Length Variation of the Nuclear Ribosomal DNA Internal Transcribed Spacer in the Genus Abies,with Reference to Its Systematic Utility in Pinaceae

The internal transcribed spacer (1TS) region (1TS1, ITS2 and 5.8S rDNA) of the nuclear ribosomal DNA (nrDNA) was amplified via PCR in 28 taxa of Abies Mill. The amplified fragments showed length polymorphism among species, with species from Central America and two species from North America having a length of approximately 2 500 base pairs (bp) and the remaining taxa having a length of approximately 1 700 bp based on 100 bp and 1 kb ladder standard markers. The complete sequencing of ITS of Abies bracteata showed that the shorter type is 1 697 bp (1TS1 is 1 296 bp, 5.8S + 1TS2 is 401 bp). For the longer one, the partial rrs1 and complete 5.8S + ITS2 sequencing revealed that thelength of 5.8S + ITS2 is the same as that of the shorter type. The length difference of ITS in Abies is mainly due to the length difference in the ITS1 region, a result similar to the previous findings in other genera of Pinaceae. Variation in ITS length seems well correlated with morphological and geographic characters in Abies, suggesting that the length variation may be a phylogenetically informative character within the genus, long ITS was also found in other genera of Pinaceae in the previous studies. The long length of ITS in the family makes the sequencing of the region and subsequent alignment of sequences among species or genera more difficult than in taxa with short ITS, such as angiosperms. Although the length variation of ITS in the genus Abies is significant, the homogenous of ITS sequence between the longer one and the shorter one is obvious if the insertion in the longer ITS is ignored.     

Comparative sequence analysis of the internal transcribed spacer 1 of Ochrobactrum species

The internal 16S/23S rDNA (rrs/rrl) internal spacer region 1 (ITS1) of 54 Ochrobactrum strains and close relatives was analysed. Separation of ITS1 containing PCR products by gel-electrophoresis, DGGE, cloning and sequencing revealed ITS1 length and sequence heterogeneity. We found up to 5 different allelic ITS1 stretches within a single strain (Ochrobactrum intermedium LMG 3301T), and 2-3 different ITS1 alleles in O. tritici. Within ITS1, ITS1c, being part of the conserved double-stranded rrn processing stem dsPS1, produced the most reliable segment tree. The overall ITS1, ITS1c and rrs phylogenetic tree topologies were generally consistent, but there was evidence for horizontal rrn (segment) transfer in O. tritici LMG 2134 (formerly O. anthropi). Good correlations were found between ITS1, ITS1c and rrs sequence similarity and DNA-DNA hybridization values indicating that phylogenetic analysis of ITS1 and ITS1c both can be used to preliminarily deduce the phylogenetic affiliation if HGT was excluded. Strains sharing > 96.19% ITS1c (> 95.11% ITS1) similarity fell within a species, and < or = 68.42% ITS1c (< or = 70.33% ITS1) similarity outside a genus. Both ITS1 and ITS1c analysis resolved microdiversity more profoundly than rrs analysis and revealed clades (genomovars) within O. anthropi that were also produced in rep cluster analysis. There was no evidence for habitat-specific ITS1 genomovars within Ochrobactrum species. Diversity of Ochrobactrum was higher in soil than at the rhizoplane below and at the species level. Isolates from soil contained only 1 rrn type whereas isolates from human clinical, animal and rhizoplane specimens could contain more.     

Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene for six Babesia spp. isolated from different geographic origins were characterized. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among these isolates. Phylogenetic analysis of the ITS1-5.8S gene-ITS2 region clearly separated the isolates into two clusters. One held an unidentified Babesia sp. transmitted by Hyalomma anatolicum anatolicum. The second held five other isolates, which were considered to be Babesia motasi. Each Babesia species cluster possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. The results showed that ITS1, ITS2 and the complete ITS1-5.8S-ITS2 region could be used to discriminate these ovine Babesia spp. effectively.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

