MBE Advance Access originally published online on August 26, 2008
Molecular Biology and Evolution 2008 25(11):2251-2254; doi:10.1093/molbev/msn188
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Letters |
Intragenomic Variation of Fungal Ribosomal Genes Is Higher than Previously Thought
* Lehrstuhl für Pharmazeutische Biologie, Universität Würzburg, Würzburg, Germany
Lehrstuhl Spezielle Botanik und Mykologie, Universität Tübingen, Tübingen, Germany
E-mail: uwe.simon{at}biozentrum.uni-wuerzburg.de.
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Abstract |
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 TOP Abstract Supplementary MaterialAcknowledgementsReferences |
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Nuclear ribosomal genes in most eukaryotes are present in multiple copies and often used for taxonomic and phylogenetic analyses. We comprehensively examined intragenomic polymorphism levels of three nuclear ribosomal loci for four important plant pathogenic fungi by polymerase chain reaction amplification and cloning. Here, we show that single nucleotide polymorphisms are present in an unexpectedly high amount. This might have implications for studies of fungal evolution, phylogenetics, and population genetics. Furthermore, our work demonstrates that the majority of all ribosomal sequences obtained from one individual and gene is identical to the majority rule consensus sequence of all detected sequence variants. Due to the large number of polymorphisms found and the fact that the polymorphism level differed markedly even between ribosomal genes of one and the same individual, we assume that nuclear ribosomal genes might not always evolve in a strictly concerted manner.
Key Words: ascomycetes • cloning • evolution • majority rule consensus sequence • intragenomic polymorphism • Taq polymerase test
Ribosomal (rDNA) genes are among the earliest and most frequently used genes for phylogenetic studies (e.g., Woese et al. 1990
, Berbee and Taylor 1999, Soltis et al. 1999, Karol et al. 2001, Medina et al. 2001, James et al. 2006). rDNA is relatively conserved allowing the reconstruction of relationships of even distantly related taxa. Yet, there are rDNA regions variable enough to discriminate between species. rDNA sequences may also exhibit variation within species, which can manifest itself by different length due to insertion or deletion (indels of single or several bases) or by differing nucleotides with no change in overall base pair number.
A number of studies hint toward intraindividual variation in the noncoding internal transcribed spacers (ITS; Gandolfi et al. 2001, Wörheide et al. 2004), which can be as high as 26 % in parthenogenic nematodes (Hugall et al. 1999). But to our knowledge, only one study has focused in depth on polymorphisms present in rDNA of individual fungi (Ganley and Kobayashi 2007).
The work presented here had been stimulated by repeatedly observing wobbles (superimposed base calls) in fungal rDNA after polymerase chain reaction (PCR) amplification and sequencing. We chose three nuclear rDNA genes coding for the small ribosomal subunit (SSU), the 5.8 S subunit plus ITS 1 and 2, and the D1–D3 region of the large subunit (LSU). We wanted to address the following questions: 1) what is the level of intragenomic polymorphism (IGP)?, 2) are IGPs equally distributed along the three loci?, and 3) does the IGP level differ between species and between genes? Additionally, we conceived a Taq test to estimate the potential contribution of Taq misreadings. We chose four important plant pathogenic ascomycetes: Mycosphaerella punctiformis, Davidiella tassiana, Teratosphaeria microspora, and Phoma exigua var. exigua. Origin of strains and GenBank accession numbers are provided in table 1. Methods are explained in the Supplementary Material online.
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Our results show that most sequences from one gene and species are identical with the majority rule consensus of all sequences obtained from this gene and species. All detected IGPs were single nucleotide polymorphisms. Almost all clones featuring one or several IGPs were unique. A few IGPs occurred in up to three clones (supplementary table 1, Supplementary Material online), and 72 out of 254 polymorphic clones (28.3%) had more than 1 IGP. For example, in D. tassiana SSU, there were 20 clones with 1, 1 with 2, 4 with 3, 1 with 4, and 1 with 6 IGPs (supplementary table 1, Supplementary Material online). When pooled for all species, IGPs were more or less equally distributed along one gene (fig. 1).
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The polymorphism level p (number of IGPs per fragment length in percent) varied between 1.7% (T. microspora, LSU) and 3.7% (D. tassiana, SSU; table 2). For the same gene, p varied considerably between species. For example, p for the D1–D3 region (roughly, the first 900 bp) of the LSU ranged from 1.7% in T. microspora to 3.6% in D. tassiana (table 2). Variation between genes within one individual was lowest in T. microspora (p = 1.7% for LSU and 2.2% for ITS) and highest in M. punctiformis (p = 2.6% for SSU and 3.6% for ITS; table 2). This is mirrored in nucleotide diversity (
) values (Nei and Li 1979; table 3).
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In M. punctiformis and T. microspora ITS 1 and 2 had more IGPs than the 5.8 S gene, and the ITS region in total was more polymorphic than either SSU or LSU. Yet, this was not the case for the other two species (supplementary table 1, Supplementary Material online). However, the whole ITS region generally had a much higher proportion of transversions and indels than both SSU and LSU (fig. 2).
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Of all 338 detected IGPs, 307 (90.8%) were caused by transitions, 25 (7.4%) by transversions of the consensus nucleotides, and 6 (1.8%) by indels. Transitions from A to G (116 = 37.8% of all transitions) and from T to C (84 = 27.4%) were much more common than transitions from G to A (58 = 18.9%) or from C to T (49 = 16%). However, these relations differed markedly between genes and species (fig. 2). Potential Taq polymerase misreadings were not observed in 55 "secondary clones" from D. tassiana and 40 secondary clones from T. microspora. Yet, Taq polymerase may work differently on genomic and plasmid DNA. Therefore, the possibility of clonal sequence variation due to Taq misreadings cannot be excluded.
