High density of long dinucleotide microsatellites in Drosophila subobscura

We isolated 96 dinucleotide repeats with five or more tandemly repeated units from a subgenomic Drosophila subobscura library. The mean repeat unit length of microsatellite clones in D. subobscura is 15, higher than that observed in other Drosophila species. Population variation was assayed in 32-40 chromosomes from Barcelona, Spain, using 18 randomly chosen microsatellite loci. Positive correlation between measures of variation and perfect repeat length measures (mean size, most common, and longest allele) is consistent with a higher mutation rate in loci with longer repeat units. Levels of microsatellite variation measured as variance in repeat number and heterozygosity in D. subobscura were similar to those of Drosophila pseudoobscura and higher than those of Drosophila melanogaster and Drosophila simulans. Our data suggest that higher levels of microsatellite variation, and possibly density, in D. subobscura compared with D. melanogaster are due to both a higher average effective population and a higher intrinsic slippage rate in the former species.     

Quantifying ascertainment bias and species-specific length differences in human and chimpanzee microsatellites using genome sequences

Surveys of variability of homologous microsatellite loci among species reveal an ascertainment bias for microsatellite length where microsatellite loci isolated in one species tend to be longer than homologous loci in related species. Here, we take advantage of the availability of aligned human and chimpanzee genome sequences to compare length difference of homologous microsatellites for loci identified in humans to length difference for loci identified in chimpanzees. We are able to quantify ascertainment bias for a range of motifs and microsatellite lengths. Because ascertainment bias should not exist if a microsatellite selected in one species is as likely to be longer as it is to be shorter than its homologue, we propose that the nature of ascertainment bias can provide evidence for understanding how microsatellites evolve. We show that bias is greater for longer microsatellites but also that many long microsatellites have short homologues. These results are consistent with the notion that growth of long microsatellites is constrained by an upper length boundary that, when reached, sometimes results in large deletions. By evaluating ascertainment bias separately for interrupted and uninterrupted repeats we also show that long microsatellites tend to become interrupted, thereby contributing a second component of ascertainment bias. Having accounted for ascertainment bias, in agreement with results published elsewhere, we find that microsatellites in humans are longer on average than those in chimpanzees. This length difference is similar among repeat motifs but surprisingly comprises two roughly equal components, one associated with the repeats themselves and one with the flanking sequences. The differences we find can only be explained if microsatellites are both evolving directionally under a biased mutation process and are doing so at different rates in different closely related species.     

Directional evolution of size coupled with ascertainment bias for variation in Drosophila microsatellites

Species-specific differences in microsatellite locus length and ascertainment bias have both been proposed to explain differences in microsatellite variability and length usually observed when loci isolated in one species are used to survey variation in a related species. Here we provide a simple algebraic approach to independently estimate the contributions of true species-specific length differences and ascertainment bias. We apply this approach to a reciprocal-isolation microsatellite study and show contributions of both ascertainment bias and a true longer average microsatellite length in Drosophila melanogaster compared with D. simulans.     

Multiple levels of single-strand slippage at cetacean tri- and tetranucleotide repeat microsatellite loci.

Between three and six tri- and tetranucleotide repeat microsatellite loci were analyzed in 3720 samples collected from four different species of baleen whales. Ten of the 18 species/locus combinations had imperfect allele arrays, i.e., some alleles differed in length by other than simple integer multiples of the basic repeat length. The estimate of the average number of alleles and heterozygosity was higher at loci with imperfect allele arrays relative to those with perfect allele arrays. Nucleotide sequences of 23 different alleles at one tetranucleotide repeat microsatellite locus in fin whales, Balaenoptera physalus, and humpback whales, Megaptera novaeangliae, revealed sequence changes including perfect repeats only, multiple repeats, and partial repeats. The relative rate of the latter two categories of mutation was estimated at 0.024 of the mutation rate involving perfect repeats only. It is hypothesized that single-strand slippage of partial repeats may provide a mechanism for counteracting the continuous expansion of microsatellite loci, which is the logical consequence of recent reports demonstrating directional mutations. Partial-repeat mutations introduce imperfections in the repeat array, which subsequently could reduce the rate of single-strand slippage. Limited computer simulations confirmed this predicted effect of partial-repeat mutations.     

Evidence for complex mutations at microsatellite loci in Drosophila.

