Phylogeny of Oedogoniales (Chlorophyceae, Chlorophyta) inferred from 18S rDNA sequences with emphasis on the relationships in the genus Oedogonium based on ITS-2 sequences

The phylogeny of Oedogoniales was investigated by using nuclear 18S rDNA sequences. Results showed that the genus Oedocladium, as a separated clade, was clustered within the clade of Oedogonium; whereas the genus Bulbochaete was in a comparatively divergent position to the other two genera. The relationship among the species of Oedogonium was discussed, focusing on ITS-2 phylogeny analyzed combining with some morphological characteristics. Our results showed that all the dioecious nannandrous taxa involved in this study were resolved into one clade, while all the monocious taxa were clustered into another clade as a sister group to the former. The report also suggests that the dioecious macrandrous taxa form a paraphyly and could be more basally situated than the dioecious nannandrous and the monoecious taxa by means of molecular phylogeny and morphotype investigations.

Phylogeny of diplozoids in five genera of the subfamily diplozoinae palombi, 1949 as inferred from ITS-2 rDNA sequences

The phylogenetic relationship of 5 genera, i.e. Diplozoon Nordmann, 1832, Paradiplozoon Achmerov, 1974, Inustiatus Khotenovsky, 1978, Sindiplozoon Khotenovsky, 1981, and Eudiplozoon Khotenovsky, 1985 in the subfamily Diplozoinae Palombi, 1949 (Monogenea, Polyopisthocotylea) was inferred from rDNA ITS-2 region using neighbour-joining (NJ), maximum likelihood (ML) and Bayesian methods. The phylogenetic trees produced by using NJ, ML and Bayesian methods exhibit essentially the same topology. Surprisingly, freshwater species of Paradiplozoon from Europe clustered together with species of Diplozoon, but separated from Chinese Paradiplozoon species. The results of molecular phylogeny and lower level of divergence (4(.)1-15(.)7%) in ITS-2 rDNA among Paradiplozoon from Europe and Diplozoon and, on the other hand, high level of divergence (45(.)3-53(.)7%) among Paradiplozoon species from Europe and China might indicate the non-monophyletic origin of the genus Paradiplozoon. Also, the generic status of European Paradiplozoon needs to be revised. The species of Paradiplozoon in China is a basal group in Diplozoinae as revealed by NJ and Bayesian methods, and Sindiplozoon appears to be closely related to European Paradiplozoon and Diplozoon. with their relationship to Eudiplozoon and Inustiatus being unresolved.

Triploid origin of the gibel carp as revealed by 5S rDNA localization and chromosome painting

5S ribosomal DNA (rDNA) was isolated and sequenced from the gibel carp Carassius auratus gibelio with 162 chromosomes and crucian carp Carassius auratus with 100 chromosomes, and fluorescent probes for chromosome localization were prepared to ascertain the ploidy origin and evolutionary relationship between the two species. Using fluorescence in-situ hybridization (FISH), major 5S rDNA signals were localized to the short arms of three subtelocentric chromosomes in the gibel carp and to the short arms of two subtelocentrics in the crucian carp. In addition, some minor signals were detected on other chromosomes of both species. Simultaneously, six chromosomes were microdissected from the gibel carp metaphase spreads using glass needles, and the isolated chromosomes were amplified in vitro by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). Significantly, when the DOP-PCR-generated probes prepared from each single chromosome were hybridized, three same-sized chromosomes were painted in each gibel carp metaphase, whereas only two painted chromosomes were observed in each crucian carp metaphase spread. The data indicate that gibel carp is of triploid origin in comparison with diploid crucian carp.

Is the genus Digramma synonymous to the genus Ligula (Cestoda : Pseudophyllidea)? Evidence from ITS and 5 ' end 28S rDNA sequences

The genus Digramma (Cestoda: Pseudophyllidea) described by Cholodkovsky in 1915 differs from the genus Ligula only by the number of the reproductive organs per proglottis. However, the occurrence of transitional forms in Digramma raises much confusion concerning its generic validity. In the present study, cestodes previously designated as Digramma and Ligula were collected from lakes in the lower and middle reaches of the Yangtze River, and also from Qinghai Lake on Qingzang plateau, China. The entire internal transcribed spacer of the ribosomal DNA (ITS rDNA) and 5' end of 28S rDNA were compared between the Digramma and Ligula specimens. The low level of nucleotide variation between the two genera may imply that cestodes in the genus Digramma are paraphyletic to the Ligula genus, and Digramma is a synonym of Ligula. However, whether previously identified Digramma cestodes represent different species in the genus Ligula requires further investigation.

