Nuclear ribosomal DNA internal transcribed spacer 1 (ITS1) in Picea (Pinaceae): sequence divergence and structure

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Abstract

The nrDNA ITS1 of Picea is 2747–3271 bp, the longest known of all plants. We obtained 24 cloned ITS1 sequences from six individuals of Picea glehnii, Picea mariana, Picea orientalis, and Picea rubens. Mean sequence divergence within these individuals (0.018 ± 0.009) is more than half that between the species (0.031 ± 0.011) and may be maintained against concerted evolution by separation of Picea 18S–26S rDNA repeats on multiple chromosomes. Picea ITS1 contains three subrepeats with a motif (5′-GGCCACCCTAGTC) that is conserved across Pinaceae. Two subrepeats are tandem, remote from the third, and more closely related and significantly more similar to one another than either is to the third subrepeat. This correlation between similarity and proximity may be the result of subrepeat duplication or concerted evolution within rDNA repeats. In inferred secondary structures, subrepeats generally form long hairpins, with a portion of the Pinaceae conserved motif in the terminal loop, and tandem subrepeats pair with one another over most of their length. Coalescence of ITS1 sequences occurs in P. orientalis but not in the other species.

Introduction

NrDNA ITS1 in Pinaceae is remarkable for its length, variability within genomes, and structural complexity. The shortest Pinaceae ITS1 is 944 bp (Pseudolarix; Vining, 1999), about 250% longer than the longest in flowering plants and ferns (Baldwin et al., 1995, Liston et al., 1996, Maggini et al., 1998), and the longest is 3271 bp (Picea abies (L.) Karst.; Maggini et al., 2000), 90% longer than the longest ITS1 of any other vascular plant (Podocarpus; Liston et al., 1996). In contrast, the longest ITS2 among Picea, Pinus, Larix, and Pseudolarix (Germano and Klein, 1999, Gernandt et al., 2001, Liston et al., 1999, Wei et al., 2003) is 245 bp, only 6% longer than the shortest (232 bp).

As a multigene family, 18S–26S rDNA is subject to concerted evolution, through which different gene copies are similar to identical within a species and yet diverge between species (Li, 1997). Concerted evolution, which is not fully understood (Elder and Turner, 1995, Li, 1997), is thought to occur primarily through unequal crossing over and gene conversion. Unlike the nrDNA of most plants, in which the repeats are uniform presumably as a result of rapid concerted evolution (Baldwin et al., 1995, Hillis and Dixon, 1991), Pinaceae nrDNA shows considerable within-individual sequence variation (Bobola et al., 1992, Bobola et al., 1996a, Bobola et al., 1996b, Germano and Klein, 1999, Gernandt and Liston, 1999, Gernandt et al., 2001, Karvonen and Savolainen, 1993). Separation of Pinaceae 18S–26S rDNA loci on numerous chromosomes (Brown et al., 1993, Brown and Carlson, 1997, Doudrick et al., 1995, Karvonen and Savolainen, 1993, Lubaretz et al., 1996, Siljak-Yakovlev et al., 2002, Vischi et al., 2003) may explain intragenomic ITS diversity in this family (Karvonen and Savolainen, 1993).

The effectiveness of concerted evolution on the ITS region in most plants makes it a primary choice for phylogeny reconstruction at lower taxonomic levels (Baldwin et al., 1995). ITS1 phylogenies in Larix, Pseudotsuga, and Tsuga were based on single sequences for each taxon or a small number of sequences for some of the taxa (Gernandt and Liston, 1999, Vining, 1999). Correspondence between ITS1 phylogenies and what is known about phylogeny from other data suggests that coalescence of ITS1 sequences occurred after speciation in these genera. Coalescence apparently occurred after divergence of the four varieties of Larix potaninii (Wei et al., 2003). This pattern does not hold for closely related taxa in Pinus subsection Cembroides, wherein different clones from the same (or different) individuals of a species are polyphyletic (Gernandt et al., 2001). The present study tests the phylogenetic utility of ITS1 in Picea with multiple, full ITS1 sequences in closely related and distantly related species.

Much of the great length and over threefold range in length of Pinaceae ITS1 is attributable to the presence of subrepeats. These blocks of similar sequence make up 10–50% of ITS1 in Pinaceae, range in number from two (Larix and Pseudolarix) to six (Pinus), and vary from 53 bp (one Picea subrepeat) to 265 bp in Tsuga (Gernandt and Liston, 1999, Gernandt et al., 2001, Maggini et al., 2000, Marrocco et al., 1996, Vining, 1999, Wei et al., 2003; this paper). These subrepeats contain a 13-bp motif, 5′-GGCCACCCTAGTC-3′, that is conserved across Pinaceae and synapomorphic for the family. This conserved motif (the “core region” of Gernandt et al., 2001) is currently known in other green plants only in the rnl exon of the mitochondrial genome of the charophyte Chaetosphaeridium globosum (Turmel et al., 2002).