帘蛤科贝类rDNA内转录间隔区序列的研究

根据18SrDNA、5．8SrDNA和28SrDNA保守序列设计引物,应用聚合酶链式反应（PCR）扩增了文蛤（Meretrix meretrix L．）、青蛤（Cyclina sinensis G）、硬壳蛤（Mercenaria mercenaria L．）和江户布目蛤（Protothaca jedoensis L．）4种帘蛤科贝类的第一内转录间隔区（ITS1）和第二内转录间隔区（ITS2）序列,并进行了测序。结果表明,文蛤、青蛤、硬壳蛤和江户布目蛤的ITS1扩增产物大小分别为978bp、663bp、757bp和942bp,GC含量分别为61．55％、60．78％、62．48％和64．86％～64．97％,其中ITS1序列长度分别为900bp、585bp、679bp和864bp,是迄今已报道双壳贝类中变化范围最大的,GC含量分别为61．67％、61．03％、63．03％和65．51％～65．62％,江户布目蛤种内ITS1序列有个体差异;ITS2扩增产物大小分别为644bp、618～620bp、593bp和513～514bp,GC含量分别为61．18％、61．29％～61．81％、62．73％和61．48％61．60％,其中ITS2序列长度分别为412bp、386～388bp、361bp和281～282bp,GC含量分别为65．29％、65．21％～66．06％、67．87％和67．38％～67．62％,青蛤和江户布目蛤种内ITS2序列有个体差异。4种蛤ITS1和ITS2序列种间差异很大,有明显的长度多态性,ITS2种间序列相似度73．0％～89．1％,与ITS1的种间序列相似度48．7％～81．5％相比略高。此外,在4种蛤ITS1和ITS2序列中各发现2个与rRNA加工有关的保守区。通过对ITS1和ITS2序列的组装获得了4种蛤5．8SrRNA基因完整序列,序列长度都是157bp,GC含量57．96％～58．60％,4种蛤5．8SrRNA基因相对保守,种间序列差异度0-6．0％,共有10个变异位点,其中转换4处,颠换6处,硬壳蛤和江户布目蛤5．8SrRNA基因序列完全相同。以ITS2序列（包含5．8SrRNA和28SrRNA基因部分序列）为标记,调用北极蛤科的Arctica islandica相应序列数据作外群,构建了帘蛤科贝类的系统发育树,其拓扑结构显示江户布目蛤与硬壳蛤亲缘关系最近,青蛤与其他3物种的亲缘关系最远。     

Genetic diversity and molecular evolution of the internal transcribed spacer (ITSs) of nuclear ribosomal DNA in the Tunisian fig cultivars (Ficus carica L.; Moracea)

Nuclear ribosomal DNAs were explored to establish genetic relationships among Ficus carica cultivars and elucidate its molecular evolution. Results suggest the occurrence of haplotypic and nucleotide diversity. The neighbour-joining dendrograms show a continuous diversity that characterize local resources. Furthermore, our results demonstrated that the ITS2 spacer is seating to a larger number of substitutions than the ITS1 spacer. Sequence analysis demonstrates that the ITS2 spacer is evolving 1.12 times faster than the ITS1 one. The ratio of transition/transversion of 0.278 suggests that the 5.8S gene is evolving 2.84 and 3.20 times less rapidly than the spacers ITS1 and ITS2, respectively. Molecular evolution analysis confirmed an explicit rejection of the null hypothesis in F. carica. ITS1, ITS2 spacers and the 5.8S gene evolved under a strictly neutral model of molecular evolution. A scenario of positive selection and recent expansion of F. carica genotypes across Tunisia seems to be retained.     

