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.     

Complex mutations in a high proportion of microsatellite loci from the protozoan parasite Plasmodium falciparum

Microsatellite loci are generally assumed to evolve via a stepwise mutational process and a battery of statistical techniques has been developed in recent years based on this or related mutation models. It is therefore important to investigate the appropriateness of these models in a wide variety of taxa. We used two approaches to examine mutation patterns in the malaria parasite Plasmodium falciparum: (i) we examined sequence variation at 12 tri-nucleotide repeat loci; and (ii) we analysed patterns of repeat structure and heterozygosity at 114 loci using data from 12 laboratory parasite lines. The sequencing study revealed complex patterns of mutation in five of the 12 loci studied. Alleles at two loci contain indels of 24 bp and 57 bp in flanking regions, while in the other three loci, blocks of imperfect microsatellites appear to be duplicated or inserted; these loci essentially consist of minisatellite repeats, with each repeat unit containing four to eight microsatellites. The survey of heterozygosity revealed a positive relationship between repeat number and microsatellite variability for both di- and trinucleotides, indicating a higher mutation rate in loci with longer repeat arrays. Comparisons of levels of variation in different repeat types indicate that the mutation rate of dinucleotide-bearing loci is 1.6-2.1 times faster than trinucleotides, consistent with the lower mean number of repeats in trinucleotide-bearing loci. However, despite the evidence that microsatellite arrays themselves are evolving in a manner consistent with stepwise mutation model in P. falciparum, the high frequency of complex mutations precludes the use of analytical tools based on this mutation model for many microsatellite-bearing loci in this protozoan. The results call into question the generality of models based on stepwise mutation for analysing microsatellite data, but also demonstrate the ease with which loci that violate model assumptions can be detected using minimal sequencing effort.     

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.     

Characterization of microsatellites and development of chromosome specific STMS markers in bread wheat

Microsatellites, or simple sequence repeats (SSRs), have become the markers of choice for genetic studies with many crop species including wheat. Currently an international effort is underway to enrich the repertoire of available sequence tagged microsatellite site (STMS) markers in wheat. As a part of this effort, we have sequenced 43 clones obtained from a microsatellite-enriched wheat genomic library; 34 clones contained 41 different microsatellites. These microsatellites (mono-, di-, tri- nucleotide repeats) were classified as 19 simple perfect, 18 simple imperfect and 4 compound imperfect types. Dinucleotide repeats were the most abundant (70%). Primer pairs for only 16 microsatellites could be designed, since the flanking sequences of the others were either too short or were otherwise not suitable for designing the microsatellite specific primers. Microsatellite loci of the expected size and polymorphism were successfully amplified from 15 of these 16 primer pairs using three wheat varieties. 14 loci detected by 12 out of the 15 functional primer pairs were assigned to 11 specific chromosomes. An erratum to this article is available at .     

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.     

Use of polymorphic short and clustered coding-region microsatellites to distinguish strains of Candida albicans

Abstract We describe the identification of polymorphic microsatellite loci in the pathogenic yeast, Candida albicans . A search for all coding-region microsatellites with more than four repeats that can be found in Candida sequences in GenBank was conducted. Nine such microsatellite sequences consisting of trinucleotide motifs were found. Three of these were perfect microsatellites while the remaining six sequences were found in one imperfect microsatellite and two compound microsatellites. Because of the close proximity of some of these repeats, all could be assayed with six PCR primer pairs. All of these microsatellite sequences were found in five nuclear genes, ZNF1, CCN1, CPH1, EFG1 , and MNT2 . Except for a single (CTT)₅ serine tract, all coded for polyglutamine tracts. Another locus with seven alleles, a region of the ERK1 protein kinase gene, was also examined, and may be a representative of a new class of highly polymorphic ‘clustered’ microsatellites. Such loci, in which several non-contiguous but closely linked microsatellites are clustered together, may be a useful source of DNA polymorphisms in microorganisms in which long microsatellite sequences are unavailable. All seven regions amplified were polymorphic, having between two and seven variable length alleles in the 11 strains of Candida albicans examined. The results of this and similar searches will facilitate epidemiological and evolutionary studies of Candida and other microorganisms.     

Characterization of microsatellite loci in Anopheles flavirostris,the principal malaria vector in the Philippines

Microsatellites were isolated and characterized from Anopheles flavirostris, the principal malaria vector in the Philippines. Fifty of the 150 positive clones sequenced contained mostly dinucleotide microsatellites and only 16 had trinucleotide repeats. We designed primers from the unique sequences flanking 18 microsatellite loci. Of these, 11 loci produced successful amplification and revealed high levels of polymorphism; 86 alleles were detected with allele number ranging from 2 to 16 at each locus. The high allelic variability will make these microsatellite loci very useful for taxonomic and population genetic studies.     