Primary sequence of nrDNA spacers translates into secondary and tertiary structures that are thought to have a role in cleaving the primary transcript to yield functional rRNAs (Hadjiolova et al., 1984, van Nues et al., 1994). Inferences of rRNA secondary structure show that the two subrepeats in Larix and Pseudolarix each folds back onto itself to form long hairpins (Gernandt and Liston, 1999, Vining, 1999). Abies, Pseudotsuga, and Tsuga have three subrepeats, the two closest of which pair with one another, while the third forms a hairpin (Gernandt and Liston, 1999, Vining, 1999). Pinus ITS1 has four or five, more or less tandem subrepeats (Gernandt et al., 2001, Marrocco et al., 1996) and an isolated subrepeat (Gernandt and Liston, 1999, Gernandt et al., 2001). In secondary structure inferences, Pinus tandem subrepeats pair with one another (Gernandt et al., 2001), and the isolated subrepeat and the fifth tandem subrepeat in Pinus species with a total of six subrepeats each forms a hairpin. In these genera, when an SSR forms a hairpin, its conserved motif is partly located in the terminal loop, and when two SSRs pair with one another, their conserved motifs partly pair.

Maggini et al. (2000) found, in addition to three subrepeats containing the conserved motif, three or four subrepeats lacking the conserved motif in P. abies. These subrepeats are longer than any of the subrepeats containing the 13-bp conserved motif and are therefore, following Maggini et al. (2000), referred to as long subrepeats (LSR). Short subrepeats containing a conserved motif are hereafter called SSRs.

We studied ITS1 in four species of Picea that span much of the phylogeny of the genus as it is currently understood (Sigurgeirsson and Szmidt, 1993; Germano, Cox, Wright, Arsenault, Klein, and Campbell, unpublished). Picea mariana (Mill.) B.S.P. and Picea rubens Sargent are closely related North American species that frequently hybridize (Manley, 1972, Perron and Bousquet, 1997). Chloroplast and nuclear DNA data place these two species together in a clade with P. omorika (Pancic) Purkyne, a native of southeastern Europe. Picea glehnii (Fr. Schmidt) Masters, which occurs on East Asian Islands, is part of a large cpDNA clade of mostly Eastern Asian species plus P. abies. Lastly, Picea orientalis (L.) Link of the Caucasus region and Northeastern Turkey belongs to another large cpDNA clade that includes mostly Asian taxa.

Objectives of this study were to: (1) study intragenomic, among-individual, and species-level sequence divergence in Picea ITS1 with multiple, full sequences of closely related species, P. mariana and P. rubens, and more distantly related species, P. glehnii and P. orientalis; (2) compare the role of conserved motifs and subrepeats in ITS1 secondary structure in Picea; (3) compare Picea ITS1 secondary structure to that of other Pinaceae—Larix and Pseudotsuga (Gernandt and Liston, 1999), Pinus (Gernandt et al., 2001, Marrocco et al., 1996), plus one new full ITS1 sequence from Abies, Pseudolarix, and Tsuga—as well as one representative of the Cupressaceae (Thuja); (4) explore phylogenetic signal in ITS1 of multiple sequences from six individuals of the four species of Picea plus a published P. abies sequence (Maggini et al., 2000); and (5) examine ITS1 of putative hybrids between P. mariana and P. rubens.

Section snippets

Plant materials

Samples from four species of Picea and one species each from Abies, Pseudolarix, and Tsuga of Pinaceae and Thuja (Cupressaceae) were collected in the wild or the Arnold Arboretum (Table 1). These samples are from single individuals of each species except for P. mariana and P. rubens, which were each represented by two individuals from separate sites. We sampled two individuals that appeared to be hybrids of P. mariana and P. rubens based on morphology (Gordon, 1976, Major, 1993, Manley, 1971)

Picea ITS1 length variation

Picea ITS1 ranges from 2747 bp (the combination of P. mariana individual SB 5′ clone 7 plus 3′ clone 7) to 3271 bp (one sequence of P. abies; Maggini et al., 2000; Table 2). Mean length is 2793 (±103) for all known Picea ITS1 sequences and 2772 (±27) excluding the long P. abies (GenBank Accession No. AJ243166). Except for the long P. abies ITS1, P. orientalis ITS1 sequences are longer (range = is 2825–2831 bp, mean = 2827 ± 3) than those of all other Picea. ITS1 of the short P. abies, P. glehnii, P.

Discussion

ITS1 of Pinaceae differs radically from that of other green plants that have been studied in length, intragenomic variability, number of 18S–26S rDNA loci in diploids, secondary structural complexity, and the presence of subrepeats containing a synapomorphic conserved motif. While ITS1 in Pinaceae undoubtedly serves the same general functions as in other plants, its organization is vastly different.

