Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Vienna, Austria
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Abstract |
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Introduction |
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Materials and Methods |
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GenBank Search
Drosophila virilis sequences were retrieved from GenBank in June 1998. Redundant sequences were removed, as were sequences that were obtained from a search for long simple sequence stretches (Tautz and Renz 1984
). Mitochondrial sequences were also excluded from the analysis. Only microsatellites with five or more repeats were counted. To test if the mean repeat number in D. virilis was significantly higher than that in D. melanogaster, we resampled (200 times) the number of microsatellite loci that we detected in D. virilis (239) from the D. melanogaster data set described in Bachtrog et al. (1999)
. We then calculated the mean repeat number of the resampled data sets. P values indicated the fraction of pseudoreplicates, which had at least as many repeats as observed in the D. virilis sample.
Cloning of Microsatellites
Genomic DNA was isolated from D. virilis and D. melanogaster by a high salt extraction method (Miller, Dykes, and Polesky 1988
). Genomic DNA was digested separately with AluI, HaeIII, and RsaI and subsequently pooled. Size-fractionated fragments 5001,100 bp were cloned into M13mp18. Clones carrying a microsatellite were identified by hybridization at 37°C with (GT)7G and (AG)7A oligonucleotides, which were end-labeled with
32P. Filters were washed 3 times in 5 x SSC, 0.1% SDS, at 37°C. Positive clones were identified by autoradiography. Filters from both species were processed together. Hence, the screening procedures were identical for both species. Positive clones were sequenced on an ABI 377 automated sequencer. (AT/TA)n repeats were not screened because of the self-complementarity and low Tm of an (AT/TA)n probe. Nevertheless, no dramatic bias in relative microsatellite density is expected, as the frequencies of (AT/TA)n microsatellites determined in GenBank surveys are very similar for D. melanogaster and D. virilis (28.9% vs. 33.9%).
Typing of Microsatellites
To determine microsatellite variability in D. virilis and D. lummei, 11 microsatellites mapping to the fourth complement were typed radioactively following standard protocols (Schlötterer 1998b
). In brief, end-labeled (
32P) PCR primers were used in a 10-µl reaction volume (1.5 mM MgCl2, 200 µM dNTPs, 1 µM of each primer, 50100 ng template DNA, and 0.5 U Taq polymerase). Initial denaturation for 4 min at 94°C was followed by 30 cycles of 1 min at 94°C, 1 min at 5056°C (depending on the primer combination), and 1 min at 72°C. We used a final extension of 72°C for 45 min to assure a quantitative terminal transferase activity of the Taq polymerase. PCR products were separated on a 7% denaturing polyacrylamide gel (32% formamide, 5.6 M urea). PCR products were sized by running a sizing ladder next to the amplified microsatellites (Schlötterer and Zangerl 1999
). Summary results of the microsatellite analysis are available on the authors' web page (http://i122server.vu-wien.ac.at/).
Statistical Analyses
Heterozygosity and variance in repeat number was determined with the software package Microsat (Minch et al. 1995
). Statistical tests were carried out with the SPSS software.
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Results |
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Microsatellite Density
We screened 4.2 x 103 D. melanogaster and 2.2 x 103 D. virilis clones carrying an insert. Eighteen positive clones were identified in D. melanogaster, and 65 were identified in D. virilis. Assuming an average insert size of 800 bp, this translates into 0.54 microsatellites per 100 kb in D. melanogaster and 3.64 microsatellites per 100 kb in D. virilis. While the microsatellite density obtained for D. melanogaster was lower than that in previous reports (England, Briscoe, and Frankham 1996
; Schug et al. 1998b
), the microsatellite density in D. virilis greatly exceeded all reported values for D. melanogaster. Consistent with our observation of an approximately sevenfold higher microsatellite density in D. virilis are experiments in which genomic DNA hybridized with microsatellite probes also detected a stronger hybridization signal in D. virilis (Pardue et al. 1987
).
Microsatellite Length in Cloned Microsatellites
In total, 10 D. melanogaster and 26 D. virilis microsatellite clones were sequenced. We used two different measurements to compare the lengths of the cloned microsatellites. First, the longest uninterrupted dinucleotide stretch was counted in each clone. Second, we allowed for one imperfection (base substitution, insertion, change in repeat type) in the microsatellite stretch and counted the total number of repeats. On average, D. virilis clones carried 12.7 uninterrupted repeats, while D. melanogaster clones had 12.2 repeats (table 1
). This difference, however, was not statistically significant (P = 0.37, Mann-Whitney U-test). Allowing for one imperfection in the microsatellite stretch, the average microsatellite lengths changed to 15.2 in D. virilis and 12.8 in D. melanogaster. The difference between both species was still not statistically significant (P = 0.15, Mann-Whitney U-test).
