The Rate and Molecular Spectrum of Spontaneous Mutations in
Arabidopsis thaliana
Stephan Ossowski
1,*
, Korbinian Schneeberger
1,*
, José Ignacio Lucas-Lledó
2,*,†
, Norman
Warthmann
1
, Richard M. Clark
3
, Ruth G. Shaw
4
, Detlef Weigel
1,†
, and Michael Lynch
2
1
Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076
Tübingen, Germany
2
Department of Biology, Indiana University, Bloomington, IN 47405, USA
3
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA
4
Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN 55108,
USA
Abstract
To take complete advantage of information on within-species polymorphism and divergence from
close relatives, one needs to know the rate and the molecular spectrum of spontaneous mutations.
To this end, we have searched for de novo spontaneous mutations in the complete nuclear
genomes of five Arabidopsis thaliana mutation accumulation lines that had been maintained by
single-seed descent for 30 generations. We identified and validated 99 base substitutions and 17
small and large insertions and deletions. Our results imply a spontaneous mutation rate of 7 × 10
−9
base substitutions per site per generation, the majority of which are G:C→A:T transitions. We
explain this very biased spectrum of base substitution mutations as a result of two main processes:
deamination of methylated cytosines and ultraviolet light–induced mutagenesis.
Most of what we know about molecular evolution comes from the comparison of biological
sequences that have survived many cycles of natural selection. In order to infer the
properties of the original source of variation and to detect the signature of natural selection
from such data sets, we need to assume that variants affecting certain types of sites, such as
the last base of fourfold redundant codons or pseudogenes, are not subject to natural
selection. This pervasive assumption is very rarely tested and difficult to avoid, because of
the slow pace of spontaneous mutagenesis. However, with the advent of high-throughput
sequencing technologies, some estimates of the rate of spontaneous mutations have begun to
appear (1–3). Here, we report a direct estimate of the spontaneous base substitution rate in
Arabidopsis thaliana, a plant species with extensive DNA methylation. As a result, we
reduce the uncertainty associated with key aspects of the evolutionary history of this species,
Copyright 2010 by the American Association for the Advancement of Science; all rights reserved.
†
To whom correspondence should be addressed. joslucas@indiana.edu (J.I.L.-L.); weigel@weigelworld.org (D.W.).
*
These authors contributed equally to this work.
Supporting Online Material
www.sciencemag.org/cgi/content/full/327/5961/92/DC1
Materials and Methods
SOM Text
Figs. S1 to S8
Tables S1 to S3
References
NIH Public Access
Author Manuscript
Science. Author manuscript; available in PMC 2014 January 02.
Published in final edited form as:
Science. 2010 January 1; 327(5961): . doi:10.1126/science.1180677.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
including the time since divergence from A. lyrata and the effect of methylation on the
probability of mutation.
We sequenced the genomes of five individuals derived by 30 generations of single-seed
descent from the reference strain Col-0 (4), for which a high-quality genome was published
in 2000 (5). We used the Illumina (Illumina, San Diego, CA) Genome Analyzer platform to
obtain a depth of sequence coverage of between 23 and 31 in each mutation accumulation
(MA) line. Sequencing reads between 36 and 43 base pairs (bp) in length were aligned to the
reference genome, from which over 1000 errors had been removed in a previous study (6).
We identified single-base substitutions using two complementary methods: a “consensus”
approach and the single-nucleotide polymorphism (SNP) caller function of SHORE (http://
1001genomes.org/ downloads/shore.html) (7).
In the consensus approach, base substitutions were called if one of the MA lines differed
from all others. We estimated a frequency of sequencing errors of ~0.3% per site per read
(8). We assumed a binomial distribution of errors to derive the probabilities of false
positives and false negatives, and we corrected our estimates accordingly (9). Because
sequencing and mapping errors are not randomly distributed among sites (3), we used strict
quality filters to exclude from analysis sites suspected to have higher error rates (8).
Between 93 and 95 million sites out of the 120 million–bp reference genome matched the
quality requirements in each line. Across all five lines, 85 single-base substitutions were
called by this method, 83 of which were confirmed by Sanger sequencing. In the other two
sites, two or three lines had a nonreference base, whereas the rest matched the reference, and
we interpret this to be a result of differential fixation of the two alleles present in ancestrally
heterozygous sites rather than as parallel mutations, although the latter cannot be ruled out.