The range in length of ITS1 in seven Pinaceae genera (Table 2) includes the ITS1 lengths of the

Acknowledgments

We thank the Arnold Arboretum and the Fay Hyland Arboretum of the University of Maine for Picea foliage, the University of Maine DNA seqeuncing facility for assistance with sequencing, David S. Gernandt, Anita S. Klein, and two anonymous reviewers for comments on a draft of this paper, and the late Craig W. Greene for inspiration. This research was supported by the Maine Agricultural and Forest Experiment Station. This is MAFES external publication 2770.

References (70)

  • M.S. Bobola et al.

    Five major nuclear ribosomal repeats represent a large and variable fraction of the genomic DNA of Picea rubens and P. mariana

    Mol. Biol. Evol.

    (1992)
  • M.S. Bobola et al.

    Using nuclear and organelle DNA markers to discriminate among Picea rubens, Picea mariana, and their hybrids

    Can. J. Forest Res.

    (1996)
  • M.S. Bobola et al.

    Hybridization between Picea rubens and Picea mariana: differences observed between montane and coastal island populations

    Can. J. Forest Res.

    (1996)
  • G.R. Brown et al.

    Preliminary karyotype and chromosomal localization of ribosomal DNA sites in white spruce using fluorescence in situ hybridization

    Genome

    (1993)
  • G.R. Brown et al.

    Molecular cytogenetics of the genes encoding 18S–5.8S–26S rRNA and 5S rRNA in two species of spruce (Picea)

    Theor. Appl. Genet.

    (1997)
  • E.S. Buckler IV et al.

    The evolution of ribosomal DNA: divergent paralogues and phylogenetic implications

    Genetics

    (1997)
  • E.S. Buckler IV et al.

    Zea systematics: ribosomal ITS evidence

    Mol. Biol. Evol.

    (1996)
  • E.S. Buckler IV et al.

    Zea ribosomal repeat evolution and substitution patterns

    Mol. Biol. Evol.

    (1996)
  • C.S. Campbell et al.

    Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae)

    Mol. Biol. Evol.

    (1997)
  • R. Cronn et al.

    PCR-mediated recombination in amplification products derived from polyploid cotton

    Theor. Appl. Genet.

    (2002)
  • J. Denduangboripant et al.

    High intraindividual variation in internal transcribed spacer sequences in Aeschynanthus (Gesneriaceae): implications for phylogenetics

    Proc. R. Soc. Lond. B

    (2000)
  • R.L. Doudrick et al.

    Karyotype of slash pine (Pinus elliottii var. elliottii) using patterns of fluorescence in situ hybridization and fluorescence banding

    J. Heredity

    (1995)
  • G.A. Dover

    Linkage disequilibrium and molecular drive in the rDNA gene family

    Genetics

    (1989)
  • J.J. Doyle et al.

    A rapid DNA isolation procedure for small quantities of fresh leaf tissue

    Phytochem. Bull.

    (1987)
  • J.F.J. Elder et al.

    Concerted evolution of repetitive DNA sequences in eukaryotes

    Quart. Rev. Biol.

    (1995)
  • J. Germano et al.

    Species-specific nuclear and chloroplast single nucleotide polymorphisms to distinguish Picea glauca, P. mariana and P. rubens

    Theor. Appl. Genet.

    (1999)
  • D.S. Gernandt et al.

    Internal transcribed spacer region evolution in Larix and Pseudotsuga (Pinaceae)

    Am. J. Bot.

    (1999)
  • A.G. Gordon

    The taxonomy and genetics of Picea rubens and its relationship to Picea mariana

    Can. J. Bot.

    (1976)
  • M. Gottschling et al.

    Secondary structure of the ITS1 transcript and its application in a reconstruction of the phylogeny of the Boraginales

    Plant Biol.

    (2001)
  • K.V. Hadjiolova et al.

    Localization and structure of endonuclease cleavage sites involved in processing of the rat 32S precursor to ribosomal RNA

    Biochem. J.

    (1984)
  • J.M. Hancock et al.

    ’Compensatory slippage’ in the evolution of ribosomal RNA genes

    Nucleic Acids Res.

    (1990)
  • S. Hartmann et al.

    Extensive ribosomal DNA genic variation in the columnar cactus Lophocereus

    J. Mol. Evol.

    (2001)
  • D.M. Hillis et al.

    Ribosomal DNA: molecular evolution and phylogenetic inference

    Quart. Rev. Biol.

    (1991)
  • P. Karvonen et al.

    Variation and inheritance of ribosomal DNA in Pinus sylvestris L. (Scots pine)

    Heredity

    (1993)
  • W.-H. Li

    Molecular Evolution

    (1997)
  • Cited by (0)

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