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。     

The internal transcribed spacers and 5.8S rRNA gene show extensive diversity among isolates of the Cryptococcus neoformans species complex

Sequences of the internal transcribed spacer (ITS) region including the 5.8S rRNA gene delineated seven genotypes within the three varieties of Cryptococcus neoformans via specific combinations of eight nucleotide differences located at positions 10, 11, 15, 19, 108 (ITS1), 221 (5.8S), 298 and 346 (ITS2). The ITS types correlated to polymerase chain reaction fingerprint/random amplification of polymorphic DNA (RAPD) molecular types: with ITS type 1 (ATACTAGC)=C. neoformans var. grubii, molecular types VNI+VNII and the serotype A allele of the AD hybrid, VNIIIA; ITS type 2 (ATATAGGC)=the serotype D allele of the AD hybrid, VNIIIB, and C. neoformans var. neoformans, VNIV; and ITS type 3 (GCGCTGGC) and ITS type 7 (ACGCTGGC)=VGI=RAPD type III, ITS type 4 (ACACTGAC)=VGII=RAPD type II, ITS type 5: (ACACTGGG)=VGIII=RAPD type I, ITS type 6 (ACACTGGC)=VGIV=RAPD type IV, all corresponding to C. neoformans var. gattii. Cloned sequences from serotype AD revealed that the hybrid serotype is diploid at the ITS1-5.8S-ITS2 locus carrying the ITS type 1 (ATACTAGC) and the ITS type 2 (ATATAGGC) alleles. ITS sequencing is a useful technique for genotyping the three C. neoformans varieties and for subtyping within C. neoformans var. gattii.     

Genotyping of Pneumocystis carinii f. sp. hominis isolates in Japan based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes

Genotyping of Pneumocystis carinii (Pc) isolated from 24 bronchoalveolar lavage (BAL) fluid specimens in Japan was examined based on nucleotide sequence variations in internal transcribed spacer regions 1 and 2 (ITS1 and ITS2, respectively) of rRNA genes. We found 11 ITS1 genotypes including 2 novel ones and 11 ITS2 genotypes including 3 new ones. Combining the ITS1 and ITS2 genotypes resulted in 30 ITS genotypes, of which 10 are newly described in this report. Two or more genotypes in ITS regions in a specimen were observed in 16 of 24 patients. Our results will be of help for the epidemiological investigation of Pc infection.     

Assessment of phylogenetic inter-relationships in the genus Cymbidium (Orchidaceae) based on internal transcribed spacer region of rDNA

Sequence data obtained from nrITS region were used to assess phylogenetic inter-relationships and infrageneric classification of ten Cymbidium species collected from north-east India. The final aligned data matrix of combined ITS 1, 5.8S and ITS 2 yielded 684 characters. The ITS 1 and ITS 2 regions showed variable sequence lengths and G + C content (%). The 5.8S region was found to be more conserved (98.71%) followed by ITS 1 (86.12%) and ITS 2 (69.40%). ITS 2 recorded highest percentage of parsimony informative sites (7.46%), high sequence divergence with indels (24.63%), high number of transitions and transversions. ITS sequence data determined the phylogeny of Asiatic Cymbidiums with high bootstrap values. All three proposed subgenera could be distinguished clearly by all four (MP, ML, NJ, and BI) phylogenetic methods. This study validates the utility of ITS rDNA region as a reliable indicator of phylogenetic relationships, especially ITS 2 as probable DNA barcode at higher levels and can serve as an additional approach for identification of broader range of plant taxa especially orchids.     

Variation of the internal transcribed spacer 1 sequence within individual strains and among different strains of Neospora caninum

Small differences have been reported in the internal transcribed spacer 1 (ITS1) region among strains of Neospora caninum. We compared ITS1 sequences among 6 N. caninum strains analyzed in our laboratory, including 2 strains that have not been examined previously (NC-Illinois and NC-Bahia). Five sequences showed 100% similarity and also were identical to 7 of 11 sequences that were previously reported by others. In contrast, initial attempts to sequence the ITS1 of NC-Bahia generated 12 nucleotide differences compared with the other 5 strains, and several ambiguous bases. However, the single band containing the ITS1 region, as observed after electrophoresis on a 2% agarose gel, became divided into 2 distinct bands when reanalyzed using 5 or 10% polyacrylamide gel electrophoresis (PAGE), and the ITS1 within these separate bands were sequenced without ambiguity. The other 5 N. caninum strains were also reexamined using PAGE, and in each strain 2 distinct bands were discovered. In comparison, 2 strains of Toxoplasma gondii continued to show only 1 band when examined using PAGE. The ITS1 sequence of NC-Bahia, from Brazil, differs in several base pairs from those of North American and European strains of N. caninum. Intrastrain variation of the ITS1 region appears to be common in N. caninum, in contrast to T. gondii.     

