绒泡菌目黏菌的ITS1-5.8S-ITS2二级结构的比较分析

为探讨核糖体DNA转录间隔区(r DNA ITS)的RNA二级结构在黏菌系统发育研究中的作用,以黏菌ITS通用引物PHYS4和PHYS5对绒泡菌目5属8种黏菌的r DNA ITS进行扩增和测序,利用RNA structure构建了ITS区的RNA二级结构模型。结果表明:ITS1在绒泡菌目黏菌中不能形成一个紧实的结构,但大部分物种都具有一段稳定的螺旋结构,可能对r RNA的成熟具有作用;5.8S r RNA的二级结构相似,由4个螺旋组成,主要为两种类型;基于5.8S r RNA和28S r RNA相互作用构建的ITS2的二级结构模型显示,它由一个封闭的多分支环和至少4个主要的螺旋组成,其中螺旋IV结构相对比较保守。由于ITS区的二级结构相比核苷酸序列更加保守,因此深入地分析其二级结构有助于认识其结构与进化的关系。     

散斑壳属Lophodermium spp.ITS区的序列分析

在国内首次对散斑壳属两个代表种的rRNA基因内转录间区(ITS区)进行了克隆测序,并与Genbank中已有的有关序列进行了比较。发现散斑壳属种间的遗传差异明显高于种内的遗传差异,这与形态学分类相符合,表明ITS区序列分析与形态学鉴定相结合的方法用于散斑壳属的分类研究是可行的。     

基于ITS2序列的獐牙菜属六种药材的分子鉴定

本文以獐牙菜属的六种药材为研究对象,采用MEGA 6.0软件确定ITS2序列的GC含量,基于K2P模型计算遗传距离,构建NJ树,ITS2 database在线分析二级结构等方法,研究ITS2条形码序列对六种药材的鉴别效果。结果表明,六种药材的ITS2序列长度范围为230~232 bp,平均GC含量为61.2%~64.0%。六种药材的种内变异均较小,其种间平均遗传距离为0.03~0.09。ITS2二级结构的茎环结构和四个臂均不同,NJ树结果显示,六种药材可分别聚为一支,呈现出明显的单系性。因此,ITS2序列可准确、有效地鉴别本研究的獐牙菜属六种药材,为这些药材的后续相关深入研究奠定了基础,也为獐牙菜属其他物种鉴定提供了参考。     

用于灵芝属10个物种快速鉴定的DNA条形码筛选与验证

灵芝栽培品种常出现同名异物或同物异名的现象,依赖于灵芝的形态学特征鉴定物种的传统分类方法不足以鉴定物种的种间和种内差异。为开发简便而准确的分子水平灵芝属物种鉴定方法,以常用于种属鉴定的核糖体内转录间隔区（ITS）、微管蛋白基因（β-tubulin）片段、大亚基核糖体DNA(LSU)片段和基因编码RNA聚合酶Ⅱ第二大亚基（RPB2）片段4个DNA条形码,对灵芝属10个物种的44份菌株进行PCR扩增和测序,比较4个条形码的扩增成功率和变异率,根据种内、种间遗传距离差异分析筛选特异性的DNA条形码间隔（DBG）,并利用单独及联合条形码构建系统发育树验证筛选的DBG鉴定灵芝属物种的准确性。研究结果表明：β-tubulin和LSU片段的PCR扩增成功率均达100%,ITS和RPB2片段分别为95.35%和90.91%;对比ITS和LSU片段,β-tubulin和RPB2片段的种内最大遗传距离分别小于各自的种间最小遗传距离,存在明显的DBG,有利于准确对物种进行种间鉴定;利用ITS、β-tubulin和RPB2片段分别构建的系统发育树均能使灵芝属10个物种的所有个体聚为1个单系分支,而LSU片段不...     

捕食线虫真菌rDNA ITS区间RFLPs分析

利用PCR-RFLP方法对捕食线虫真菌进行了系统发育研究。以ITS1和ITS4为引物对3属14种16个菌株的核糖体DNA转录间区（ITS）进行了PCR扩增,4种内切酶（AluI、HaeIII、HpaII、TaqI）酶切,结果表明不同属的ITS区长度没有明显差异,其长度范围在585～695之间。酶切图谱种间差别明显,种内基本一致,同属菌株图谱没有特异性,暗示传统的分属可能过细,某些属的成立还有待商榷,PCR-RFLP对确定疑难种的地位有重要意义,但不适用于种下水平的系统学研究。     