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Data Bank Survey
We conducted a GenBank search for dinucleotide microsatellites in D. virilis. Like in D. melanogaster (Schug et al. 1998b
; Bachtrog et al. 1999
), (GT/CA)n was the most frequent repeat type (45.6%) in D. virilis, followed by (AT)n (33.9%) and (GA/CT)n (19.7%). Only two (GC)n microsatellites with five repeats each were detected.
Microsatellite Density
Two hundred thirty-nine D. virilis dinucleotide microsatellites with five or more repeats were identified in approximately 0.5 Mb of nonredundant genomic sequence. Hence, the microsatellite density would be about 48 microsatellites per 100 kb, which is about twice as high as that in D. melanogaster (22 microsatellites per 100 kb; Bachtrog et al. 1999
). The microsatellite density in D. melanogaster was determined from large contiguous sequences. In contrast, for D. virilis, we concatenated short GenBank entries. Therefore, coding sequences were overrepresented in the D. virilis data set, which may explain why the GenBank survey detected only a twofold higher microsatellite density as compared to the sevenfold higher density obtained from the direct cloning experiments.
Microsatellite Length
The mean length of D. virilis microsatellites was 7.69 repeats. Compared with D. melanogaster, which has a mean length of 6.75 repeats, D. virilis microsatellites are longer. This difference is statistically significant (P < 0.005). In contrast to the estimated microsatellite density, this result is not expected to be biased by a higher representation of coding sequence in the data set. From the length distribution of microsatellites in the D. melanogaster and D. virilis genome (fig. 1
), it is apparent that short microsatellites are less abundant in D. virilis, while longer ones are slightly more frequent.
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Discussion |
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It is widely accepted that estimates of microsatellite length distributions from population data suffer from the problem of ascertainment bias (Ellegren, Primmer, and Sheldon 1995
), with the problem being that PCR primers are preferentially designed for those loci with more repeats, as they are normally more polymorphic. Direct cloning is therefore often regarded as a more objective way to characterize the length distribution of microsatellites. Hence, it is surprising that we obtained a large difference between the mean repeat number obtained from cloning experiments compared with those from GenBank surveys (table 3
). Such a difference could be caused by a cloning bias in favor of longer microsatellites. Since longer microsatellites are less abundant, the estimated density of microsatellites will be lower. In addition, heterochromatic regions of the genome, which are depauperate of microsatellite DNA (Pardue et al. 1987
), could further reduce the density of microsatellites as estimated by direct cloning.
It should be noted, nevertheless, that the mean length of a microsatellite is strongly affected by the definition of a microsatellite. We set the minimum length of a microsatellite to five repeats, which resulted in a mean length of 7.69 repeats for D. virilis microsatellites. Increasing the lower boundary to six repeats changes the mean repeat number to 8.77. Furthermore, the length distribution of microsatellites will also be affected when imperfections in the microsatellite structure are permitted. Very often, reports about the cloning of microsatellites also include interruptions in the repeat structure. GenBank surveys, however, only report the number of uninterrupted repeats. Therefore, our data suggest that microsatellite lengths should not be regarded as absolute values which can easily be compared.
Regardless of these differences, our comparison of the two species D. melanogaster and D. virilis consistently showed the same trend, strongly supporting the existence of a real biological phenomenon.
Microsatellites Are Longer in D. virilis than in D. melanogaster
In GenBank surveys, we observed that microsatellites in D. virilis are longer than those in D. melanogaster. Also, direct cloning experiments (table 3
) found a higher mean microsatellite length in D. virilis. While the latter result was not statistically significant, it has to be noted that the difference in mean repeat number between D. melanogaster and D. virilis, as inferred from the GenBank survey, is about one repeat unit. Given the large variance in repeat number (table 1
), a large sample size is required to obtain statistical significance for such a small difference in mean repeat number. Thus, we obtained a statistically significant difference only for our GenBank survey.
While a difference in mean microsatellite repeat number of less than one repeat unit may appear small given the large variance, the comparison with human data indicates that such a difference has important implications. Previously, we used 33 Mb of nonredundant genomic sequence in humans and calculated the mean repeat number of all dinucleotide microsatellites with five or more repeat units (Harr and Schlötterer 2000
). Interestingly, the obtained mean repeat number of human microsatellites is 7.64, which is almost identical to the mean repeat number observed in D. virilis (7.69). Based on the genomic length distribution of microsatellites, Kruglyak et al. (1998)
recently calculated that the slippage rate (per repeat unit) of human microsatellites is about 21-fold higher than that in D. melanogaster if the same base substitution rate is assumed. Hence, it may be expected that D. virilis microsatellites should have a higher mutation rate than D. melanogaster loci.
