Secondary structure of nrDNA Internal Transcribed Spacers as a useful tool to align highly divergent species in phylogenetic studies.

Loreta B Freitas,Geraldo Mäder,Priscilla M Zamberlan,Sandro L Bonatto,Gustavo A Silva-Arias,Giovanna C Giudicelli

doi:10.1590/1678-4685-gmb-2016-0042

Abstract

Recently, it has been suggested that internal transcribed spacer (ITS) sequences are under selective constraints to preserve their secondary structure. Here, we investigate the patterns of the ITS nucleotide and secondary structure conservation across the Passiflora L. genus to evaluate the potential use of secondary structure data as a helpful tool for the alignment in taxonomically complex genera. Considering the frequent use of ITS, this study also presents a perspective on future analyses in other plant groups. The ITS1 and ITS2 sequences presented significant differences for mean values of the lowest energy state (LES) and for number of hairpins in different Passiflora subgenera. Statistical analyses for the subgenera separately support significant differences between the LES values and the total number of secondary structures for ITS. In order to evaluate whether the LES values of ITS secondary structures were related to selective constraints, we compared these results among 120 ITS sequences from Passiflora species and 120 randomly generated sequences. These analyses indicated that Passiflora ITS sequences present characteristics of a region under selective constraint to maintain the secondary structure showing to be a promising tool to improve the alignments and identify sites with non-neutral substitutions or those correlated evolutionary steps.

Highlights

Ribosomal DNA consists of one of the largest multigenic families in eukaryotic genomes and is present at one or several locations in arrays of tandem elements (Hillis and Dixon, 1991)
Each unit is composed of three rRNA gene regions (18S, 5.8S, and 26S) that are preceded by an external transcribed spacer, and the two internal transcribed spacers ITS1 and ITS2 separate the genes respectively (Buckler-IV et al, 1997)
Decaloba showed the longest length for ITS1 (336 base pairs - bp), as did Passiflora for ITS2 (239 bp)

Summary

Introduction

Ribosomal DNA (rDNA) consists of one of the largest multigenic families in eukaryotic genomes and is present at one or several locations in arrays of tandem elements (Hillis and Dixon, 1991). The rDNA exons are highly conserved across eukaryotic organisms, whereas the ITS regions present length variability due to point mutations and insertions/deletions (indels) (Baldwin et al, 1995; Álvarez and Wendel, 2003). In the Passiflora genus, ITS regions are the most commonly used marker for estimating the phylogenetic relationships among subgenera and species (Muschner et al, 2003; Krosnick and Freudenstein, 2005; Lorenz-Lemke et al, 2005; Koehler-Santos et al, 2006; Mäder et al, 2010; Cazé et al, 2013; Krosnick et al, 2013; Ramaiya et al, 2014), this is usually combined with other sequences in these studies

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