Factors affecting germline mutations in a hypervariable microsatellite: A comparative analysis of six species of swallows (Aves: Hirundinidae)

https://doi.org/10.1016/j.mrfmmm.2011.01.006Get rights and content

Abstract

Microsatellites mutate frequently by replication slippage. Empirical evidence shows that the probability of such slippage mutations may increase with the length of the repeat region as well as exposure to environmental mutagens, but the mutation rate can also differ between the male and female germline. It has been hypothesized that more intense sexual selection or sperm competition can also lead to elevated mutation rates, but the empirical evidence is inconclusive. Here, we analyzed the occurrence of germline slippage mutations in the hypervariable pentanucleotide microsatellite locus HrU10 across six species of swallow (Aves: Hirundinidae). These species exhibit marked differences in the length range of the microsatellite, as well as differences in the intensity of sperm competition. We found a strong effect of microsatellite length on the probability of mutation, but no residual effect of species or their level of sperm competition when the length effect was accounted for. Neither could we detect any difference in mutation rate between tree swallows (Tachycineta bicolor) breeding in Hamilton Harbour, Ontario, an industrial site with previous documentation of elevated mutation rates for minisatellite DNA, and a rural reference population. However, our cross-species analysis revealed two significant patterns of sex differences in HrU10 germline mutations: (1) mutations in longer alleles occurred typically in the male germline, those in shorter alleles in the female germline, and (2) male germline mutations were more often expansions than contractions, whereas no directional bias was evident in the female germline. These results indicate some fundamental differences in male and female gametogenesis affecting the probability of slippage mutations. Our study also reflects the value of a comparative, multi-species approach for locus-specific mutation analyses, through which a wider range of influential factors can be assessed than in single-species studies.

Introduction

Microsatellites are tandemly repeated short nucleotide sequences. Due to their high polymorphism in numbers of repeated units, microsatellites have become popular markers in population genetics with a broad spectrum of applications [1]. The high polymorphism is due to the relatively high mutation rate (10−2–10−6 per meiotic event) as compared with non-repetitive DNA [2], which makes them useful for the study of mutational processes also in non-model species breeding in the wild. Here we investigate how the mutation rates in a highly polymorphic microsatellite, the HrU10 locus [3], vary within and among six species of swallows (Aves: Hirundinidae), and we try to identify key factors explaining this variation.

The most frequent mechanism for microsatellite mutagenesis is the process of replication slippage [4]. Replication slippage is initiated by proofreading errors during replication [5], and includes a gain or loss of one or more repeated units, compatible with a stepwise mutation model [6]. Several factors may contribute to replication slippage, including motif size [7], [8], chromosomal location [9], G/C-content [10], stabilization pattern within the microsatellite [11], effectiveness of mismatch repair systems [12], [13], potential to create secondary structures [14], [15], environmental conditions [16] and sex [17], [18], [19], [20]. Because of the increased probability to introduce slippage events in long tracts of tandemly repeated units, there is generally a positive relationship between microsatellite mutation rate and allele size [17], [18], [19], [21], [22], [23], [24].

One unresolved issue is whether there is a sex-biased transmission of mutations during meiosis, as contradicting results exist in the literature (e.g. [25], [26]). Most authors have argued for a male driven mutation bias, because males have more germline cell-divisions than females and most mutations originate as replication errors (see [27], [28], [29] for reviews). However, this can hardly be true as a general, genome-wide explanation, as there is also evidence of female-biased mutation rates at specific loci, including the HrU10 [18], [30]. Thus, more information about germline-specific mutation patterns may provide new insights to sex-related factors causing slippage mutations during gametogenesis.

It has also been postulated that intense sexual selection may lead to elevated mutation rates [31], [32]. The logic of this idea is that sexual selection, for example mediated through female mating preferences, may give a high selective premium on mutations that are beneficial (i.e. preferred by females), even when most mutations are not beneficial and will be selected against. This idea may be one solution to the “lek paradox”, which refers to the puzzle of how genetic variation can be maintained in characters subject to sexual selection [33]. Positive relationships between genetic variation and indicators of strong sexual selection have been reported in several studies [34], [35], [36]. In particular, a positive relationship between mutation rates in DNA minisatellites and the frequency of extrapair paternity (EPP) has been claimed for socially monogamous birds [35], [37], but the evidence has recently been challenged [38]. Whether a similar relationship exists for microsatellites has not yet been tested, despite their extensive use in avian parentage studies.

