体基因转录间隔区(ITS)通用引物,PCR扩增了大豆疫霉根腐病菌具有差异的17个菌株的ITSI与ITS2,经过与DL2000的标准分子量DNA进行比较,得到了大约800~1000bp左右的片段,并对PCR产物进行了序列测定。以USA为外类群利用最大简约法构建了大豆疫霉根腐病菌的系统发生树,并分析了菌株之间的遗传进化关系。结果表明:不同菌株ITS1和ITS2在碱基构成上有很大差异,17个菌株大致分为4个谱系中,且来自于同一地区的菌株大都分布在同一谱系中,显示出地理上的差异。     

The First Internal Transcribed Spacer (ITS-1) of Ribosomal DNA as a Molecular Marker for Phylogenetic and Population Analyses in Crustacea

The objective of the present study is to explore the feasibility of using the first internal transcribed spacer (ITS-1) of ribosomal DNA as a molecular marker for studying the interspecific and intraspecific genetic variations among crustaceans. We designed primers that could amplify ITS-1 from a majority of taxonomic groups of crustaceans. The gene was found to exhibit a high degree of length polymorphism among different groups, ranging from 182 bp in the barnacle Balanus amphitrite to approximately 820 bp in the spiny lobster Panulirus japonicus. With respect to differences between congeneric species, it was found that the ITS-1 sequences of 3 mitten crabs, Eriocheir sinensis, Eriocheir leptognathus, and Eriocheir formosa, exhibit 5.4% to 16.3% nucleotide divergence, suggesting that ITS-1 is informative for phylogenetic analysis at the species level. Yet there are extensive (0.9%–2.3%) variations within individual E. formosa, so that phylogenetic analyses could be obscured. ITS-1 was found to vary between 2 geographical populations of the shrimp Penaeus japonicus. The variations involved substitutions as well as insertions/deletions between shrimp from Australia and South China Sea. These results show that ITS-1 is highly divergent among different crustaceans and could be an appropriate marker for molecular systematic studies at the species and population levels, although the presence of intragenomic variation needs to be taken into consideration. Received August 15, 2000; accepted February 9, 2001     

The Genus Artemisia and its Allies: Phylogeny of the Subtribe Artemisiinae (Asteraceae, Anthemideae) Based on Nucleotide Sequences of Nuclear Ribosomal DNA Internal Transcribed Spacers (ITS)

Abstract: Sequences of the internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA were analysed for 44 Artemisia species (46 populations) representing all the five classical subgenera and the geographical range of the genus, 11 species from 10 genera closely related to Artemisia, and six outgroup species from five other genera of the Anthemideae. The results definitely support the monophyly of the genus Artemisia in its broadest sense (including some taxa segregated as independent genera, like Oligosporus and Seriphidium ). Eight main clades are established in this molecular phylogeny within Artemisia; they agree in part with the classical subdivision of the genus, but they also suggest that some infrageneric groups must be redefined, especially the subgenus Artemisia. The subgenera Tridentatae and Seriphidium are independent from each other. Some of the satellite genera are clearly placed within Artemisia ( Artemisiastrum, Filifolium, Mausolea, Picrothamnus, Sphaeromeria, Turaniphytum ), whereas some others fall outside the large clade formed by this genus (Brachanthemum, Elachanthemum, Hippolytia, Kaschgaria). Our results, correlated to other data such as pollen morphology, allow us to conclude that the subtribe Artemisiinae as currently defined is a very heterogeneous group. Affinities of the largest genus of the subtribe and tribe, Artemisia, and of other genera of the subtribe to some genera from other subtribes of the Anthemideae strongly suggest that subtribe Artemisiinae needs a deep revision and redefinition. Phylogenetic utility of region trnL-F of the plastid DNA in the genus Artemisia and allies was also evaluated: sequences of the trnL-F region in Artemisia do not provide phylogenetic information.     

Implications of High Molecular Divergence of Nuclear rRNA and Phylogenetic Structure for the Dinoflagellate Prorocentrum (Dinophyceae,Prorocentrales)

The small and large nuclear subunit molecular phylogeny of the genus Prorocentrum demonstrated that the species are dichotomized into two clades. These two clades were significantly different (one‐factor ANOVA, p < 0.01) with patterns compatible for both small and large subunit Bayesian phylogenetic trees, and for a larger taxon sampled dinoflagellate phylogeny. Evaluation of the molecular divergence levels showed that intraspecies genetic variations were significantly low (t‐test, p < 0.05), than those for interspecies variations (> 2.9% and > 26.8% dissimilarity in the small and large subunit [D1/D2], respectively). Based on the calculated molecular divergence, the genus comprises two genetically distinct groups that should be considered as two separate genera, thereby setting the pace for major systematic changes for the genus Prorocentrum sensu Dodge. Moreover, the information presented in this study would be useful for improving species identification, detection of novel clades from environmental samples.