Extensive 5.8S nrDNA polymorphism in <Emphasis Type="Italic">Mammillaria</Emphasis> (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions

The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Non-concerted ITS evolution, early origin and phylogenetic utility of ITS pseudogenes in Pyrus

Molecular studies of 19 species of the genus Pyrus L. revealed different degrees of intra-individual polymorphism of the internal transcribed spacer (ITS 1, 5.8S rDNA and ITS 2) region due to the existence of putative non-functional copies (pseudogenes), putative recombinants and non-concerted evolution among functional copies. Different types of ITS pseudogenes displaying lower GC content and unstable secondary structure were preferentially amplified under normal PCR conditions. Functional ITS copies were successfully obtained in all investigated accessions under the modified PCR conditions. All pseudogenes were highly divergent from their corresponding functional copies and formed a monophyletic group in the phylogenetic tree based on all paralogs, indicating they were of relatively early origin. Functional ITS copies led to confused and poorly resolved phylogeny as a result of low sequence divergence, existence of unidentified ancient recombinants and a high degree of intra-individual functional ITS polymorphism, while certain types of pseudogenes and some relict pseudogenes offered more credible clues for the evolutionary history of Pyrus species.     

The rDNA ITS Region in the Lessepsian Marine Angiosperm <Emphasis Type="BoldItalic">Halophila stipulacea</Emphasis> (Forssk.) Aschers. (Hydrocharitaceae): Intragenomic Variability and Putative Pseudogenic Sequences

Halophila stipulacea is a dioecious marine angiosperm, widely distributed along the western coasts of the Indian Ocean and the Red Sea. This species is thought to be a Lessepsian immigrant that entered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869). Previous studies have revealed both high phenotypic and genetic variability in Halophila stipulacea populations from the western Mediterranean basin. In order to test the hypothesis of a Lessepsian introduction, we compare genetic polymorphism between putative native (Red Sea) and introduced (Mediterranean) populations through rDNA ITS region (ITS1-5.8S-ITS2) sequence analysis. A high degree of intraindividual variability of ITS sequences was found. Most of the intragenomic polymorphism was due to pseudogenic sequences, present in almost all individuals. Features of ITS functional sequences and pseudogenes are described. Possible causes for the lack of homogenization of ITS paralogues within individuals are discussed.     

云南山茶(Camellia reticulata) nrDNA ITS序列多态性分析

对云南山茶 (Camellia reticulata) 8个品种的核核糖体DNA内转录间隔区(nrDNA ITS)进行克隆测序,将获得的ITS序列进行GC含量、 5.8S区二级结构的稳定性、替代模式、核苷酸多样性及系统发育关系的相关分析.实验结果显示,云南山茶8个品种的ITS序列存在丰富的基因组内多态性,同时包含中性进化的假基因,表明其ITS序列逃离了一致性进化.云南山茶ITS序列多态性的原因可能来自广泛的种间杂交,以及rDNA位点在基因组中有不确定的物理位置.ITS假基因为品种的物种形成研究提供了更全面的遗传证据,同时也提示了利用ITS假基因进行系统发育分析可能会对其真实的系统关系造成影响.     

枇杷属内栽培种和部分野生种ITS序列分析

对不同栽培区的25种普通枇杷品种以及7种枇杷属野生种的ITS序列进行扩增并测序,采用邻接法和最大简约法进行系统发育树的构建并对枇杷属内不同种间的遗传关系进行了分析。结果表明:枇杷属植物ITS序列ITS1+5.8S rDNA+ITS2总长度为592 bp或594 bp,长度变化发生在ITS2。所有样本的ITS1和5.8S rDNA长度一样,都是223 bp和168 bp;而ITS2为201 bp或203 bp。5种枇杷属野生种的ITS序列长度为594 bp,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;其余2种枇杷属野生种(麻栗坡枇杷、小叶枇杷)和普通枇杷栽培种的ITS序列长度都为592 bp。所有样本ITS序列的GC含量为64.2%~64.5%,其中ITS1为64.1%~65.5%,ITS2为68.1%~72.6%。对所有样本的ITS序列比对产生44个可变位点,其中38个为简约信息位点,其中11个位于ITS1,5个位于5.8S rDNA,22个位于ITS2。最大的种间序列差异为7.7%,最小的种间差异发生在麻栗坡枇杷和小叶枇杷之间,仅为0.2%。普通枇杷种内的ITS序列差异很低,25种普通枇杷栽培种之间的序列差异为0~1.5%。所研究的枇杷属植物可分为3个分支。分支Ⅰ包括所有普通枇杷品种,分支Ⅱ包含5种野生枇杷种,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;分支Ⅲ由2个野生枇杷种(麻栗坡枇杷、小叶枇杷)组成。该研究结果表明ITS序列对枇杷种间鉴定和系统发育分析具有一定意义,但对普通枇杷栽培种间的鉴定作用不大。     

