Abstract
BackgroundMicrosatellites are frequently used genetic markers in a wide range of applications, primarily due to their high length polymorphism levels that can easily be genotyped by fragment length analysis. However, the mode of microsatellite evolution is yet not fully understood, and the role of interrupting motifs for the stability of microsatellites remains to be explored in more detail. Here we present a sequence analysis of mutation events and a description of the structure of repeated regions in the hypervariable, pentanucleotide microsatellite locus HrU10 in barn swallows (Hirundo rustica) and tree swallows (Tachycineta bicolor).ResultsIn a large-scale parentage analysis in barn swallows and tree swallows, broods were screened for mutations at the HrU10 locus. In 41 cases in the barn swallows and 15 cases in the tree swallows, mutations corresponding to the loss or gain of one or two repeat units were detected. The parent and mutant offspring alleles were sequenced for 33 of these instances (26 in barn swallows and 7 in tree swallows). Replication slippage was considered the most likely mutational process. We tested the hypothesis that HrU10, a microsatellite with a wide allele size range, has an increased probability of introductions of interruptive motifs (IMs) with increasing length of the repeated region. Indeed, the number and length of the IMs was strongly positively correlated with the total length of the microsatellite. However, there was no significant correlation with the length of the longest stretch of perfectly repeated units, indicating a threshold level for the maximum length of perfectly repeated pentanucleotide motifs in stable HrU10 alleles. The combination of sequence and pedigree data revealed that 15 barn swallow mutations (58%) produced alleles that were size homoplasic to other alleles in the data set.ConclusionOur results give further insights into the mode of microsatellite evolution, and support the assumption of increased slippage rate with increased microsatellite length and a stabilizing effect of interrupting motifs for microsatellite regions consisting of perfect repeats. In addition, the observed extent of size homoplasy may impose a general caution against using hypervariable microsatellites in genetic diversity measures when alleles are identified by fragment length analysis only.
Highlights
Microsatellites are frequently used genetic markers in a wide range of applications, primarily due to their high length polymorphism levels that can be genotyped by fragment length analysis
"interrupting motifs" (IMs) that deviate in sequence from the repeated motif and mutations in the flanking regions may contribute to the observed length polymorphism [3]
Sequencing of 33 mutated and 66 parental HrU10 alleles was consistent with the hypotheses that longer alleles tend to be more instable due to increased slippage rate
Summary
Microsatellites are frequently used genetic markers in a wide range of applications, primarily due to their high length polymorphism levels that can be genotyped by fragment length analysis. Microsatellites consist of tandemly repeated sequence motifs, no more than 6 bases long. They are scattered throughout most eukaryotic genomes and are extensively used as tools for a wide range of applications, such as e.g. molecular forensics, parentage testing, analysis of genetic structure of populations and the assessment of phylogenetic relationships [1]. The major characteristic that makes microsatellites a useful and powerful genetic tool is the extensive length polymorphism that first of all reflects allelic variation in the number of the tandemly arranged perfect repeats [2]. Mutation rates in microsatellites are affected by stabilization patterns and potential secondary structures [13,21]
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