The nature of microsatellite mutagenesis is difficult to study empirically, especially within natural populations of non-model organisms. Large and correct pedigrees are required in the form of true identification of both genetic parents for a large number of offspring, which may be challenging for both field-work logistics and the availability of markers with sufficiently high mutation rates. On the other hand, parentage analyses have so far been conducted in more than 150 studies of more than 130 bird species [39], and consequently a large body of suitable pedigree data exist from many species. Furthermore, particular microsatellites have high variability and are frequently used as heterologous markers in parentage across a range of species [40], and can thus be specifically targeted for cross-species comparisons. One such marker is the avian microsatellite pentanucleotide HrU10 [3]. This microsatellite marker is applicable in several bird species, especially within Hirundinidae, and it has been used in several parentage studies [41], [42], [43], [44], [45], [46]. HrU10 has one of the highest mutation rates ever reported and the mutation rates of this marker have also been shown to differ between species [18], [30]. Dawson et al. [47] linked this marker to chromosome 18 in the chicken (Gallus gallus) genome. Moreover, Anmarkrud et al. [30] identified an increased HrU10 mutation rate in tree swallows (Tachycineta bicolor) compared to barn swallows (Hirundo rustica), and the tree swallow is a species with a substantially higher level of EPP than the barn swallow [41], [42]. Together, this makes HrU10 a promising candidate marker to test the hypothesis of a positive association between microsatellite mutation rates and the frequency of EPP in birds. Here we expand the previous data set on tree swallows and barn swallows [30] with mutation data from four other hirundine species and an additional population of tree swallows, for which the frequency of EPP is known.

Microsatellite mutation rates might also be affected by the presence of mutagenic substances in the environment, such as radioactive isotopes or polycyclic aromatic hydrocarbons (PAHs). Ellegren et al. [16] documented increased slippage mutation rates at two microsatellite loci in barn swallows breeding in Chernobyl, Ukraine, and exposed to radioactive isotopes released into the local environment in the 1986 accident. Similarly, exposure to PAHs seems to elevate mutation rates in minisatellite DNA in gulls [48] and mice [49] in a polluted industrial area in Hamilton Harbour, Ontario, Canada, though it is unknown whether the same effect applies to microsatellite DNA.

Here, we report the analysis of 100 germline mutations in the HrU10 marker assembled from six species of swallow (Aves: Hirundinidae), whose populations have previously been subject to parentage analysis. We ask whether the rate of germline mutations differs among the species, and if so, whether this variation can be attributed to variation among species in size range of the microsatellite and/or their level of EPP. We also wanted to test whether the previously reported female mutation bias in tree swallows and barn swallows [30] were upheld in the additional species, and whether we could detect any sex-specific mutation patterns across the entire data set. Finally, we ask whether a study population of tree swallows in the polluted area of Hamilton Harbour, Ontario, showed signs of an elevated mutation rate in this microsatellite as compared to a rural reference population studied previously near Lake Opinicon, Ontario [30].

Section snippets

General methods

A critical assumption underlying analyses of germline mutations is the correct identification of both genetic parents. We therefore restricted our analysis to data sets containing offspring for which both genetic parents had been identified with a robust parentage system (microsatellites or DNA fingerprints/minisatellites) and for which the population frequency of EPP had been assessed. Our six study species belong to two main clades within the Hirundinidae family: core martins (purple martin

Number of mutations and mutation rates

A total of 100 HrU10 mutations were uncovered among 3604 meiotic events (parent-offspring transmitted alleles) (1802 young), equivalent to an overall mutation rate (μ) of 2.8% (Table 1). The mutation rate varied significantly among the six study species alleles (GLZ; χ2 = 38.01, P < 0.001) and ranged from 0.6% in house martins to 10.8% in sand martins. The six species also differed in median allele size (Table 1) and there was a positive correlation between mutation rates and median allele sizes at

Discussion

The 100 germline mutations in our data set of genotypes at the HrU10 microsatellite, assembled from the six swallow species, typically showed either a loss or gain of one repeat unit (5 bp), which is consistent with the mutational process of replication slippage [4]. The probability of mutation clearly increased with allele size. When the effect of allele size was accounted for, there was no residual variation in mutation rate among species that could be attributed to the risk of sperm

Conflict of interest

The authors declare that there are no conflicts of interests.

Acknowledgements

We thank all who assisted with the field- and lab-work. The research was approved by the Animal Care Committees of Environment Canada and Queen's University, and the Directorate for Nature Management in Norway. This study was supported by grants from the Norwegian Research Council and Natural History Museum, University of Oslo (PhD fellowship JAA), as well as funding through Environment Canada (Ecotoxicology and Wildlife Health).

References (83)

  • A.P. Møller et al.