Sequence analysis of the ribosomal DNA internal transcribed spacer region in some scallop species (Mollusca: Bivalvia: Pectinidae).

The internal transcribed spacer (ITS) region of the ribosomal DNA from the European scallops Aequipecten opercularis, Mimachlamys varia, Hinnites distortus, and Pecten maximus was PCR amplified and sequenced. For each species, three or five clones were examined. The size ranged between 636 and 713 bp (ITS1, 209-276 bp; 5.8S rRNA gene, 157 bp; ITS2, 270-294 bp) and GC content ranged between 47 and 50% (ITS1, 43-49%; 5.8S rRNA gene, 56-57%; ITS2, 44-49%). Variation within repeats was minimal; only clones from M. varia and P. maximus displayed a few variable sites in ITS2. Among scallops, including Chlamys farreri whose ITS sequence appears in databases, significant variation was observed in both ITS1 and ITS2. Phylogenetic analysis using ITS1, ITS2, or both spacer sequences always yielded trees with similar topology. Aequipecten opercularis and P. maximus grouped in one clade and the other three scallops (C. farreri, M. varia, and H. distortus) in another, where M. varia and H. distortus are the more closely related species. These results provide new insights into the evolutionary relationships of scallop species and corroborate the close evolutionary relationship between the tribes Aequipectinini and Pectinini previously deduced from 18S rDNA sequences.     

Sequence heterogeneity in the internal transcribed spacers of two rat ribosomal DNA clones

The sequence of one cloned rat rDNA segment is determined, encompassing the 3'-terminal part of 18 S rDNA (231 bp), ITS 1 (1072 bp), 5.8 S rDNA (156 bp), ITS 2 (771 bp) and the 5'-terminal part of 28 S rDNA (210 bp). Comparison with a distinct clone of the same rDNA segment reveals the identity of the rRNA gene sequences and considerable heterogeneity in both ITS 1 and ITS 2. The heterogeneities include mainly single base-pair changes (23 deletions or insertions and 14 substitutions) distributed all along the ITS sequences.     

Discordance in Variation of the ITS Region and the Mitochondrial COI Gene in the Subterranean Amphipod Crangonyx islandicus

The amphipod Crangonyx islandicus is a recently discovered species endemic to Iceland. Populations of C. islandicus are highly structured geographically and genetically. The COI and 16S mitochondrial genes confine six monophyletic groups which have diverged for up to 5 million years within Iceland, and may present two cryptic species. To investigate the potential cryptic species status we analyse here the internal transcribed spacers (ITS1 and ITS2) and compare its variation with the patterns obtained with the mtDNA. The ITS regions present much less divergence among the geographic regions in comparison with the mtDNA, distances based on ITS1 are correlated with the COI distances as well as with geographic distances, but most of the variation is observed within individuals. The variation in the ITS region appears to have been shaped both by homogenization effect of concerted evolution and divergent evolution. A duplication of 269 base pairs is found in the ITS1 of all individuals from the southern populations, its divergence from its paralog appears to predate the split of the different groups within Iceland but some evidence point to rapid diversification after the split. This duplication does not affect the secondary structures found in the 3' and 5' ends of the sequence, suggested to have a role in the excision of the ITS1. Compensatory base changes within the ITS2 sequences which have been suggested to be a species indicator were not detected.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

PCR-SSCP技术在假丝酵母属菌种鉴别中的应用

选取中国工业微生物菌种保藏管理中心(CICC)保藏的假丝酵母属的7个种30株菌, 对其rDNA的ITS1区及ITS2区进行了PCR-SSCP指纹图谱分析, 结果表明在假丝酵母属种水平的区分鉴定中, ITS1区与ITS2区的PCR-SSCP图谱均能对本研究所选7个种的菌株进行显著区分, 比较两个区段的PCR-SSCP图谱及鉴别效果, 发现ITS2区的应用效果要优于ITS1区。     

Unusual compact rDNA gene arrangements within some members of the Ascomycota: evidence for molecular co-evolution between ITS1 and ITS2.