Sequence conservation in the internal transcribed spacers and 5.8S ribosomal RNA of parasitic scuticociliates Miamiensis avidus (Ciliophora, Scuticociliatia)

The scuticociliate Miamiensis avidus is a histophagous parasite that causes high mortality in cultured marine fishes, with clinical signs of severe ulcers and hemorrhages in the skeletal muscle. The internal transcribed spacer (ITS) region, which is widely used in taxonomy and molecular phylogeny because of a high degree of variation, was compared for 21 cloned strains of M. avidus (Ciliophora, Scuticociliatia). These strains were isolated from olive flounder Paralichthys olivaceus, ridged-eye flounder Pleuronichthys cornutus and spotted knifejaw Oplegnathus fasciatus in Korea and Japan. The ITS1 (140 bp), ITS2 (236 bp) and 5.8S (119 bp) regions from 21 strains were identical, indicating that these regions are highly conserved in M. avidus. Phylogenic analysis of ITS2 shows that the ciliate should be included in the Philasterida with a close relationship to Pseudocohnilembus hargisi. This study exhibits the first detailed analysis of the ITS1, 5.8 S and ITS2 rRNA regions of M. avidus.     

Application of ITS sequences of nuclear rDNA in phylogenetic and evolutionary studies of angiosperms

Nuclear rRNA genes (rDNA) in angiosperms are arranged in long tandem repeat ing units, much like those of other higher eukaryotes. Owing to rapid concerted evolution, the repeat units have homogenized or nearly so in most species. The internal transcribed spacer (ITS) of nuclear rDNA is composed of ITS1 and ITS2, which are seperated by 5.8S rDNA. The two spacers, ITS1 (187～298 bp) and ITS2 (187～252 bp), can be readily amplified by PCR and sequenced using universal primers. The sequences contain many vari able sites and potential informative sites among related species, and have been proven to be a useful molecular marker in phylogenetic and evolutionary studies of many angiosperm taxa. It can be used not only in classification and phylogenetic inferences at the levels of family, subfamily, tribe, genus and section, but also in reconstruction of reticulate evolution and de tection of the speciation via hybridization and polyploidization. But this region may not be useful for resolving phylogenetic relationships among families or taxa of higher hierarchy ow- ing to the rapid variation of the ITS sequences.     

Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan

Endosymbiotic green algae of Japanese Paramecium bursaria were phylogenetically analyzed based on DNA sequences from the ribosomal DNA operon (18S rDNA, ITS1, 5.8S rDNA, and ITS2). Phylogenetic trees constructed using 18S rDNA sequences showed that the symbionts belong to the Chlorella sensu stricto (Trebouxiophyceae) group. They are genetically closer to the C. vulgaris Beijerinck group than to C. kessleri Fott et Nováková as proposed previously. Branching order in C. vulgaris group was unresolved in 18S rDNA trees. Compared heterogeneities of 18S rDNA, ITS1, 5.8S r, and ITS2 among symbionts and two Chlorella species, indicated that the ITS2 region (and probably also ITS1) is better able to resolve phylogenetic problems in such closely related taxa. All six symbiotic sequences obtained here (approximately 4000-bp sequences of 18S rDNA, ITS1, 5.8S rDNA, and ITS2) were completely identical in each, strongly suggesting a common origin.     

米尔顿姬小蜂rDNA ITS1和ITS2的序列分析及其分子鉴定

本研究测定了米尔顿姬小蜂Anselmella miltoni Girault的rDNA ITS1和ITS2序列,以探讨其分子鉴定方法。米尔顿姬小蜂的ITS1和ITS2侧翼区（18S和5.8S）序列相对稳定,ITS1和ITS2序列存在种间差异。根据18S rDNA部分序列,利用DNAMAN的Maximum Likelihood方法构建了与膜翅目其它科的系统发育树。根据米尔顿姬小蜂ITS1和ITS2序列设计了特异性引物,应用特异性引物对样品进行了PCR扩增,扩增效果理想,采用上述特异性引物可从单头米尔顿姬小蜂稳定地扩增出明显的目的DNA条带。因此,可以采用ITS1和ITS2区的特异性对米尔顿姬小蜂进行快速的分子鉴定。     

Primers are designed for amplification and direct sequencing of ITS region of rDNA from Myxomycetes

Four new primers were designed, based on comparison of Physarum polycephalum sequences retrieved from Genbank (primers PHYS-5 and PHYS-4) and our own sequences (primers PHYS-3 and PHYS-2), to amplify the ITS regions of rDNA, including the 5.8S gene segment from Lamproderma species. Sequencing analysis shows that Lamproderma contains ITS1-5.8S-ITS2 regions of approximately 900 bp, which is similar in size to most eukaryotes. However, the corresponding region in another common myxomycete, Fuligo septica, is more than 2000 bp due to the presence of large direct-repeat motifs in ITS1. Myxomycete rDNA ITS regions are interesting both as phylogenetic markers in taxonomic studies and as model sequences for molecular evolution.     

Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia,Asteraceae): Additional information from internal transcribed spacer sequences of nuclear ribosomal DNA

The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.     

Unbiased analyses of ITS folding motifs in a taxonomically confusing lineage: Anagnostidinema visiae sp. nov. (cyanobacteria)

Cyanobacteria are diverse prokaryotic, photosynthetic organisms present in nearly every known ecosystem. Recent investigations around the world have recovered vast amounts of novel biodiversity in seldom sampled habitats. One phylogenetically significant character, the secondary folding structures of the 16S–23S ITS rDNA region, has allowed an unprecedented capacity to erect new species. However, two questions arise: Is this feature as informative as is proposed, and how do we best employ these features? Submerged sinkholes with oxygen-poor, sulfur-rich ground water in Lake Huron (USA) contain microbial mats dominated by both oxygenic and anoxygenic cyanobacteria. We sought to document some of this unique cyanobacterial diversity. Using culture-based investigations, we recovered 45 strains, of which 23 were analyzed employing 16S–23S rDNA sequences, ITS folding patterns, ecology, and morphology. With scant morphological discontinuities and nebulous 16S rDNA gene sequence divergence, ITS folding patterns were effective at articulating cryptic biodiversity. However, we would have missed these features had we not folded all the available motifs from the strains, including those with highly similar 16S rDNA gene sequences. If we had relied solely on morphological or 16S rDNA gene data, then we might well have missed the diversity of Anagnostidinema. Thus, in order to avoid conformation basis, which is potentially common when employing ITS structures, we advocate clustering strains based on ITS rDNA region patterns independently and comparing them back to 16S rDNA gene phylogenies. Using a total evidence approach, we erected a new taxon according to the International Code of Nomenclature for Algae, Fungi, and Plants: Anagnostidinema visiae.     

普通小麦基因组最可能的4个供体的ITS1和ITS2序列及其亲缘关系

对普通小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）基因组（ＡＡＢＢＤＤ）最可能的供体－Ｔ．ｕｒａｔｒｔｕＴｈｕｍ．（ＡＡ）、Ｔ．ｍｏｎｏｃｃｕｍｖａｒ．ｂｏｅｏｔｉｃｕｍ（Ｂｏｉｓｓ．）ＭＫ（ＡＡ）、ＡｅｇｉｌｏｐｓｓｐｅｌｔｏｉｄｅｓＴａｕｓｃｈ．和Ａｅ．ｔａｕｓｃｈｉｉ（Ｃｏｓｓ．（ＤＤ）的核糖体ＲＮＡ基因ＩＴＳ区进行了ＰＣＲ扩增和克隆,并测定了ＩＴＳ１和ＩＴＳ２的ＤＮＡ序列,讨论和纠正了前人     

四照花亚属的nrDNA ITS致同进化不完全

四照花亚属(Cornus subg.Syncarpea)隶属于山茱萸科山茱萸属(Cornus),我国该亚属共有5种8亚种。为探讨四照花亚属nrDNA ITS序列的致同进化不完全现象及假基因产生的可能原因,分析了该亚属4种(每种1~2个居群)共21个个体的nrDNA ITS序列。结果表明,这些类群的nrDNA ITS存在多态性,通过分析这些nrDNA ITS克隆序列的G+C含量、5.8S保守基序和二级结构最小自由能,推测其可能存在假基因。系统发育研究结果显示所有nrDNA ITS序列分成5个分支,同一个体的不同拷贝被分别置于两个甚至多个分支中,且不同分支显示了不同种间关系。四照花亚属物种个体内部存在nrDNA ITS不完全致同进化,可能归咎于不完全的世系分选(incomplete lineage sorting)、种间杂交或多倍化等进化事件,从而导致基因组内nrITS区序列出现多态性,同时也导致难以通过外部形态来划分亚属内种间界限。     