In agreement with the prediction of the model of Kruglyak et al. (1998)
, we found microsatellites in D. virilis to be more variable than those in D. melanogaster. The interpretation of this result, however, is complicated by the fact that the effective population size and the mutation rate determine microsatellite variability. Presuming a constant base substitution rate in D. melanogaster and D. virilis, the population sizes of both species are very similar (Hilton and Hey 1996, 1997
; Vieira and Charlesworth 1999
). Hence, it could be concluded that the higher variability of D. virilis microsatellites is the result of a higher mutation rate. Nevertheless, it has previously been shown that microsatellite mutation rate is positively correlated with repeat number (Goldstein and Clark 1995
; Schlötterer et al. 1998
; Schug et al. 1998a
; Harr and Schlötterer 2000
). Hence, longer microsatellites are more variable than short ones. In our data set, the microsatellites of D. virilis were longer on average than those of D. melanogaster (table 3 ). Accordingly, we can not determine whether the slippage rate per repeat unit of D. virilis microsatellites is higher than that in D. melanogaster.
Our observation of longer D. virilis microsatellites fits the general trend for longer repetitive sequences in this species. The neurogenic gene mastermind has high levels of cryptic simplicity in its coding region. As described for cryptic simple regions in general (Tautz, Trick, and Dover 1986
), mastermind also has a high frequency of indels between species (Newfeld, Smoller, and Yedvobnick 1991
). The comparison between D. melanogaster and D. virilis indicates a longer mastermind protein in D. virilis (1,596 vs. 1,655 amino acids). Further support for a general trend of longer repetitive DNA in D. virilis comes from the segmentation gene hunchback. Similar to mastermind, hunchback contains regions of high cryptic simplicity, and the hunchback protein in D. virilis is also longer (816 vs. 758 amino acids in D. melanogaster) (Treier, Pfeifle, and Tautz 1989
).
Consistent with the observation that the general level of repetition seems to be related to genome size (Hancock 1996
), D. virilis has a larger genome size than D. melanogaster (0.340.38 pg per haploid genome compared with 0.180.21 pg in D. melanogaster) (Powell 1997
). Even if only the euchromatic genome sizes are compared, the D. virilis genome is still approximately 36% larger than the D. melanogaster genome. The length difference between D. virilis and D. melanogaster microsatellites, however, is too small to account for the difference in genome size. More likely, size differences in long introns are an important factor in determining the genome sizes of both species (Moriyama, Petrov, and Hartl 1998
).
Why Does the Distribution of Microsatellites Differ Between Species?
Recent results indicate that the intraspecific size distribution of microsatellites is not constrained by selection, but through a size-dependent mutation mechanism: long microsatellite alleles have a downward mutation bias, while shorter alleles do not show such a trend (Schlötterer 1998a
; Harr and Schlötterer 2000
). Interestingly, this mutation pattern has been described for a wide range of species, including yeast (Wierdl, Dominska, and Petes 1997
), D. melanogaster (Schlötterer et al. 1998a
; Harr and Schlötterer 2000
), and humans (Ellegren 2000
; Xu, Peng, and Fang 2000
). Although D. melanogaster and humans share the allele-specific mutation spectrum described above, the two species differ in their mean microsatellite lengths. Harr and Schlötterer (2000)
recently suggested that each species has a characteristic (species-specific) microsatellite length at which the mutation spectrum changes. Rather than no mutation bias or a slightly upward mutation bias, as is typical for short alleles, those alleles, which are longer than the critical length, have a downward mutation bias. Assuming that different species have characteristic lengths at which the mutation behavior changes, the differences in microsatellite length distribution could be explained. This model is fully applicable to the observed difference between D. virilis and D. melanogaster. Given that long microsatellite alleles are described for both D. melanogaster and D. virilis, their mutation spectra could be compared to verify the model of Harr and Schlötterer (2000)
.
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Acknowledgements |
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Footnotes |
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1 Keywords: microsatellites
Drosophila virilis,
Drosophila melanogaster,
genome evolution
2 Address for correspondence and reprints: Christian Schlötterer, Institut für Tierzucht und Genetik, Josef-Baumann Gasse 1, 1210 Vienna, Austria. E-mail: christian.schloetterer{at}vu-wien.ac.at
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