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A secondary structural common core in the ribosomal ITS2 (internal transcribed spacer) of Culexspecies from diverse geographical locations.

ABSTRACT: In the present study, sequence and structural analysis of ITS2 region (the spacer segment between 5.8S and 28S rRNA of mature rRNA sequences) of 7 Culex species belonging to 5 different geographical locations was carried out. Alignment of the ITS2 sequence from the 7 species revealed 8 homologous domains. Four species namely C. vishnui, C. annulus, C. pipiens, C. quiquefasciatusshowed high sequence (98-100%) and RNA secondary structure similarity. The ITS2 similarity among different species is high despite their varying geographical locations. Several common features of secondary structure are shared among these species, with some of them supported by compensatory changes, suggesting the significant role by ITS2 as an RNA domain during ribosome biogenesis.

SUBMITTER: Bhargavi R

PROVIDER: S-EPMC1891629 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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A secondary structural common core in the ribosomal ITS2 (internal transcribed spacer) of Culexspecies from diverse geographical locations.

Bhargavi Ryavarapu R Vishwakarma Siddharth S Murty Upadhyayula Suryanarayana US

Bioinformation 20051005 2

In the present study, sequence and structural analysis of ITS2 region (the spacer segment between 5.8S and 28S rRNA of mature rRNA sequences) of 7 Culex species belonging to 5 different geographical locations was carried out. Alignment of the ITS2 sequence from the 7 species revealed 8 homologous domains. Four species namely C. vishnui, C. annulus, C. pipiens, C. quiquefasciatusshowed high sequence (98-100%) and RNA secondary structure similarity. The ITS2 similarity among different species is h ...[more]

PMID: 17597853

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Project description:Molecular-based taxonomy, specifically DNA barcoding, has streamlined organism identification. For land plants, the recommended 2-locus barcode of rbcL and matK is not suitable for all groups, thus the second subunit of the nuclear internal transcribed spacer (ITS2) has received attention as a possible alternative. To date, evaluations of ITS2 have mostly been limited in scope to specific plant orders/families and single source material. Prior to using ITS2 to routinely characterize land plants present in environmental samples (i.e., DNA metabarcoding), a wet lab protocol optimized for bulk sample types is needed. To address this gap, in this study we determined the broad recoverability across land plants when using published ITS2 primer pairs, and subsequently optimized the PCR reaction constituents and cycling conditions for the best two performing primer pairs (ITS2F/ITSp4 and ITSp3/ITSu4). Using these conditions, both primer pairs were used to characterize land plants present in 17 diverse soils collected from across the US. The resulting PCR amplicons were prepared into libraries and pooled for sequencing on an Illumina® MiniSeq. Our existing bioinformatics workflow was used to process raw sequencing data and taxonomically assign unique ITS2 plant sequences by comparison to GenBank. Given strict quality criteria were imposed on sequences for inclusion in data analysis, only 43.6% and 7.5% of sequences from ITS2F/ITSp4 and ITSp3/ITSu4 respectively remained for taxonomic comparisons; ~7-11% of sequences originated from fungal co-amplification. The number of orders and families recovered did differ between primer pairs, with ITS2F/ITSp4 consistently outperforming ITSp3/ITSu4 by >15%. Primer pair bias was observed in the recovery of certain taxonomic groups; ITS2F/ITSp4 preferentially recovered flowering plants and grasses, whereas ITSp3/ITSu4 recovered more moss taxa. To maximize data recovery and reduce potential bias, we advocate that studies using ITS2 to characterize land plants from environmental samples such as soil use a multiple primer pair approach.

Project description:BackgroundMost phylogenetic studies using current methods have focused on primary DNA sequence information. However, RNA secondary structures are particularly useful in systematics because they include characteristics, not found in the primary sequence, that give "morphological" information. Despite the number of recent molecular studies on octocorals, there is no consensus opinion about a region that carries enough phylogenetic resolution to solve intrageneric or close species relationships. Moreover, intrageneric morphological information by itself does not always produce accurate phylogenies; intra-species comparisons can reveal greater differences than intra-generic ones. The search for new phylogenetic approaches, such as by RNA secondary structure analysis, is therefore a priority in octocoral research.ResultsInitially, twelve predicted RNA secondary structures were reconstructed to provide the basic information for phylogenetic analyses; they accorded with the 6 helicoidal ring model, also present in other groups of corals and eukaryotes. We obtained three similar topologies for nine species of the Caribbean gorgonian genus Eunicea (candelabrum corals) with two sister taxa as outgroups (genera Plexaura and Pseudoplexaura) on the basis of molecular morphometrics of ITS2 RNA secondary structures only, traditional primary sequence analyses and maximum likelihood, and a Bayesian analysis of the combined data. The latter approach allowed us to include both primary sequence and RNA molecular morphometrics; each data partition was allowed to have a different evolution rate. In addition, each helix was partitioned as if it had evolved at a distinct rate. Plexaura flexuosa was found to group within Eunicea; this was best supported by both the molecular morphometrics and combined analyses. We suggest Eunicea flexuosa (Lamouroux, 1821) comb. nov., and we present a new species description including Scanning Electron Microscopy (SEM) images of morphological characteristics (sclerites). Eunicea flexuosa, E. pallida, E. laxispica and E. mammosa formed a separate clade in the molecular phylogenies, and were reciprocally monophyletic with respect to other Eunicea (Euniceopsis subgenus, e.g. E. tourneforti and E. laciniata) in the molecular morphometrics tree, with the exception of E. fusca. Moreover, we suggest a new diagnostic character for Eunicea, also present in E. flexuosa: middle layer sclerites > 1 mm in length.ConclusionITS2 was a reliable sequence for intrageneric studies in gorgonian octocorals because of the amount of phylogenetic signal, and was corroborated against morphological characters separating Eunicea from Plexaura. The ITS2 RNA secondary structure approach to phylogeny presented here did not rely on alignment methods such as INDELS, but provided clearly homologous characters for partition analysis and RNA molecular morphometrics. These approaches support the divergence of Eunicea flexuosa comb. nov. from the outgroup Plexaura, although it has been considered part of this outgroup for nearly two centuries because of morphological resemblance.

Dataset Information

A secondary structural common core in the ribosomal ITS2 (internal transcribed spacer) of Culexspecies from diverse geographical locations.

Publications

A secondary structural common core in the ribosomal ITS2 (internal transcribed spacer) of Culexspecies from diverse geographical locations.

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