Molecular variation of Bothriocephalus acheilognathi Yamaguti, 1934 (Cestoda : Pseudophyllidea) in different fish host species based on ITS rDNA sequences

The molecular variation in Bothriocephalus acheilognathi Yamaguti, 1934 from 11 species of freshwater fish collected in Australia, China, the Czech Republic, England and Hawaii was investigated by determining the nucleotide sequences of the internal transcribed spacer region. The length of the first and second internal transcribed spacer sequences of multiple individuals ranged from 553 to 571 bp and 553 to 615 bp, and the G + C content from 53.1 to 53.5%. The percentage sequence divergence varied between 0 and 0.9% in the ITS1 and 0 and 6.6% in the ITS2, respectively, indicating the occurrence of intraspecific variation. It is demonstrated that the fragment length variation resulted primarily from microsatellite polymorphisms present in the ITS region, especially in the ITS2 region. Phylogenetic analyses revealed that B. acheilognathi examined in this study consisted of three closely related genotypes with certain degrees of host-specificity, and the genotype representing isolates from Cyprinus carpio L. was the most common and diverse form within the species B. acheilognathi.

Characteristics of rDNA intergenic spacer region from a waterbloom cyanobacteria species Oscillatoria sp.

Sequence of rDNA intergenic spacer region (ISR) from a waterbloom cyanobacterial species Oscillatoria sp, was determined and analyzed. The results of sequence comparison showed that the spacer had a high level sequence divergence, suggesting the sequence may be a target sequence for developing cyanobacteria genus- and species-specific oligonucleotide probes. In addition, a 20bp sequence of rDNA ISR was found highly conserved in all species of cyanobacteria, which was not found in other eubacteria. This conserved sequence within a variable region indicates that it might be a functional oligonucleotide in the processing of the rRNA precursor.

贾第虫rDNA的研究

典型的真核生物有四种rRNA（18S、5.8S、28S和5SrRNA）。一般18S、5.8S和28S的基因分别由转录间隔区（ITS）隔开而位于同一个转录单位上构成一个rRNA基因拷贝，多个rRNA基因拷贝串联形成rDNA。rDNA聚集在一起构成核仁组织区（NOR），成为核仁发生的位置。5SrRNA基因除在少数真核生物（如：酵母）中是和18S、28S rRNA基因位于同一个转录单位上外，一般是处在核仁以外的区域。贾第虫一度被认为是现存最原始的真核生物。支持这一观点的一个重要证据之一就是它还不具核仁结构。那么它的rDNA与典型真核生物的相比会有怎样的特点呢？本文在基因组的水平上对贾第虫的rDNA进行了全面调查分析，并对5S rRNA及其相关蛋白进行重点研究，得到如下结果和结论： 1）贾第虫的18S rRNA（1448bp）基因和28S rRNA（2300bp）基因比其他一些真核生物的（一般为1800bp和3400bp）要小的多，甚至比一些原核生物的相应的rRNA基因还要小。不仅如此，其5.8S rRNA基因和28SrRNA基因之间的转录间隔区（ITS2）比典型真核生物的对应区域也要短得多（只有54bp），且GC含量较高。结构预测表明该间隔区不能形成在许多真核生物中所能形成的保守的二级结构。更特别的是，贾第虫基因组中的rRNA基因序列大部分都是不完整的，并且不按照18S-5.8S-28S rRNA基因顺序排列，也没有多个完整拷贝顺序排列的区域。这提示贾第虫rRNA基因可能是以一种不同于典型真核生物的方式聚集的。因此本文认为以上这些特点可能与贾第虫不能形成典型核仁结构有关。 2）本文从贾第虫基因组中鉴定出了5S rRNA基因，并实验验证了其表达及其完整基因序列所编码的5S rRNA具有典型真核生物的T型二级结构，且具有绝大多数保守位点。RT-PCR表明该基因具有转录活性。该结果否定了前人的贾第虫没有5S rRNA的实验结果。并表明贾第虫尽管很原始，但其5S rRNA基因仍然是独立存在的和单独转录的。贾第虫基因组中总共有8个5S rRNA基因拷贝（且其中还有一个拷贝具有15个bp的异常插入）这大大低于一般真核生物的拷贝数。这些5S rRNA基因也不形成串联排列的区域。 我们还在贾第虫中鉴定出在真核生物中唯一与5S rRNA接触的核糖体蛋白L5蛋白并验证了其表达，该序列与其他真核生物的L5蛋白相似性很高，这提示贾第虫在5S rRNA基因转录出核后与L5蛋白结合形成5S RNP的过程可能与典型的真核生物是一致的。此外，我们从贾第虫中鉴定不出符合典型真核生物TFIIIA因子特征的蛋白，这提示贾第虫5S rRNA的转录起始以及转录后出核的机制可能与典型真核生物不同。过去对贾第虫的研究表明高等真核生物里RNA聚合酶III所独有的四个亚基在贾第虫中找不到同源物，而这样不完整的RNA聚合酶III已经可以在贾第虫中完成5S rRNA的转录了，这表明RNA聚合酶III所独有的这些亚基可能是为了完成其他功能而进化出来的。