基于ITS2和16S rRNA的西施舌群体遗传差异分析

目前,我国西施舌群体分子遗传差异研究结果存在争议。分析我国南北沿海（9个群体）、与广西北海毗邻的越南（1个群体）西施舌核DNA的内转录间隔区2（ITS2）和线粒体DNA的16S rRNA基因（16S）片段核苷酸序列及其二级结构,为解决争议问题提供分子生物学资料。扩增获得西施舌ITS2片段和16S序列,其长度分别为389-402 bp和306 bp,加之下载序列共147条;序列分析显示,74个ITS2序列共有17种基因型,73个16S序列有15种单倍型,其中,长乐（CL）群体独享9种ITS2基因型和5种16S单倍型,非长乐群体（nCL）多数为群体间交叉共享1种或几种基因（单倍）型;基因（单倍）型核苷酸变异位点占5.7%（ITS2）和11.8%（16S）;基于ITS2和16S的CL群体和nCL群体间的遗传距离与群体内遗传距离之比分别为2.42和11.08,nCL群体间的平均遗传距离均为0.007;二级结构显示CL群体ITS2的9种基因型和16S的5种单倍型均区别于nCL群体,nCL的ITS2和16S二级结构分别相似;ITS2和16S基因的系统发育分析显示,CL西施舌形成支持率很高（98,96）的单系支,而nCL群体则交叉聚为另一支（98,96）。研究结果揭示,福建西施舌是腔蛤蜊属（Coelomactra）的一个新种。     

节丛孢属rDNA ITS区RFLPs分析

利用PCR朢FLP方法对捕食线虫真菌节丛孢属进行了系统发育研究。以ITS1和ITS4为引物对该属10种12个菌株的核糖体DNA转录间区（ITS）进行了PCR扩增,4种内切酶（AluI、HaeIII、HpaII、TaqI）酶切。结果表明该属的ITS区长度没有明显差异,其长度范围在658～705之间,酶切图谱能体现不同种间的多态性,根据4种内切酶酶切结果,利用UPGMA法构建的节丛孢属分子系统树,基本体现了属内不同种间的亲缘关系,从分子水平证明了节丛孢属形态分类上的合理性,同时对该属属征的扩大提出疑议。     

我国赫坎按蚊复合体成员种的rDNA-ITS2区序列差异及系统发育分析

测定了我国赫坎按蚊复合体 9成员种的核糖体DNA第二内转录间隔区 (rDNA ITS2 )序列 ,根据序列差异分析各蚊种间的系统发育关系。结果显示 :( 1 )ITS2区序列最长的是中华按蚊 ( 4 6 8bp) ,最短的是克劳按蚊和赫坎按蚊 ( 4 36bp) ;GC含量为 4 4 9%～ 4 6 8% ;( 2 )发现该复合体 4成员种的ITS2区序列存在种内个体间差异 ,幅度为 0～ 3 8% ,明显小于种间差异 ;( 3)将各蚊种的ITS2区序列进行同源排序比较 ,发现其变异大多是简单重复单元的拷贝数不同 ;种间差异性最大的是克劳按蚊与嗜人按蚊( 32 3% ) ,最小的是贵阳按蚊与凉山按蚊 ( 9 0 % )平均差异率为 2 2 3% ;( 4 )根据ITS2区序列特征 ,用 3种方法构建的树状图拟合一致。以上结果表明赫坎按蚊复合体各成员种rDNA ITS2序列在种内非常保守 ,以种间序列差异分析为基础的分子鉴别技术是甄别蚊种分类地位混淆和错误的有效方法。     

南方灵芝ITS、EF1-α和RPB2序列多态性研究

内转录间隔区（ITS）、延伸因子1-α（EF1-α）与RNA聚合酶II第二大亚基（RPB2）是真菌系统学研究中常用的基因片段。已有研究发现,在同一个体内ITS也可能存在差异,但EF1-α和RPB2是否也存在同样的现象却鲜有报道。本研究通过克隆测序,分析了南方灵芝Ganoderma australe的ITS、EF1-α和RPB2在同一个体中的序列差异,并探讨这种序列差异是否会影响分子鉴定的准确性。结果表明,在南方灵芝供试样品中,ITS、EF1-α和RPB2序列都有可能存在个体内变异,但个体内变异程度不尽相同。基于供试样品和克隆测序结果发现,ITS序列在同一个体内的变异最高可达2.3%,而RPB2序列在同一个体内的差异仅0.5%,EF1-α序列在同一个体内序列差异也可高达1.8%。ITS和EF1-α序列在同一个体内的差异可能超过了灵芝属部分物种间的差异,对于部分灵芝属物种来说直接利用克隆序列进行分子鉴定或系统发育分析可能存在一些问题。     

