Copyright Ó 2007 by the Genetics Society of America DOI: 10.1534/genetics.107.076067 An Unusually Low Microsatellite Mutation Rate in Dictyostelium discoideum,an Organism With Unusually Abundant Microsatellites Ryan McConnell, Sara Middlemist, Clea Scala, Joan E. Strassmann and David C. Queller1 Department of Ecology and Evolutionary Biology, Rice University, Houston, Texas 77005 Manuscript received May 18, 2007 Accepted for publication September 4, 2007 ABSTRACT 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: 1 3 10À6 for 10 dinucleotide loci and 6 3 10À6 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. ICROSATELLITES, also known as simple se- In humans, certain triplet repeats that occur in or M quence repeats, are long stretches of a short near coding regions are subject to expansions that (1–6 bp), tandemly repeated DNA unit, such as the directly cause genetic diseases (Ashley and Warren motif CAA repeated 20 times. Microsatellites are com- 1995; Cummings and Zoghbi 2000). Whether D. dis- mon throughout eukaryotic genomes and their lengths coideum experiences such deleterious effects from its are often highly polymorphic, making them powerful many coding-region repeats is unknown. However, un- markers for use in genetic mapping (Weber 1990; published work shows that these exonic microsatellites Dietrich et al. 1994; Dib et al. 1996; Roder et al. 1998), are highly variable (C. Scala,N.Mehdiabadi,J. population genetics (Jarne and Lagoda 1996; Di Strassmann and D. Queller, unpublished results), Rienzo et al. 1998; Goldstein and Schlotterer 1999; suggesting that they are not tightly controlled by Thuillet et al. 2002), and determination of kinship selection. However, selection ought to be potent in D. (Queller et al. 1993). discoideum. It has a large geographic range ½eastern The social amoeba Dictyostelium discoideum has the North America and part of eastern Asia (Swanson et al. highest density of microsatellite repeats of any sequenced 1999) and therefore should have a large effective organism, making up .11% of its genome (Eichinger population size. Molecular evidence suggests that it is et al. 2005). As is usual (Ellegren 2004), the noncoding typical of unicellular eukaryotes to have a population regions are richest in microsatellites, because they are size (estimated as Nem) large enough to make selection a less functional. However, there is also an exceptional very potent force relative to drift (Lynch and Conery number of long triplet repeat loci within genes, resulting 2003). This makes it harder to explain the persistence of in large numbers of homopolymer amino acid strings. large numbers of apparently functionless, or even The most common are polyasparagine and polyglut- deleterious, microsatellites. amine; 2091 of the 13,541 predicted genes have tracts of Mutational changes in the number of repeats occur $20 consecutive repeats, and some of these have multiple during DNA replication when the two DNA strands tracts (Eichinger et al. 2005). Microsatellites occur on temporarily dissociate and then realign out of register, average every 724 bp in exons and encode 3.3% of all creating an unpaired repeat loop on one of the strands amino acids (Eichinger et al. 2005). Other eukaryotic (Streisinger et al. 1966; Levinson and Gutman 1987; genomes also have amino acid repeats, although at a Schlo¨tterer and Tautz 1992; Strand et al. 1993). much lower density (Marcotte et al. 1998; Li et al. 2004). Primary replication slippage occurring on the template strand deletes repeat units, while slippage on the nascent strand creates additional repeats. The alter- 1Corresponding author: Department of Ecology and Evolutionary Bio- mith logy, MS-170, Rice University, 6100 Main St., Houston, TX 77005. native hypothesis of unequal crossing over (S E-mail: [email protected] 1973; Sia et al. 1997) is not supported by research that Genetics 177: 1499–1507 (November 2007) 1500 R. McConnell et al. experimentally restricted most forms of recombination We estimated D. discoideum microsatellite mutation in Escherichia coli (Levinson and Gutman 1987) and rates using a mutation accumulation experiment. In yeast (Henderson and Petes 1992) without lowering such experiments, lines are repeatedly passed through microsatellite instability. single-cell bottlenecks to fix mutations randomly. The Kruglyak et al. (1998) proposed a mutation model cell divisions between the single-cell bottlenecks pro- suggesting that higher mutation rates result in more vide some opportunity for strong selection to have microsatellites and a shift toward