In addition to this conservative approach, we used SHORE to detect single-base
substitutions, short insertions and deletions (indels) of up to 3 bp, and long deletions. The
algorithms implemented in SHORE are more sensitive (8), and between 98.8 and 100.9
million sites in each line had sufficient read information for calling either a mutation or the
reference base. We detected 99 single-base substitutions (98 of which were confirmed by
Sanger sequencing, and 1 was rejected because the reference base was revealed), 9 short
deletions (8 confirmed, 1 rejected), 5 short insertions of 1 bp (all confirmed), and 8 long
deletions covering11 toover 5000bp (4confirmed, 2 ambiguous, and 2 rejected). A 2-bp
deletion was shown to be present in two lines, suggesting that it was heterozygous in the
ancestral line. The chromosomal positions of all validated mutations are shown in Fig. 1.
Both false positives and false negatives are expected to be absent from the final set of simple
base-substitution mutations (8). Fifteen sites where all MA lines had a common
composition, but different from the reference, including 13 single-base substitutions and two
deletions of 1 and 2 bp, were interpreted as fixed mutations in the ancestral line.
We estimated the overall mutation rate to be 5.9 × 10
−9
± 0.6 × 10
−9
base substitutions per
site per generation according to the consensus approach and 6.5 × 10
−9
± 0.7 × 10
−9
according to SHORE. In addition, joint maximum-likelihood estimates of the overall
mutation rate and the sequencing error frequency were obtained following a recently
developed method (9). With this approach, a slightly higher mutation rate of 7.1 × 10
−9
±
0.7 × 10
−9
at a slightly lower error frequency of 0.2% was estimated. Mutations were evenly
distributed among MA lines (Table 1). Within chromosomes, a significantly higher base-
substitution mutation rate for intergenic regions was observed closer to the centromere
(within 3.0 × 10
6
bp, for example) than farther away (Fisher’s exact test, P value = 0.01, for
nonmethylated sites).
Ossowski et al.
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The estimated rates of 1- to 3-bp deletions and insertions are 0.6 × 10
−9
± 0.2 × 10
−9
and 0.3
× 10
−9
± 0.1 × 10
−9
per site per generation, respectively. Out of the 13 short indels that we
observed, 6 were found in complex sequences, corresponding to a mutation rate of 4.0 ×
10
−10
± 1.6 × 10
−10
indels per site per generation, or about 0.05 ± 0.02 indels per haploid
genome per generation, excluding homopolymers and micro-satellites. This estimate should
be considered a lower bound, given the unknown level of false negatives among indels. The
short-read sequencing approach is less well-suited for the analysis of dinucleotide repeats,
because the most frequent class of dipolymers (AT/TA) has low read coverage, most likely
resulting from the sequencing library construction protocol (10) (fig. S1). Deletions larger
than 3 bp occurred at a frequency of 0.5 × 10
−9
± 0.2 × 10
−9
per site per generation and
removed on average 800 ± 1900 bp per event.
The preceding estimates, together with the recently published rate of mutation at
dinucleotide microsatellites (11), give an almost complete view of the spectrum of
spontaneous mutations in A. thaliana (Table 2).Our estimate of themutation rate is close to
the lower bound of an indirect estimate based on the divergence between monocots and
dicots (12). If we assume that only nonsynonymous mutations and indels affecting coding
regions are likely to affect fitness, the diploid genomic rate of mutations affecting fitness
would be 0.2 ± 0.1 per generation, which is not significantly different from previous
estimates based on the evaluation of fitness components in MA lines of A. thaliana (13, 14).
Alternatively, the proportion of deleterious mutations among mutations in coding regions
can be estimated from sequence comparisons between A. thaliana and A. lyrata (8, 15).
Using that information, the estimated genomic deleterious mutation rate is 0.14 ± 0.04 per
generation.
