紫芝基因组rDNA序列特征及ITS2二级结构比较

紫芝栽培品种‘紫芝S2’(武芝2号)的ITS序列与NCBI数据库中5个紫芝菌株/分离株相似度高达99.79%-100%,在系统进化树上相聚成一类。本研究预测‘紫芝S2’基因组与参考基因组中的rRNA基因簇,分析rDNA结构及各构件序列间的多态性。从高质量‘紫芝S2’基因组中挖掘得到完整rDNA,序列全长40.377 kb,由4组串联重复的(18S、5.8S、28S、5S) rRNA基因簇组成,并含有完整的基因内间隔区(ITS1、ITS2)和基因间间隔区(IGS1、IGS2)。在紫芝S2的rDNA中,高度保守的28S rRNA基因间出现3个SNP和2个插入(1 bp,10 bp)位点;虽然第4条ITS2中有1个SNP位点,但紫芝S2的4条ITS2在二级结构上的分子形态高度一致,与ITS2数据库中其他紫芝菌株仅存在螺旋区间夹角的微小差异。由‘紫芝S2’基因组rDNA的ITS2生成的DNA条形码与二维码,可以作为该栽培品种鉴定与同源物种其他菌株鉴别的分子标记。     

基于核核糖体DNA ITS序列的真藓科系统发育新证据

主要是利用核基因组的核糖体DNA内转录间隔区（nrDNA ITS）序列数据探讨了Buck和Goffinet（2000）限定的真藓科内各属间及属内种间的系统发育关系。ITS序列的最大简约法（MP）及贝叶斯推论（BI）分析结果显示：不支持Cox（1999）利用rps4/trnL-trnF数据分析将丝瓜藓属移至提灯藓科的结论。ITS数据分析与Holyoak（2007）利用trnL-F,trnG和atpB-rbcL序列数据分析将Bryum algovicum、B.caespiticium、B.capillare、B.pseudotriquetrum和Plagiobryum zierii划归Ptychostomum的结论一致,同时,为Spence(2005)依据形态学特征将B.pallescen、B.pallens、B.uliginosum、B.amblyodon和B.lonchocaulon划归Ptychostomum提供ITS分子证据支持。基于以上研究,建议将以上提到的真藓属种和平蒴藓移至Ptychostomum。     

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。     

丹皮药材及其伪品、混淆品核糖体rDNA ITS2的序列分析

分析安徽道地药材丹皮（Paeonia suffruticosa Andr）及其伪品、混淆品核糖体DNA第二内转录间隔区（ITS2）的序列。分析丹皮的分子遗传多样性,为丹皮的真品药材基原鉴定提供分子依据。采用DNA克隆测序技术对从不同市场购得的丹皮样品及其伪品、混淆品的ITS2基因进行测序分析研究。丹皮与其伪品川赤芍、亳州芍药的rDNA-ITs2序列长度均为412bp。排序比较发现不同市场上的丹皮样品rDNA-ITs2序列变异位点丰富。鉴定出与丹皮真品（黄沙与白沙）及伪品的rDNA-ITS2序列完全相同的样品。其他样品的rDNA-ITs2序列与丹皮真品的差异明显。通过丹皮ITS2序列差异比较分析。ITS2序列可以作为鉴别丹皮药材真伪的一种手段。     

用核糖体DNA的ITS序列探讨中国柽柳科植物系统分类中的几个 …

采用ＰＣＲ直接测序的方法对中国柽柳科３属１０种代表植物的ＩＴＳ序列进行了比较研究,结果表明,属内片段长度、ＧＣ含量和位点变异量上均比较一致;ＩＴＳ－１长度为２４９￣２６９,ＩＴＳ－２长度为２２４￣２５３,ＧＣ含量４６％￣６３％,信息位点占总位点的３０％。运用ＰＡＵＰ软件分析得到了单个最简约树,总步长为３９５步,一致性指数ＣＩ＝０．９１６,保持性指数ＲＩ＝０．８８６。柽柳属的６个类群县１００％的强支     

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。     

杂交稻亲本核糖体DNA的ITS序列比较

以典型粳稻(O．sativa ssp．japonicn)秋光作为外类群,典型籼稻(O．sativa ssp．indica)南京11号作为对照,对两优培9、培两优288、汕优63三个籼型组合的6个亲本的核糖体基因(nrDNA)内转录间隔区(ITS)的全序列进行了比较分析,结果表明：供试材料的ITS序列有较丰富的遗传差异,以ITS序列信息构建的系统树能反映出品种间的亲缘关系,3个组合亲本间遗传距离以超级稻两优培九的亲本9311与培矮64S遗传距离最远．     

药用野生稻复合体ITS1和ITS2序列变异及其系统进化分析

通过PCR扩增并测序分析稻属药用野生稻复合体5个野生稻种基因组完整的ITS区及5.8S区,并与栽培稻ITS序列进行比较,构建分子系统进化树,探讨了稻属药用野生稻复合体内不同种间的亲缘关系和系统进化.结果表明,ITS1和ITS2均有较高的G/C含量,ITS1序列的长度多态性相对较高,ITS2序列的碱基突变频率较高.药用野生稻和高秆野生稻亲缘关系很近,而与栽培稻亲缘关系较远;短药野生稻、斑点野生稻、澳洲野生稻与药用野生稻亲缘关系渐近.处于进化的过渡阶段.     