Fifteen lines each of Drosophila melanogaster, D. simulans, and D. sechellia were scored for 19 microsatellite loci. One to four alleles of each locus in each species were sequenced, and microsatellite variability was compared with sequence structure. Only 7 loci had their size variation among species consistent with the occurrence of strictly stepwise mutations in the repeat array, the others showing extensive variability in the flanking region compared to that within the microsatellite itself. Polymorphisms apparently resulting from complex nonstepwise mutations involving the microsatellite were also observed, both within and between species. Maximum number of perfect repeats and variance of repeat count were found to be strongly correlated in microsatellites showing an apparently stepwise mutation pattern. These data indicate that many microsatellite mutation events are more complex than represented even by generalized stepwise mutation models. Care should therefore be taken in inferring population or phylogenetic relationships from microsatellite size data alone. The analysis also indicates, however, that evaluation of sequence structure may allow selection of microsatellites that more closely match the assumptions of stepwise models.     

Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times, which cause their genome-wide underrepresentation

Microsatellites are short tandemly repeated DNA sequence motifs that are highly variable in most organisms. In contrast to mammals, long microsatellites (>15 repeats) are extremely rare in the Drosophila melanogaster genome. To investigate this paucity of long microsatellites in Drosophila, we studied 19 loci with exceptionally long microsatellite alleles. Inter- and intraspecific analysis showed that long microsatellite alleles arose in D. melanogaster only very recently. This lack of old alleles with many repeats indicated that long microsatellite alleles have short persistence times. The size distribution of microsatellite mutations in mutation-accumulation lines suggests that long alleles have a mutation bias toward a reduction in the number of repeat units. This bias causes the short persistence times of long microsatellite alleles. We propose that species-specific, size-dependent mutation spectra of microsatellite alleles may provide a general mechanism to account for the observed differences in microsatellite length between species.     

Drosophila virilis has long and highly polymorphic microsatellites

Comparative genomics is a powerful approach to inference of the dynamics of genome evolution. Most information about the evolution of microsatellites in the genus Drosophila has been obtained from Drosophila melanogaster. For comparison, we collected microsatellite data for the distantly related species Drosophila virilis. Screening about 0.5 Mb of nonredundant genomic sequence from GenBank, we identified 239 dinucleotide microsatellites. On average, D. virilis dinucleotides were significantly longer than D. melanogaster microsatellites (7.69 repeats vs. 6.75 repeats). Similarly, direct cloning of microsatellites resulted in a higher mean repeat number in D. virilis than in D. melanogaster (12.7 repeats vs. 12.2 repeats). Characterization of 11 microsatellite loci mapping to division 40-49 on the fourth chromosome of D. virilis indicated that D. virilis microsatellites are more variable than those of D. melanogaster.     

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.     

A Phylogenetic Perspective on Sequence Evolution in Microsatellite Loci

We examined the evolution of the repeat regions of three noncoding microsatellite loci in 58 species of the Polistinae, a subfamily of wasps that diverged over 140 million years ago. A phylogenetic approach allows two new kinds of approaches to studying microsatellite evolution: character mapping and comparative analysis. The basic repeat structure of the loci was highly conserved, but was often punctuated with imperfections that appear to be phylogenetically informative. Repeat numbers evolved more rapidly than other changes in the repeat region. Changes in number of repeats among species seem consistent with the stepwise mutation model, which is based on slippage during replication as the main source of mutations. Changes in repeat numbers can occur even when there are very few tandem repeats but longer repeats, especially perfect repeats led to greater rates of evolutionary change. Species phylogenetically closer to the one from which we identified the loci had longer stretches of uninterrupted repeats and more different motifs, but not longer total repeat regions. The number of perfect repeats increased more often than it decreased. However, there was no evidence that some species have consistently greater numbers of repeats across loci than other species have, once ascertainment bias is eliminated. We also found no evidence for a population size effect posited by one form of the directionality hypothesis. Overall, phylogenetic variation in repeat regions can be explained by adding neutral evolution to what is already known about the mutation process. The life cycle of microsatellites appears to reflect a balance between growth by slippage and degradation by an essentially irreversible accumulation of imperfections. Received: 13 April 1999 / Accepted: 8 September 1999     

Microsatellite variation among divergent populations of stalk-eyed flies, genus Cyrtodiopsis

