Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Austria
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Abstract |
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Introduction |
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A very useful approach to the study of microsatellite evolution is to map sequence changes in the microsatellite onto a phylogenetic tree. Because uninterrupted microsatellites are on average more polymorphic than interrupted ones (Weber 1990
; Goldstein and Clark 1995
), there is a preference to select loci with an uninterrupted repeat in the focal species (Ellegren, Primmer, and Sheldon 1995
). Caused by this ascertainment bias, the generally emerging picture from cross-species comparisons is that microsatellite loci seem to decay with increasing phylogenetic distance through the accumulation of point mutations in the repeat array. Here, we demonstrate that interruptions of the repeat motif can also be removed, thus providing a mechanism that counteracts the decay of microsatellites.
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Material and Methods |
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Molecular Biology
PCR primers were designed from D. melanogaster sequences. Identical PCR conditions were used for all species. Primer sequences and annealing temperatures are given in table 1
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For DNA sequencing, 50 µl PCR reactions with 100 ng of genomic DNA, 1.5 mM MgCl2, 200 µM dNTPs, 1 µM of each primer, and 2.5 U Taq polymerase were gel-purified and directly sequenced in both directions using the BigDye sequencing chemistry on an ABI 377 automated sequencer. The sequences have been deposited in GenBank (accession numbers AJ246181AJ246213).
Data Analysis
To test for orthology, we amplified the microsatellite with an alternative set of primers and sequenced the PCR product. In all cases, the sequences obtained with both primer sets were identical for each species.
Sequences were aligned using CUSTAL W (Thompson, Higgins, and Gibson 1994
). A parsimony criterion was used for the alignment of the repetitive microsatellite structure. Considering that base substitutions in the microsatellite repeat are less frequent than slippage mutations, the alignment of the microsatellite region was constructed by reducing the number of base substitutions in the repeat while allowing for indels to account for variation in repeat number (which is generated by DNA slippage). For one locus (DMTOR), sufficient flanking sequence was available to reconstruct the phylogeny of this locus. This alignment is available from the authors' webpage (http://i122server.vu-wien.ac.at). The microsatellite itself (i.e., positions 5191 of the D. melanogaster sequence AJ246186) and additional repetitive sequences in the flanking region that showed length variability across species (i.e., positions 247298, 360369, 408416, and 468478 of the D. melanogaster sequence AJ246186) were excluded from the analysis. The phylogenetic tree was obtained by applying the maximum-likelihood criterion using the Puzzle, version 4.0.1 (Strimmer and von Haeseler 1996
), software and the Tamura-Nei model for sequence evolution (Tamura and Nei 1993
). The obtained phylogenetic grouping was largely consistent with another data set based on rDNA sequences (Pélandakis and Solignac 1993
). As the locus DMTOR did not amplify in the species Drosophila ananassae and Drosophila kikkawai, we included these species in the tree according to the rDNA phylogeny (fig. 1 ).
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Results |
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Locus DMTOR
To determine whether an interruption of a microsatellite sequence represents an ancestral or derived state, a reliable phylogeny is required. We used the flanking sequence at DMTOR to reconstruct the phylogenetic grouping of the species in which DMTOR could be amplified (fig. 1
). The obtained species phylogeny of locus DMTOR is largely consistent with a previously published phylogeny (Pélandakis and Solignac 1993
).
The (CA)n repeat motif of locus DMTOR is conserved in the melanogaster subgroup and some more distantly related species (fig. 2
). At character 27, the (CA)n microsatellite is interrupted by a T (fig. 2
). Figure 3A
shows the character evolution of this substitution. Within the melanogaster subgroup, all species except D. melanogaster harbor a T at position 27, indicating that T represents the ancestral character state. Thus, D. melanogaster has lost the T substitution and gained an uninterrupted stretch of microsatellite repeats. Sequencing of four D. melanogaster alleles with different repeat numbers always revealed an uninterrupted microsatellite. Sequence analysis of Drosophila mauritiana (N = 4) showed that the AG substitution at position 37 was not fixed; two individuals did not carry this interruption. Most likely, this is not the result of a purification event in the microsatellite, but a recent substitution which is specific to D. mauritiana. The microsatellite region of more distantly related species cannot be aligned unambiguously (homologous positions could not be identified with certainty) in the microsatellite region; therefore, no statement about gain or loss of interruptions in the microsatellite can be made. It should be noted, however, that the most distantly related species, Drosophila eugracilis and Drosophila elegans, have a relatively long stretch of seven or nine uninterrupted CA repeats, respectively, possibly indicating that some mechanisms are counteracting base substitutions in the microsatellite.