longer microsatellites. effects, but weakly selected mutations will be repre- High mutation rates could also account for the mainte- sented nearly randomly. nance of high variability. So one possible explanation for the high number, long length, and variability of microsatellites in D. discoideum is that this species could MATERIALS AND METHODS have an unusually high mutation rate for microsatel- lites. It is this hypothesis that we test in this report. Mutation accumulation: We started each of 90 mutation accu- Microsatellites mutate at rates much higher than the mulation lines from a common ancestor, the lab-maintained À9 axenic D. discoideum AX4 clone. The lines grew on SM agarose usual base-pair substitution rate of 10 /locus/gener- plates (10 g glucose, 10 g bactopeptone, 1 g yeast, 1 g MgSO , llegren uschiazzo emmell 4 ation (E 2000b; B and G 1.9 g KH2PO4, 0.6 g K2HPO4, 20 g agar, 1 liter H2O) with the 2006). Drosophila microsatellites have the lowest reported bacterium Klebsiella aerogenes used as a food source for the mutation rates: in the 10À6–10À4 range (Schlo¨tterer amoebas. To obtain our mutation accumulation lines, we plated et al. 1998; Schug et al. 1998; Harr and Schlo¨tterer out the ancestral clone and selected a single plaque to serve as azquez the ancestor (perfectly circular clearings or plaques in the 2000; V et al. 2000). Mammalian mutation rates, bacterial lawn derive from a single cell). This single cell line À5 À2 including that of humans, fall between 10 and 10 was plated out clonally, 10 single plaques were selected, and (Serikawa et al. 1992; Weber and Wong 1993; Dietrich the process was repeated to obtain 10 plaques from each of et al. 1994; Brinkmann et al. 1998; Sajantila et al.1999; these. Xu et al. 2000), as do rates reported for plants (Udupa From the resulting 100 lines, 90 were used as mutation aum huillet igouroux accumulation lines and the remaining 10 were control lines and B 2001; T et al.2002;V et al. that are not part of this report. Each mutation accumulation 2002). line was put though a series of 70 single-cell bottlenecks, Slippage rates in vitro are 100- to 1000-fold higher separated by 48-hr episodes of growth on plates as described than in vivo rates (Strand et al. 1993) because func- above. The single-cell bottlenecks were accomplished by tional mismatch repair systems maintain drastically randomly selecting a clonal plaque at the end of 48 hr and transferring cells from that plaque to the next plate. lower rates in the latter. Only those slippage mutations We estimate that the 48-hr growth periods encompassed an overlooked by the mismatch repair system are propa- average of 14.18 cell generations. This figure is the un- gated in successive replication events. Mutations in the weighted average of estimates for the ancestral clone (14.12 mismatch repair system destabilize microsatellite DNA 6 SD 0.62, an average of eight estimates) and the 90 mutation in E. coli (Levinson and Gutman 1987), yeast (Strand accumulation clones at the end of the experiment (14.24 6 SD ierdl olodner 0.71, n ¼ 90). Each estimate was obtained by collecting and et al. 1993; W et al. 1997), and humans (K counting the cells from a single clonal plaque after 48 hr and 1996). Observed microsatellite mutation rates thus taking the base 2 logarithm. Thus, each line went through reflect a balance between primary replication slippage 14.18 3 70 ¼ 1007 cell generations. and mismatch repair efficiency. We extracted DNA from all 90 lines at the completion of the Mutation rates are not uniform even within a ge- 10th and the 70th bottleneck. D. discoideum has a multicellular fruiting stage and we extracted DNA from the spore masses nome. Most strikingly, rate of slippage increases with with 150 ml of a 5% chelex solution. The thousand generations microsatellite length (Weber and Wong 1993; Kroutil of the experiment were all in the single-cell vegetative stage. et al. 1996; Wierdl et al. 1997; Brinkmann et al. 1998; Microsatellite selection, amplification, and genotyping: We Schlo¨tterer 2000; Ellegren 2004), as there are more downloaded the genomic DNA sequence of all six D. discoideum sites where slippage can occur and the conformational chromosomes from the online Dictyostelium database (http:// www.dictybase.org). A modified version of the program Sputnik entropy of slippage is 2 kcal/mol more destabilizing (http://espressosoftware.com/pages/sputnik.jsp) was used to for long direct repeats than for shorter repetitive runs compile a list of all microsatellites containing at least five perfect (Harvey 1997).