Microsatellite primers are often developed in one species and used to assess neutral variability in related species. Such analyses may be confounded by ascertainment bias (i.e. a decline in amplification success and allelic variability with increasing genetic distance from the source of the microsatellites). In addition, other factors, such as the size of the microsatellite, whether it consists of perfect or interrupted tandem repeats, and whether it is autosomal or X-linked, can affect variation. To test the relative importance of these factors on microsatellite variation, we examine patterns of amplification and allelic diversity in 52 microsatellite loci amplified from five individuals in each of six populations of Cyrtodiopsis stalk-eyed flies that range from 2.2 % to 11.2% mitochondrial DNA sequence divergence from the population used for microsatellite development. We find that amplification success and most measures of allelic diversity declined with genetic distance from the source population, in some cases an order of magnitude faster than in birds or mammals. The median and range of the repeat array length did not decline with genetic distance. In addition, for loci on the X chromosome, we find evidence of lower observed heterozygosity compared with loci on autosomes. The differences in variability between X-linked and autosomal loci are not adequately explained by differences in effective population sizes of the chromosomes. We suggest, instead, that periodic selection events associated with X-chromosome meiotic drive, which is present in many of these populations, reduces X-linked variation.     

Microsatellite length differences between humans and chimpanzees at autosomal Loci are not found at equivalent haploid Y chromosomal Loci

When homologous microsatellites are compared between species, significant differences in mean length are often noted. A dominant cause of these length differences is ascertainment bias due to selection for maximum repeat number and repeat purity when the markers are being developed. However, even after ascertainment bias has been allowed for through reciprocal comparisons, significant length differences remain, suggesting that the average microsatellite mutation rate differs between species. Two classes of mechanism have been proposed: rapid evolution of enzymes involved in the generation and repair of slippage products (enzyme evolution model) and heterozygote instability, whereby interchromosomal events at heterozygous sites offer extra opportunities for mutations to occur (heterozygote instability model). To examine which of these hypotheses is most likely, we compared ascertainment bias and species length differences between humans and chimpanzees in autosomal and Y chromosomal microsatellites. We find that levels of ascertainment bias are indistinguishable, but that interspecies length differences are significantly greater for autosomal loci compared with haploid Y chromosomal loci. Such a pattern is consistent with predictions from the heterozygote instability model and is not expected under models of microsatellite evolution that do not include interchromosomal events such as the enzyme evolution model.     

Microsatellite evolution--a reciprocal study of repeat lengths at homologous loci in cattle and sheep

The application of microsatellites in evolutionary studies requires an understanding of the patterns governing their evolution in different species. The finding that homologous microsatellite loci are longer, i.e., containing more repeat units, in human and in other primates has been taken as evidence for directional microsatellite evolution and for a difference in the rate of evolution between species. However, it has been argued that this finding is an inevitable consequence of biased selection of longer-than-average microsatellites in human, because cloning procedures are adopted to generate polymorphic and, hence, long markers. As a test of this hypothesis, we conducted a reciprocal comparison of the lengths of microsatellite loci in cattle and sheep using markers derived from the bovine genome as well as the ovine genome. In both cases, amplification products were longer in the focal species, and loci were also more polymorphic in the species from which they were originally cloned. The crossing pattern that we found suggests that interspecific length differences detected at homologous microsatellite loci are the result of biased selection of loci associated with cloning procedures. Hence, comparisons of microsatellite evolution between species are flawed unless they are based on reciprocal analyses or on genuinely random selection of loci with respect to repeat length.      

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.      

Development of microsatellite loci for the endangered species Pityopsis ruthii (Asteraceae)

? Premise of the study: Microsatellite loci were developed for the endangered species Pityopsis ruthii and will permit genetic and conservation studies of the species. ? Methods and Results: A microsatellite-enriched library was used to develop 12 polymorphic microsatellite loci for P. ruthii. The loci amplified perfect and imperfect repeats with three to seven alleles per locus. Observed heterozygosity ranged from 0.05 to 0.80 and expected heterozygosity ranged from 0.23 to 0.75. ? Conclusions: These microsatellite loci provide a sufficient set of markers for further investigation of population genetics of P. ruthii.     