石斛内生炭角菌DNA提取方法优化及分子鉴定

本文对石斛内生炭角菌DNA提取方法进行优化,并基于ITS序列及其ITS2二级结构对其进行分子鉴定。比较试剂盒法及不同浓度（2%、3%、4%）CTAB法的DNA提取效果并对CTAB法进行优化;进行ITS序列的扩增及ITS2二级结构的预测。结果表明：4%的CTAB提取效果最佳,优化的CTAB法大大缩短了DNA提取时间;ITS序列与炭角菌属3个种的相似度均为99%,ITS2二级结构仅与Xylaria arbuscula的二级结构100%相似。本研究所优化的CTAB法能为后续相关研究提供便利;结合ITS序列及ITS2二级结构能对石斛内生炭角菌进行准确的分子鉴定。     

Molecular characterization of pouched amphistome parasites (Trematoda: Gastrothylacidae) using ribosomal ITS2 sequence and secondary structures

Members of the family Gastrothylacidae (Trematoda: Digenea: Paramphistomata) are parasitic in ruminants throughout Africa and Asia. In north-east India, five species of pouched amphistomes, namely Fischoederius cobboldi, F. elongatus, Gastrothylax crumenifer, Carmyerius spatiosus and Velasquezotrema tripurensis, belonging to this family have been reported so far. In the present study, the molecular phylogeny of these five gastrothylacid species is derived using the second internal transcribed spacer (ITS2) sequence and secondary structure analyses. ITS2 sequence analysis was carried out to see the occurrence of interspecific variations among the species. Phylogenetic analyses were performed for primary sequence data alone as well as the combined sequence-structure information using neighbour-joining and Bayesian approaches. The sequence analysis revealed that there exist considerable interspecific variations among the various gastrothylacid fluke species. In contrast, the inferred secondary structures for the five species using minimum free energy modelling showed structural identities, in conformity with the core four-helix domain structure that has been recently identified as common to almost all eukaryotic taxa. The phylogenetic tree reconstructed using combined sequence-structure data showed a better resolution, as compared to the one using sequence data alone, with the gastrothylacid species forming a monophyletic group that is well separated from members of the other family, Paramphistomidae, of the amphistomid flukes group. The study provides the molecular characterization based on primary sequence data of the rDNA ITS2 region of the gastrothylacid amphistome flukes. Results also demonstrate the phylogenetic utility of the ITS2 sequence-secondary structure data for inferences at higher taxonomic levels.     

Analysis of nrDNA polymorphism in closely related diploid sexual, tetraploid sexual and polyploid agamospermous species

Nuclear sequences of ITS1-5.8S-ITS2 region of rDNA may be an important source of phylogenetically informative data provided that nrDNA is cloned and the character of sequence variation of clones is properly analyzed. nrDNA of selected Taraxacum sections was studied to show sequence variation differences among diploid sexual, tetraploid sexual and polyploid agamospermous species. We examined nucleotide characteristics, substitution pattern, secondary structure, and the phylogenetic utility of ITS1-5.8S-ITS2 from 301 clones of 32 species representing 11 sections. The most divergent sequences of ITS1&2 differed by 17.1% and in 5.8S only by 3.7%. The ITS1-5.8S-ITS2 characteristics, integrity and also stability of secondary structures confirmed that pseudogenes are not responsible for the above variation. The within-individual polymorphism of clones implies that the concerted evolution of ITS cistron of agamospermous polyploid Taraxacum is remarkably suppressed. Sequences of ITS clones proved to be a useful tool for mapping pathways of complex reticulation (polyploid hybridity) in agamospermous Taraxacum.     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

The phylogeny of Schistidium (Bryophyta, Grimmiaceae) based on primary and secondary structure study of nuclear rDNA internal transcribed spacers

The phylogeny of Schistidium (Bryophyta, Grimmiaceae) was studied on the basis of nucleotide sequences of internal transcribed spacers ITS1-2 of nuclear DNA and trnT-trnD region of chloroplast DNA. The consistency of phylogenetic trees constructed from nuclear and chloroplast sequences was shown. A basal grade and two large clades were resolved on the phylogenetic trees. Morphological characteristics specific for these clades were described. ITS1 and ITS2 secondary structures of Schistidium species were modeled using thermodynamic criteria. Four different structures of the longest ITS1 hairpin were identified. Possible paths of Schistidium evolution were considered based on the four types of ITS1 secondary structure and phylogenetic trees.     

