Gene 249 (2000) 115–125 www.elsevier.com/locate/gene Comparative analysis of intra-individual and inter-species DNA sequence variation in salmonid ribosomal DNA cistrons Kent M. Reed *, Jeremiah D. Hackett, Ruth B. Phillips Department of Biological Sciences, University of Wisconsin–Milwaukee, Milwaukee, WI 53211, USA Received 17 December 1999; accepted 24 March 2000 Received by M. Schartl Abstract This study examines sequence divergence in three spacer regions of the ribosomal DNA (rDNA) cistron, to test the hypothesis of unequal mutation rates. Portions of two transcribed spacers (ITS-1 and 5∞ ETS) and the non-transcribed spacer (NTS) or intergenic spacer (IGS ) formed the basis of comparative analyses. Sequence divergence was measured both within an individual lake trout ( Salvelinus namaycush) and among several related salmonid species ( lake trout; brook trout, Salvelinus fontinalis; Arctic char, Salvelinus alpinus; Atlantic salmon, Salmo salar; and brown trout, Salmo trutta). Despite major differences in the length of the rDNA cistron within individual lake trout, minimal sequence difference was detected among cistrons. Interspecies comparisons found that molecular variation in the rDNA spacers did not conform to the predicted pattern of evolution (ITS spacers<ETS spacers<IGS). Specifically, the IGS contains a region that appears to be as highly, or more conserved than the ITS-1. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Intergenic spacer (IGS); Molecular evolution; Ribosomal DNA (rDNA); Salmon; Trout 1. Introduction intergenic spacer [(IGS) or non-transcribed spacer (NTS)] lies between the 28S and 18S coding regions of The theory of concerted evolution predicts that the consecutive cistrons. The extreme ends of the IGS are processes of mutation, recombination and gene conver- part of the transcriptional unit and are also referred to sion will change tandemly duplicated DNA sequences as the external transcribed spacers (5∞ ETS and 3∞ ETS). into new but homogeneous sequences. The multicopy Because the rDNA cistron is present in multiple copies, ribosomal DNA (rDNA) is a special case of gene variation in the sequence of individual cistrons will duplication and has been useful as a model for testing occur if the rate of gene conversion is insufficient to hypotheses of concerted evolution (Hillis et al., 1991). produce homogeneous copies of the rDNA cistron The ribosomal DNA of vertebrates is present in tandem across the genome. For example, studies of the tandem arrays of hundreds to thousands of copies. Each copy arrays of 5S rDNA have shown the rate of concerted of the rDNA cistron contains three rRNA coding evolution is insufficient to homogenize fully these regions (18S, 28S and 5.8S) that are separated from sequences ( Kellogg and Appels, 1995; Sajdak et al., each other by spacers. The internal transcribed spacers 1998). (ITS-1 and ITS-2) occur between the 18S and 5.8S, and Variation in the rate of molecular evolution is the 5.8S and 28S coding regions respectively. The larger observed across the rDNA cistron, with non-coding regions evolving faster than coding regions [reviewed in Gerbi (1985) and Hillis and Dixon (1991)]. This discrep- Abbreviations: ETS, external transcribed spacer; IGS, intergenic ancy occurs because mutations in non-coding regions spacer; ITS, internal transcribed spacer; NOR, nucleolus organizer region; NTS, non-transcribed spacer; PCR, polymerase chain reaction. are not likely to affect normal rRNA function, whereas * Corresponding author. Present address: Program on Comparative mutations in the coding regions could have deleterious Genomics, Department of Veterinary PathoBiology, 295 ASVM, 1988 effects for the individual. Although rRNA coding Fitch Ave, University of Minnesota, St Paul, MN 55108 USA. regions are not constrained by the necessity of maintain- Fax: +1-612-625-0204. E-mail address: reedx054@tc.umn.edu ( K.M. Reed ) ing a functional reading frame as seen for protein 0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0378-1119(00)00156-6