Microsatellite evolution at two hypervariable loci revealed by extensive avian pedigrees

Genealogies generated through a long-term study of superb fairy-wrens (Malurus cyaneus) were used to investigate mutation within two hypervariable microsatellite loci. Of 3,230 meioses examined at the tetranucleotide locus (Mcy micro 8), 45 mutations were identified, giving a mutation rate of 1.4%. At the dinucleotide locus (Mcy micro 4) 30 mutations were recorded from 2,750 meioses giving a mutation rate of 1.1%. Mutations at both loci primarily (80%; 60/75) involved the loss or gain of a single repeat unit. Unlike previous studies, there was no significant bias toward additions over deletions. The mutation rate at Mcy micro 8 increased with allele size, and very long alleles (>70 repeats) mutated at a rate of almost 20%. The length of the mutating allele and allele span, however, were strongly correlated so it was not possible to isolate the causative factor. Allele size did not appear to affect mutation rate at Mcy micro 4, but the repeat number was considerably lower at this locus. The gender of the mutating parent was significant only at Mcy micro 8, where mutations occurred more frequently in maternal alleles. However, at both loci we found that alleles inherited from the mother were on average larger than those from the father, and this in part drove the higher mutation rate among maternally inherited alleles at Mcy micro 8.     

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.      

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

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.      

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.     

Mutation in microsatellite repeats of DNA and embryonal death in humans

In the analysis of tetranucleotide DNA repeats inheritance carried out in 55 families with a history of spontaneous miscarriages and normal karyotypes in respect to 21 loci located on seven autosomes, 8 embryos (14.5%) demonstrating 12 cases of the presence of alleles absent in both parents were described. The study of chromosome segregation using other DNA markers permitted highly probable exclusion of false paternity as well as uniparental disomy as the reasons for parent/child allele mismatches. The high probability of paternity together with the presence of a "new" allele at any offspring locus points to the mutation having occurred during game-togenesis in one of the parents. Examination of mutation in spontaneous abortuses revealed an increased number of tandem repeat units at microsatellite loci in three cases and an decreased number of these repeats in six cases. In two abortuses, a third allele absent in both parents, which resulted from a somatic mutation that occurred during embryonic development, was observed. The prevalence of the male germline mutations, revealed during investigation of the mutation origin, was probably associated with an increased number of DNA replication cycles in sperm compared to the oocytes. In spontaneous abortuses, the mean mutation rate of the tetranucleotide repeat complexes analyzed was 9.8 x 10(-3) per locus per gamete per generation. This was about five times higher than the spontaneous mutation rate of these STR loci. It can be suggested that genome instability detected at the level of repeated DNA sequences can involve not only genetically neutral loci but also active genomic regions crucial for embryonic viability. This results in cell death and termination of embryonic development. Our findings indicate that the death of embryos with normal karyotypes in most cases is associated with an increased frequency of germline and somatic microsatellite mutations. The data of the present study also provide a practical tool for the quantitative evaluation of this phenomenon and for the analysis of the reasons for miscarriages and embryonic death in certain families.     

Polarity of mutations in tumor-associated microsatellite instability

Many factors have been implicated in influencing the rate of microsatellite mutations, including the length and base composition of the repeat motif, number of repeats, base composition of flanking sequences and, perhaps most importantly, degree of perfection of the repeats. The latter is of clinical relevance, since in both spino-cerebellar ataxia and fragile X syndrome, alleles with imperfect repeats appear to be much more stable than perfect ones. As yet, the relative importance of increased replication slippage and decreased mismatch repair efficiency in the preference of mutations to occur within perfect repeats has not been fully determined. D13S308E is an asymmetric trinucleotide repeat microsatellite with the sequence (CAT)₃CAC(CAT)CAC(CAT)₂CAC(CAT)CAC(CAT) ₁₅ , thus containing two parts: an 11-repeat imperfect portion (underlined above) and a 15-repeat perfect one (bold). We sequenced eight new mutant alleles of D13S308E from three human gastric tumors with instability in this and other microsatellites. In all mutations the size variation occurred exclusively in the perfect part of the microsatellite. These results constitute direct evidence that the molecular basis of microsatellite alterations seen in normal cells is similar to those that occur in human tumors with extensive microsatellite instability. The investigation of mechanisms involved in microsatellite mutations has been handicapped by the fact that they are rare events. The microsatellite instability observed in malignant tumors provides us with a useful general system to study these mechanisms. Received: 24 June 1997 / Accepted 7 October 1997     

An analysis of microsatellite loci in Arabidopsis thaliana: mutational dynamics and application

Microsatellite loci are among the most commonly used molecular markers. These loci typically exhibit variation for allele frequency distribution within a species. However, the factors contributing to this variation are not well understood. To expand on the current knowledge of microsatellite evolution, 20 microsatellite loci were examined for 126 accessions of the flowering plant, Arabidopsis thaliana. Substantial variability in mutation pattern among loci was found, most of which cannot be explained by the assumptions of the traditional stepwise mutation model or infinite alleles model. Here it is shown that the degree of locus diversity is strongly correlated with the number of contiguous repeats, more so than with the total number of repeats. These findings support a strong role for repeat disruptions in stabilizing microsatellite loci by reducing the substrate for polymerase slippage and recombination. Results of cluster analyses are also presented, demonstrating the potential of microsatellite loci for resolving relationships among accessions of A. thaliana.     