基于ITS条形码序列的刺桐姬小蜂分子鉴定

【目的】刺桐姬小蜂Quadrastichus erythrinae Kim体型小,传统的形态学鉴定方法难以快速准确识别。【方法】本研究测定了刺桐姬小蜂的rDNA ITS1和ITS2序列,根据18S rDNA部分序列,利用MEGA的最大相似法(Maximum Likehood)构建系统发育树。根据刺桐姬小蜂ITS1和ITS2序列设计了特异引物,应用特异引物对单只刺桐姬小蜂进行PCR扩增,可稳定地扩增出明显的目的DNA条带。【结果】研究表明,基于ITS基因的DNA条形码技术可以用于刺桐姬小蜂的快速准确鉴定。【结论】因此,采用ITS1和ITS2区的特异性引物可对刺桐姬小蜂进行快速分子鉴定。     

Nucleotide sequences of the 5.8S rRNA gene and internal transcribed spacer regions in carrot and broad bean ribosomal DNA

Summary Nucleotide sequences of the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of ribosomal DNA (rDNA) from two dicot plants, carrot and broad bean, were determined. These sequences were compared with those of rice, a monocot plant, and other eukaryotic organisms. Both types of ITS region in some species of Angiospermae were the shortest among all eukaryotes so far examined and showed a wide range of variation in their G+C content, in contrast to a general trend toward very high G+C content in animals. Phylogenetic relationships of plants with animals and lower eukaryotes were considered using the nucleotide sequences of carrot and broad bean 5.8S rDNA that were determined in the present study, together with that of wheat 5.8S rRNA, which has been reported previously.     

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。     

Measuring rDNA diversity in eukaryotic microbial systems: how intragenomic variation, pseudogenes, and PCR artifacts confound biodiversity estimates

Molecular approaches have revolutionized our ability to study the ecology and evolution of micro-organisms. Among the most widely used genetic markers for these studies are genes and spacers of the rDNA operon. However, the presence of intragenomic rDNA variation, especially among eukaryotes, can potentially confound estimates of microbial diversity. To test this hypothesis, bacterially cloned PCR products of the internal transcribed spacer (ITS) region from clonal isolates of Symbiodinium, a large genus of dinoflagellates that live in symbiosis with many marine protists and invertebrate metazoa, were sequenced and analysed. We found widely differing levels of intragenomic sequence variation and divergence in representatives of Symbiodinium clades A to E, with only a small number of variants attributed to Taq polymerase/bacterial cloning error or PCR chimeras. Analyses of 5.8S-rDNA and ITS2 secondary structure revealed that some variants possessed base substitutions and/or indels that destabilized the folded form of these molecules; given the vital nature of secondary structure to the function of these molecules, these likely represent pseudogenes. When similar controls were applied to bacterially cloned ITS sequences from a recent survey of Symbiodinium diversity in Hawaiian Porites spp., most variants (approximately 87.5%) possessed unstable secondary structures, had unprecedented mutations, and/or were PCR chimeras. Thus, data obtained from sequencing of bacterially cloned rDNA genes can substantially exaggerate the level of eukaryotic microbial diversity inferred from natural samples if appropriate controls are not applied. These considerations must be taken into account when interpreting sequence data generated by bacterial cloning of multicopy genes such as rDNA.     

Heterogeneity of the yeasts Zygowilliopsis californica: Z. californica var. dimennae comb. nov., stat. nov. and Z. californica var. fukushimae comb. nov., stat. nov.

A significant heterogeneity of the species Zygowilliopsis californica was revealed using RFLP-analysis of the PCR-amplified rDNA fragment spanning the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. Phylogenetic analysis of the nucleotide sequences of ITS1 and ITS2 rDNA differentiated three varieties: Z. californica var. californica, Z. californica var. dimennae, and Z. californica var. fukushimae. The most variable was the ITS2 region, where 7–26 nucleotide substitutions were revealed. The varieties formed semisterile hybrids with meiotic segregation of control markers. The limits of the phylogenetic species concept are discussed.     

基于核rDNA的ITS序列在种子植物系统发育研究中的应用

种子植物核rDNA是高度重复的串联序列,由于同步进化的力量．大多数物种中这些重复单位间已发生纯合或接近纯合。5．8S rDNA把核rDNA的内转录间隔区分为ITS1和ITS2两部分．在被子植物中ITS1的长度为165～298bp,ITS2的长度为177～266bp,而在裸子植物中ITS片段较长。且其长度变化主要由ITS1的长度变异所致。可对这两个片段PCR产物进行直接测序或克隆测序。由于ITS序列变异较快．能够提供较丰富的变异位点和信息位点,已成为被子植物较低分类阶元的系统发育和分类研究中的重要分子标记,为探讨多倍体复合体网状进化关系,异源多倍体的起源提供了重要的系统学信息．但它一般不适合科以上水平的系统学研究。裸子植物中ITS片段较长,重复序列间的纯合程度不同,测序比较困难．因此对探讨裸子植物系统发育和分类受到了一定的限制,但近年来有所发展。