荔枝nrDNA ITS序列多态性分析

为了解荔枝nrDNA ITS序列多态性,对荔枝4个品种的nrDNA ITS序列进行克隆测序,分析其序列特征、核苷酸多态性、遗传距离、5.8S保守基序、二级结构最小自由能、系统发育树及rRNA二级结构等。结果表明,荔枝nrDNA ITS序列在品种间、品种内均存在多态性,同时包含假基因序列,表明其ITS序列逃离了致同进化。分子方差分析（AMOVA）显示,荔枝nrDNA ITS序列遗传变异主要来源于品种内。自然杂交可能是荔枝ITS序列产生多态性的主要原因。     

Non-concerted ITS evolution in Mammillaria (Cactaceae)

Molecular studies of 21 species of the large Cactaceae genus Mammillaria representing a variety of intrageneric taxonomic levels revealed a high degree of intra-individual polymorphism of the internal transcribed spacer region (ITS1, 5.8S rDNA, ITS2). Only a few of these ITS copies belong to apparently functional genes, whereas most are probably non-functional (pseudogenes). As a multiple gene family, the ITS region is subjected to concerted evolution. However, the high degree of intra-individual polymorphism of up to 36% in ITS1 and up to 35% in ITS2 suggests a non-concerted evolution of these loci in Mammillaria. Conserved angiosperm motifs of ITS1 and ITS2 were compared between genomic and cDNA ITS clones of Mammillaria. Some of these motifs (e.g., ITS1 motif 1, 'TGGT' within ITS2) in combination with the determination of GC-content, length comparisons of the spacers and ITS2 secondary structure (helices II and III) are helpful in the identification of pseudogene rDNA regions.     

PCR产物直接测序还是克隆测序?——密叶杉属rDNA ITS序列的测定方法

通过PCR产物直接测序和克隆测序对三种密叶杉属 (Athrotaxis)植物rDNA内转录间隔区 (ITS)及5 .8SrDNA序列进行了测定与分析。实验表明A .selaginoidesrDNA重复序列间的纯合程度很高 ,对PCR产物直接测序就可以测定其ITS区序列。而A .laxifolia、A .cupressoides的ITS1重复序列间的纯合程度较低 ,各重复单位间序列存在插入 /缺失 ,只有对PCR产物进行克隆测序才能确定其序列。A .laxi folia、A .cupressoides的ITS2区尽管也存在多态性 ,但不同重复序列的浓度比较平均 ,对PCR产物直接测序就可确定重复序列间的变异情况。本实验表明尽管是同一属的三种植物 ,但其rDNA重复序列间的纯合程度不同 ,同一植物ITS的不同区域 ,其重复序列间的纯合程度也不同 ,针对不同的ITS片段可采用不同的方法以测定其序列。     

苏铁珊瑚根共生念珠藻ITS序列多拷贝研究

目的:研究苏铁珊瑚根共生念珠藻16SrDNA-23SrDNA基因间隔序列(ITS)拷贝数。方法:提取苏铁珊瑚根共生念珠藻全DNA,PCR扩增其16SrDNA-23SrDNA基因间隔序列(ITS)克隆并测序;采用Cluster X 1.83对所得序列进行比对,用DNAman软件对比对结果进行人工校正;采用割胶回收电泳检测、菌液PCR及混合模板PCR扩增三种方法验证ITS-L2。结果:ITS-S(420 bp)不含任何氨基酸编码序列,ITS-L1(676 bp)包含一个异亮氨酸和一个丙氨酸编码序列;ITS-L1和ITS-L2在琼脂糖凝胶电泳检测时长度分别为676 bp和1000 bp,而谱带经克隆测序后结果表明ITS-L1和ITS-L2碱基序列和长度几乎相同,仅有两个碱基发生变异。结论:ITS-L2为假阳性,可能是由ITS-S和ITS-L1形成的异源双链;苏铁珊瑚根共生念珠藻含有两种类型的ITS拷贝,一种是含有tRNAIle和tRNAAla编码序列的ITS,另一种是不含tRNAIle和tRNAAla编码序列的ITS。     

Evolutionary Diversification Indicated by Compensatory Base Changes in ITS2 Secondary Structures in a Complex Fungal Species, <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prospects for discriminating Zingiberaceae species in India using DNA barcodes