The internal transcribed spacers of the ribosomal DNA tandem repeat were examined in members of the ascomycetous genus Sphaeronaemella. Species of Sphaeronaemella and its mitotic counterpart Gabarnaudia, have a compact rDNA gene arrangement due to unusually short internal transcribed spacer (ITS) regions. Examination of these regions from phylogenetically related taxa, Cornuvesica, Gondwanamyces, and Ceratocystis, showed that their ITS1 and ITS2 regions could be folded into central hairpin-like structures with the size reduction in species of Sphaeronaemella being due to length reduction of the main-hairpin and the loss of smaller hairpin-like structures that emanate from the main hairpin. A databank compilation, combined with newly obtained sequences, provided an ITS data set that includes sequences of 600 species belonging to the Ascomycota. Correlation analysis revealed that the sizes of ITS1 and ITS2 show a strong positive correlation, suggesting that the 2 rDNA regions have co-evolved. This supports biochemical evidence indicating that the ITS1 and ITS2 segments interact to facilitate the maturation of the rRNA precursor.     

降香黄檀的DNA条形码鉴定研究

DNA条形码技术是利用基因组中一段短的标准序列进行物种的鉴定并探索其亲缘进化关系。本研究对采自海南不同地区降香黄檀五个居群24份样品的psbA-trnH,rbcL,核ITS及ITS2序列进行PCR扩增和测序,比较各序列扩增和测序效率。种间和种内变异,采用BLAST1和邻接 (NJ) 法构建系统聚类树方法评价不同序列的鉴定能力。结果表明ITS2在所研究的材料中具有最高的扩增和测序效率,而ITS扩增效率较低。ITS2完整序列在区分黄檀属不同种间差异具有较大优势。因此可利用ITS2从分子水平区分降香黄檀与其他混伪种。     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.     

GC balance in the internal transcribed spacers ITS 1 and ITS 2 of nuclear ribosomal RNA genes

Summary The internal transcribed spacer (ITS) 1 and 2, the 5.8S rRNA gene, and adjacent 18S rRNA and 25S rRNA coding regions of two Cucurbitaceae (Cucurbita pepo, zucchini, ITS 1: 187 bp, and ITS 2: 252 bp in length, andCucumis sativus, cucumber, ITS 1: 229 bp, and ITS 2: 245 bp in length) have been sequenced. The evolutionary pattern shown by the ITSs of these plants is different from that found in vertebrates. Deletions, insertions, and base substitutions have occurred in both spacers; however, it is obvious that some selection pressure is responsible for the preservation of stem-loop structures. The dissimilarity of the 5 region of ITS 2 found in higher plants has consequences for proposed models on U3 snRNA-ITS 2 interaction in higher eukaryotes.The two investigated Cucurbitaceae species show a G+C content of ITS 1 that nearly equals that of ITS 2. An analysis of the ITS sequences reveals that in 19 out of 20 organisms published, the G+C content of ITS 1 nearly equals that of ITS 2, although it ranges from 20% to 90% in different organisms (GC balance). Moreover, the balanced G+C content of the ITSs in a given species seems to be similar to that of so-called expansion segments (ESs) in the 25/28S rRNA coding region. Thus, ITSs show a phenomenon called molecular coevolution with respect to each other and to the ESs. In the ITSs of Cucurbitaceae the balanced G+C composition is at least partly achieved by C to T transitions, via deamination of 5-methylcytosine. Other mutational events must be taken into account. The appearance of this phenomenon is discussed in terms of functional constraints linked to the structures of these spacers.