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models

Among ciliates, Paramecium has become a privileged model for the study of “species problem” particularly in the case of the “Paramecium aurelia complex” that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5′LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.     

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.     

The ITS region of groundwater amphipods: length,secondary structure and phylogenetic information content in Crangonyctoids and Niphargids

The phylogenetic analysis of groundwater amphipods is challenging due to the lack of suitable morphological characters. However, molecular phylogenies based on the 18S and 28S nuclear genes of two Crangonyctoidea species endemic to Iceland, Crymostygius thingvallensis and Crangonyx islandicus, support the taxonomy of these species on the basis of morphological characters. Molecular analyses suggest that the genus Crangonyx is paraphyletic, with the species that is found in Eurasia being highly divergent genetically from the species present in North America and Iceland. Studies of the phylogenetic relationships within the genus Niphargus also warrant further work. The nuclear ITS2 region has recently been proposed as a barcoding marker for plants and animals. In addition, ITS2 has been used to build phylogenies at high taxonomic levels by including its secondary structure. In this study, we want to evaluate the applicability of the ITS region for this group of species and describe its characteristics. The taxonomy of C. thingvallensis, as well as the paraphyly of the genus Crangonyx, is supported herein by phylogenies based on the ITS2 variation. The secondary structure and the length of the ITS2 sequences of the Crangonyctoidea and the Niphargidae species studied are highly variable and are characterized by duplications. The ITS2 sequence of Niphargus plateaui is the longest metazoan sequence deposited in the ITS2 database so far. Although saturation was observed in the nucleotide variation of this marker, the addition of the secondary structure information for the reconstruction of the phylogeny did not add support to the phylogenetic trees. The ITS1 region, which is known to be more variable than ITS2 and bears a large duplication within C. islandicus, was found to be less useful for phylogenetic reconstruction.     

Thysanophora penicillioides includes multiple genetically diverged groups that coexist respectively in Abies mariesii forests in Japan

We investigated intraspecific diversity and genetic structures of a saprotrophic fungus--Thysanophora penicillioides--based on sequences of nuclear ribosomal internal transcribed spacer (ITS) in 15 discontinuous Abies mariesii forests of Japan. In such a well-defined morphological species, numerous unexpected ITS variations were revealed: 12 ITS sequence types detected in 254 isolates collected from 15 local populations were classified into five ITS sequence groups. Maximally, four ITS groups consisted of seven ITS types coexisting in one population. However, group 1 was dominant with approximately 65%; in particular, one haplotype, 1a, was most dominant with approximately 60% in respective populations. Therefore, few differences were recognized in genetic structure among local populations, implying that the gene flow of each lineage of the fungus occurs among local populations without geographic limitations. However, minor haplotypes in some ITS groups were found only in restricted areas, suggesting that they might expand steadily from their places of origin to neighboring A. mariesii forests. Aggregating sequence data of seven European strains and four North American strains from various substrates to those of Japanese strains, 18 ITS sequence types and 28 variable sites were recognized. They were clustered into nine lineages by phylogenetic analyses of the beta-tubulin and combined ITS and beta-tubulin datasets. According to phylogenetic species recognition by the concordance of genealogies, respective lineages correspond to phylogenetic species. Plural phylogenetic species coexist in a local population in an A. mariesii forest in Japan.     

Genealogical analyses of multiple loci of litostomatean ciliates (Protista, Ciliophora, Litostomatea)

The class Litostomatea is a highly diverse ciliate taxon comprising hundreds of free-living and endocommensal species. However, their traditional morphology-based classification conflicts with 18S rRNA gene phylogenies indicating (1) a deep bifurcation of the Litostomatea into Rhynchostomatia and Haptoria+Trichostomatia, and (2) body polarization and simplification of the oral apparatus as main evolutionary trends in the Litostomatea. To test whether 18S rRNA molecules provide a suitable proxy for litostomatean evolutionary history, we used eighteen new ITS1-5.8S rRNA-ITS2 region sequences from various free-living litostomatean orders. These single- and multiple-locus analyses are in agreement with previous 18S rRNA gene phylogenies, supporting that both 18S rRNA gene and ITS region sequences are effective tools for resolving phylogenetic relationships among the litostomateans. Despite insertions, deletions and mutational saturations in the ITS region, the present study shows that ITS1 and ITS2 molecules can be used to infer phylogenetic relationships not only at species level but also at higher taxonomic ranks when their secondary structure information is utilized to aid alignment.