核rDNA-ITS序列在昆虫学研究上的应用

核糖体DNA内部转录间隔区(internal transcribed spacer,ITS)作为线粒体DNA信息的补充,在昆虫学的研究中越来越受到重视.本文描述了ITS序列的结构和特点,总结了其在品系鉴定、亲缘关系和系统发育、物种进化及扩散、昆虫与环境的关系方面的应用.品系鉴定多集中于形体学无法区分的种类;亲缘关系和系统发育的研究目的是了解物种是如何起源和进化的;而与环境的关系主要针对社会性昆虫和寄生性昆虫.最后讨论了ITS序列在应用中所存在的问题,以及造成这些问题的可能原因.

核糖体rDNA序列分析在丛枝菌根真菌研究中的应用

丛枝菌根真菌(AMF)是一类古老、专性活体营养的共生菌物,尚未获得纯培养,在一定程度上限制了人们对AMF的深入研究。以DNA分析技术为基础的分子生物学技术增加了AMF检测的敏感性与特异性,rDNA序列的同源性和变化性可更真实地反映物种之间的亲缘关系及进化地位,因而被广泛应用于AMF分类、鉴定、遗传、生态及物种多样性等研究中。本文简要综述了rDNA序列分析技术在AMF系统发育、分子检测及群落结构特征研究中的应用现状。

Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca)

Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of Crassostrea: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)-the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)-the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of Crassostrea. This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.

Sequence analysis of the ITS region and 5.8S rDNA of Porphyra haitanensis

The sequences of the ITS (internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli (NB, PT and ST) were amplified, sequenced and analyzed. In addition, the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated. The results are as follows: the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains. The sequences of ITS l were 331 by to 334 bp, while those of the 5.8S rDNA were 158 by and the sequences of ITS2 ranged from 673 by to 681 bp. The sequences of the ITS had a high level of homology (up to 99.5%) with that of P. haitanensis (DQ662228) retrieved from GenBank, but were only approximately 50% homologous with those of other species of Porphyra. The results obtained when a phylogenetic tree was constructed coincided with the results of the homology analysis. These results suggest that the three cultivated strains of P. haitanensis evolved conservatively and that the ITS showed evolutionary consistency. However, the sequences of the ITS and 5.8S rDNA of different Porphyra species showed great variations. Therefore, the relationship of Porphyra interspecies phyletic evolution could be judged, which provides the proof for Porphyra identification study. However, proper classifications of the subspecies and the populations of Porphyra should be determined through the use of other molecular techniques to determine the genetic variability and rational phylogenetic relationships.

Phylogenetic relationships of five species of Dorippinae (Crustacea, Decapoda) revealed by 16S rDNA sequence analysis

A molecular phylogeny is presented for the subfamily Dorippinae (including 9 individuals, representing 5 species and 4 genera), based on the sequence data from 16S rRNA gene. Two-cluster test between lineages in these phylogenetic trees has been performed. On the basis of rate constancy, the rate of nucleotide substitutions of 16S rDNA sequence data is estimated as 0.27% per million years. The analysis strongly supports the recognition of the Dorippinae as a monophyletic subfamily. Phylogenetic tree indicates that the subfamily Dorippinae is divided into two main clades, and genus Dorippe appears basal in the subfamily, diverging from other species 36.6 Ma ago. It is also clear that the Heikea is closely related to the genus Neodorippe. The divergence time between them is 15.8 Ma.

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo. (C) 2008 Elsevier B.V. All rights reserved.