Germline mutations of tetranucleotide DNA repeats in families with normal children and reproductive pathology

We have previously reported a high rate of tetranucleotide DNA repeat mutations, including mutations of both germline and somatic origin, in spontaneous human abortuses. To analyze in more detail mutational microsatellite (MS) variability in meiosis and its possible association with disturbed embryonic development, we have conducted a comparative study of mutation rates of a complex of 15 autosomal tetranucleotide MSs in 55 families with healthy children and in 103 families that have had spontaneous abortuses with normal karyotypes. In the families with miscarriage, the gametic MS mutation rate was higher than in the families with normal reproductive function (4.36 x 10(-3) versus 2.32 x 10(-3) per locus per gamete per generation), but this difference was statistically nonsignificant (P = 0.25). No association of MS mutations with familiar miscarriage was found. Mutations at the MS loci studied were recorded almost 3 times as often in spermatogenesis as in oogenesis, which is likely to result from a greater number of DNA replication cycles in male germline cell precursors than in female ones. Mutations increasing and reducing the MS sequence length appeared at virtually the same rate. Changes in MS DNA sequence length per one repeated element, i.e., single-step mutations (93% of cases) exceeded all other events of allele length change. The highest number of mutations (81.2%) was found in longer alleles. This distribution of mutations by size, direction, and parental origin corresponds to the multistep mutation model of their emergence via mechanism of DNA strand slippage during replication.     

Human Microsatellites: Mutation and Evolution

Microsatellites (MSs) are short tandem DNA repeats with the repetitive motif of two to six nucleotides, forming tracts up to hundreds of nucleotides long. Notwithstanding the active use of MSs in genetic studies of various biological problems, the reasons for their wide occurrence in the genome, their possible functions, and mutational behavior are still unclear. The mutation rate in MS repeats is on average several orders of magnitude higher than in the remaining DNA, which allows for direct estimation of evolutionary transformation rate in nucleotide sequences of the genome. Mutation process in MSs is highly heterogeneous, with distinct differences between species; furthermore, within a species it differs among loci with different repeat size, among alleles of one locus, and among individuals of different sex and age. Most MS mutations are caused by DNA slippage during replication but the probability of this event depends on the locus. In this review, a number of models of MS evolution are discussed, which account for the relationship between mutation rate and allele size, different mutation direction in alleles of different size, and the appearance of point mutations within repeat tracts restricting allele size. The MS evolution is considered mainly in the context of selective neutrality, although there is evidence showing functional significance of some variants of tandem repeats and thus their possible selective value.     

Isolation of polymorphic microsatellite markers for Begonia sutherlandii Hook. f.

Seven polymorphic microsatellite loci have been characterized for investigating population structure in the patchily distributed herb Begonia sutherlandii. Two loci (BSU3 and BSU4) exhibited population specific null alleles; primer redesign and allele sequencing for one of these loci showed two transition mutations in the original primer site. Two loci exhibited imperfect repeat polymorphisms due to single base pair indels in the flanking region (locus BSU6) and in the microsatellite region itself (BSU7). Transversion mutations were also found in the microsatellite region of locus BSU7. The remaining three loci amplified in all individuals tested and appeared to conform to a simple stepwise mutation pattern.     

Nine polymorphic microsatellite loci for the northern leopard frog (Rana pipiens)

We describe the cloning and characterization of nine microsatellite loci from the northern leopard frog. Seven loci consist of tetranucleotide repeats, one locus consists of a dinucleotide repeat and one locus consists of a GT repeat juxtaposed with a GATA repeat. In a sample of 36 frogs from a natural population, polymorphism at these loci ranged from two to 13 alleles per locus with expected heterozygosities ranging from 0.5 to 0.91. These loci will be useful to researchers since this species is used for a broad range of studies.     

Conservation of a dinucleotide simple sequence repeat locus in sharks

Recent studies indicate that the flanking region and repeat motif structure of conserved microsatellite loci are useful for phylogenetic inference. Most comparative studies of microsatellite loci involve relatively closely related species, however, primarily because primers developed for one species often amplify only related species. We describe an analysis of a microsatellite locus in lamniform sharks that we estimate has been conserved for a billion years. Combined analysis of the flanking sequence and repeat motif structure resulted in a gene tree comparable to those reported from similar analyses of other genes. The conservation of the simple sequence repeat (SSR), and of the sequence flanking the SSR, is explained by a low substitution rate in sharks coupled with the possibility that mutations which interrupt perfect repeats are lost by replication slippage.     