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A second interruption in the microsatellite (an insertion of AG at positions 45 and 46) is conserved across the melanogaster and takahashii subgroup. While this interruption is fixed in the melanogster subgroup, Drosophila mimetica and D. prostipennis have lost it. Mapping this character on the phylogenetic tree (fig. 3D ) suggests that this interruption has been lost twice (in D. mimetica and in D. prostipennis). Most important, phylogenetic analysis of the flanking sequence of microsatellite locus DS06335b resulted in the same topology for the suzuki/takahashii group (not shown). Thus, it can be excluded that the repeated loss of an interruption in the microsatellite is an artifact of an inaccurate phylogenetic assumption (i.e., that the gene tree of DMTOR differs from the gene tree of DS06335b).
For comparison, we also included the sequences of two more distantly related Drosophila species, D. ananassae and D. kikkawai. Drosophila kikkawai, the most distantly related species, has a long microsatellite with eight uninterrupted CA repeats at this locus. Interestingly, only the focal species, D. melanogaster, carries a longer uninterrupted stretch of repeats.
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Discussion |
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While the obtained high bootstrap support values may suggest that noncoding sequences, such as flanking regions of microsatellites, are potentially useful for phylogenetic reconstruction, determination of the correct alignment of these regions can be quite challenging.
How General Is the Phenomenon of Microsatellite Purification?
In this study, we report evidence for microsatellite purification at three different loci. These data were extracted from a survey of 53 microsatellites in the melanogaster group (unpublished data). In total, approximately 300 locus/species combinations were amplified and sequenced. It is difficult to infer the frequency of purification events from this data set without certainty about the ancestral state of the imperfection in the microsatellite. Very often, this proof was not possible, because either the relevant species did not amplify or the homologous positions could not be unambiguously identified, particularly for more diverged taxa. Thus, we did not include them in this report.
Purification of imperfections in a microsatellite repeat was also described in other studies. The analysis of a dinucleotide (DQCAR) in a phylogenetic framework identified the loss of an interruption at the allele DQB1 *0202 (Jin et al. 1996
; Taylor, Durkin, and Breden 1999
). Laken et al. (1997)
described the loss of an interrupting base substitution in a mononucleotide run in the APC (adenomatous polyposis coli) gene, which was associated with colorectal cancer. Similarly, two reports which studied the evolution of an interrupted microsatellite in a plasmid in yeast also identified mutational events which restored a pure microsatellite stretch (Petes, Greenwell, and Dominska 1997
; Maurer, O'Callaghan, and Livingston 1998
). Hence, purification of an imperfect microsatellite is apparently a general phenomenon of microsatellite DNA and needs to be considered for the long-term evolution of microsatellite DNA.
Mechanisms of Microsatellite Purification
Average rates of DNA slippage mutation in D. melanogaster are on the order of 10-6 (Schug, Mackay, and Aquadro 1997
; Schlötterer et al. 1998
), which is about 100 times as frequent as base substitutions (Powell 1997) and 1,000 times as frequent as deletions (Petrov and Hartl 1998
). Hence, the high frequency of DNA slippage makes this mutation process a good candidate for the removal of interruptions in a microsatellite array. Purification may occur through the following mechanism: the template strand slips back before the interruption is synthesized on the new DNA strand, and the interruption is placed upstream of the 3' end of the nascent DNA strand (fig. 7
). When DNA synthesis continues on the new DNA strand, this strand loses the interruption in the microsatellite. Experimental evidence from microsatellites with imperfections strongly suggests that microsatellite interruptions are lost by an intramolecular process, such as DNA slippage (Petes, Greenwell, and Dominska 1997
; Maurer, O'Callaghan, and Livingston 1998
). Nevertheless, the important difference between the plasmid-based studies and our observations in D. melanogaster is that the plasmids carried a large number of repeats (>25 repeats). While large deletions are common in plasmid systems with long microsatellites (mean deletion size of 12.8 repeats [Petes, Greenwell, and Dominska 1997
] or 51.5 repeat units [Maurer, O'Callaghan, and Livingston 1998
]), they are not expected to be a major evolutionary factor in D. melanogaster, because the microsatellites in this species are, on average, shorter than the deletions observed in the plasmid studies (Schug et al. 1998b
; Bachtrog et al. 1999
).