Survey and analysis of microsatellites in the silkworm, Bombyx mori: frequency, distribution, mutations, marker potential and their conservation in heterologous species

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of >/=15 bases of mononucleotide repeats and >/=5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2-14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama.     

Microsatellite evolution: polarity of substitutions within repeats and neutrality of flanking sequences.

Though extensively used in a variety of disciplines, the evolutionary pattern of microsatellite sequences is still unclear. We addressed several questions relating to microsatellite evolution by analysing historically accumulated mutation events in a large set of artiodactyl (CA)n repeats, through sequence analysis of orthologous bovine and ovine loci. The substitution rate in microsatellite flanking sequences was not different from that in intron sequences, suggesting that if intron sequences in general are selectively neutral, sequences close to microsatellites are similarly so. This observation thus does not support the idea that successful heterologous amplification of microsatellites across distantly related taxa would be due to flanking sequences generally being under some form of selection. Interestingly, the substitution rate at the first nucleotide positions flanking repeats was significantly higher than in sequences further away. Moreover, the substitution rate in repeat units in the very end of microsatellites was significantly higher than that in the middle of repeat regions. Together these observations suggest a relative instability close to the boundary between repetitive and unique sequences. We present three models that potentially could explain such a feature, all involving inefficiency of mismatch repair systems.     

Simultaneous estimation of all the parameters of a stepwise mutation model.

Minisatellite and microsatellite are short tandemly repetitive sequences dispersed in eukaryotic genomes, many of which are highly polymorphic due to copy number variation of the repeats. Because mutation changes copy numbers of the repeat sequences in a generalized stepwise fashion, stepwise mutation models are widely used for studying the dynamics of these loci. We propose a minimum chi-square (MCS) method for simultaneous estimation of all the parameters in a stepwise mutation model and the ancestral allelic type of a sample. The MCS estimator requires knowing the mean number of alleles of a certain size in a sample, which can be estimated using Monte Carlo samples generated by a coalescent algorithm. The method is applied to samples of seven (CA)n repeat loci from eight human populations and one chimpanzee population. The estimated values of parameters suggest that there is a general tendency for microsatellite alleles to expand in size, because (1) each mutation has a slight tendency to cause size increase and (2) the mean size increase is larger than the mean size decrease for a mutation. Our estimates also suggest that most of these CA-repeat loci evolve according to multistep mutation models rather than single-step mutation models. We also introduced several quantities for measuring the quality of the estimation of ancestral allelic type, and it appears that the majority of the estimated ancestral allelic types are reasonably accurate. Implications of our analysis and potential extensions of the method are discussed.SINCE the discovery that a large number of loci with tandemly repeated sequences in human and many eukaryote species are highly polymorphic because of copy number variation of the repeats in different individuals (Jeffreys 1985; Litt and Luty 1989; Weber and May 1989), allele size data from such loci are rapidly becoming the dominant source of genetic markers for genome mapping, forensic testing, and population studies. Loci with repeat sequences longer than 5 bp are generally referred to as minisatellite or variable number tandem repeat loci, and those with repeat sequences between 2 to 5 bp are referred to as microsatellite or short tandem repeat loci (Tautz 1993). Because mutations change the copy number of such loci in a stepwise fashion, rapid accumulation of population samples from minisatellite and microsatellite loci has resurrected the interest of the stepwise mutation model (SMM), which was popular in the 1970s.     

Polymorphism,monomorphism, and sequences in conserved microsatellites in primate species

Dimeric short tandem repeats are a source of highly polymorphic markers in the mammalian genome. Genetic variation at these hypervariable loci is extensively used for linkage analysis, for the identification of individuals, and may be useful for interpopulation and interspecies studies. In this paper, we analyze the variability and the sequences of a segment including three microsatellites, first described in man, in several species of primates (chimpanzee, orangutan, gibbon, and macaque) using the heterologous primers (man primers). This region is located on the human chromosome 6p, near the tumor necrosis factor genes, in the major histocompatibility complex. The fact that these primers work in all species studied indicates that they are conserved throughout the different lineages of the two superfamilies, the Hominoidea and the Cercopithecidea, represented by the macaques. However, the intervening sequence displays intraspecific and interspecific variability. The sites of base substitutions and the insertion/ deletion events are not evenly distributed within this region. The data suggest that it is necessary to have a minimal number of repeats to increase the rate of mutation sufficiently to allow the development of polymorphism. In some species, the microsatellites present single base variations which reduce the number of contiguous repeats, thus apparently slowing the rate of additional slippage events. Species with such variations or a low number of repeats are monomorphic. These microsatellite sequences are informative in the comparison of closely related species and reflect the phylogeny of the Old World monkeys, apes, and man.Correspondence to: B. Crouau-Roy     