Human microsatellites: mutation and evolution

Microsatellites (MSs) are short tandem DNA repeats with the repetitive motif of two to six nucleotides, forming tracts up to hundreds of nucleotides long. Notwithstanding the active use of MSs in genetic studies of various biological problems, the reasons for their wide occurrence in the genome, their possible functions, and mutational behavior are still unclear. The mutation rate in MS repeats is on average several orders of magnitude higher than in the remaining DNA, which allows for direct estimation of evolutionary transformation rate in nucleotide sequences of the genome. Mutation process in MSs is species-specific; furthermore, within a species it differs among loci with different repeat size, among alleles of one locus, and among individuals of different sex and age. Most MS mutations are caused by DNA slippage during replication but the probability of this event depends on the locus. In this review, a number of models of MS evolution are discussed, which account for the relationship between mutation rate and allele size, different mutation direction in alleles of different size, and the appearance of point mutations within repeat tracts restricting allele size. The MS evolution is considered mainly in the context of selective neutrality, although there is evidence showing functional significance of some variants of tandem repeats and thus their possible selective value.     

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.     

Allelic variation,fragment length analyses and population genetic models: a case study on Drosophila microsatellites*

The allelic variation of 16 microsatellite loci from selected species of the Drosophila melanogaster and D. obscura group was determined. Intra‐ and interspecific sequence comparisons allowed discrimination of mutations affecting the repetitive microsatellite from those affecting the flanking regions. The hypotheses that slippage needs a minimum number of repeats in order to become efficient with respect to microsatellite variability, and of an increased mutation rate with increased length of the microsatellite are supported by the results of our analyses. There is in particular at the interrupted complex microsatellite locus BICOID in the species of the D. obscura group, extensive variation in the flanking regions in addition to length and sequence variation of the repetitive microsatellite. The allelic variation at this locus can hardly be explained by slippage alone. Estimates of microsatellite variability by fragment length analyses will pick up only a minor fraction of allelic variation at such loci, and conclusions that are based on the stepwise mutation model will not hold.     

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     

Characteristics of loci and individuals are associated with germline microsatellite mutation rates in lesser kestrels (Falco naumanni)

Although microsatellites are one of the most popular tools in genetic studies, their mutational dynamics and evolution remain unclear. Here, we apply extensive pedigree genotyping to identify and analyze the patterns and factors associated with de novo germline mutations across nine microsatellite loci in a wild population of lesser kestrels (Falco naumanni). A total of 10 germline mutations events were unambiguously identified in four loci, yielding an average mutation rate of 2.96x10(-3). Across loci, mutation rate was positively correlated with locus variability and average allele size. Mutations were primarily compatible with a stepwise mutation model, although not exclusively involved single-step changes. Unexpectedly, we found an excess of maternally transmitted mutations (male-to-female ratio of 0.1). One of the analyzed loci (Fn2.14) resulted hypermutable (mutation rate=0.87%). This locus showed a size-dependent mutation bias, with longer alleles displaying deletions or additions of a small number of repeat than shorter alleles. Mutation probability at Fn2.14 was higher for females and increased with parental (maternal) age but was not associated with individual physical condition, multilocus heterozygosity, allele length or allele span. Overall, our results do not support the male-biased mutation rate described in other organisms and suggest that mutation dynamics at microsatellite loci are a complex process which requires further research.     

Isolation and characterization of polymorphic tetranucleotide microsatellite loci in the pelagic perciform fish Pomatomus saltatrix (Linnaeus, 1766) from South Africa

Eight polymorphic microsatellite loci, containing simple tetranucleotide repeats, were isolated de novo from a Pomatomus saltatrix partial genomic library using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. These loci were further characterized in 100 individuals from two putative populations off the South African east coast. The loci are highly polymorphic with 18-37 alleles (on average 24 alleles/locus) and the observed heterozygosity in both populations was high (0.79). These loci will be used to assess population structuring in P. saltatrix along the southern African coast with consideration of implications for future management of this important linefish species.     

Evolution of Microsatellite Alleles in Four Species of Mice (Genus Apodemus)

Microsatellite length variation was investigated at a highly variable microsatellite locus in four species of Apodemus. Information obtained from microsatellite allele sequences was contrasted with allele sizes, which included 18 electromorphs. Additional analysis of a 400-bp unique sequence in the flanking region identified 26 different haplotype sequences or ``true' alleles in the sample. Three molecular mechanisms, namely, (1) addition/deletion of repeats, (2) substitutions and indels in the flanking region, and (3) mutations interrupting the repeat, contributed to the generation of allelic variation. Size homoplasy can be inferred for alleles within populations, from different populations of the same species, and from different species. We propose that microsatellite flanking sequences may be informative markers for investigating mutation processes in microsatellite repeats as well as phylogenetic relationships among alleles, populations, and species. Received: 3 November 1999 / Accepted: 2 May 2000