We did not detect any difference between the “unpolarized” (8) spectrum of base-
substitution mutations and the genome-wide spectrum of polymorphisms at synonymous
sites surveyed in natural populations by two independent studies (6, 16) (fig. S2; Fisher’s
exact test, P = 0.4 and P = 0.7, respectively). Transitions were 2.4 times more frequent than
transversions, and G:C→A:T transitions, most of which are silent at the third codon
position, were by far the most frequent type of mutations (Fig. 2). Under the observed
mutational spectrum, the base-composition equilibrium achieved only by mutation would be
85% A+T, which is far from the current 68% observed in intergenic and intronic regions and
from the 65% in fourfold redundant coding sites across the A. thaliana genome. Whether
selection is preventing a further increase in A+T content, or whether the genome is still
evolving toward a higher A+T content, is not known.
Spontaneous deamination of methylated cytosine, which leads to thymine substitution (17–
19), is thought to be a major source of mutations. Thus, we exploited a single base–
resolution methylation map of the A. thaliana genome (20) to test whether cytosine
methylation can account for the overabundance of G:C→A:T transitions. G:C sites where
the cytosine has been reported to be at least partially methylated had a higher probability of
mutation to A:T than nonmethylated sites (Fisher’s exact test, P = 3.2 × 10
−7
). However,
91% of G:C sites in A. thaliana were not reported to be methylated, and they too had a
higher rate of transition (but not transversion) than A:T sites (Fisher’s exact test, P = 1.2 ×
10
−8
). G:C sites in CpG contexts are known to be more frequently methylated (20, 21).
However, transitions at G:C sites not known to be methylated do not happen in CpG
contexts more often than expected by chance (Fisher’s exact test, P = 0.6). This suggests
that factors in addition to methylation contribute to the high rate of transitions atG:C sites.
In both prokaryotes and eukaryotes, most of the mutations caused by ultraviolet (UV) light
are G: C→A:T transitions at sites where the C is adjacent to another C or to a T
(dipyrimidine sites) (22). Among the 33 observed transition mutations at nonmethylated G:C
Ossowski et al.
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sites, 31 were in dipyrimidine contexts, which is more than expected by chance at the P =
0.02 level (Fisher’s exact test). Thus, we conclude that the increased rate of transitions at
G:C sites, relative to A:T sites, can be largely explained by the combined effect of UV-
induced mutagenesis and deamination of methylated cytosines. This implies that the
mutation rate in nature could be higher than that reported here, becauseUVradiation during
the MA experiment was probably lower than in natural conditions.
We used the Arabidopsis Information Resource (TAIR) 8 annotation (www.arabidopsis.org)
to group all analyzed sites into the functional classes: intergenic, intronic, untranslated
region (UTR), coding, pseudogene, mobile element, and noncoding. There is no deficit of
nonsynonymous mutations (G test, P = 0.4), supporting the notion that themutation rate we
observed is not affected by selection. We did, however, observe an excess of intergenic
mutations, relative to mutations in coding regions, introns, and UTRs (fig. S3). These
differences were still significant after taking into account the effects of base composition
and methylation (G test, P = 0.00025). To test whether the lack of mutations in genic regions
was due to undetected levels of selection, we compared the intergenic mutation rate with the
rate at synonymous sites and introns, which are less likely to be under strong selection,
andwe still detected a significant deficit of mutations in the latter (Fisher’s exact test,
P=0.001, for nonmethylated sites). We attribute the deficit of genic mutations to our
observation of a higher mutation rate in pericentromeric regions (see above), where gene
density is lower (5), although transcription-coupled DNA repair could also contribute to the
pattern. Lastly, the finding of a higher mutation rate in pericentromeric regions provides an
explanation of the Arabidopsis-specific pattern of higher polymorphism levels near the
centromeres (16, 23), although the underlying mechanism of such a mutational bias remains
to be explained.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank C. Lanz for generating the Illumina data, S. E. Jacobsen for providing the methylation data, and P. Tiffin
for valuable comments. Funded by the Deutsche Forschungsgemeinschaft (DFG) (ERA-PG ARelatives), a
Gottfried Wilhelm Leibniz Award (DFG), the Max Planck Society (D.W.), NIH grant GM36827 to M. L. and W.
Kelly Thomas, Pioneer Hi-Bred International to E. Darmo, and NSF grants DEB 9629457 and 9981891 to R.G.S.
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