Microsatellite allele frequencies in humans and chimpanzees, with implications for constraints on allele size

The distributions of allele sizes at eight simple-sequence repeat (SSR) or microsatellite loci in chimpanzees are found and compared with the distributions previously obtained from several human populations. At several loci, the differences in average allele size between chimpanzees and humans are sufficiently small that there might be a constraint on the evolution of average allele size. Furthermore, a model that allows for a bias in the mutation process shows that for some loci a weak bias can account for the observations. Several alleles at one of the loci (Mfd 59) were sequenced. Differences between alleles of different lengths were found to be more complex than previously assumed. An 8-base-pair deletion was present in the nonvariable region of the chimpanzee locus. This locus contains a previously unrecognized repeated region, which is imperfect in humans and perfect in chimpanzees. The apparently greater opportunity for mutation conferred by the two perfect repeat regions in chimpanzees is reflected in the higher variance in repeat number at Mfd 59 in chimpanzees than in humans. These data indicate that interspecific differences in allele length are not always attributable to simple changes in the number of repeats.      

Microsatellite variation in a social insect

We undertook a study to explore the potential of microsatellite loci as genetic markers for investigating kinship patterns in a social waspParachartergus colobopterus. A plasmid library with small inserts was screened for several oligonucleotide repeat motifs. Positive clones were sequenced and several were selected for further work. PCR primers were constructed that would amplify the tandem repeat region and a number of female wasps were scored for variation in the number of tandem repeats at these loci. The five amplified loci were far more variable than allozymes, with an average heterozygosity of 0.35. This work was supported by NSF Grants BSR-9021514 and IBN-9210051.     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species

A search of nearly 2000 sequences from Solanaceae species in the EMBL and Genbank databases yielded 220 microsatellites. Among these were 80 microsatellites from 675 Lycopersicon entries. Dinucleotide repeats, as well as (CAA)_n and (TAA)_n repeats, were over-represented in non-coding DNA. The other trinucleotide repeats were predominantly found in exonic DNA. PCR analysis of 44 of the microsatellite-containing Lycopersicon loci identified 36 primer pairs that yielded well-scorable fragments, or groups of fragments, in L. esculentum cultivars and accessions of Lycopersicon species. Twenty-nine of these amplified bands that were polymorphic among the four Lycopersicon species. Ten primer pairs generated polymorphic bands among seven tomato cultivars. Upon examining the number of microsatellites and the degree of polymorphisms in relation to the repeat type and motif, the type of DNA the microsatellite resided in, the length of the microsatellite, and the presence of imperfections in the microsatellite, only two significant correlations were found. (i) Imperfect repeats were less polymorphic among species than perfect repeats. (ii) The percentage of loci polymorphic among cultivars increased from 6% for the shortest loci (with eight or less repeat units) to 60% for the group with the longest repeats (12 repeat units or longer). Among the species, however, all length classes contained about 83% polymorphic loci. In general, 2–4 alleles were found for each locus among the samples of the test set. In a few cases, up to eight alleles were found. A combination of these microsatellite loci can therefore be useful in distinguishing cultivars of tomato, which are genetically very closely related to each other. Received: 9 August 1996 / Accepted: 23 August 1996     

Rapid divergence of microsatellite abundance among species of Drosophila

Among major taxonomic groups, microsatellites exhibit considerable variation in composition and allele length, but they also show considerable conservation within many major groups. This variation may be explained by slow microsatellite evolution so that all species within a group have similar patterns of variation, or by taxon-specific mutational or selective constraints. Unfortunately, comparing microsatellites across species and studies can be problematic because of biases that may exist among different isolation and analysis protocols. We present microsatellite data from five Drosophila species in the Drosophila subgenus: D. arizonae, D. mojavensis, and D. pachea (three cactophilic species), and D. neotestacea and D. recens (two mycophagous species), all isolated at the same time using identical protocols. For each species, we compared the relative abundance of motifs, the distribution of repeat size, and the average number of repeats. Dimers were the most abundant microsatellites for each species. However, we found considerable variation in the relative abundance of motif size classes among species, even between sister taxa. Frequency differences among motifs within size classes for the three cactophilic species, but not the two mycophagous species, are consistent with other studied Drosophila. Frequency distributions of repeat number, as well as mean size, show significant differences among motif size classes but not across species. Sizes of microsatellites in these five species are consistent with D. virilis, another species in the subgenus Drosophila, but they have consistently higher means than in D. melanogaster, in the subgenus Sophophora. These results confirm that many aspects of microsatellite variation evolve quickly but also are subject to taxon-specific constraints. In addition, the nature of microsatellite evolution is dependent on temporal and taxonomic scales, and some variation is conserved across broad taxonomic levels despite relatively high rates of mutation for these loci.