Motif mismatches in microsatellites: insights from genome-wide investigation among 20 insect species

We present a detailed genome-wide comparative study of motif mismatches of microsatellites among 20 insect species representing five taxonomic orders. The results show that varying proportions (∼15–46%) of microsatellites identified in these species are imperfect in motif structure, and that they also vary in chromosomal distribution within genomes. It was observed that the genomic abundance of imperfect repeats is significantly associated with the length and number of motif mismatches of microsatellites. Furthermore, microsatellites with a higher number of mismatches tend to have lower abundance in the genome, suggesting that sequence heterogeneity of repeat motifs is a key determinant of genomic abundance of microsatellites. This relationship seems to be a general feature of microsatellites even in unrelated species such as yeast, roundworm, mouse and human. We provide a mechanistic explanation of the evolutionary link between motif heterogeneity and genomic abundance of microsatellites by examining the patterns of motif mismatches and allele sequences of single-nucleotide polymorphisms identified within microsatellite loci. Using Drosophila Reference Genetic Panel data, we further show that pattern of allelic variation modulates motif heterogeneity of microsatellites, and provide estimates of allele age of specific imperfect microsatellites found within protein-coding genes.     

Microsatellites in the Sand Lizard (Lacerta agilis): Description, Variation, Inheritance, and Applicability

We developed microsatellite markers for the sand lizard (Lacerta agilis) to enable investigations of the genetic variability within and among populations with a heterogeneous spatial distribution in Sweden. The populations, which could not be characterized by variation in allozymes or mitochondrial DNA, had a substantial level of variability in microsatellite loci. However, the variability in Swedish populations was limited compared to a large, outbred Hungarian population. In the sand lizard, the number of (GT/CA) _n repeats was approximately three times higher than that for (CT/GA) _n. The number of repeats and the frequency of microsatellites were within the range reported for other species. Three of nine microsatellite loci showed alleles that could not be amplified, which is in agreement with recent reports describing microsatellite “null alleles” as a common occurrence. We discuss the caution which this calls for when calculating paternity probabilities and when estimating between-population allelic differentiation. A potential problem with different mutation rates for alleles within the same locus is discussed.     

Trinucleotide microsatellites in Norway spruce ( Picea abies): their features and the development of molecular markers

Trinucleotide microsatellites have proven to be the markers of choice in human genetic analysis because they are easier to genotype than dinucleotides. Their development can be more time-consuming due to their lower abundance in the genome. We isolated trinucleotide microsatellites in Norway spruce ( Picea abies K.) using an enrichment procedure for the genomic-library construction. Here we report on the characterisation of 85 ATC microsatellite-containing clones, from which 39 markers were developed. Many of the clones showed the occurrence of tandem repeats of higher order than the trinucleotide ones, often resembling minisatellite repeats. The sequencing of a sample of the alleles at one of the loci revealed size homoplasy due to base substitutions within the microsatellite region. The presence of ATC motifs within repetitive sequence families was observed. We found a significant relationship between the level of polymorphism and the length of the microsatellite. The levels of variability for ATC trinucleotide markers were lower than those for dinucleotides, both when tested on all loci in a set of six individuals and on a subset of loci in four natural populations. This difference is most likely attributable to lower mutation rates for trinucleotide than for dinucleotide loci. The availability of markers with different mutation rates allows one to select the proper marker set to investigate population processes on different time scales.     

Rate and pattern of mutation at microsatellite loci in maize

Microsatellites are important tools for plant breeding, genetics, and evolution, but few studies have analyzed their mutation pattern in plants. In this study, we estimated the mutation rate for 142 microsatellite loci in maize (Zea mays subsp. mays) in two different experiments of mutation accumulation. The mutation rate per generation was estimated to be 7.7 x 10(-4) for microsatellites with dinucleotide repeat motifs, with a 95% confidence interval from 5.2 x 10(-4) to 1.1 x 10(-3). For microsatellites with repeat motifs of more than 2 bp in length, no mutations were detected; so we could only estimate the upper 95% confidence limit of 5.1 x 10(-5) for the mutation rate. For dinucleotide repeat microsatellites, we also determined that the variance of change in the number of repeats (sigma(m)2) is 3.2. We sequenced 55 of the 73 observed mutations, and all mutations proved to be changes in the number of repeats in the microsatellite or in mononucleotide tracts flanking the microsatellite. There is a higher probability to mutate to an allele of larger size. There is heterogeneity in the mutation rate among dinucleotide microsatellites and a positive correlation between the number of repeats in the progenitor allele and the mutation rate. The microsatellite-based estimate of the effective population size of maize is more than an order of magnitude less than previously reported values based on nucleotide sequence variation.     