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In principle, the slippage process leading to the removal of an interruption in the microsatellite repeat does not differ from a slippage event, which merely alters the repeat number. Comparisons of microsatellite mutation rates for interrupted and pure microsatellites indicate that interrupted microsatellites have a lower mutation rate (Weber 1990
; Jin et al. 1996
; Petes, Greenwell, and Dominska 1997
). One possible explanation for this phenomenon is that microsatellite mutation rates are length- dependent (Jin et al. 1996
; Wierdl, Dominska, and Petes 1997
; Brinkmann et al. 1998
; Schlötterer et al. 1998
) and that the mutation rate increases more than would be expected by a linear function of microsatellite length (Wierdl, Dominska, and Petes 1997
; Brinkmann et al. 1998
). Because the interruption of a microsatellite is often asymmetric, the mutation generates two arrays of different lengths. Studies of interrupted microsatellites showed that most of the slippage events occur in the longer stretch of the subdivided microsatellite (Loridon et al. 1998
; Schlötterer and Zangerl 1999
). Thus, the lower mutation rate of interrupted microsatellites and the predominance of slippage mutations in its longer stretch strongly suggest that an interruption in the microsatellite poses a significant barrier to DNA slippage. Hence, despite the general similarity to normal slippage events, it can be assumed that purification events occur more rarely than per-repeat-unit slippage events in an uninterrupted microsatellite.
Implications for the Long-Term Evolution of Microsatellites
The simplest model of microsatellite evolution assumes that a random mutation process generates short "proto" microsatellites. With an increasing repeat number, the probability of a DNA slippage event increases. Under the conservative assumption that the gain of repeat units is as likely as their loss, there is a certain probability that DNA slippage can generate a long stretch of microsatellite DNA. Similarly, DNA slippage may also reduce the length to a few repeat units. Theoretical studies using general models of microsatellite evolution indicated that the persistence time of microsatellites can be quite large (Tachida and Iizuka 1992
; Stephan and Kim 1998
).
With increasing persistence times, base substitutions in the microsatellite array become an important evolutionary factor. Provided that interruptions in a microsatellite repeat have a dramatic impact on its mutational behavior, base substitutions in a microsatellite repeat have been regarded as an essential component determining their long-term evolution. While several studies have assumed that selection maintains microsatellites below a certain size, it has recently been suggested that base substitution in the microsatellite array would also prevent infinite growth of microsatellites (Bell and Jurka 1997
; Kruglyak et al. 1998
). Hence, the length distribution of microsatellites is assumed to be an equilibrium state affected by the combination of two mutation processes occurring at different rates: DNA slippage and base substitutions. Even if a biased mutation model is accepted, under which the gain of repeat units is more likely than the loss of repeat units, computer simulations have shown that interruptions in the microsatellite are an effective mechanism for prevention of infinite growth of microsatellites (Palsbøll, Bérubé, and Jørgensen 1999
).
Under the models used by Bell and Jurka (1997)
, Kruglyak et al. (1998)
, and Palsbøll, Bérubé, and Jørgensen (1999)
, interruptions in the microsatellite can be lost by back mutations only. Provided that the rate of base substitutions or non-DNA slippage-based indels is much lower than DNA slippage rates, interruptions of a microsatellite are effectively unidirectional if slippage-based removal of microsatellite interruptions is not considered. A recent study put an even stronger emphasis on the consequences of an interruption in a microsatellite (Taylor, Durkin, and Breden 1999
). Based on known phylogenies, Taylor, Durkin, and Breden (1999)
used seven loci to map changes in the microsatellite stretch on these phylogenies and concluded that once an interruption of a microsatellite occurred, the locus degenerated quickly. This model of interruption-induced microsatellite decay provides an interesting hypothesis, even though it remains unknown whether the persistence time would have been longer without the initial interruption.
Consistent with previous observations, we also detected several interruptions in the microsatellite. Our data, however, indicate that an interruption is often a transitory state in the evolution of a microsatellite locus and does not always result in its degeneration. Interestingly, even though the orthologous microsatellite sequence from distantly related species (such as D. elegans [DMTOR] or D. kikkawai [DS06335b]) was not completely pure, parts of uninterrupted microsatellite DNA of lengths similar to that found in the focal species D. melanogaster could still be observed.
The evolutionary importance of purging interruptions in a microsatellite array is essentially dependent on their frequency. Based on our data set, we were able to demonstrate that the phenomenon exists, but whether this is a rare or frequent event remains open to further investigations.
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Acknowledgements |
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Footnotes |
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1 Keywords: Drosophila
microsatellite
interruption
slippage
2 Address for correspondence and reprints: Christian Schlötterer, Institut für Tierzucht und Genetik, Veterinärplatz 1, 1210 Wien, Austria. E-mail: christian.schloetterer{at}vu-wien.ac.at
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