Our observations contrast with results obtained by whole-genome shotgun sequencing, which yielded very low polymorphism levels (Ganley and Kobayashi 2007). Accordingly, the authors concluded that rDNA repeats evolve in a strictly concerted way. Among the five fungi studied there, Aspergillus nidulans is most closely related to the species analyzed in our work. We have therefore examined 46 clones of the D1–D3 region of the LSU in A. nidulans using the same strain (FGSC-A4). We found 16 IGPs for a 917-bp fragment corresponding to p = 1.7%. One IGP was present in two clones. Only transitions were found: 11 between A and G and 5 between C and T. This is interesting because Ganley and Kobayashi (2007) reported 10 IGPs for the complete LSU and calculated a nucleotide diversity of = 0.00007 for rDNA of A. nidulans—10-fold below the value we obtained for the D1–D3 region of the LSU ( = 0.00079). Furthermore, 8 of the 11 high-confidence polymorphisms they observed in all rDNA repeats were indels, whereas only 3 were transitions. Possibly, a moderate coverage (the number of reads per nucleotide) might not be sensitive enough for a reliable detection of IGPs occurring in only one or few arrays. Wendl and Yang (2004) have estimated that a coverage of at least 13 is needed to reliably assemble eukaryote genomes, whereas the highest level reached by Ganley and Kobayashi (2007) was 7.7. Indeed, IGP levels may be considerably higher than observed in our clone samples as suggested by extrapolations using EstimateS (Colwell [2005]; see supplementary table 2, Supplementary Material online).
The relatively high IGP levels and values observed in all four species and rDNA regions as well as the intraindividual differences in p between different genes suggest that nuclear ribosomal arrays might not always evolve in a strictly concerted manner. This has already been assumed for arbuscular mycorrhizal fungal spores (Pawlowska and Taylor 2004). According to Ganley and Kobayashi (2007), rDNA repeats normally experience "rapid homogenization." This means that mutations are either deleted or multiplied during continual unequal recombination until one variant finally becomes dominant. In consequence, most of the unique polymorphisms found here should have arisen relatively recently (before homogenization). On the other hand, almost one-third of the polymorphic variants showed more than one IGP, indicating that repeats may also accumulate point mutations and, therefore, avoid homogenization to a certain degree.
High IGP levels in rDNA have been found in pro- and eukaryotes (Cilia et al. 1996, Campbell et al. 1997). In fungi, rare or absent sexual events and a "history of lab cultivation" may diminish gene variation (Ganley and Kobayashi 2007). The number of rDNA loci within the genome might influence polymorphism levels as could the absolute number of rDNA repeats. In ascomycetes, copy number differs widely. Aspergillus nidulans displays about 45 copies (Ganley and Kobayashi 2007), whereas Neurospora crassa has some 200 (Maleszka and Clark-Walker 1990). Genes coding for rRNA may form one cluster as in Saccharomyces cerevisiae or two as in Schizosaccharomyces pombe (Pasero and Marilley 1993). Furthermore, Maleszka and Clark-Walker (1990) demonstrated that slow growing variants of the ascomycetous yeast Kluyveromyces lactis had fewer rDNA copies than fast growing ones. They observed that a long vegetative growth phase of a slowly proliferating mycelium triggered the development of faster growing variants with higher copy numbers. The IGP level of rDNA in fungi could also be influenced by the karyotic state: diploid versus haploid (e.g., in yeasts), monokaryotic (vegetative mycelium of Ascomycota) versus dikaryotic (Basidiomycota), or multinucleate (as in Glomeromycota).
Extrachromosomal rDNA (EX rDNA) might contribute to gene variation. In several fungi, linear or circular plasmids have been found, usually located within mitochondria and mostly coding for polymerases (Jabaji-Hare et al. 1994, Katsuja et al. 1997). However, in ascomycetous yeasts, EX rDNA was found. It has been calculated that Saccharomyces carlsbergensis has no more than 0.5–2 such plasmids per cell (Meyerink et al. 1979). However, in S. cerevisiae, their number seems to be much higher and apparently increases exponentially with cell age, so that plasmid rDNA content could equal total yeast DNA in old cells (Sinclair and Guarente 1997). This has not been shown for filamentous fungi, but it cannot be ruled out that EX rDNA partly contributes to the observed IGPs.
One of our objectives was to test whether superimposed base calls in sequences obtained directly from PCR products had been due to polymorphisms. This did not seem to be the case. Either the relevant IGPs have not been detected or these base calls originate from errors during cycle sequencing.
In summary, our results show that the number of IGPs in fungal nuclear ribosomal genes is unexpectedly high in the species tested, which indicates that concerted evolution does not always work as strictly as hitherto assumed for these loci.
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Supplementary Material |
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Supplementary tables 1 and 2, figure 1, and the methods section are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractSupplementary MaterialAcknowledgementsReferences |
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We are grateful to Diana Kuhn for assisting with PCR. U.K.S. has been funded by the German Research Foundation Deutsche Forschungsgemeinschaft. We thank two anonymous reviewers for helpful suggestions and critical advice. U.K.S. planned and performed the experiments and M.W. conceived the test for the Taq polymerase. Both authors wrote the manuscript.
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Footnotes |
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Geoffery McFadden, Associate Editor
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