    Impaired swimming behaviour and morphology of sperm from barn swallows Hirundo rustica in Chernobyl

    Mutat. Res.

    (2008)
  • B. Brinkmann et al.

    Mutation rate in human microsatellites: influence of the structure and length of the tandem repeat

    Am. J. Hum. Genet.

    (1998)
  • J. Ortego et al.

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

    Mutat. Res.

    (2008)
  • K. Labib et al.

    Is the MCM2-7 complex the eukaryotic DNA replication fork helicase?

    Curr. Opin. Genet. Dev.

    (2001)
  • D.B. Goldstein et al.

    Microsatellites: Evolution and Applications

    (1999)
  • Y.C. Li et al.

    Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review

    Mol. Ecol.

    (2002)
  • C.R. Primmer et al.

    New microsatellites from the pied flycatcher Ficedula hypoleuca and the swallow Hirundo rustica genomes

    Hereditas

    (1996)
  • G. Levinson et al.

    Slipped-strand mispairing: a major mechanism for DNA sequence evolution

    Mol. Biol. Evol.

    (1987)
  • M. Strand et al.

    Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair

    Nature

    (1993)
  • M. Kimura et al.

    Stepwise mutation model and distribution of allelic frequencies in a finite population

    Proc. Natl. Acad. Sci. USA

    (1978)
  • T.J. Anderson et al.

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

    Mol. Ecol.

    (2000)
  • R. Chakraborty et al.

    Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci

    Proc. Natl. Acad. Sci. USA

    (1997)
  • C. Schlötterer et al.

    High mutation rate of a long microsatellite allele in Drosophila melanogaster provides evidence for allele-specific mutation rates

    Mol. Biol. Evol.

    (1998)
  • K.A. Eckert et al.

    Mutation rate and specificity analysis of tetranucleotide microsatellite DNA alleles in somatic human cells

    Mol. Carcinog.

    (2002)
  • J.D. Hawk et al.

    Variation in efficiency of DNA mismatch repair at different sites in the yeast genome

    Proc. Natl. Acad. Sci. USA

    (2005)
  • C.E. Pearson et al.

    Interruptions in the triplet repeats of SCA1 and FRAXA reduce the propensity and complexity of slipped strand DNA (S-DNA) formation

    Biochemistry

    (1998)
  • H. Ellegren et al.

    Fitness loss and germline mutations in barn swallows breeding in Chernobyl

    Nature

    (1997)
  • N.R. Beck et al.

    Microsatellite evolution at two hypervariable loci revealed by extensive avian pedigrees

    Mol. Biol. Evol.

    (2003)
  • J. Brohede et al.

    Heterogeneity in the rate and pattern of germline mutation at individual microsatellite loci

    Nucleic Acids Res.

    (2002)
  • H. Ellegren

    Heterogeneous mutation processes in human microsatellite DNA sequences

    Nat. Genet.

    (2000)
  • W.E. Hoekert et al.

    Multiple paternity and female-biased mutation at a microsatellite locus in the olive ridley sea turtle (Lepidochelys olivacea)

    Heredity

    (2002)
  • Y. Lai et al.

    The relationship between microsatellite slippage mutation rate and the number of repeat units

    Mol. Biol. Evol.

    (2003)
  • O. Paun et al.

    Evolution of hypervariable microsatellites in apomictic polyploid lineages of Ranunculus carpaticola: directional bias at dinucleotide loci

    Genetics

    (2006)
  • J.C. Whittaker et al.

    Likelihood-based estimation of microsatellite mutation rates

    Genetics

    (2003)
  • C.R. Primmer et al.

    Unraveling the processes of microsatellite evolution through analysis of germ line mutations in barn swallows

    Mol. Biol. Evol.

    (1998)
  • H.B. Bohossian et al.

    Unexpectedly similar rates of nucleotide substitution found in male and female hominids

    Nature

    (2000)
  • K.D. Makova et al.

    Strong male-driven evolution of DNA sequences in humans and apes

    Nature

    (2002)
  • H. Ellegren

    Characteristics, causes and evolutionary consequences of male-biased mutation

    Proc. R. Soc. B

    (2007)
  • J.A. Anmarkrud et al.

    Microsatellite evolution: Mutations, sequence variation, and homoplasy in the hypervariable avian microsatellite locus HrU10

    BMC Evol. Biol.

    (2008)
  • M. Petrie et al.

    Sexual selection and the evolution of evolvability

    Heredity

    (2006)
  • S. Cotton et al.

    Male mutation bias and possible long-term effects of human activities

    Conserv. Biol.

    (2010)
  • View full text