We evaluated nine plastid(matK, rbcL, rpoC1, rpoB,rpl36-rps8, ndhJ, trnL-F, trnH-psbA, accD) and two nuclear(ITS and ITS2) barcode loci in family Zingiberaceae by analyzing 60 accessions of 20 species belonging to seven genera from India.Bidirectional sequences were recovered for every plastid locus by direct sequencing of polymerase chain reaction(PCR)amplicons in all the accessions tested. However, only 35(58%)and 40 accessions(66%) yielded ITS and ITS2 sequences,respectively, by direct sequencing. In different bioinformatics analyses, matK and rbcL consistently resolved 15 species(75%)into monophyletic groups and five species into two paraphyletic groups. The 173 ITS sequences, including 138 cloned sequences from 23 accessions, discriminated only 12 species(60%), and the remaining species were entered into three paraphyletic groups. Phylogenetic and genealogic analyses of plastid and ITS sequences imply the possible occurrence of natural hybridizations in the evolutionary past in giving rise to species paraphyly and intragenomic ITS heterogeneity in the species tested. The results support using matK and rbcL loci for barcoding Zingiberaceae members and highlight the poor utility of ITS and the highly regarded ITS2 in barcoding this family, and also caution against proposing ITS loci for barcoding taxa based on limited sampling.     

Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis

In a search for specific molecular markers for population analysis of Puccinia striiformis f. sp. tritici, the ribosomal DNA (rDNA) intergenic spacer (IGS) 1 region (rDNA-IGS1, between the 28S and the 5S rDNA genes) was amplified, cloned, and sequenced. It was found to exhibit multiple bands and length polymorphism. Surprisingly, single isolates were found to possess between three to five different IGS1 haplotypes. Bands were cloned and sequenced, and two highly variable regions (α and β) were found between conserved regions, with repeat units interspersed in both types of regions. There were 14 different repeat units, and these were sometimes grouped further into four combinations of repeat units, with a few individual nucleotides (A or C) inserted between the repeats. Among three geographically dispersed isolates, the variable region α was divided into eight types, and the variable region β was divided into two types based on repeat units. Most of the 14 repeat units were shared by the variable and the conserved regions. Among the three isolates, there were a total of 12 IGS1 haplotypes, but some of these were shared between isolates such that there were only eight unique haplotypes. The occurrence of multiple haplotypes within single isolates may be useful for analyzing the population structure, tracking the origin of annual epidemics and providing insights into evolutionary biology of this pathogen.     

Ribosomal ITS Sequences Allow Resolution of Freshwater Sponge Phylogeny with Alignments Guided by Secondary Structure Prediction

Freshwater sponges include six extant families which belong to the suborder Spongillina (Porifera). The taxonomy of freshwater sponges is problematic and their phylogeny and evolution are not well understood. Sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) of 11 species from the family Lubomirskiidae, 13 species from the family Spongillidae, and 1 species from the family Potamolepidae were obtained to study the phylogenetic relationships between endemic and cosmopolitan freshwater sponges and the evolution of sponges in Lake Baikal. The present study is the first one where ITS1 sequences were successfully aligned using verified secondary structure models and, in combination with ITS2, used to infer relationships between the freshwater sponges. Phylogenetic trees inferred using maximum likelihood, neighbor-joining, and parsimony methods and Bayesian inference revealed that the endemic family Lubomirskiidae was monophyletic. Our results do not support the monophyly of Spongillidae because Lubomirskiidae formed a robust clade with E. muelleri, and Trochospongilla latouchiana formed a robust clade with the outgroup Echinospongilla brichardi (Potamolepidae). Within the cosmopolitan family Spongillidae the genera Radiospongilla and Eunapius were found to be monophyletic, while Ephydatia muelleri was basal to the family Lubomirskiidae. The genetic distances between Lubomirskiidae species being much lower than those between Spongillidae species are indicative of their relatively recent radiation from a common ancestor. These results indicated that rDNA spacers sequences can be useful in the study of phylogenetic relationships of and the identification of species of freshwater sponges.     

Recent hybrid speciation in Cardamine (Brassicaceae) – conversion of nuclear ribosomal ITS sequences in statu nascendi

 The very recent allopolyploid speciation of Cardamine insueta and Cardamine schulzii is well documented. We used this system for a further understanding of the evolution of the internal transcribed spacers (ITS) of nuclear ribosomal DNA in recently formed hybrids. The ITS sequencing of the two parent species and the alloploid offspring suggests a synopsis of the types of ITS evolution, reported so far in the literature. We detected homogenization to one parental ITS type with a very strong bias to the maternal sequence. Nevertheless, maintainance of both parental ITS sequences in the alloploids was also recorded. Our data suggest: (1) rapid genomic change in newly formed alloploids, and (2) a multiple origin of C. insueta and C. schulzii. Received: 24 August 1998 / Accepted: 8 September 1998