An unusually low microsatellite mutation rate in Dictyostelium discoideum, an organism with unusually abundant microsatellites

The genome of the social amoeba Dictyostelium discoideum is known to have a very high density of microsatellite repeats, including thousands of triplet microsatellite repeats in coding regions that apparently code for long runs of single amino acids. We used a mutation accumulation study to see if unusually high microsatellite mutation rates contribute to this pattern. There was a modest bias toward mutations that increase repeat number, but because upward mutations were smaller than downward ones, this did not lead to a net average increase in size. Longer microsatellites had higher mutation rates than shorter ones, but did not show greater directional bias. The most striking finding is that the overall mutation rate is the lowest reported for microsatellites: approximately 1 x 10(-6) for 10 dinucleotide loci and 6 x 10(-6) for 52 trinucleotide loci (which were longer). High microsatellite mutation rates therefore do not explain the high incidence of microsatellites. The causal relation may in fact be reversed, with low mutation rates evolving to protect against deleterious fitness effects of mutation at the numerous microsatellites.     

Microsatellite Variation in Honey Bee (Apis Mellifera L.) Populations: Hierarchical Genetic Structure and Test of the Infinite Allele and Stepwise Mutation Models

Samples from nine populations belonging to three African (intermissa, scutellata and capensis) and four European (mellifera, ligustica, carnica and cecropia) Apis mellifera subspecies were scored for seven microsatellite loci. A large amount of genetic variation (between seven and 30 alleles per locus) was detected. Average heterozygosity and average number of alleles were significantly higher in African than in European subspecies, in agreement with larger effective population sizes in Africa. Microsatellite analyses confirmed that A. mellifera evolved in three distinct and deeply differentiated lineages previously detected by morphological and mitochondrial DNA studies. Dendrogram analysis of workers from a given population indicated that super-sisters cluster together when using a sufficient number of microsatellite data whereas half-sisters do not. An index of classification was derived to summarize the clustering of different taxonomic levels in large phylogenetic trees based on individual genotypes. Finally, individual population X loci data were used to test the adequacy of the two alternative mutation models, the infinite allele model (IAM) and the stepwise mutation models. The better fit overall of the IAM probably results from the majority of the microsatellites used including repeats of two or three different length motifs (compound microsatellites).     

Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis

Homoplasy has recently attracted the attention of population geneticists, as a consequence of the popularity of highly variable stepwise mutating markers such as microsatellites. Microsatellite alleles generally refer to DNA fragments of different size (electromorphs). Electromorphs are identical in state (i.e. have identical size), but are not necessarily identical by descent due to convergent mutation(s). Homoplasy occurring at microsatellites is thus referred to as size homoplasy. Using new analytical developments and computer simulations, we first evaluate the effect of the mutation rate, the mutation model, the effective population size and the time of divergence between populations on size homoplasy at the within and between population levels. We then review the few experimental studies that used various molecular techniques to detect size homoplasious events at some microsatellite loci. The relationship between this molecularly accessible size homoplasy size and the actual amount of size homoplasy is not trivial, the former being considerably influenced by the molecular structure of microsatellite core sequences. In a third section, we show that homoplasy at microsatellite electromorphs does not represent a significant problem for many types of population genetics analyses realized by molecular ecologists, the large amount of variability at microsatellite loci often compensating for their homoplasious evolution. The situations where size homoplasy may be more problematic involve high mutation rates and large population sizes together with strong allele size constraints.     

Evolution of microsatellites in Arabis petraea and Arabis lyrata, outcrossing relatives of Arabidopsis thaliana

We examined microsatellite variation in two diploid, outcrossing relatives of Arabidopsis thaliana, Arabis petraea and Arabis lyrata. The primer sequences were derived from A. thaliana. About 50% (14 loci) of the A. thaliana primers could successfully amplify microsatellites in the related species. Analysis of microsatellite structure in the related species showed that there had been large changes in the microsatellites: there were large differences in repeat numbers and many of the A. thaliana simple repeats were shorter in the related species. For the loci we compared, the related species had a much lower level of variability at the microsatellites than Japanese wild populations of A. thaliana. This is presumably related to the different microsatellite structures, because allozyme data showed that the outcrossing relatives were highly polymorphic compared to other outcrossing herbaceous species. Use of microsatellites in assessing variability or phylogenetic relationships between different species requires caution, because changes in microsatellite structure may alter evolutionary rates.      

Microsatellites in Zea - variability,patterns of mutations,and use for evolutionary studies

To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees. Received: 10 February 2001 / Accepted: 21 May 2001